bacterial attachment to micro- and nano- structured surfaces - swinburne research … ·...

___________________________________________________________________________

___________________________________________________________________________

Bacterial attachment to micro- and nano-

structured surfaces

Submitted in total fulfilment of the requirements for the degree of

Doctor of Philosophy

by

Natasa Mitik – Dineva

Environmental and Biotechnology Centre

Faculty of Life and Social Sciences

Swinburne University of Technology

February 2009

i

Abstract

Abstract

The ongoing interest in bacterial interactions with various surfaces, followed by

attachment and subsequent biofilm formation, has been driven by the importance of

bacterial activities in number of medical, industrial and technological applications.

However, bacterial adhesion to surfaces has not been completely understood due to the

complexity of parameters involved.

The study presented herein investigates the attachment pattern of nine medically

and environmentally significant bacteria belonging to different taxonomic lineages:

Firmicutes - Bacillus, Gammaproteobacteria, Alphaproteobacteria and Bacteriodetes.

Physicochemical assessment techniques such as contact angle and surface charge

measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM),

confocal microscopy (CLSM), as well as X-ray photoelectron spectroscopy (XPS),

X-ray fluorescence spectroscopy (XRF) and time-of-flight secondary ion mass

spectroscopy (ToF-SIMS) analysis were all employed in order to attain better insight

into the factors that influence bacterial interactions with surfaces. Bacterial surface

characteristics such as surface wettability and charge in addition to substratum surface

wettability, tension, charge and chemistry were also considered. However due to the

recent interest in designing micro-textured surfaces with antibacterial and/or antifouling

effects the prime was given to the influence of micro- and nano-meter scale surface

textures on bacterial adhesion.

The interactions between selected bacteria and glass, polymer and optical fibre

surfaces were studied. Carefully designed methods for surface modification allowed

alteration of the topography of glass, polymer and optical fibre surfaces while

maintaining other surface parameters near constant. This allowed isolated assessment of

only the effects of surface roughness on bacterial adhesion.

Obtained results indicated consistent cellular inclination towards the smoother

surfaces for all of the tested species. Enhanced bacterial presence on the smoother

surfaces was also accompanied by changes in the bacterial metabolic activity as

ii

Abstract

indicated by the elevated levels of secreted extracellular polymeric materials (EPS) and

modifications in the cells morphology. The results indicate that nano-scale surface

roughness exert greater influence on bacterial adhesion than previously believed and

should therefore be considered as a parameter of primary interest alongside other well-

recognized factors that control initial bacterial attachment.

iii

Acknowledgements

Acknowledgements

Swinburne University Honours graduate Sarah Murphy reproduced structural changes in the poly (tert-butyl methacrylate) chemistry after UV exposure by molecular modelling. She also probed the attachment behaviour of Escherichia coli and Alivibrio fischeri on the native and modified P(t)BMA surfaces. Swinburne University Honours candidate Vi Khanh Truong provided surface tension analysis for the glass, polymer and optical fibre surfaces. Swinburne University PhD candidate Daniel White assisted in preparation of glass and optical fibre surfaces by treatment with buffered hydrofluoric acid. Swinburne University PhD candidate Radu Codrin Mocanasu assisted in preparation and modification of polymer, P(t)BMA, surfaces.

Copy-editing and proofreading was provided by Campbell Aitken - Express Editing

Writing and Research. Editorial assistance improved grammar, sentence construction

and styling, however it did not affect the content and the quality of the analysis

provided.

I would like to express my sincere gratitude to Dr Paul Stoddart and Dr Russell

Crawford for co-supervising this project. Their technical knowledge, constant support

and encouragement were of great importance through the whole study.

I am also grateful to Dr James Wang and Hans Brinkies who guided me through the

wonders of AFM and SEM into the wonderful world of “nano” science.

My deepest and most sincere gratitude goes to my mentor Professor Elena Ivanova,

initially for being a friend and only after for her supervision and guidance in completion

of this research. Her contributing knowledge and dedication to the project were

remarkable and beyond my expectations. She gave me confidence, inspired me and

challenged me along the way and I am truly grateful.

This project would not have been complete without the technical support of Ngan,

Soula, Sheila, Savithri and Chris and I am most thankful for their assistance.

iv

Acknowledgements

I enjoyed the stimulating and fun environment at Swinburne University and for this

would like to thank my student colleagues Jacque B, Kerrie, Jacque M, Daniel, Pete,

Shee Ping, Abi, Sarah, Yuri, Barbara, Khanh, Kiran, Stave, Elisabeth, Paul, Natalie and

Mark.

For giving me the opportunity and making Melbourne a very special place for me I

would like to thank Krole and Čile.

It is tempting to individually thank all of my friends who shared the good and bad

moments of life with me; however due to the probability of leaving someone out, I will

simply say “THANK YOU ALL”.

I would like to thank my mother and father for teaching me that everything is

‘reachable’ and my sister for showing me that stubbornness can sometimes be virtue.

Your constant encouragement and unconditional love meant everything throughout my

education and I am endlessly grateful for that.

Finally I would like to thank and dedicate this thesis to mu loving husband Milan who

unselfishly followed me across the globe so I can fulfil my dream.

I may not say it enough, but you know I mean it: “THANK YOU”

v

Declaration

Declaration

I Natasa Mitik-Dineva declare that this thesis is my original work and contains no

material that has been accepted for the award of Doctor of Philosophy, or any other

degree or diploma, except where due reference is made.

I declare that to the best of my knowledge this thesis contains no material previously

published or written by other person accept where due reference has been made.

Wherever contributions of others were involved every effort has been made to

acknowledge contribution of the respective workers or authors.

I also declare that this theses has been professionally edited, however the extend of the

editing only affected the grammar and style of the thesis and not its substantive content.

Signature_______________________________________________________________

vi

List of publications


Book chapters

N Mitik-Dineva, PR Stoddart, JR Crawford, EP Ivanova, Bacterial cell interactions

with optical fiber surfaces In: “Fiber Lasers: Research, Technology and Applications”

to be published by Nova Science Publishers, Inc. (in press)

N Mitik-Dineva, PR Stoddart, R Crawford, EP Ivanova, Adhesion of Bacteria In:

“Wiley Encyclopedia of Biomedical Engineering”, 6-Volume Set M Akay (Ed), John

Wiley & Sons, Inc. 2006

N Mitik-Dineva, PR Stoddart, Applications of Atomic Force Microscopy in

Topographic Imaging In: "The surface structure and properties of microbial cells on a

nanometer scale" published by Nova Science Publishers, Inc.2006

Peer-reviewed papers

N Mitik-Dineva, J Wang, VK Truong, P Stoddart, F Malherbe, RJ Crawford, EP

Ivanova, Marine bacteria interactions with nano-smooth glass surfaces, Biofouling,

2008 (under revision). N Mitik-Dineva, J Wang, VK Truong, RP Stoddart, F Malherbe, RJ Crawford, EP

Ivanova, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus

attachment pattern on nano-scale rough glass surfaces, Current Microbiology, 2009 58,

268–273 (Published on-line Nov 2008).

EP Ivanova, N Mitik-Dineva, CR Mocanasu, S Murphy, J Wang, G van Reissen, JP

Crawford, Vibrio fischeri and Escherichia coli tendencies towards

photolithographically modified nanosmooth poly (tert-butyl methacrylate) polymer

surfaces, Nanotechnology, Science and Applications, 2008, 1, 33-44

vii


EP Ivanova, N Mitik-Dineva, J Wang, KD Pham, JP Wright, DV Nicolau, CR

Mocanasu, JP Crawford, Staleya guttiformis attachment on poly(tert-

butylmethacrylate) polymeric surfaces, Micron, 2008, 39, 1197-1224.

N Mitik-Dineva, EP Ivanova, J Wang, RC Mocanasu, PR Stoddart, RJ Crawford,

Impact of nano-topography on bacterial attachment, Biotechnology Journal, 2008, 3,

536-544

EP Ivanova, JP Bowman, R Christen, NV Zhukova, AM Lysenko NM Gorshkova, N

Mitik-Dineva, AF Sergeev, VV Mikhailov. Salegentibacter flavus sp. nov. IJSEM,

2006, 56, 583-586

Peer-reviewed conference proceedings

N Mitik-Dineva, J Wang, PR Stoddart, JR Crawford, EP Ivanova, Nano-structured

surfaces control bacterial attachment, 2008, ICONN – Conference Proceedings, 113-

117.

Conference presentations with published abstracts

N Dineva-Mitik, DK Pham, JP Wright, P Sawant, DV Nicolau, EP Ivanova, Study on

Staleya guttiformis attachment to poly(tert-butylmethacrylate) and polystyrene maleic

acid polymeric surfaces and optical imaging fibre, 2nd FEMS Congress of European

Microbiologist, Madrid, July 4-8 2006

N Mitik-Dineva, J Wang, RC Mocanasu, PR Stoddart, EP Ivanova, Impact on nano-

scale roughness on bacterial adhesion, ASM Annual Meeting, Adelaide 2007

viii


N Mitik-Dineva, J Wang, VK Truong, P Stoddart, F Malherbe, RJ Crawford, EP

Ivanova, Marine bacteria interactions with abiotic environment: nano-structured glass

surfaces ASM Annual Meeting, Melbourne 2008

Conference presentations

N Mitik-Dineva, RC Mocanasu, S Murphy, EP Ivanova, JR Crawford, V. fischeri and

E. coli adhesion tendencies towards photolitographically modified nano-smooth

poly(tert-butyl methacrylate) polymer surfaces, 26th Colloid and surface student

conference, 2008, Warrnambool

___________________________________________________________________ _______ __

Table of contents

ix

Table of contents

ABSTRACT I ACKNOWLWDGEMENTS III DECLARATION V LIST OF PUBLICATIONS VI TABLE OF CONTENTS VIII LIST OF TABLES XIII LIST OF FIGURES XV

CHAPTER 1 – ITRODUCTION 1 1.1 OVERVIEW 2 1.2 AIMS OF THE STUDY 4

CHAPTER 2 – LITERATURE REVIEW 6 2.1 OVERVIEW 7 2.2 BACTERIAL ATTACHMENT 7 2.2.1 REVERSABLE ATTACHMENT 8 2.2.2 IRREVERSABLE ATTACHMENT 9

2.3 BIOFILMS – THE SUBSEQUENT EFFECT OF BACTEIAL ADHESION 9 2.3.1 OVERVIEW 9 2.3.2 STAGES IN THE BIOFILM DEVELOPMENT 10

2.3.2.1 BIOFILM INITIATION 11 2.3.2.2 BIOFILM MATURATION 11 2.3.2.3 BACTERIAL DETECHMENT FROM THE BIOFILM SURFACE 13

2.3.3EFFECTS RESOLVING FROM THE BIOFILM PRESENCE 14

2.4 THEORETICAL APPROACHES IN UNDERSTANDING THE BASIC PRINCIPLES OF CELL‐SURFACE INTERACTIONS 16 2.4.1 DLVO THEORY 17 2.4.2 THERMODYNAMIC THEORY 17 2.4.3 TENTATIVE SCENRIO FOR INITIAL BACTERIAL ADHESION AT NANOMETER PROXIMITY 18 2.4.4 APPLICATION OF THE ADHESION THEORIES 19

2.5 BIOLOGICAL ASPECTS OF BACTERIAL ADHESION 20 2.5.1 OVERVIEW 20 2.5.2 ADHESINS 21 2.5.3 EXTRACELLULAR BIO‐PRODUCTS 23

2.6 PHYSICOCHEMICAL ASPECTS OF BACTERIAL ADHESION 24 2.6.1 ENVIRONMENTAL PARAMETERS THAT INFLUENCECELL‐SUBSTRATE INTERACTIONS 24 2.6.2 BACTERIAL SURFACE CHARACTERISTICS THAT INFLUENCE CELL‐SUBSTRATE INTERACTIONS 25

___________________________________________________________________ _______ __

Table of contents

x

2.6.2.1 CELL SURFACE WETTABILITY 25 2.6.2.2 CELL SURFACE CHARGE 26

2.6.3 SUBSTRATUM SURFACE CHARACTERISTICS THAT INFLUENCE CELL‐SUBSTRATE INTERACTIONS 28

2.6.3.1 SUBSTRATUM SURFACE WETTABILITY 28 2.6.3.2 SUBSTRATUM SURFACE CHARGE 30 2.6.3.3 SURFACE TENSION 31

2.7 EFFECTS OF SURFACE TOPOGRAPHY ON BACTERIAL ADHESION 33 2.8 TECHNIQUES FOR STUDYING BACTERIAL ADHESION 35 2.9 BACTERIAL ATTACHMENT TO GLASS SURFACES 38 2.10 BACTERIAL ATTACHMENT TO POLYMER SURFACES 39 2.11 BACTERIAL ATTACHMENT TO OPTIC FIBRES 42

CHAPTER 3 – METHODOLOGY 47 3.1 OVERVIEW 47 3.2 BACTERIA 48 3.2.1 NON‐MARINE BACTERIA 48

3.2.2.1 ESCHERICHIA COLI K12 48 3.2.1.2 PSEUDOMONAS AERUGINOSA ATCC 9027 49 3.2.1.3 STAPHYLOCOCCUS AUREUS CIP 68.5 49

3.2.2 MARINE BACTERIA 50 3.2.2.1 COBETIA MARINA DSM 4741T 50 3.2.2.2 PSEUDOALTEROMONAS ISSACHENKONII KMM 3549T 50 3.2.2.3 SALEGEBTIBACTER FLAVUS CIP 107843T 51 3.2.2.4 STALEYA GUTTIFORMIS DSM 11458T 51 3.2.2.5 SULFITOBACTER MEDITERRANEUS ATCC 700865T 52 3.2.2.6 ALIVIBRIO FISCHERI DSM 507T 53 3.2.3 CULTURE CONDITIONS ATTACHMENT EXPERIMENTS AND STAINING PROTOCOLS 53 3.2.3.1 CULTURE CONDITIONS 53 3.2.3.2 BACTERIAL ATTACHMENT EXPERIMENTS 54

•BACTERIAL ADSORPTION ON NANO‐STRUCTURED GLASS SURFACES (AS‐RECEIVED AND CHEMICALY MODIFIED) 54 •BACTERIAL ADSORPTION ON NANO‐STRUCTURED P(t)BMA POLYMER SURFACES (NATIVE AND PHOTOLITHOGRAPHYCALLY MODIFIED) 54 •BACTERIAL ADSORPTION ON OPTICAL FIBRES (AS‐RECEIVED AND CHEMICALLY MODIFIED) 55

3.2.3.3 FLUORESCENT LABELLING OF PRODUCED EPS AND VIABLE CELLS 55

3.3 SURFACES 57 3.3.1 GLASS 57 3.3.2 POLYMER 57

3.3.2.1 OVERVIEW 57 3.3.2.2 POLYMER FILM PREPARATION 58 3.3.2.3 PHOTOLITHOGRAPHY 59

3.3.3 OPTICAL FIBRES 59 3.3.3.1 OVERVIEW 59 3.3.3.2 SURFACE PREPARATION 60

___________________________________________________________________ _______ __

Table of contents

xi

3.3.3.3 SURFACE MODIFICATION 60

3.4 QUALITATIVE ANALYSES OF THE ABIOTIC AND BIOLOGICAL SURFACES 62 3.4.1 CONTACT ANGLE MEASUREMENTS 62

3.4.1.1 BACTERIAL SURFACE WETTABILITY 62 3.4.1.2 SUBSTRATUM SURFACE WETTABILITY 63

3.4.2 SURFACE FREE ENERGY 64 3.4.3 SURFACE CHARGE MEASUREMENTS 64

3.4.3.1 BACTERIAL SURFACE CHARGE 64 3.4.3.2 SUBSTARTUM SURFACE CHARGE 65

3.4.4 AFMCHARACTERIZATION OF THE SURFACES 66 3.4.5TIME OF FLIGHT SECONDARY ION MASS SPECTROMETRY (ToF SIMS) 66 3.4.6 X‐RAY PHOTOELECTRON SPECTROSCOPY (XPS) 67 3.4.7 X‐RAY FLUORESCENCE SPECTROSCOPY (XRF) 68 3.4.8 SCANNING ELECTRON MICROSCOPY (SEM) 68 3.4.9 CONFOCAL SCANING LASER MICROSCOPY (CSLM) 69

CHAPTER 4 – THE EFFECTS OF NANO‐STRUCTURED GLASS SURFACES ON BACTERIAL ATTACHMENT 70 4.1 BACTERIAL SURFACE CHARACTERISTICS 71 4.1.1 OVERVIEW 71 4.1.2 CELL SURFACE WETTABILITY 71 4.1.3 CELL SURFACE CHARGE 73

4.2 SUBSTRATUM SURFACE CHARACTERISTICS 75 4.2.1 OVERVIEW 75 4.2.2 SUBSTRATUM SURFACE WETTABILITY AND SURFACE TENSION 75 4.2.3 SUBSTRATUM SURFACE CHARGE 76 4.2.4 XPS ANALYSIS OF THE AS‐RECEIVED AND THE MODIFIED GLASS SURFACES 77 4.2.5 XRF ANALYSIS OF THE AS‐RECEIVED AND THE MODIFIED GLASS SURFACES 81 4.2.6 AFM ANALYSIS OF THE AS‐RECEIVED AND THE MODIFIED GLASS SURFACES 82 4.2.7 SEM OF THE AS‐RECEIVED AND THE MODIFIED GLASS SURFACES 84

4.2.7.1 OVERVIEW 84 4.2.7.2 EVALUATION OF CONTROL GLASS SURFACES 85

4.3 INVESTIGATION OF BACTERIAL ADHESION ON NANO‐SMOOTH GLASS SURFACES 86 4.3.1 ATTACHMENT OF ESCHERICHIA COLI CELLS ON AS‐RECEIVED AND MODIFIED GLASS SURFACES 86 4.3.2 ATTACHMENT OF PSEUDOMONAS AERUGINOSA CELLS ON AS‐RECEIVED AND MODIFIED GLASS SURFACES 90 4.3.3 ATTACHMENT OF STAPHYLOCOCCUS AUREUS CELLS ON AS‐RECEIVED AND MODIFIED GLASS SURFACES 96 4.3.4 ATTACHMENT OF COBETIA MARINA CELLS ON AS‐RECEIVED AND MODIFIED GLASS SURFACES 99 4.3.5 ATTACHMENT OF PSEUDOALTEROMONAS ISSACHENKONII CELLS ON AS‐RECEIVED AND MODIFIED GLASS SURFACES 104 4.3.6 ATTACHMENT OF SALEGENTIBACTER FLAVUS ON AS‐RECEIVED AND MODIFIED GLASS SURFACES 107

___________________________________________________________________ _______ __

Table of contents

xii

4.3.7 ATTACHMENT OF STALEYA GUTTIFORMIS CELLS ON AS‐RECEIVED AND MODIFIED GLASS SURFACES 111 4.3.8 ATTACHMENT OF SULFITOBACTER MEDITERRANEUS CELLS ON AS‐RECEIVED AND MODIFIED GLASS SURFACES 114 4.3.9 ATTACHMENT OF ALIVIBRIO FISCHERI CELLS ON AS‐RECEIVED AND MODIFIED GLASS SURFACES 120

8.4 CONCLUSION 125

CHAPTER 5 – THE EFFECTS OF NANO‐STRUCTURED P(t)BMA POLYMER SURFACES ON BACTERIAL ATTACHMENT 126 5.1 OVERVIEW 127 5.2 BACTERIAL SURFACE CHARACTERISTICS 127 5.3 P(t)BMA SURFACE CHARACTERISTICS 128 5.3.1 SURFACE WETTABILITY AND TENSION 128 5.3.2 SURFACE CHARGE 130 5.3.3 XPS SURFACE ANALYSIS 131 5.3.4 AFM ANALYSIS 134 5.3.5 SEM ANALYSIS 136

5.3.5.1 OVERVIEW 136 5.3.5.2 CONTROL P(t)BMA SURFACES 137

5.4 INVESTIGATION OF BACTERIAL ADHESION ON NANO‐SMOOTH P(t)BMA SURFACES 138 5.4.1 ATTACHMENT OF ESCHERICHIA COLI CELLS ON NATIVE AND MODIFIED P(t)BMA SURFACES 138 5.4.2 ATTACHMENT OF PSEUDOMONAS AERUGINOSA CELLS ON NATIVE AND MODIFIED P(t)BMA SURFACES 142 5.4.3 ATTACHMENT OF STAPHYLOCOCCUS AUREUS CELLS ON NATIVE AND MODIFIED P(t)BMA SURFACES 145 5.4.4 ATTACHMENT OF COBETIA MARINA CELLS ON NATIVE AND MODIFIED P(t)BMA SURFACES 149 5.4.5 ATTACHMENT OF PSEUDOALTEROMONAS ISSACHENKONII CELLS ON NATIVE AND MODIFIED P(t)BMA SURFACES 154 5.4.6 ATTACHMENT OF SALEGENTIBACTER FLAVUS CELLS ON NATIVE AND MODIFIED P(t)BMA SURFACES 158 5.4.7 ATTACHMENT OF STALEYA GUTTIFORMIS CELLS ON NATIVE AND MODIFIED P(t)BMA SURFACES 162 5.4.8 ATTACHMENT OF SULFITOBACTER MEDITERRANEUS CELLS ON NATIVE AND MODIFIED P(t)BMA SURFACES 169 5.4.9 ATTACHMENT OF ALIVIBRIO FISCHERI CELLS ON NATIVE AND MODIFIED P(t)BMA SURFACES 173

5.5 CONCLUSION 178

CHAPTER 6 – BACTERIAL CELLS INTERACTIONS WITH THE SURFACE OF MICRO‐NANO‐STRUCTURED OPTIC FIBRES 179

___________________________________________________________________ _______ __

Table of contents

xiii

6.1 BACTERIAL SURFACE CHARACTERISTICS 180 6.2 SUBSTRATUM SURFACE CHARACTERISTICS 180 6.2.1OVERVIEW 180 6.2.2 SUBSTRATUM SURFACE WETTABILITY AND TENSION178 6.2.3 ToF‐SIMS ANALYSIS 181 6.2.4 AFM ANALYSIS 184 6.2.5 SEM ANALYSIS 185

6.2.5.1 OVERVIEW 185 6.2.5.2 CONTROL FIBRE SURFACES 186

6.3 OBSERVED BACTERIAL ADHESIVE BEHAVIOUR ON MICRO‐NANO STRUCTURED FIBRE SURFACES 187 6.4 CONCLUSION 192

CHAPTER 7 – DISCUSSION 193 7.1 OVERVIEW 194 7.2 BACTERIAL ATTACHMENT ON THE AS‐RECEIVED AND MODIFIED SURFACES AND SURFACE WETTABILITY 196 7.2.1 OVERVIEW 196 7.2.2 THE EFFECTS OF CELL SURFACE WETTABILITY ON BACTERIAL ADHESION TO GLASS, POLYMER AND FIBRE SURFACES 197 7.2.3THE EFFECTS OF SUBSTRATUM SURFACE WETTABILITY ON BACTERIAL ADHESION 202

7.3 BACTERIAL ATTACHMENT ON THE AS‐RECEIVED AND MODIFIED SURFACES AND SURFACE CHARGE 204 7.3.1 OVERVIEW 204 7.3.2THE EFFECTS OF CELL SURFACE CHARGE ON BACTERIAL ADHESION TO GLASS, POLYMER AND FIBRE SURFACES 204 7.3.3THE EFFECTS OF SUBSTRATUM SURFACE CHARGE ON BACTERIAL ADHESION 208

7.4 BACTERIAL ATTACHMENT ON THE AS‐RECEIVED AND MODIFIED SURFACES AND SURFACE ROUGHNESS 208 7.5 GENERAL DISCUSSION 214

CHAPTER 8 – CONCLUSION AND FUTURE DIRECTIONS 216 8.1 SUMMARY 217 8.2 FUTURE DIRECTIONS 219 8.3 CLOSE 220

LIST OF REFERENCES 222

________________________________________________________________________

xvi

List of figures

Figure 3.1: SEM images of the as-received optic fibre surfaces, scale bar

250µm on image (a) and 1µm on image (b)

60

Figure 3.2: SEM images of the optic fibre after exposure to the etching

solution for 20min. Scale bar equals 250µm on image (a)

and 1µm on image (b)

61

Figure 4.1: Advancing water contact angles measured on the as-received

(a) and on the modified (b) glass surface

75

Figure 4.2: Regional and wide spectra collected from the modified (a, c,

e, g, i, k) and the as-received glass surface (b, d, f, h, j, l)

80

Figure 4.3: Typical AFM images of the as-received (a) and modified (b)

glass surfaces. Imaged areas represent 5 × 5 µm2 and 5 × 6

µm2, respectively

83

Figure 4.4: Typical SEM images of glass surfaces. The scale bar

observed on all images is equal to 1µm. (a) Modified glass

surface (b) modified glass surface with marine broth 2216

(c) as-received glass surface (d) as-received glass surface

with marine broth

85

Figure 4.5: Typical SEM representing the attachment pattern of E. coli

cells after 12 h incubation on the as-received glass surface

(a and b), and on the modified glass surface (c and d)

87

Figure 4.6: Selected AFM images representing the morphology and

surface topography of E. coli cells after 12 h of incubation

on the as-received glass (a), and on the modified (b) glass

surfaces

98

Figure 4.7: Typical CLSM images showing the EPS production (a, d)

and the viable (b, e) E. coli cells after 12 h of incubation on

as-received (a, b, c) and modified (d, e) glass surfaces. Scale

bar on image (a),(b), (d) and (e) is 10 µm and 2 µm on

image (c)

90

Figure 4.8: Typical SEM images showing the attachment behaviour of

Pseudomonas aeruginosa cells after 12 h incubation on the

as-received (a) and (b), and on the modified glass surface (c)

and (d). Scale bar represents 10 µm on (a), (c) and (e) and 1

µm on (b) and (d)

92

Figure 4.9: Selected AFM representing the morphology and surface

topography of Pseudomonas aeruginosa cells after 12h

incubation on the as-received(a), and on the modified glass

surface (b and c)

93

Figure 4.10: Selection of CLSM images representing the viability (viable

cells are red stained) and the EPS production (produced

EPS are green stained) of Pseudomonas aeruginosa cells

after 12h incubation on as-received glass surface (a and b)

and the modified glass surface

95

________________________________________________________________________

xvii

Figure 4.11: Typical SEM images showing the attachment behavior of

Staphylococcus aureus cells after 12h incubation on the as-

received (a and b), and on the modified glass surface (c and

d). Scale bar indicates 10µm on image (a) and(c), and 1µm

on (b) and (d

97

Figure 4.12: Selected of AFM representing the morphology and surface

topography of Staphylococcus aureus cells after 12h

incubation on the as-received glass surface (a), and on the

modified glass surface (b

98

Figure 4.13: Typical CLSM images of Staphylococcus aureus cells

attaching to the as-received (a and b) and to the modified (c

and d) glass surface after 12h incubation. Scale bar on all

images is 10 um

99

Figure 4.14: Typical SEM images showing the attachment behaviour of

C. marina cells after 12h incubation on the as-received (a)

and (b), and on the modified glass surface (c) and (d). Scale

bar on all images represents 2 µm

100

Figure 4.15: Typical AFM images of C. marina cells attaching to the as-

received (a) and to the modified (b) glass surface after 12h

incubation. Scanned areas approximately 3.0µm x 3.0µm

and 4.5µm x 4.5µm, respectively

101

Figure 4.16: Typical CLSM images of C. marina cells attaching to the as-

received (a) (b) and to the modified (c) and (d) glass surface

after 12h incubation. Scale bar on all images is 10 um

103

Figure 4.17: Typical SEM images of Pseudoalteromonas issachenkonii

cells attaching to the as-received (a) and (b) and to the

modified (c) and (d) glass surface after 12h incubation

105

Figure 4.18: Selected AFM images of Pseudoalteromonas issachenkonii

cells attaching to the as-received (a) and to the modified (b)

glass surface after12h incubation

105

Figure 4.19: Selected CLSM images of Pseudoalteromonas issachenkonii

cells attaching to the as-received (a) and (b) and to the

modified (c) and (d) glass surface after 12h incubation.

Scale bar on all images is 2 um

106

Figure 4.20: Typical SEM images of Salegentibacter flavus cells attaching

to the as-received (a) and (b) and to the modified (c) and (d)

glass surface after 12h incubation. Scale bar represents

10µm on image (a) and (c) and 1 µm on image (b) and (d)

108

Figure 4.21: Selected AFM images of Salegentibacter flavus cells

attaching to the as-received (a, scanned area 50µmx50µm),

(b, scanned area 4.0µmx4.0µm) and to the modified (c,

scanned area 35µmx35µm), (d, scanned area 4.5µmx4.5µm)

glass surfaces after 12h incubation

110

Figure 4.22: Typical CLSM images of Salegentibacter flavus cells

attaching to the as-received (a) and (b) and to the modified

(c) and (d) glass surface after 12 h of incubation. Scale bar

111

________________________________________________________________________

xviii

on all images is 2 um

Figure 4.23: Typical SEM images of Staleya guttiformis cells attaching to

the “as-received’ (a) and (b) and to the modified (c) and (d)

glass surface after 12 h of incubation. Scale bar represents

10µm on image (a) and (c) and 1 µm on image (b) and (d)

112

Figure 4.24: Selected AFM images of Staleya guttiformis cells attaching

to the as-received (a) and to the modified (b) after 12 h of

incubation. Scanned areas 4.0µm x 4.0µm and 7.0µm x

7.0µm, respectively.

113

Figure 4.25: Typical CLSM images of Staleya guttiformis cells attaching

to the as-received (a) and (b) and to the modified (c) and (d)

glass surface after 12 h of incubation. Scale bar on all

images represents 10 µm

114

Figure 4.26: Selected SEM showing the attachment behaviour of

Sulfitobacter mediterraneus cells after 12 h incubation on

the as-received glass surface (a) and (b), and on the

modified glass surface (c) and (d). Scale bar represents

10µm on images (a) and (c), and 1µm on image (b) and (d).

116

Figure 4.27: Selected AFM images of Sulfitobacter mediterraneus cells

attaching to the as-received ((a), scanned area 4.5x4.5µm)

and to the modified ((b), scanned area 4.5x4.5µm) glass

surface after 12 h of incubation. Image (c) represents the

appearance of Sulfitobacter mediterraneus cells adsorbed to

the modified glass surface after 18 h incubation (scanned

area 14x14µm).

118

Figure 4.28: Selected CLSM images of Sulfitobacter mediterraneus cells

attaching to the as-received (a) and (b) and to the modified

(c) and (d) glass surface after 12 h of incubation. Scale bar

on all images is 2 µm

119

Figure 4.29: Selected SEM showing the attachment behaviour of A.

fischeri cells after 12 h incubation on the as-received glass

surface (a) and (b), and on the modified glass surface (c)

and (d). Scale bar on all images represents 2 µm

121

Figure 4.30: Selected AFM images of A. fischeri cells attaching to the as-

received (a, 3.5x3.5µm) and to the modified (b, 7.0x7.0µm)

glass surface after 12h incubation. Image (c) presents

transverse profile of the EPS deposited on the modified glass

surface

123

Figure 4.31: Typical CLSM images of A. fischeri cells attaching to the as-

received (a) and (b) and to the modified (c) and (d) glass

surface after 12 h of incubation. Scale bar on all images is 2

µm

124

Figure 5.1: Static water contact angles measured on the native (a) and

on the modified (b) polymer surfaces

127

Figure 5.2: Reaction scheme for formation of activated P(t)BMA. Image

adopted from journal article, Ivanova et al. (Ivanova et al.,

128

________________________________________________________________________

xix

2006c)

Figure 5.3: Regional and wide spectra collected from the modified (a, c,

e, g, i, k) and the native polymer surfaces (b, d, f, h, j, l).

132

Figure 5.4: The structural re-arrangement undertaken by the P(t)BMA

monomer through photolithographic treatment is visualized

by the use of molecular modelling. Oxygen molecules are

indicated by red sections, hydrogen molecules are indicated

by blue sections and carbons are indicated by grey sections.

Figure adopted from Murphy’s honours report (Murphy,

2007)

133

Figure 5.5: Typical 3D AFM images of the native (a) and modified (b)

P(t)BMA surfaces Scanned areas represent 7.0µm x 7.0µm

134

Figure 5.6: Negative control SEM images of the P(t)BMA. Scale bar

equals 2µm on all images. (a) Native P(t)BMA (b) Native

P(t)BMA with marine broth (c) Modified P(t)BMA) (d)

Modified P(t)BMA with marine broth.

136

Figure 5.7: Selection of SEM representing the attachment behaviour of

E. coli cells after 12h incubation on the native P(t)BMA, (a)

and (b), and on the modified P(t)BMA surface (c) and (d).

Scale bar represents 10 µm on image (a) and (c), 2 µm on

(b) and (d)

138

Figure 5.8: Selection of AFM representing the morphology and surface

topography of E. coli cells after 12h incubation on the: (a)

native P(t)BMA surface and (b): on the modified P(t)BMA

surface. Image (c) represents transverse profile of the extra-

cellular deposits on the modified P(t)BMA

139

Figure 5.9: Selection of CLSM images representing the EPS production

(a, c) and the viability (b, d) of E. coli cells after 12h

incubation on native (a, b) and modified (c, d) P(t)BMA

surface. Scale bar represents 5µm on all images

140


Pseudomonas aeruginosa cells after 12h incubation on the

native images (a) and (b), and on the modified P(t)BMA

surface, images (c) and (d). Scale bar represents 10 µm on

image (a) and (c), 2 µm on (b) and (d)

142


topography of Pseudomonas aeruginosa cells and produced

EPS after 12h incubation on the native (a) and modified (b)

P(t)BMA surface

142


(a, c) and the viability (b, d) of Pseudomonas aeruginosa

cells attaching to the native (a, b) and to the UV-exposed (c,

d), P(t)BMA polymer surface after 12h incubation. Scale bar

on all images represents 2µm

143


Staphylococcus aureus cells after 12h incubation on the

144

________________________________________________________________________

xx

native P(t)BMA, (a) and (b), and on the modified P(t)BMA

surface (c) and (d). Scale bar represents 10 µm on (a) and

(c), 1 µm on (b) and (d)


topography of Staphylococcus aureus cells after 12h

incubation on the native (a) and modified (b) P(t)BMA

surface.

145


(a, c) and the viability (b, d) of Staphylococcus aureus cells

attaching to the native (a and b) and to the UV-exposed (c

and d) P(t)BMA polymer surface after 12h incubation. Scale

bar on all images is 2um

147


C. marina cells after 12h incubation on the native P(t)BMA,

(a) and (b), and on the modified P(t)BMA surface (c) and

(d). Scale bar represents 10 µm on image (a) and (c), 2 µm

on image (b) and (d).

149


topography of C. marina cells and produced EPS after 12h

incubation on the native (a) and modified (b) P(t)BMA

surfaces. Image (c) represents transverse profile of the

overall height of cells and EPS adsorbed on the modified

P(t)BMA

151


(b, d) and the viability (a, c) of C. marina cells attaching to

the native (a, b) and to the UV-exposed (c,d)) P(t)BMA

polymer surface after 12h incubation. Scale bar on all

images is 2um

152


Pseudoalteromonas issachenkonii cells after 12h incubation

on the native P(t)BMA, (a) and (b), and on the modified

P(t)BMA surface, (c) and (d). Scale bar represents 10 µm on

images (a) and (c), 1 µm on images (b) and (d).

154


topography of Pseudoalteromonas issachenkonii cells and

produced EPS after 12h incubation on the native (a) and

modified (b) P(t)BMA surfaces

155


(a, c) and the viability (b, d) of Pseudoalteromonas

issachenkonii cells attaching to the native (a, b) and to the

UV-exposed (c, d) P(t)BMA polymer surface after 12h

incubation. Scale bar on all images is 10µm

157


Salegentibacter flavus cells after 12h incubation on the

native P(t)BMA, (a) and (b), and on the modified P(t)BMA

surface (c) and (d). Scale bar represents 2 µm on all images

158

________________________________________________________________________

xxi

Figure 5.23: Selection of AFM representing the native and modified

P(t)BMAS surface topography after 12h incubation in

Salegentibacter flavus culture medium.

160


(a, b) of Salegentibacter flavus cells attaching to the native

(a) and to the UV-exposed (b) P(t)BMA polymer surface

after 12h incubation. Scale bar on all images is 2µm

160


Staleya guttiformis cells after 12h incubation on the native

P(t)BMA, (a) and (b), and on the modified P(t)BMA surface

(c) and (d). Scale bar represents 10 µm on images (a) and

(c), 2 µm on images (b) and (d).

163

Figure 5.26: Selection of AFM images representing the morphology and

surface topography of Staleya guttiformis cells after 12h

incubation on the native P(t)BMA surface

163

Figure 5.27: Typical high-resolution AFM topographical images (non-

contact mode) of Staleya guttiformis cells; (a) cell attached

to the native P(t)BMA surface and a lose granular EPS

surrounding the cell; (b) zoomed area on the surface of the

cell showing cell surface topography.

164

Figure 5.28: A typical AFM topographical image of the loose granular

EPS on the native P(t)BMA surface; (a) high resolution

image obtained in the non-contact mode; (b) a transverse

profile of granular EPS in a nano-meter scale. Similar

images were obtained in different regions of at least two

different samples

165

Figure 5.29: Selection of AFM images representing the morphology and

surface topography of Staleya guttiformis cells after 12h

incubation on the modified P(t)BMA surface; image (c)

represents transverse profile of the overall height of EPS

deposited on the surface

166


(a, c) and the viability (b, d) of Staleya guttiformis cells after

12h incubation on native (a, b) and modified (c, d) P(t)BMA

surface. Scale bar indicates 10µm on image a, b, c and d.

Face contrast images of Staleya guttiformis cells attached to

the native (e) and to the modified (f) P(t)BMA surface

representing the overall cell distribution and the presence od

EPS on the cell surface

168

Figure 5.31: Selection of SEM images representing the attachment

behaviour of Sulfitobacter mediteraneus cells after 12h

incubation on the native P(t)BMA, (a) and (b), and on the

modified P(t)BMA surface (c) and (d). Scale bar represents

10 µm on images (a) and (c), 2 µm on images (b) and (d.)

170

Figure 5.32: Selection of AFM images representing the attachment

behaviour of Sulfitobacter mediteraneus cells after 12h

171

________________________________________________________________________

xxii

incubation on the native P(t)BMA, (a) and on the

modified(b) P(t)BMA surface)


(a, c) and the viability (b, d) of Sulfitobacter mediteraneus

cells after 12h incubation on native (a, b) and modified (c, d)

P(t)BMA surface. Scale bar represents 1um

172

Figure 5.34: Selection of SEM images representing the attachment

behaviour of A. fischeri cells after 12h incubation on the

native P(tBMA), (a) and (b), and on the modified P(t)BMA

surface (c) and (d). Scale bar represents 10 µm on images

(a) and (c), 2 µm on images (b) and (d.)

174

Figure 5. 35: AFM images of A. fischeri cells attaching to the native (a)

and to the modified (b) P(t)BMA surface after 12h

incubation

174


(a, c) and the viability (b, d) of A. fischeri cells after 12h

incubation on native (a, b) and modified (c, d) P(t)BMA

surface. Scale bar represents 10um on all images.

176

Figure 6.1: Images representing measured water contact angles on the

as-received (a) and on the eroded (b) fibre surface

179

Figure 6.2: ToF-SIMS scans from the (a) as-received and (b) eroded

fiber surface

180

Figure 6.3: Positive (a, b) and negative (c, d) spectra collected from the

as-received (a, c) and the eroded (b, d) fibre surface

182

Figure 6.4: Surface topography of the as-received and the eroded fibre

as inferred from AFM

182

Figure 6.5: Control SEM images of the as-received fibre surfaces

without (a) and with marine broth (b) and the chemically

eroded fibre surface without (c) and with marine broth (d).

Scale bar on all images is 10µm.

184

Figure 6.6: SEM images of the attachment pattern of E. coli,

Pseudomonas aeruginosa, Staphylococcus aureus, C.

marina, Pseudoalteromonas issachenkonii and Staleya

guttiformis on the as-received fibre surface

187

Figure 6.7: CLSM image representing the EPS production of E. coli (a),

Pseudomonas aeruginosa (b), Staleya guttiformis (c) and

Pseudoalteromonas issachenkonii (d) after 12h incubation

on the as-received fibre surfaces

188

Figure 6.8: CLSM image representing the EPS production of E. coli (a),

Pseudomonas aeruginosa (b), Staphylococcus aureus (c), C.

marina (d), Pseudoalteromonas issachenkonii (e) and

Staleya guttiformis (f) after 12h incubation on the modified

fibre surface

189

Figure 7.1: Evaluation of the attachment pattern of E. coli (ec),

Sulfitobacter mediterranues (sm), Pseudomonas aeruginosa

(pa), Salegentibacter flavus (sf), Pseudoalteromonas

194

________________________________________________________________________

xxiii

issachenkonii (pi), Staleya guttiformis (sg), Staphylococcus

aureus (sa), C. marina (cm) and A. fischeri (af) on the as-

received and modified glass surfaces: number of attached

cells versus bacterial surface wettability





aureus (sa), C. marina (cm) and A. fischeri (af) on the as-

received and modified P(t)BMA surfaces: number of

attached cells versus bacterial surface wettability

196


Pseudomonas aeruginosa (pa), Pseudoalteromonas

issachenkonii (pi), Staleya guttiformis (sg) and C. marina

(cm) on the ‘as received’ fibre surfaces: number of the

attached cells versus bacterial surface wettability

197

Figure 7. 4: Evaluation of the attachment pattern of E. coli (ec),




aureus (sa), C. marina (cm) and A. fischeri (af)on both glass

and polymer surfaces: number of attached cells versus

substratum surface wettability

198

Figure 7.5: Evaluation of the attachment pattern of Pseudomonas

aeruginosa (pa), Salegentibacter flavus (sf), C. marina (cm),

Staphylococcus aureus (sa), Pseudoalteromonas

issachenkonii (pi), A. fischeri (af), E. coli (ec), Sulfitobacter

mediterraneus (sm) and Staleya guttiformis (sg)_to the “as

received” and modified glass surfaces: number of the

attached cells versus bacterial surface charge

201

Figure 7.6: Evaluation of the attachment pattern of Pseudomonas

aeruginosa (pa), Salegentibacter flavus (sf), C. marina (cm),

Staphylococcus aureus (sa), Pseudoalteromonas

issachenkonii (pi), A. fischeri (af), E. coli (ec), Sulfitobacter

mediterraneus (sm) and Staleya guttiformis (sg) to the “as

received” and modified P(t)BMA surfaces: number of the


202

Figure 7.7: Evaluation of bacterial attachment pattern to the “as

received” and modified fibre surfaces: number of the


203

Figure 7.8: Variations in the length of E. coli (ec), Pseudomonas

aeruginosa (pa), Staphylococcus aureus (sa),

Pseudoalteromons issachenkonii (pi), C. marina (cm),

Salegentibacter flavus (sf), Staleya guttiformis (sg),

Sulfitobacter mediterraneus (sm) and A. fischeri (sf) cells

after attaching to the as-received surfaces and their modified

208

________________________________________________________________________

xxiv

equivalents

Figure 7.9: Conversion of vegetative cells of S. mediterraneus ATCC

700856T into coccoid forms after attachment to Pt BMA, 24

h. Top: Vegetative cells with subpolar flagella; middle:

initial step towards coccoid body formation; bottom: coccoid

form of S. mediterraneus ATCC 700856T(Ivanova et al.,

2002a)…

209

_______________________________________________________________________

xiv

List of tables

Table 3.1: Table 3.1: Surface tensions and its parameters (mJ/m2) of

common solvent in the measurement of contact angles

64

Table 4.1: Water contact angles of bacterial cell surfaces 72

Table 4.2: Electrophoretic mobility and calculated zeta potential values

on bacterial cell surfaces

74

Table 4.3: Substratum surface wettability and surface free energy before

and after modification

76

Table 4.4: Glass surface charge as inferred from zeta potential

measurement

77

Table 4.5: Relative atomic concentration of the chemical elements

presented at the glass surfaces as determined by XPS analysi

78

Table 4.6: Relative contributions of different chemical states assigned to

the XPS peaks

81

Table 4.7: Detection limits and percentages of all detected components in

the as-received and the modified glass surfaces

82

Table 4.8: Glass surfaces roughness parameters 84

Table 4.9: Pseudomonas aeruginosa cells surface parameters after

attachment on the as-received and modified glass surfaces

94

Table 4.10: C. marina cell surface roughness on selected 0.5µmx0.5µm

areas on top of the cells attached to the as-received and

modified glass surface

102

Table 5.1: Observed water contact angle values for native and modified

P(t)BMA.

127

Table 5.2: Substratum surface wettability and surface free energy before

and after modification

128

Table 5.3: Polymer surface charge as inferred from zeta potential

measurements

129

Table 5.4 Relative contributions of different chemical states assigned to

the XPS peaks.

130

Table 5.5: Surface roughness parameters of the P(t)BMAbefore and after exposure

to UV light as inferred from the AFM measurements

135

Table 5.6: Roughness parameters taken from the surface of C. marina

cells attached to the native ad modified P(t)BMA surface

149

Table 5.7: Roughness parameters taken from the surface of

Pseudoalteromonas issachenkonii cells attached to the native

ad modified P(t)BMA surface

155

Table 5.8: Dimensions of Pseudoalteromonas issachenkonii cells

attached to the native ad modified P(t)BMA surface

156

Table 5.9: Roughness parameters taken from the surface of Staleya

guttiformis cells attached to the native P(t)BMA surface and

from the polymer surface itself

165

Table 5.10: Roughness parameters taken from the surface of Staleya 167

_______________________________________________________________________

xv

guttiformis cells attached to the modified P(t)BMA surface and

from the polymer surface itself.

Table 5.11: Roughness parameters taken from the surface of A. fischeri

cells attached to the native ad modified P(t)BMA surface

175

Table 5.12: Dimensions of A. fischeri cells attached to the native ad

modified P(t)BMA surface

175

Table 6.1: Surface wettability and surface tensions of the as-received and

the modified fibre surfaces…

179

Table 6.2: Roughness parameters from the as-received and the eroded

fibre surface as inferred from AFM

183

Table 6.3: Numbers of attached cells per surface area (mm2) on the as-

received fibre

186

Table 7.1: Numbers of bacteria/cm2 attached to all tested surfaces and

their modified equivalents

192

Chapter 1:

Introduction

______________________________________________________________________

1

CHAPTER 1

INTRODUCTION

Chapter 1:

Introduction

______________________________________________________________________

2

1.1 Overview

Bacterial attachment and the factors that influence the process have been the focus

of intensive studies over the past few decades. The interest in surface-attached bacteria

is driven by the importance of cellular activity on many surfaces with significant

environmental, biotechnological, medical and industrial applications. A great deal of

effort has gone into developing easily cleanable surfaces with the potential to resist

cellular aggregation and biofilm formation. Such surfaces would be extremely useful in

numerous industrial, medical and research applications (Rozhok and Holz, 2005).

This research has allowed a greater insight into the forces involved in bacterial

adhesion. Despite continuing discussions about the relative significance of the factors

that influence bacterial adhesion, one conclusion appears to be inevitable: bacterial

attachment to surfaces is a complex phenomenon that is further complicated by factors

associated with the characteristics of both the bacteria and the surfaces onto which the

attachment takes place. Numerous studies have shown that bacterial adhesion depends

on several physicochemical, biological and environmental parameters (Wong et al.,

2002, Palmer et al., 2007, Dong et al., 2002, Vogler, 1998, Pringle and Fletcher, 1986,

Danese et al., 2000, Camesano and Logan, 1998, Eboigbodin et al., 2005).

The mechanisms that control bacterial adhesion have been addressed on various

levels: theoretical approaches such as the DLVO theory, developed by Derjaguin,

Landau, Verwey and Overbeek, and thermodynamic theories have revealed some of the

basic physicochemical aspects of bacterial adhesion (Bruinsma et al., 2001, Cao et al.,

2006), such as the influence of surface charge and tension on long range cell-substratum

interactions and the effects of surface hydrophobicity in short-range interactions

(Pereira et al., 2000, Castellanos et al., 1997, Bos et al., 1999, Bos et al., 2000).

Bacterial studies, on the other hand, have provided useful information regarding the role

that bacterial surface characteristics such as cell shape and size, surface protrusions,

production of extracellular polymeric substances (EPS) and surface physicochemical

properties play in the attachment process. Nevertheless, the most extensively explored

cell surface characteristics in terms of their relation to surface adhesion are bacterial

wettability and charge (Mandlik et al., 2008, O'Toole and Kolter, 1998a, Benito et al.,

1997).

Chapter 1:

Introduction

______________________________________________________________________

3

Apart from the cell surface characteristics, it is now generally accepted that a wide

range of substratum surface properties such as morphology, wettabilty, charge and

surface chemistry can all exert strong influence over the tendency of bacteria to attach

to different surfaces (Shellenberger and Logan, 2002, Hazan et al., 2006, Riedewald,

2006, Satriano et al., 2006, de Kerchove and Elimelech, 2005). The effects of

substratum surface wettability and charge have long been considered factors of primary

interest in cell-surface interactions; however, it is also true that much attention has been

given to the effects of surface topography, roughness and porosity on bacterial adhesion

(Sharon, 2006, Whitehead and Verran, 2006, Emerson et al., 2006). Nevertheless,

surface topography, roughness and porosity have generally been considered factors of

secondary importance that operate in conjunction with more crucial parameters such as

surface wettability, charge and surface tension (Advincula et al., 2007, Li and Logan,

2004)

A majority of recent approaches towards developing new and more reliable, non-

adhesive, anti-bacterial materials have moved in one of two directions. One approach

attempts to define some general pattern of cellular response towards different surfaces

based on bacterial or substratum surface physicochemical characteristics; however, the

ability to accurately predict bacterial adhesive behaviour based solely on these factors

has proven to be elusive. The second approach is the development of new non-adhesive

materials with increased antimicrobial characteristics and lower contamination risks.

This approach is predominantly based on modifying substrata adhesiveness by changing

their wettability, charge or topography (Mei et al., 2005).

A substantial amount of work has been directed towards producing surfaces with

well-defined surface characteristics. The amount of published research on cellular

responses to surfaces with different topographies has increased considerably in the last

two decades. Numerous studies have suggested that surfaces with appropriate

topography can not only alter bacterial adhesion but also affect the settlement of algae

(Hoipkemeier-Wilson et al., 2004, Callow et al., 2002) and deter colonization of

invertebrate shells (Carman et al., 2006), (Scheuerman et al., 1998) by prohibiting

attachment (Carman et al., 2006). Although surface micro and nano-texture is generally

accepted to be an important factor in cell-surface interactions, the significance of

Chapter 1:

Introduction

______________________________________________________________________

4

surface irregularities has not been systematically addressed (Shellenberger and Logan,

2002). The effects of surface roughness on bacterial adhesion have been studied over a

wide range of physical scales, but it has not been shown that surface roughness on a

scale much smaller that the bacterium might be a major determinant of the success of

the initial course of bacterial attachment (Sharon, 2006, Whitehead and Verran, 2006,

Emerson et al., 2006, Bruinsma et al., 2001, Hoipkemeier-Wilson et al., 2004, Carman

et al., 2006).

1.2 Aims of the study

Improved understanding of the effects of surface roughness on bacterial adhesion

will greatly contribute towards a better understanding of cell-surface interactions, which

will in turn facilitate the design and manufacture of surfaces with potential cyto-

repellent characteristics.

The uniqueness of this study – and the area in which it makes a new contribution to

knowledge – is in the investigation of the impact of micro and nano-scale surface

texture on bacterial attachment as an isolated phenomenon. Nine bacterial strains

belonging to different taxa were studied in order to reveal their strategies of attachment

to three chemically and structurally diverse surfaces. A large number of bacteria were

tested, giving a solid overview of the influence of surface roughness on bacterial

adhesion. Glass and poly(tert-butyl methacrylate (P(t)BMA) polymer surfaces were

selected as sample substrates for studying the effects of nano-scale surface roughness,

whereas optical fibre cores were selected for evaluating the effects of micro-scale

surface roughness on bacterial adhesion. These surfaces were selected for the study

based on their extensive biotechnological, clinical and industrial applications.

The principal aspect in which the results of this study add to the body of knowledge

gained from recent similar research into the effect of surface texture on bacterial

attachment is in the meticulously designed experimental procedure that allowed data to

be obtained under carefully controlled environmental conditions. Briefly, each of the

evaluated surfaces was progressively modified in a manner that significantly altered the

surface topography while leaving all other surface parameters unchanged. The

Chapter 1:

Introduction

______________________________________________________________________

5

modification procedures allowed a direct comparison of the attachment strategies of all

bacteria over six surfaces, both in their ‘as-received’ and modified forms. This allowed

a greater insight into the effects of surface topography on bacterial attachment whilst

minimising the influence of other parameters.

Contrary to generally accepted wisdom that nano-smooth surfaces represent a ‘less

adhesive’ surface, the results of this study suggest that smoother surfaces might actually

exhibit a stimulating effect on bacterial adhesion. The results presented in this thesis

also show that even nano-scale changes in surface roughness can provoke considerable

metabolic and morphologic changes in cellular response that greatly influence their

tendency for attachment onto substrate surfaces.

After beginning with a brief overview of novel techniques that have recently been

applied to studies of bacterial and substratum surface characteristics, this study will

address the general principles that apply to the process of attachment of bacteria to

substrate surfaces in greater detail. In addition, the driving forces that control bacterial

adhesion and subsequent biofilm formation, as well as the impact of the surface

characteristics of the substrate surface on bacterial attachment, their metabolic activity

and their morphology will also be discussed (Filloux and Vallet, 2003). Particular

emphasis will be given to the effects of micro and nano-scale surface topographies on

bacterial adhesion.

Chapter 2:

Literature review

______________________________________________________________________

6

CHAPTER 2

LITERATURE REVIEW

Chapter 2:

Literature review

______________________________________________________________________

7

2.1 Overview

This chapter aims to provide a clear and concise understanding of the biological

and physicochemical factors that influence bacterial adhesion and subsequent biofilm

formation. The literature contains conflicting evidence about the factors that influence

bacterial adhesion. Therefore general aspects of bacterial adhesion and biofilm structure

as well as detailed analysis of bacterial and substratum surface characteristics will be

reviewed. Theoretical considerations such as the DVLO and the thermodynamic theory

and their limitations in predicting trends in bacterial adhesion will also be addressed.

Particular emphasis will be placed on the impact of micro and nanometer roughness on

bacterial adhesion.

2.2 Bacterial attachment

The word ‘adhesion’ derives from the Latin adhaesio, or adhaerere, i.e. ‘to stick

to’, and has a wide range of definitions depending on its usage. In the context of

bacterial adhesion, the term can be defined as the discrete and sustained association

between a bacterium and a surface (or substratum); it is synonymous with the English

word ‘attachment’ as something that attaches one thing to another (Mittelman, 1996).

Bacterial adhesion to surfaces is an important biological process that plays a pivotal

role in natural, industrial and clinical environments. The interactions between bacterial

cell and substratum appear to be mediated by a range of physical and chemical factors

related to both bacteria and substrata. Despite being the focus of intense study over past

decades, bacterial adhesion resists simple categorisation and remains poorly understood

due to the enormous diversity and complexity of parameters involved (Beech and

Sunner, 2004, Parsek and Singh, 2003, Simoni et al., 2000, Teixeira and Oliveira, 1999,

Benito et al., 1997, Whittaker et al., 1996, An and Friedman, 1998). Numerous studies

conducted over past decades concluded that the attachment process is complex and

becomes further complicated by subsequent changes in the cell metabolism or the

surface itself (biological substrates) (Bruinsma et al., 2001, Roosjen et al., 2006, Pham

Chapter 2:

Literature review

______________________________________________________________________

8

et al., 2003, Pratt and Kolter, 1998, Joseph and Wright, 2004, Fletcher, 1996, Palmer et

al., 2007).

Although it is clear that the surface characteristics of both, the substrate and

bacteria play a role in the attachment process, studies have tended to focus on the effects

of one component of this essentially binary system. Apart from substratum surface and

bacterial surface characteristics, environmental effects can also have a substantial

influence. Nevertheless, progress has been made in understanding the factors that

influence bacterial adhesion on biological and non-biological surfaces and the properties

of bacteria that facilitate their attachment. It is now a well-accepted fact that cell-

substrate interaction is a two-step process – initial reversible attachment followed by

irreversible attachment.

2.2.1 Reversible attachment

The first step in cell-substrate interaction, reversible attachment, drives bacteria in

close proximity (approximately 100 nm) to the target surface in order for the initial

attachment to occur. Mechanisms by which bacteria are transported in close proximity

to the surface include Brownian movement, gravity-specific sedimentation and

convective mass transport. The main characteristic of this stage of bacterial adhesion is

sustained in the name itself; reversible attachment indicates that the existing cell-

substratum adhesive forces are not strong enough to resist the effects of fluid shear

forces, and cells can be easily removed from the surface by rinsing (Palmer et al., 2007,

Marshall et al., 1971). This step in cell-substrate interaction is dependent on the

existence and strength of electrostatic forces,

hydrophobic interactions and van der Waals forces (van Loosdrecht et al., 1987a). It is

believed that two factors are required for reversible attachment to occur; first,

conditioning of the target surface, and second, transport of bacteria to the surface. The

surface conditioning phase involves deposition of organic molecules - proteins in

particular - on the solid-liquid interface. Although contradictory opinions on the

importance of the surface conditioning phase exist, it is believed that the presence of

these molecules has the capability to alter some of the surface physicochemical

Chapter 2:

Literature review

______________________________________________________________________

9

properties such as charge, wettability, surface free energy, etc. (Palmer et al., 2007,

Fletcher and Floodgate, 1973, Whitehead and Verran, 2006).

2.2.2 Irreversible attachment

The second stage in bacterial adhesion - irreversible attachment - includes physical

transport as well as active cellular motion by means of flagella and pili. The existence of

flagella, pili and/or curli, as well as the strain-specific capability to produce extra-

cellular products (mainly exopolysaccharides - EPS), is the basis for the irreversible

attachment in the cell-substrate interactions. In this stage cells form anchoring,

irreversible bonds with the surface and much stronger forces are needed in order to

remove bacteria (Palmer et al., 2007). Several studies involving wild types and mutant

strains that lacked the existence of cellular motility extensions indicated that the

necessity of cellular motility for initial interaction with the surface is most likely strain-

specific, and in some circumstances can be facilitated by environmental conditions such

as the chemical composition of the surrounding medium (O'Toole and Kolter, 1998a,

Pratt and Kolter, 1998). A detailed understanding of irreversible cell adhesion is crucial

for developing a greater understanding and possibly control over biofilm development.

Better understanding of the fundamental nature of irreversible bacterial adhesion with

particular emphasis on the effects of EPS will be addressed in this study. (More detail

regarding the effects of bacterial motion extensions is presented in Chapter 2.4.2.)

2.3 Biofilms –the subsequent effect of bacterial adhesion

2.3.1 Overview

The vast majority of our present knowledge regarding bacteria reflects the average

properties and behaviour of very large assemblies of cells attached to surfaces. Indeed,

if bacterial adhesion is followed by colonisation, then the eventual result is the

establishment of structured communities referred to as biofilm. The colonization of

Chapter 2:

Literature review

______________________________________________________________________

10

biological and non-biological surfaces and the specific role of bacterial biofilms have

received considerable attention over the last decade (Beech and Sunner, 2004, Bayles,

2007, Bruhn et al., 2007, Beech et al., 2002, Chmielewski and Frank, 2003, Busscher

and van der Mei, 1997, Davey and O'Toole, 2000, Donlan, 2002, Hall-Stoodley et al.,

2004).

Biofilms are highly organised microbial aggregates encased in a protective and

adhesive matrices that resist unfavourable environmental influences (Mandlik et al.,

2008). Biofilm development is influenced by the underlying substrate and the

surrounding microenvironment. The purpose of such communities is to promote the

survival of bacteria by enabling them to perform specialised metabolic functions that

they might not be capable of in planktonic form (Eboigbodin et al., 2005). Although

biofilm structures have been recognized for some time, we are just beginning to

understand the formation process at the molecular level. Recent studies indicate that

biofilms are constantly changing, three dimensional, multi-cellular communities

containing primitive circulatory system structures. A biofilm does not necessarily

involve one bacterial type; there are generally a few bacterial types involved, and each

of them plays a separate role in order to maintain a stable community. Apart from

bacteria, biofilm aggregates may also contain fungi, protozoa, debris, corrosion

products, etc. Several steps are involved in biofilm development, and these are

described in the following section.

2.3.2 Stages in the biofilm development

During their existence biofilms pass through several stages: initiation, maturation,

maintenance and dissolution. In order to form a three-dimensional structure, bacteria

must be able to attach to, move on and sense the surface. Environmental stimuli and

multiple genetic pathways that vary among bacteria regulate biofilm formation (O'Toole

et al., 2000, Pratt and Kolter, 1999). It is now clear that the natural assemblages of

bacteria in the biofilm itself function as a cooperative system with extremely complex

control mechanisms (Davey and O'Toole, 2000, Sauer and Camper, 2001).

Chapter 2:

Literature review

______________________________________________________________________

11

2.3.2.1 Biofilm initiation

The initiation process begins at a point when bacteria sense and respond to

favourable environmental conditions, such as nutrients, iron, oxygen, specific

osmolarity, temperature or even the surface texture. In their natural habitat most bacteria

alternate between free-swimming (planktonic) and sessile forms depending on various

environmental stimuli, including the availability of nutrients. However, environmental

perturbations can trigger the transition from a planktonic bacterial form to a complex

biofilm (Figure 2.1).

Figure 2.1: Graphic representation of the

initial stages of biofilm development. Image

reproduced from the Centre for Biofilm

Engineering website (Engineering, 2008).

Biofilm growth will continue as long as fresh nutrients are provided. Whilst some

bacteria - such as Vibrio cholerae or Escherichia coli - demand very specific nutrient

media, others - like P. aeruginosa or Pseudomonas fluorescens - can form biofilm under

any conditions. Planktonic bacteria that can easily be triggered into forming biofilm

under appropriate conditions are called wild type, whilst those that are unable to form

biofilm are described as surface attachment defective (SAD) types. Some of the SAD

mutants undergo initial attachment normally, yet they are incapable of completing

further stages in the biofilm development process (O'Toole and Kolter, 1998a).

Chapter 2:

Literature review

______________________________________________________________________

12

2.3.2.2 Biofilm maturation

In order to maintain a newly established biofilm, bacteria undergo further

maturation into characteristic biofilm architectures that are often associated with the

production of signalling molecules. These signalling molecules include short peptides,

cyclic dipeptides (CDP), fatty acid derivates, together with the commonly reported acyl-

homoserine lactones (HSL) and exopolysaccharides (EPS). Acyl homoserine lactones

(the so called quorum-sensing molecules) are synthetised after the initial adhesion and

are essential for cell-cell communication, which is in turn important for establishing a

well-organized surface community (Figure 2.2).

Figure 2.2: Graphic presentation of

inter-cell communication mechanism in a

biofilm. Image reproduced from the

Centre for biofilm engineering website

(Engineering, 2008).

Many studies have explored the effects of HSLs on bacterial attachment and

subsequent biofilm development (Cha et al., 1998, Aizawa et al., 2000, Llamas et al.,

2005, Donabedian, 2003). One study on P. aeruginosa biofilm formation showed that

the removal of the genes necessary for synthesis of HSL will result in biofilm forming

without the usual well-spaced microcolonies (Miller and Bassler, 2001). This means

that the HSL deprived mutants are capable of establishing early cell-substrate

interactions, but are unable to create the specific architecture essential for biofilm

sustainability.

Like the acyl-HSL, EPS seem to play a specific role in establishing stable

interactions between cells and surfaces and between cells themselves. For example, a

strain of V. cholerae defective in EPS synthesis can hardly establish initial attachment,

and even those cells that do successfully attach are unable to develop architecture

specific to the wild type. The EPS complex provides strong adhesive forces between the

Chapter 2:

Literature review

______________________________________________________________________

13

cell and the attachment surface; it also provides protection from unfavourable

environmental influences. It is believed that the protective EPS shelter most likely slows

down the penetration of antibiotics or enzymes from surface predators such as protozoas

and microalgae (O'Toole et al., 2000).

Every microbial biofilm is believed to be unique; however some general, structural

characteristics can still be regarded as universal. For instance each biofilm structure is

believed to contain fluid-filled channels for nutrients and oxygen supply and a centrally

located ‘voids’ for removal of metabolic waste. Typical biofilm structure of

‘mushroom’ type is presented in Figure 2.3.

Figure 2.3: Graphic representation of

biofilm maturation stage. Image

reproduced from the Centre for Biofilm

Engineering website (Engineering, 2008).

Nevertheless biofilms are heterogeneous, constantly changing structures as result of the

influence of various external and internal perturbations.

2.3.2.3 Bacterial detachment from the biofilm surface

Bacterial participation in biofilm populations will continue while sufficient

amounts of nutrients and stable environmental conditions such as pH, temperature,

wettability, etc. are no longer available. At the point at which the quantities of available

nutrients are being depleted to a level too low to sustain bacteria they will detach from

the biofilm and once again transform into their planktonic form (Figure 2.4) (Power et

al., 2007, Bos et al., 1999, Fletcher, 1996, Lynch and Robertson, 2008).

Chapter 2:

Literature review

______________________________________________________________________

14

Figure 2.4: Graphic representation

of cellular detachment from the

biofilm structure and returning to

planktonic form. Image reproduced

from the Centre for Biofilm

Engineering website(Engineering,

2008).

Some authors believe that the loss of EPS or the production of enzymes such as

alginate or lyase may also play a role in detachment; however, despite intensive study,

very little is known about the pathways involved in this process (O'Toole and Kolter,

1998a, O'Toole and Kolter, 1998b, O'Toole et al., 2000, Pratt and Kolter, 1999,

Engineering, 2008). A better understanding of the processes of bacterial detachment

will contribute significantly towards our ability to prevent undesirable bacterial

presence and thereafter activity.

2.3.3 Effects resolving from the biofilm presence

The consequences of bacterial adhesion to surfaces can be beneficial or deleterious

depending on the situation (Figure 2.5).

Figure 2.5: Environmental, industrial

and medical impacts of biofilm

formation. Image reproduced from the

Centre of Biofilm Engineering website

(Engineering, 2008).

Chapter 2:

Literature review

______________________________________________________________________

15

For instance, bioreactor and biofilter systems rely on the adhesion, growth and

aggregation of bacterial cells to support materials for effective operation. Biofilms are

also needed for effective wastewater treatment or for degradation of soluble waste

(Fernández et al., 2007). Some of the newly developed areas of biofilm use include

biomining, where biofilms are used for recovery and extraction of metals from primary

concentrates (Rawlings and Johnson, 2007), inhibition of metal corrosion (Zuo, 2007)

and bioremediation of hydrocarbons and heavy metals (Singh et al., 2006). On the other

hand, bacterial aggregation followed by subsequent biofilm growth poses serious risks

to the safe and efficient functioning of man-made structures. For instance, biofilm

presence can lead to corrosion or fouling of heat exchangers and ship’s hulls (Qian et

al., 2007), hence resulting in significant economic losses and environmental damage

(Howell and Behrends, 2006).

Significant negative consequences of bacterial adhesion can also arise with respect

to medical devices and instruments or biological surfaces – for example, deposition of

dental plaque, infections of tissue or prosthetic implants, etc (Figure 2.6) (Whittaker et

al., 1996, Parsek and Singh, 2003, Benito et al., 1997).

(a) (b) (c)

Figure 2.6: Examples of the medical impact (a-deposition of dental plaque, b-infection

of artificial heart valves, c- increased antimicrobial resistance) from biofilm presence.

Image reproduced from the Centre for Biofilm Engineering website (Engineering,

2008).

In recent years biofilms have been identified as the main cause of a several severe

infectious disorders, particularly those involving the use of indwelling medical devices

Chapter 2:

Literature review

______________________________________________________________________

16

(Lynch and Robertson, 2008). Despite the recent discovery and release of novel and

powerful antimicrobials, bacterial infections due to biofilm presence are still the most

common cause for biomaterial implant failure (Gottenbos et al., 2001). Bacterial

adhesion on medical implants such as voice prostheses, orthopaedic prostheses, contact

lenses, and artificial heart valves is followed by biofilm formation resulting in organised

consortia covered by “glycocalyx” (most likely EPS products), in which cell are

embedded and protected from unfavourable environmental influences including

antimicrobials (Davey and O'Toole, 2000). Enhanced bacterial resistance to various

antimicrobials is due to bacterial phenotypic changes within the biofilm and suppression

of antibiotic activity by the extracellular enzymes produced within the biofilm itself.

Bacterial aggregation and biofilm formation can also increase antimicrobial resistance,

most likely as a result of exchange of the genetic elements bearing antibiotic resistance

traits between bacteria within the biofilm community (Lynch and Robertson, 2008).

In addition to causing serious infections, bacteria within biofilm produce antigens

that stimulate the host immune system to produce antibodies. While bacteria residing in

the biofilm are often resistant to these mechanisms, the immune response may cause

damage to surrounding tissue. Bacteria such as S. aureus, P. aeruginosa, Klebsiella

pneumoniae, Candida spp., E. coli, Enterococci are considered to be the pathogens that

most frequently stimulate the immune system in this way.

2.4 Theoretical approaches to understanding the basic

principles of cell-substrate interactions

Microbial cell surfaces are chemically and structurally complex and heterogeneous.

For this reason, a physicochemical approach to explaining the microbial adhesive

behaviour and producing a better understanding of cell-substrate interactions has proven

elusive. The process is further complicated by the fact that the biological drivers of

bacterial adhesion are often quite specific to the structure of the individual bacterium

and are therefore difficult to generalize. Nevertheless, two physicochemical models

Chapter 2:

Literature review

______________________________________________________________________

17

have been developed: the DLVO approach and the thermodynamic theory (Busscher

and van der Mei, 1997, Power et al., 2007, Palmer et al., 2007).

2.4.1 DLVO theory

Contact between bacteria and different surfaces may occur through reversible and

irreversible interactions. In describing the interaction energies between the two surfaces,

the classical DLVO theory developed by Derjaguin, Landau, Verwey, and Overbeek

considers the final outcome of the interchange between long-range, attractive van der

Waals and repulsive electrostatic interactions (Korenevsky and Beveridge, 2007).

According to the DVLO theory, there are two distances of separation. At larger

separation distances, the bacterium is only weakly held near the surface by van der

Waals forces. At this stage closer approach is inhibited by electrostatic repulsion and

shear forces can easily remove bacteria from the surface. Once this repulsion is

overcome by the bacterium, it may be bound at the closer separation distance, where the

attractive forces are strong and adhesion becomes irreversible. One crucial parameter in

the DLVO theory is the importance of the ionic strength of the solution in which cells

are being resuspended. As suggested by Rijnaarts et al. in low ionic strength solutions

long-range electrostatic repulsion forces control bacterial adhesion (Rijnaarts et al.,

1999). On the other hand in high ionic strength solutions steric interactions (i.e., cell

surface hydrophobicity) dominate. The importance of the ionic strength of the solution

is demonstrated by the significantly different numbers of cells that attach under high

and low ionic conditions. It has also been demonstrated that attachment numbers

increase with the increasing ionic strength and hydrophobicity of the medium (Busscher

and Norde, 2000, Power et al., 2007). The main disadvantage of the DLVO theory -

with which many researchers now agree - is the fact that it was used to explain the

impact of electrolyte concentration and net surface charge on bacterial adhesion in

laboratory experiments. DLVO theory does not consider bacterial cell surfaces as non-

inert, highly dynamic particles capable of responding to environmental perturbations

such as changes of pH, temperature and ionic strength.

Chapter 2:

Literature review

______________________________________________________________________

18

In order to provide a more reasonable explanation of cell-substrate interactions, the

classical DLVO theory has been extended to include short-range, steric and Lewis acid-

base interactions (Busscher and van der Mei, 1997, Van Oss et al., 1988). The acid-base

interactions are based on electron-donating and electron-accepting interactions between

polar moieties in aqueous solutions. These interactions have an enormous influence

compared to the electrostatic and van der Waals interactions; however, they are also

short-range interactions (less than 5 nm), which means they only become operative

when the interacting surfaces are in close proximity.

2.4.2 Thermodynamic theory

The second theoretical approach that has been used to explain and/or predict the

mechanism of bacterial adhesion is based on thermodynamic principles. This theory

interprets bacterial attachment as a spontaneous decrease of the free energy in a system.

It also considers attachment as an equilibrium process that is quantified by the

interfacial free energies of the interacting surfaces (Power et al., 2007, Bos et al., 1999).

The disadvantage of this method is the need for sophisticated software and the existence

of already measured/published contact angles in order to accurately calculate surface

tensions.

To confirm the thermodynamic concept, contact angle measurements of liquids on

test substrata and bacterial cell surfaces have been carried out in laboratory experiments

(Fletcher and Marshal, 1982). The results have shown that microbial adhesion can

generally be classified as weak reversible secondary minimum adhesion which can

develop into irreversible primary minimum adhesion. As expected, the progression from

weak reversible secondary minimum adhesion to irreversible primary minimum

adhesion is much easier in high ionic strength suspensions and when the

microorganisms involved have surface appendages (Bos et al., 1999).

Chapter 2:

Literature review

______________________________________________________________________

19

2.4.3 Tentative scenario for initial bacterial adhesion at nanometer

proximity

On approaching a surface, a bacterium experiences a sequential chain of

interactions. The first long-range interactions - which are function of the distance and

the surface free energy - occur at distances of more than 100 nm. At this range weak

attractive van der Waals and repulsive electrostatic forces are believed to be the main

drivers of initial adhesion.

Electrostatic forces play a major role in initial bacterial adhesion at distances of 10-

20 nm. Electrostatic interactions are attractive when the surfaces have opposite net

surface charges and repulsive when surfaces have like charges. In this respect, bacteria

bearing net negative charge are expected to be attracted to positively charged surfaces;

however, in many natural environments, repulsive forces are reduced as the ionic

strength of the medium increases. For example, the electrolyte concentration of

seawater is sufficient to eliminate the electrostatic repulsion barrier.

Interfacial water may be a barrier to adhesion at distances of 0.5-2 nm, though it

can be removed by hydrophobic interactions (Goertz et al., 2007)). Furthermore, at

separation distances of less than 1 nm, hydrogen bonding, cation bridging, and receptor-

ligand interactions become important (Mittelman, 1996, Bos et al., 1999).

2.4.4 Application of the adhesion theories

Difficulties in the applicability of both the thermodynamic and DLVO theories in

bacterial adhesion have been suggested. The structural and chemical diversity of

bacterial cells, together with the lack of finite boundaries because of the existence of

fimbriae, stalks, flagella or long chain biopolymers frustrates the use of a reductionist,

theoretical approach. During adhesion bacteria employ several stages of interaction,

each involving different molecular components of the bacterial or substratum surfaces.

Non-polar (hydrophobic) groups on fimbriae, EPS, LPS or outer membrane proteins

may all assist the bacterium in approaching the surface more closely. This may cause

conformational changes in other bacterial surface polymers, thereby exposing further

Chapter 2:

Literature review

______________________________________________________________________

20

functional groups for stronger attractive interactions. The production of additional

adhesive polymers can also be triggered at this time. This is an important issue for the

thermodynamic theory, which can only be applied to processes in an equilibrium

situation. Bacterial production of extracellular biopolymers is irreversible at the initial

stages of adhesion and therefore disturbs the necessary equilibrium.

At this point in time there is sufficient evidence available to confirm that some

aspects of bacterial adhesion can be described by physicochemical approaches.

Nevertheless, as the complexity of the cell surface appendages increases, the

applicability of these approaches and the final outcomes from them become

compromised (Bos et al., 1999, Busscher and van der Mei, 1997).

2.5 Biological aspects of bacterial adhesion

2.5.1 Overview

Due to the importance of bacterial attachment and subsequent biofilm formation for

marine industries, medicine, and the food-processing industry (among others),

considerable effort has been invested into revealing, understanding and manipulating

the factors that influence this process. Many studies of bacterial attachment and biofilm

formation have been conducted, each revealing bits and pieces of this complicated

puzzle (Gallardo-Moreno and Calzado-Montero, 2006, Busscher and Norde, 2000,

Roosjen et al., 2006, van Merode et al., 2007, Emerson et al., 2006, Bakker et al., 2002,

Etzler, 2006, Bruinsma et al., 2001, Korenevsky and Beveridge, 2007, Li and Logan,

2004, Liu et al., 2008, Advincula et al., 2007, Cao et al., 2006, Arnold and Bailey, 2000,

Öner and McCarthy, 2000, Riedewald, 2006). Numerous studies over the last three

decades have focused on two major aspects of bacterial attachment - the

physicochemical and biological factors. Physicochemical aspects of bacteria adhesion

focus on the effects of cell surface characteristics such as wettability and charge, and

will be described in the next section; whereas this section addresses biological aspects

Chapter 2:

Literature review

______________________________________________________________________

21

of bacterial adhesion, such as the presence and composition of cellular appendages and

extra-cellular deposits.

Bacteria as a biological surface are capable of subtle and complex responses to

environmental stimuli which can lead to a rich phenomenology of adhesive interactions.

Depending on whether the whole cell surface or just one specific molecular group is

interacting with the surface, bacterial cell adhesion can be considered as specific or non-

specific; in both cases, cell appendages play an important role in attaching and sensing

the proximity to the surface (Mandlik et al., 2008, Ton-That and Schneewind, 2003).

Non-specific bacterial adhesion is typically involved in the colonization of inert

surfaces, as opposed to specific bacterial adhesion, which is typical for attachment to

biological surfaces and is less affected by environmental factors such as temperature,

humidity and pH (An and Friedman, 1998). In the case of specific bacterial adhesion,

only certain highly localised stereochemical molecular groups on the microbial surface

interact and make contact with the surroundings. In bacterial adhesion to biological

surfaces, it is essential that the host cells also play an active role in the process through

the activation of specific genes, as they are not inert surfaces (Triandafillu et al., 2003,

Laarmann and Schmidt, 2003).

The factors, which depend on the particular characteristics of individual biological

systems, are discussed in this section.

2.5.2 Adhesins

Bacterial surface appendages (adhesins) enable microorganisms to sense their

surrounding environment and respond to changes. Adhesins are located on the microbial

surface and are responsible for recognising and binding to specific receptor sights on the

host cells (Soto and Hultgren, 1999). Adhesins can be assembled into hair-like

appendages, known as fimbria or pili, that extend out from the bacterial surface or can

be directly associated with the microbial cell surface - so-called non-pilus adhesions

(Soto and Hultgren, 1999). Some examples of bacterial appendages that are involved in

cellular adhesion include:

Chapter 2:

Literature review

______________________________________________________________________

22

• P pili and type 1 pili - observed on the surface of Escherichia coli and

Enterobacteriaceae. The expression and assembly of P pili requires eleven genes, and

type 1 pili seven genes. These organelles mediate binding to mannose-oligosaccharides

and represent important virulence determinants.

• Type IV pili - implicated in a variety of functions such as adhesion to host-cell

surfaces, twitching motility, modulation of target cell specificity and bacteriophage

adsorption. They are found on bacteria such as P. aeruginosa, pathogenic Neisseria,

Moraxella bovis, V. cholerae, and enteropathogenic E. coli.

• Curli - thin, irregular and highly aggregated surface structures mainly found in E.

coli and Salmonella enteritidis. They mediate binding to a variety of host proteins

including fibrinogen, plasminogen, and human contact proteins.

• Flagella - large complex protein assemblages extending out from the bacterial wall.

Recent studies showed that bacteria express a wide variety of adhesins in order to

establish adhesion to host cells (Whittaker et al., 1996, Ubbinka and Schär-

Zammarettib, 2005, Soto and Hultgren, 1999, Laarmann and Schmidt, 2003, Dupres et

al., 2005, Bhosle et al., 1998). The type of adhesin used by bacteria is dependent on the

strain, environmental factors and the receptors expressed by the host cell. These

adhesive interactions are of particular importance in symbiotic and pathogenic

relationships to ensure recognition of certain surfaces and binding sites via the

signalling system of surface components or receptors. The importance of these cellular

appendages in the initial cellular response to host cell or substratum was explored by

Waar et al. (Waar et al., 2002). Waar et al explored the adhesion of five different strains

of Enterococcus faecalis on polyethylene (PE), fluoro-ethylene-propylene (FEP) and

silicone rubber (SR) biopolymeric surfaces. Their results suggested that bacterial

surface proteins (non-pillous adhesions) are not necessary and do not have significant

impact in the initial deposition of E. faecalis, but are of outmost importance in the

stationary phase and the end-point phase. To be specific, the number of cells present on

the polymer surface at the latest stages of the attachment is influenced by the expression

of gene-specific surface adhesions that can stimulate the adhesion of more cells. It is

believed that attached bacteria can diminish the effects of antagonistic sites existing on

Chapter 2:

Literature review

______________________________________________________________________

23

the polymer surface. They can also raise the number of new adhesion sites - a

mechanism known as positive cooperativity (Waar et al., 2002) .

2.5.3 Extracellular bio-products

Extracellular polymeric substances - or extracellular biopolymers - are microbial

products synthesised during their growth phase. Extracellular biopolymers comprise

extracellular polysaccharides that can interact with hydrophilic surfaces,

lipopolysaccharides that can interact with hydrophobic surfaces and proteins that often

react in specific attachment mechanisms (Ryu et al., 2004). The majority of

extracellular biopolymers are of polysaccharide nature and are therefore referred to as

‘extracellular polysaccharides’, thus the same abbreviation - EPS - can be applied for

both. EPS synthesis is now accepted as the key mechanism facilitating irreversible

bacterial attachment to inanimate surfaces in particular (Beech et al., 2005).

The chemical composition of EPS varies considerably with their function. For

instance, gel-like EPS surrounding the cell usually have a protective role, contrary to the

‘free-EPS’ released into the culture medium whose role is mostly related to irreversible

cell adhesion (Beech et al., 1999). Depending on their location in terms of proximity to

the cell wall, EPS can be classified as capsules, sheaths or slimes (Beveridge and

Graham, 1991). Beech at al. managed to isolate three different types of EPS (capsular,

free EPS from the culture medium and EPS associated with biofilm) from Pseudomonas

sp, and reported differences between the three exopolymers - namely, less uronic acid

was detected in the capsular exopolymers compared to the other two (Beech et al.,

1999). Also, O- and N-acetylation were found in greater quantities in the biofilm

exopolymer compared to the free exopolymer, which had an increased beta-sheet

component and a reduced unordered component when compared to the biofilm and

capsular exopolymers. Beech at al. came to conclude that although the three types of

EPS shared some of their chemical components, the overall physicochemical

characteristics of the isolated exopolymers is influenced by their function and the

cellular mode of growth (Beech et al., 1999).

Chapter 2:

Literature review

______________________________________________________________________

24

The chemical composition of EPS also depends on the bacterial habitat. For

example, bacterial alginate (β-1, 4-D-mannuronic and L-guluronic acids) produced by

the opportunistic pathogen P. aeruginosa during its colonization of lungs has a binding

capacity to facilitate attachment to the epithelial cells of the respiratory tract. In

contrast, the alginate produced by the river-dwelling Pseudomonas fluorescens inhibits

adhesion. These physicochemical properties of the alginates are due to different degrees

of acetylation and different ratios of mannuronic to guluronic acid (Mitik-Dineva et al.,

2006 ).

The distinction between different types of EPS is difficult and imprecise, mainly

due to the limited quantities produced and the tight bonds between them and the cellular

surface or the substratum surface.

2.6 Physicochemical aspects of bacterial adhesion

It is now well accepted that bacterial attachment to surfaces is a complex process

influenced not only by cell surface characteristics, but also by a range of environmental

and substratum surface parameters. A more detailed description of the effects each of

these parameters may have on bacterial adhesion is provided in this section.

2.5.1 Environmental parameters that influence cell-substrate interactions

Environmental parameters such as temperature, the pH of the surrounding medium,

humidity, the length of exposure, presence of antibiotics, cell concentration, availability

of nutrients and chemical treatment are all believed to be important influences on cell-

substrate interactions. For example, the number of attached cells tends to decrease as the

temperature is reduced from room temperature down to approximately 3˚C. On the

other hand, pH changes between 4 and 9 do not significantly affect the adhesion

process. More information on this topic is presented in a several book chapters in

highly-regarded book by Costerton and Lappin-Scott (Costerton and Lappin-Scott,

1995b, Brading et al., 1995, Costerton and Lappin-Scott, 1995a).

Chapter 2:

Literature review

______________________________________________________________________

25

2.6.2 Bacterial surface characteristics that influence cell-substrate

interactions

2.6.2.1 Cell surface wettability

Hydrophobicity is an interfacial phenomenon that has been suggested as one of the

crucial parameters in cell-substrate interactions. Cell surface wettability varies among

species and is influenced by factors such as bacterial age, cell concentration, growth

medium and most importantly by the cell surface structure. Studies conducted over past

decades managed to reveal some of the factors that control cell surface wettability, yet

there is no general agreement on ‘if’ and ’how’ important it is in predicting bacterial

susceptibility towards certain surfaces. The role of cell surface wettability in microbial

adhesion has attracted the attention of not only microbiologists, but also physicists,

engineers and chemists (Gallardo-Moreno and Calzado-Montero, 2006, Benito et al.,

1997). It is believed that the presence of molecules such as proteins and lipids on the

cell surface are the key factors in determining cell surface wettability (Palmer et al.,

2007). Experiments in which cells were treated with proteolytic enzymes confirmed that

cell surface wettability decreased as a result of cell surface conformation changes

(Palmer et al., 2007). Conducted X-ray photoelectron spectroscopy (XPS) analysis of

bacterial surfaces have revealed that high cell surface hydrophobicity levels are

frequently accompanied by a high nitrogen/carbon ratio, contrary to cell surface

hydrophilicity where the oxygen/carbon ratio is higher (Palmer et al., 2007).

Korenevsky and Beveridge studied the relationship between cellular surface

characteristics and bacterial adhesion. They concluded that the presence of rough LPS

and capsular polysaccharides on the outer membrane increased the cells’ hydrophobicity

(Korenevsky and Beveridge, 2007).

Various methods for measuring cell surface wettability have been developed - the

MATH test (microbial adherence to hydrocarbons), the HIC (hydrophobic interaction

chromatography), and the water contact angle measurements method, which was

adopted for the purpose of this study. They all have their advantages and disadvantages;

for instance the MATH test is influenced by electrostatic interactions (Busscher et al.,

1999), while HIC is affected significantly by the cell’s attachment to the

Chapter 2:

Literature review

______________________________________________________________________

26

chromatography column as opposed to the stationary phase (which in turn is affected by

the ionic strength of the surrounding medium). The disadvantage of the contact angle

measurements method on the other hand is the complicated sample preparation

technique which requires lawns of partially dehydrated cells. The complexity arises

from the fact that defining accurate drying time is difficult and partially subjective, yet

it has been suggested that this is probably the only method accurate enough to measure

such cell-substrate characteristics (Gallardo-Moreno and Calzado-Montero, 2006).

Groups which have studied cell-substrate interactions vary widely in their

assessments of the magnitude of the impact of cell surface wettability. Van Loosdrecht

et al. (Palmer et al., 2007, van Loosdrecht et al., 1990) believe that cell surface

wettability is the key mechanism in determining bacterial propensity towards certain

surfaces. They also suggest that bacterial adhesiveness increases with increasing cell

surface hydrophobicity, and vice versa (Van Loosdrecht et al., 1987b, Basson et al.,

2007). In contrast, a study conducted by Li and Logan (Li and Logan, 2004) concluded

that bacterial adhesion is not significantly influenced by cell surface wettability;

similarly, Benito et al. concluded that there is no linear relationship between

hydrophobicity and bacterial attachment (Benito et al., 1997) .

The most detached assessment of the role of cell surface hydrophobicity in

determining bacterial adhesion comes from van der Mei at al., who summarised the

literature about contact angles tested with a wide variety of microbial strains (total of

142 isolates). As they have indicated, no generalisations can be made about microbial

cell surface physicochemical characteristics and the way they behave with respect to

different surfaces (van der Mei et al., 1998). Nevertheless, a generally accepted rule

does exist; hydrophobic cells attach better than hydrophilic cells. Hydrophobic cells

also tend to attach better to hydrophobic surfaces, in contrast to hydrophilic cells who

prefer hydrophilic surfaces (Bayoudh et al., 2006).

2.6.2.2 Cell surface charge

Alongside cell surface wettability, cell surface charge is the most explored

bacterial surface characteristic in terms of its involvement with bacterial adhesion.

Contrary to non-biological particles, direct measurement methods for determination of

Chapter 2:

Literature review

______________________________________________________________________

27

‘soft’ particle surface charge are not yet available, therefore indirect measurements

(electrokinetic assays) are being used to quantify bacterial surface electrical contribution

(Gallardo-Moreno and Calzado-Montero, 2006). As indicated by Galarrdo-Moreno et

al., a good indication of this contribution can be inferred from zeta potential value (ζ)

(Gallardo-Moreno and Calzado-Montero, 2006). The zeta potential of a surface is

indirectly inferred from electrophoretic mobility (EPM), which represents the ratio

between the velocity of a particle under applied electric field and the value of its electric

strength (Gallardo-Moreno and Calzado-Montero, 2006). The relationship between zeta

potential and EPM is established through the Helmholtz-Smoluchowski equation:

ζ = (4πη/ε) EPM

where η and ε represent viscosity and dielectric permittivity of the liquid in which

particles are being resuspended.

The vast majority of bacterial cells bear negative net surface charge (van der Mei and

Busscher, 2001), yet positively charged organisms with potential tendency to adhere to

negatively charged surfaces have been reported (Jucker et al., 1996). The magnitude of

surface charge varies among species and is influenced by several factors such as the

growth medium in which cells are being resuspended, its pH and ionic strength (EPM

decreases with increasing ionic strength due to shielding of the bacterial surface (de

Kerchove and Elimelech, 2005, Eboigbodin et al., 2006)), bacterial age and cultural

conditions, and most of all by the cellular surface structure. Surface charge - just like

cell surface wettability - derives from the structure and composition of the cellular outer

membrane, in particular in the presence of membrane proteins, sialic acids and surface

EPS and/or LPS (Eboigbodin et al., 2006).

Many attempts to associate cell surface charge and cellular adhesive behaviour

have been made. No general agreement exists; studies that support and oppose the

existence of a correlation between cell surface charge and cellular adhesive behaviour

have been reported (van Loosdrecht et al., 1987a, Van Loosdrecht et al., 1987b,

Castellanos et al., 1997). When it is considered that bacterial surface charge can vary

due to the possibility of cellular morphological transformations in response to

environmental perturbations, it seems unlikely that bacterial adhesion can be correlated

with bacterial surface charge. Nevertheless, study by Li and Logan suggested that

Chapter 2:

Literature review

______________________________________________________________________

28

bacterial surface charge can be negatively correlated with bacterial adhesion (van

Loosdrecht et al., 1990, Li and Logan, 2004).

2.6.3 Substratum surface characteristics that influence cell-substrate

interactions

2.6.3.1 Substratum surface wettability

Numerous researchers have attempted to correlate bacterial adhesion with the

physicochemical properties of substrata. It was found that bacterial attachment is more

effective on hydrophobic surfaces as shown by the number of attached cells, rate of

attachment and strength of binding (Busscher et al., 1990, Doyle and Rosenberg, 1990).

The ‘hydrophobic effect’ refers to the proclivity of one nonpolar molecule for another

nonpolar molecule over water. When two hydrophobic surfaces approach one another in

an aqueous environment, inverse ordered layers of water are displaced. The entropy

increase during the displacement process creates energetically favourable conditions for

adhesion (van Loosdrecht et al., 1990). On the other hand hydrophilic surfaces that are

highly hydrated can be more resistant to bacterial adhesion because of adsorbed water

that must be displaced before adhesion can occur. As a result, the effectiveness of the

adhesion will depend on the difference between the bacterium ↔ substratum attraction

forces and the adsorbed water ↔ substratum attraction forces.

Nevertheless, studies that suggest similar adhesion on hydrophilic and hydrophobic

surfaces or even better adhesion on hydrophilic substrata have also been reported

(Mittelman, 1996)). Li and Logan (Li and Logan, 2005) studied the attachment

behaviour of seven bacterial strains (E. coli JM109, E. coli D21, E. coli D2, P.

aeruginosa PA01, P. aeruginosa PDO300, Burkholderia cepacia G4, Burkholderia

cepacia Env435 and Bacillus subtilis ATCC7003) on two metal oxide-coated surfaces

(TiO2 and SnO2), both exposed to UV light (wavelength 254nm). Exposing the TiO2 and

SnO2 surfaces to UV light resulted in decreased surface hydrohobicity which reduced

the adhesion of all tested strains by 10 to 43%, depending on the strain. Greater

reduction in adhesion for E. coli, B. cepacia and B. subtilis, or the ‘less-sticky’ strains,

Chapter 2:

Literature review

______________________________________________________________________

29

as Li and Logan (Li and Logan, 2005) referred to them, was also observed. Contrary to

this P. aeruginosa and some E. coli strains known to be EPS and/or LPS overproducers

were affected in lesser extent by the changes in the surface hydrophobicity, thus

pointing to the importance of these extra-cellular products in initial cell-substrate

interactions.

In general there appear to be two different attachment mechanisms depending on

the substratum surface characteristics. Adsorption on hydrophobic surfaces is rapid with

strong binding forces; on the other hand, adhesion to hydrophilic surfaces can be

reversible and irreversible as proposed by Marshall (Marshall, 1992) and can be

described by the DLVO theory. Initially, a weak and reversible stage of the adhesion is

observed at a separation distances of several nanometres, at which point the bacterium

can be removed by shear forces or desorbed spontaneously. At a later stage, this

attachment can be converted into irreversible adhesion by synthesis of extracellular

biopolymers or by stabilisation of conformational changes in existing polymers. These

polymers bridge separation distances of less than 1 nm, displacing the adsorbed water

and /or neutralising the electrostatic repulsion.

Lately particular attention has been given to the so-called superhydrophobic

surfaces. A superhydrophobic surface is defined as having a water contact angle of more

than 150º and roll-off angle lower than 5º (Michielsen and Lee, 2007). It is generally

accepted that the surface contact angle and thus surface hydrohobicity can be influenced

by changes in surface chemistry and surface roughness (Bormashenko et al., 2006a,

Bormashenko et al., 2006b, Suzuki et al., 2007, Abdelsalam et al., 2005). Two different

models that explain the effects of surface roughness on surface wettability have been

proposed. One is the Cassie-Baxter model, according to which the air trapped in surface

irregularities below the water droplet increases surface hydrophobicity. This is due to

the fact that the water droplet will partially sit on the air trapped in so-called air pockets.

The other model is proposed by Wenzel, who suggests that the increased surface

hydrophobicity is due to the overall increase of the surface area with increased

roughness (Bormashenko et al., 2006a).

As a general rule, increasing the roughness of a surface with a contact angle greater

than 90° will result in an increased contact angle, and increasing the roughness of a

surface which has a contact angle less than 90° will result in a lower contact angle

Chapter 2:

Literature review

______________________________________________________________________

30

(Wenzel, 1936).. Hierarchical micro and nano-scale surface textures can reinforce both

surface hydrophilicity and surface hydrophobicity (Michielsen and Lee, 2007)

(Bormashenko et al., 2007, Bormashenko et al., 2006b) 06); however, the relationship

between surface roughness and surface hydrophobicity has attracted more attention than

surface hydrophilicity. The interest in superhydrophobic surfaces is based on their

potential to mimic the ‘lotus’ phenomenon, where ‘self-cleaning’ water droplets can

easily roll off from the surface and wash off any dirt from it. Surfaces with such

characteristics are of significant interest in fundamental research and in many industrial,

electrochemical and medical applications (Li et al., 2007).

2.6.3.2 Substratum surface charge

The correlations between bacteria, their surface properties and substratum charge

have been investigated at length over the years (Rozhok and Holz, 2005, Simoni et al.,

2000, Li and Logan, 2004, Gottenbos et al., 2001). Considering that the majority of

bacteria bear net negative surface charge, and in agreement with basic physic principles

it is generally accepted that bacterial adhesion is discouraged on negatively charged

surfaces and promoted on positively charged (Gottenbos et al., 2001). A study

conducted by Rozhok and Holz that addressed the adhesive potential of negatively

charged E. coli K12 cells towards negative, neutral and positive charged surfaces

supported this assumption (Rozhok and Holz, 2005). Negatively charged E. coli K12

cells were found to be most attracted to the positively charged gold surfaces. Cells

managed to attach in greater numbers on positively charged gold surfaces, less on

neutral and in minimal numbers on negatively charged surfaces. Since E. coli K12 cells

bear negative net surface charge, as do the majority of bacteria, greater attachment to

the positively charged surfaces was expected.

Chapter 2:

Literature review

______________________________________________________________________

31

Figure 2.7: AFM images of E. coli K-12 bacterial cells bound to: (A) neutral, (B)

positively, and (C) negatively charged bare gold substrates. Figure adopted from

journal article (Rozhok and Holz, 2005).

Although negatively charged, cells were also found on the negatively charged gold

surfaces; attachment of negatively charged cells to negatively charged surfaces was

attributed to the accompanying cellular transformation (Figure 2.7). Rozhok and Holz

(2005) found that while attaching to the unfavourable negatively charged surface, the

LPS located on the E. coli K12 surface were being transformed. It is believed that the

transformation of the surface LPS results in exposure of areas with positive build-up

charge on the cell surface which can interact with the negatively charged surface. The

areas bearing positive surface charge are most likely destroyed negatively charged O-

chains of LPS molecules that shield positively charged core regions.

Although bacterial susceptibility to positively charged surfaces has been reported,

the relationship between bacterial adhesion and substratum surface charge remains

unclear. Studies suggesting that no correlation exists between bacterial adhesion and

substratum charge have also been reported (Li and Logan, 2004).

2.6.3.3 Surface tension

The free energy at the interface between solid surfaces and aqueous solutions gives

a direct measure of the interfacial attractive forces. In this light, modifying the surface

Chapter 2:

Literature review

______________________________________________________________________

32

energy of various materials/substrates is believed to be a potential method of

minimising cellular adhesion (Liu and Zhao, 2005).

The most recent stand for estimate of the solid’s free energy (γi) derive from contact

angles of several diagnostic liquids measured on the particular solid surface. There have

been several attempts to elucidate the association between contact angle of pure liquid

on solid surface and the surface tension. One of them is presented through the Young

equation:

γl/v cosθ = γs/v – γs/l

where θ is the contact angle and:

γl/v is the surface tension or surface free energy of liquid to air;

γs/v is the surface tension or surface free energy of solid to air;

γs/l is the surface tension or surface free energy of interface between solid and

liquid.

According to Van Oss et al. the surface free energy (γi) is the sum of the Lifshitz-

van der Waals a-polar component (γLW

) and the Lewis acid-base polar component (γAB

)

(Etzler, 2006),where:

γi = γiLW

+ γiAB

(1)

The Lifshitz-van der Waals a-polar component (γLW

) can be formulated according to

Good (Good, 1952) as below:

γijLW

= [(γiLW

)1/2

-(γjLW

)1/2

]2 (2)

γijLW

= γiLW

+ γjLW

– 2(γiLW

γjLW

)1/2

(3)

Whereas the Lewis acid-base polar (γAB

) component of the equation can be divided into

two components: an electron donor (γi-) and an electron acceptor (γi

+) (Van Oss et al.,

1988), where:

γiAB

= 2(γi+γi

-)1/2

(4)

In which case the definition of the Lewis acid-base interactions between two substances

in the condensed state will be as follows (van Oss, 1994):

γs/lAB

= 2(γs+γl

-)1/2

+ 2(γl+γs

-)1/2

(5)

Therefore, (γsLW

) can be evaluated from contact angles between solid and liquid.

Coming back to the Young equation; according to van Oss (Van Oss et al., 1988), this

equation

Chapter 2:

Literature review

______________________________________________________________________

33

γlLW

cosθ = γsLW

– γs/lLW

(6)

can be derived as follows:

1 + cosθ = 2(γSLW

/ γLLW

)1/2

(7)

in which case the complete Young Equation is:

γl/v(1+cosθ) = 2 [(γsLW

γlLW

)1/2

+ (γs+ γl

-)1/2

+ (γs- γl

+)1/2

] (8)

Surface energy is believed to be the most important physicochemical characteristic

of solid surfaces (Liu and Zhao, 2005). Numerous studies over recent decades have

investigated the effects of surface energy on bacterial adhesion. The results described so

far have been inconsistent; studies indicating that bacterial adhesion decreases with

decreasing substrate surface energy or increases with increasing surface energy have

been reported (Bakker et al., 2003, Liu and Zhao, 2005). In contrast, studies reporting

that bacterial adhesion increases with decreasing free energy have also been published

(Li and Logan, 2004, Pereira et al., 2000).

2.7 Effects of surface topography on bacterial adhesion

As defined by the British Standard, surface roughness represents irregularities in

the surface texture which are inherent in the production process but exclude waviness

and errors of form (Whitehead and Verran, 2006). The roughness of the substrate is

known to play a significant role in bacterial attachment to different surfaces, yet it was

never considered a factor of primary interest; most research attention was directed

towards the effects of surface wettability, charge and surface free energy.

Existing knowledge about the effects of surface roughness on cell-substratum

interactions is also controversial and far from settled. Results suggesting bacterial

adhesion is encouraged on rougher surfaces do exist, the hypothesis being that surface

pits, cracks, grooves and abrasion defects can provide shelter for attached bacteria from

unfavourable environmental factors (Verran and Boyd, 2001, Verran et al., 1980,

Whitehead and Verran, 2006, Taylor et al., 1998, Messing and Oppermann, 1979).

Chapter 2:

Literature review

______________________________________________________________________

34

Surface imperfections also allow time for cells to establish stable, irreversible

attachment following the initial reversible physicochemical attachment (Taylor et al.,

1998). There is still disagreement over whether there is a threshold below and above

which surface roughness can promote or prevent bacterial adhesion. It is believed that

surface irregularities comparable to the size of bacteria (1-1.5µm in diameter) are

capable of retaining more cells than smoother surfaces. Adhesion is increased on such

surfaces on account of increased contact area between the cell and its surrounding. One

of the recently developed concepts in understanding cell-substrate interactions is the

“attachment point” theory (Howell and Behrends, 2006). According to the attachment

point theory, organisms smaller than the scale of the surface micro-texture can take

advantage of multiple attachment points on the surface and will attach in relatively large

numbers. They will also have greater adhesion strength when compared to micro-

organisms that are of scale larger than the surface roughness (Callow et al., 2002,

Verran and Boyd, 2001, Chae et al., 2006, Shellenberger and Logan, 2002). They will

also be well protected from hydrodynamic shear forces in microscopic shelters on the

textured surface (Scardino et al., 2006). Several research projects studying the

relationship between surface roughness and attachment of organisms (such as barnacle

cyprids and algal spores) has supported the applicability of the attachment points theory

(Hoipkemeier-Wilson et al., 2004, Callow et al., 2002, Petronis et al., 2000, Berntsson

et al., 2000). On the other hand, only a few studies have observed the effects of surface

topography on the adhesive behaviour of smaller micro-organisms such as bacteria,

despite the fact that they are believed to be the initial colonizers of many surfaces and to

be necessary for further biofilm development and macro-fouling colonisation (Scardino

et al., 2006).

Research stating that smoother surfaces enhance bacterial adhesion has also been

reported. A study by Bruinsma et al. (Bruinsma et al., 2002) explored the attachment

behaviour of P. aeruginosa cells to contact lenses as an important initial step in the

development of microbial keratitis. After a short period of wear, a so-called

conditioning film consisting of lipids, salts, components from the lens care solutions,

proteins, etc. forms on the lens surface. Depending on the time of exposure (i.e., time of

wear) the chemical composition of the conditioning film as well as the lens’s

physicochemical characteristics will change and it will act as a base for bacterial

Chapter 2:

Literature review

______________________________________________________________________

35

attachment. As Bruinsma et al.’s results indicated, wearing contact lenses for a period

of 10 days resulted in increased surface hydrophobicity and surface roughness (5 nm),

in contrast to the over-wear (50 days) when the lenses’ surface wettability decreased

resulting in hydrophilic surface and increased surface roughness (10 nm). According to

this study, the numbers of attached P. aeruginosa cells decreased after 10 days of wear

and dropped even further after over-wear of 50 days, indicating smoother surfaces

might have an enhancing effect on bacterial adhesion. This is an indication of the

controlling effects surface roughness might have on bacterial adhesion, and also means

that balancing the effects of surface roughness versus wearing comfort should be taken

into account when developing novel surfaces for application in ophthalmology.

The majority of studies exploring the effects of surface topography on bacterial

adhesion have focused on the effects of micro-textured surfaces (Shellenberger and

Logan, 2002, Pereira et al., 2000, Taylor et al., 1998, Palmer et al., 2007, Emerson et

al., 2006, Whitehead and Verran, 2006, Sharon, 2006, Li and Logan, 2004). These and

other researchers addressed this issue as a factor of secondary importance alongside

other crucial parameters in cell-substrate interactions, such as surface chemistry, charge

and wettability.

2.8 Techniques for studying bacterial adhesion

The study of microbial surfaces and the changes they undergo upon interacting with

different surfaces are of utmost importance in medicine and biotechnology. In recent

years several new and sophisticated microscopy techniques have provided a way to

bypass the limitations of single-cell observations. Adhesion studies have been facilitated

by the emergence of several experimental techniques that can probe the interactions of

single cells with their neighbouring surfaces or other cells. Techniques such as confocal

scanning laser microscopy (CSLM), transmission electron microscopy (TEM), scanning

electron microscopy (SEM) and a variety of scanning probe microscopies have been

developed into powerful nanotechnology tools (Mitik-Dineva and Stoddart, 2006,

Ubbink and Schär-Zammaretti, 2005).

Chapter 2:

Literature review

______________________________________________________________________

36

Of all the scanning probe microscopies, the AFM has rapidly become an

irreplaceable tool for small-scale imaging. The advantage of AFM over other similar

techniques is its ability to perform real-time, high-resolution imaging of the native

bacterial cell surface in three dimensions, without pre-imaging preparation such as

staining or shadowing that might affect shape. In addition to allowing observation of

cell morphology, AFM can provide cross sections and measurements of bacterial

dimensions. Previous methods for establishing bacterial size were mainly based on

epifluorescence microscopy (EFM), but the analysis of images taken by EFM in order

to determine cell size proved to be very time-consuming (Nishino et al., 2004).

Nevertheless, the AFM has its disadvantages as well. Because the cantilever cannot

determine the edge of a surface accurately it needs a certain margin of error. This error

will eventually result in overestimation and production of an image that is larger than

actual object. The error when imaging biological samples can also be attributed to the

cellular dependence on environmental parameters such as the surrounding humidity and

temperature. For this reason the measurements of cell size - height in particular -

obtained by AFM should always be treated with caution.

The AFM operates by mechanically scanning a sharp tip mounted on a flexible

cantilever over the sample surface. There are three AFM operational modes - contact,

intermittent (tapping) and non-contact. Apart from imaging of the surface topography of

bacteria or substrata, the AFM can be also used for force measurements. Razatos et

al.(Razatos et al., 1998, Razatos, 2001) developed an AFM-based methodology for

directly measuring the interaction forces between AFM cantilevers with standard silicon

nitride (Si3N4) tips and bacterial lawns immobilized onto flat glass substrates. When

microspheres are glued onto standard tips, the probes can be modified to study the

interaction of bacteria with various surfaces of interest, e.g. mica, hydrophilic glass,

hydrophobic glass, polystyrene, and Teflon. For the purposes of the study presented

herein the AFM was used for imaging and quantitative analyses of the bacterial and

substratum surface topography.

Confocal scanning laser microscopy (CSLM) has become a standard technique for

obtaining high-resolution images at various depths in a sample; it allows 3-D images to

be reconstructed from successive focal planes. The CSLM method requires minimal

sample preparation and thus enables the researcher to study the organism itself, the

Chapter 2:

Literature review

______________________________________________________________________

37

surrounding environment, metabolic activity and genetic control within the biofilm. The

SCLM transforms the optical microscope into an analytical spectrofluorimeter

(Caldwell et al., 1992). In this research project the CSLM was used to visually present

EPS produced by cells during the 12 h incubation period on each of the tested surfaces.

X-ray photoelectron spectroscopy (XPS) has also found applications in recent

studies (van der Mei et al., 2000). This technique provides semi-quantitative

information on the elemental composition of the outer 1-5 nm of a surface, together

with some basic chemical information - for example, the relative concentration of an

element in different functional groups. Some modern XPS instruments provide a

chemical mapping capability with 3-5 micron resolution. The data are often useful for

indicating the relative concentration of elements or functional groups on a surface after

exposure to different experimental conditions (Waar et al., 2002). Sample preparation

requires some care, as they must be vacuum compatible and free of surface

contamination.

Scanning electron microscopy (SEM) has revolutionised surface research. SEM has

been commercially available since 1965, although the theory of electron optical systems

was developed decades before. In the 21st century, SEM is firmly established as an

irreplaceable tool in industrial and research laboratories for investigation of surface

textures. SEM projects a high-energy beam of electrons that interact with the atoms

located on the sample surface, producing signals that contain information about the

sample's surface composition, topography and conductivity. The advantage of SEM is

its ability to examine and analyse specimens, including bacteria, at magnifications from

5x to 500,000x. The disadvantage of using SEM for imaging biological samples is the

need for sample preparation; all samples must have electrically conductive surfaces in

order to prevent accumulation of electrostatic charge. For this reason, all biological

samples are usually protected with an ultra-thin coating of electrically-conducting

material, deposited on the sample either by low-vacuum sputter coating or by high-

vacuum evaporation. The most frequently used conductive materials are platinum and

gold, which was also used study presented herein. A gold coating prevents surface

charging during electron irradiation and can also maximize the signal and improve

spatial resolution. This requirement can be relaxed somewhat in the “environmental”

SEM but at the cost of some resolution (Stoddart and Brack, 2007).

Chapter 2:

Literature review

______________________________________________________________________

38

2.9 Bacterial attachment to glass surfaces

Soda-lime glass or ‘commercial glass’ as it is frequently referred to, is the most

common and inexpensive glass available on the market. Its main chemical constituent is

silica which represents approximately 70% of its structure, followed by soda and lime at

approximately 10% each, then low percentages of other materials such as alumina and

magnesium. Soda-lime glass has specific physicochemical characteristics such as high

light transmission and a smooth and inert surface suitable for modifications; these and

its low cost make it the most appropriate material for a wide variety of industrial and

commercial applications, such as production of window panels, bottles, glass

containers, laboratory glassware, jars and many other household articles. Instruments

such as XPS, XRF, ToF-SIMS (Time-of-Flight Secondary Ion Mass Spectrometry),

AFM, SEM have been employed to reveal details of its composition and surface texture

(Vadillo-Rodríguez and Logan, 2006). Due to the extensive use of soda-lime glass in

the production of domestic, laboratory, medical and industrial glassware, its

physicochemical characteristics as well its susceptibility towards biological and non-

biological colonization have been intensively studied (Wong et al., 2002, Verran et al.,

1980, Shellenberger and Logan, 2002, Gottenbos et al., 2000, Gallardo-Moreno and

Gonzalez-Martin, 2002, Burks et al., 2003). One of the great advantages of soda-lime

glass substrates is the ease with which it can be modified. Techniques such as polymer

spin-coating, metal thin-film deposition or chemical surface abrasion have all been

employed to modify the surface of glass slides for research purposes.

Due to all the above-mentioned reasons, soda-lime glass was selected as the

hydrophilic model surface for studying the adhesive behaviour of Escherichia coli, P.

aeruginosa, S. aureus, C. marina, P. issachenkonii, Saligentibactr flavus, S. guttiformis,

S. mediterraneus and A. fischeri. For the purpose of this study the surface of standard

soda-lime glass microscopy slides were chemically eroded by treatment with buffered

hydrofluoric acid.

Chapter 2:

Literature review

______________________________________________________________________

39

2.10 Bacterial attachment to polymer surfaces

Polymers have been intensively used as biomaterials in a variety of industrial and

medical applications due to their specific and easily modifiable physicochemical

characteristics. Bacterial adhesion to biopolymers has been the focus of many research

projects, yet the exact mechanisms of bacterial adhesion to polymeric surfaces, and the

initial steps of adhesion in particular, have not been described definitively.

Understanding the peculiarities of cell interaction with biopolymer surfaces is a topic of

particular interest, because bacterial susceptibility to treatment is significantly decreased

in the presence of polymeric devices (Speranza et al., 2004, Gottenbos et al., 2000).

The current study employed a polymer, P(t)BMA, that is commonly used in

biomedical applications to investigate the factors that may control the non-specific

attachment of nine taxonomically diverse bacterial species. P(t)BMA substrates were

selected as hydrophobic sample surfaces for studying the attachment behaviour of

Escherichia coli, P. aeruginosa, S. aureus, C. marina, P. issachenkonii, S. flavus, S.

guttiformis, S. mediterraneus and A. fischeri because of their exceptional mechanical

and optical characteristics. P(t)BMA has high transparency and stiffness, low water

absorption and high abrasion resistance (Ivanova et al., 2006), and as a result it has been

frequently used as a positive photoresist.

Conventional lithography involves the interaction of an incident beam with a solid

substrate (Chen and Pepin, 2001). Several methods of lithography have been developed

including laser DWW, electron beam, ion bean, X-Ray lithography and UV

photolithography (Jaeger, 2002). UV photolithography involves the exposure of

polymer substrates to UV light as a means of creating a desired pattern on the polymer

surface. It is the simplest form of lithography that can chemically functionalise the

surface molecules of a photoresist polymer (Weibel et al., 2007). UV photolithography

has typically been applied to SiOx (Mooney et al., 1996, Hickman et al., 1994, Bhatia et

al., 1993) or glass and fused silica substrates (Dulcey et al., 1991, Stenger et al., 1992),

and was the first technique used to pattern a self assembled monolayer (Senaratne et al.,

2005). It is also frequently used for semiconductor fabrication of integrated circuits

(Weibel et al., 2007). The techniques of lithography and photolithography have

previously shown their potential in producing micron to nano-sized features required for

Chapter 2:

Literature review

______________________________________________________________________

40

immobilization of proteins and cells (Veiseh et al., 2002, Mooney et al., 1996, Hickman

et al., 1994, Bhatia et al., 1993). Nevertheless, there are still some major challenges with

respect to high-output array fabrication, reproducibility and the possibility of creating

nano-scale features (Senaratne et al., 2005).

When exposed to UV light, the chemical structure of a positive resist is changed so

that the polymer is weakened and becomes more soluble in the photoresist developer,

meaning the resist is washed away from the area of exposure to the light and a positive

image is transferred to the resist layer. A negative photoresist reacts in an opposite

manner and becomes relatively insoluble to the photoresist developer, meaning the

resist that is not exposed to UV light is washed away and a negative image is transferred

to the resist (Figure 2.8) (Jaeger, 2002, Murphy, 2007).

Figure 2.8: Schematic representation of a negative and positive photoresist. Image

adopted from (Murphy, 2007).

P(t)BMA was originally designed as a positive photoresist in which chemical

contrast is readily induced through exposure to deep UV irradiation (Osaki and Carsten,

Chapter 2:

Literature review

______________________________________________________________________

41

2003). In its native state P(t)BMA is typically hydrophobic due to the presence of

methyl (CH3) groups on the polymer backbone and the tert-butyl ester as seen in Figure

3.9 (Raczkowska et al., 2004).

Figure 2.9: Molecular structure of native P(t)BMA. Image adopted from journal article

(Raczkowska et al., 2004).

Ivanova et al. (Ivanova et al., 2006) used a similar UV-exposure process to that

employed by Raczkowska et al. (Raczkowska et al., 2004) to modify P(t)BMA and

consequently observed alterations on the polymer surface, achieving moderately less

hydrophobic characteristics than the commercially obtained polymer. This was thought

to be due to the formation of carboxylic acid groups on the polymer surface (Ivanova et

al., 2006). A proposed scheme for UV photolithographic modification is shown in

Figure 3.10.

Chapter 2:

Literature review

______________________________________________________________________

42

Figure 2.10: Proposed reaction scheme for P(t)BMA when undergoing the UV

photolithography. The native P(t)BMA polymer displays methyl (CH3) groups, whereas

the treated polymer displays carboxyl (COOH) groups. Image adopted from (Ivanova et

al., 2006).

Currently there is no complete model that explains the attachment of bacterial cells

from different taxonomic groups to a variety of substrates. With this in mind, the

attachment pattern of selected bacteria was tested on two types of polymers surfaces, the

native P(t)BMA and the modified P(t)BMA; the latter surface was developed from the

native P(t)BMA by exposing it to UV light (photolithography).

2.11 Bacterial attachment to optical fibres

Optical fibres can be defined as glass or plastic fibre structures capable of guiding

light throughout their length. Modern optical fibres fabricated from high purity silica are

capable of transmitting optical signals over large distances with low losses. Other

advantages of optical fibres include the reduced need for free space optics with their

Chapter 2:

Literature review

______________________________________________________________________

43

associated alignment and maintenance difficulties, a large spectral bandwidth that can

be exploited and a general immunity to electromagnetic interference.

Following the initial development of the principles of fibre optics by Tyndall in the

1840s, interest in optical fibres continued to develop, leading to new technologies and

applications in industries such as television, telecommunications, laser machining,

dentistry medicine and many other fields (Bates, 2001). Optical fibres can be used as

environmental sensors - gauging temperature or pressure - for down-hole measurements

in the oil and gas industry, and for structural health monitoring in the civil engineering

and aerospace industries (Udd, 1995). In medicine, fibre optic technology is exploited

in the design of instruments such as endoscopes for minimally invasive exploratory or

surgical procedures (Gannot and Ben-David, 2003). Optical fibres are now commonly

used in optical spectroscopy, to couple a light source to a remote measurement position

and to couple the resulting signal to a spectrometer (Gannot and Ben-David, 2003),

thereby avoiding the need for hazardous free-space beams and tedious optical

alignments. Spectroscopic imaging can be performed with an imaging optical fibre,

which is composed of fibres (or ‘pixels’) fused together in a coherent arrangement so

that each fibre maintains its relative position throughout the length of the bundle (Dubaj

et al., 2002). Optical fibre probes have been used to perform absorption, fluorescence

and Raman spectroscopic measurements in a wide range of biomedical applications

(Marazuela and Moreno-Bondi, 2002, Potyrailo et al., 1998).

Raman spectroscopy is an inelastic light scattering process, usually implemented

with a laser in the visible, near infrared, or near ultraviolet range (Petry et al., 2003,

Carey, 1999, Keller et al., 2006). The light from the laser interacts with vibrational

excitations in the system, resulting in characteristic shifts in the energy of the scattered

light. Raman spectroscopy is capable of providing specific identification of the

molecular composition of a sample (chemical/structural fingerprinting), as well as

information concerning conformation and bond structure (Keller et al., 2006). The main

advantages of Raman spectroscopy over other vibrational spectroscopic techniques lie

in the fact that it is relatively insensitive to water and requires no special sample

preparation. Due to these robust characteristics, Raman spectroscopy has been used

extensively in many different biomedical testing applications, such as the evaluation of

skin composition, quantification of blood components (glucose, cholesterol and urea),

Chapter 2:

Literature review

______________________________________________________________________

44

estimation of protein structure and cancer and pre-cancer diagnosis (Carey, 1999, Petry

et al., 2003). The technique is increasingly used to identify single bacteria (Schuster et

al., 2000, Gessner et al., 2002, Rosch et al., 2005, Xie et al., 2005). In comparison with

current tests based on bacterial cultures, the rapid identification of single bacteria by

Raman scattering can help to avoid production downtime in pharmaceutical clean rooms

and reduce health hazards in clinical situations and food processing.

Surface-enhanced Raman scattering (SERS), initially observed by Fleishman et al.

in 1974 (Fleischman et al., 1974), allows a significant increase in sensitivity compared

to normal Raman scattering. This is achieved through an enhancement of the Raman

signal by a factor of up to 1014

, provided the sample is in close proximity to a

nanostructured metal surface (primarily gold, silver or copper). The metal also serves to

quench fluorescence, thus opening the door to the development of a number of new

applications. Over the years there has been increased interest in applying SERS in many

fields such as forensic science, homeland security, biochemistry and medicine (Haynes

et al., 2005). The spectrum obtained by SERS is the result of an analyte’s molecular

structure, which is useful for real-time detection of certain compounds in biofluids at

sub-nanomolar concentrations. These characteristics of SERS allow in-vivo

measurements, highlighting its advantages over other similar analytical techniques.

Attempts to use SERS as a ‘fingerprinting’ tool for the detection of various analytes

have already been reported. The development of biosensors for environmental as well as

in-vivo measurements has been undertaken by Murphy et al. (Murphy et al., 2000) and

Stuart et al. (Stuart et al., 2006), who have developed laboratory-based SERS systems

for monitoring sea-water pollution and glucose levels, respectively. It has been shown

that SERS is particularly sensitive to biochemicals in the immediate vicinity of the

metal surface, such as flavins that are associated with the cell wall (Zeiri and Efrima,

2005).

Despite its promising capabilities, SERS has been slow to reach the commercial

marketplace. The main reason for the delay in more widespread use of the technique lies

in the difficulty of producing uniform, reproducible substrates with high sensitivity (Vo-

Dinh and Stokes, 2002). The production of SERS substrates can be achieved by the

fabrication of surfaces with precise nanometre-scale structures (Vo-Dinh, 1998); this

has emerged as an important application of nanotechnology. A particular challenge

Chapter 2:

Literature review

______________________________________________________________________

45

involves the fabrication of SERS substrates on the tips of optical fibres, so that the

technique can derive additional benefit from the advantages of optical fibre technologies

(White and Stoddart, 2005, White et al., 2007, Polwart et al., 2000).

There have been several noteworthy recent developments in bionanotechnology

based on the employment of optical fibre sensors of nanometre size suitable for in-vivo

monitoring of biological processes in the living cell or nano-environments (Stoddart and

Brack, 2007). Fibre optic nanosensors can be defined as nano-scale measurement

devices that consist of a biologically or chemically sensitive layer (Vo-Dinh and Kasili,

2005). The tip of the bio-sensor probe can be functionalized with biomolecules such as

proteins, enzymes, antibodies or biological systems such as cells or whole organisms,

thus fabricating a ‘whole-cell’ biosensor (Biran et al., 2003, D'Souza, 2001). Previously,

whole cell biosensing devices measured the change in the metabolic rate of the cell, and

this was interpreted as the analytical signal. More recent biosensing devices are also

based on the cells’ ability to respond to environmental perturbations by their expression

of specific genes (Biran et al., 2003).

As an alternative to fibre optic sensors, Gessner et al. used a SERS substrate on the

submicron tip of a tapered optical fibre to record the spectra of monolayers of a yeast

with a spatial resolution of 200-500 nm (Gessner et al., 2002). This approach can be

extended further by combining SERS with scanning near-field optical microscopy

(SNOM), and has been used to image DNA fragments with 100 nm resolution on a

silver island film SERS substrate (Deckert et al., 1998). The SERS effect can also be

induced directly by applying a thin layer of silver islands to the tip of a tapered optical

fibre. The probe can be placed in intimate contact with almost any type of surface

because of the small size of the tip (Stokes et al., 2004).

Notwithstanding recent advances in the technology, the fabrication of whole-cell

biosensing devices has proven extremely challenging, mainly because the

immobilisation of live cells onto the fibres - or the insertion of the probe into the cell -

frequently results in cellular death and impaired sensitivity. Investigation of the cell-

optical fibre surface interactions is therefore a key issue in the design of biosensing

devices (Vo-Dinh and Kasili, 2005, Vo-Dinh et al., 2002, White and Stoddart, 2005,

White et al., 2007, Polwart et al., 2000, Stoddart and Brack, 2007, Biran et al., 2003,

D'Souza, 2001, Vo-Dinh, 1998). The present study aims to determine the influence of

Chapter 2:

Literature review

______________________________________________________________________

46

surface characteristics and chemistry on the attachment of three medically and six

environmentally significant microorganisms on the surface of optical imaging fibres

used in biomedical applications.

Chapter 3: Methodology

______________________________________________________________________

47

CHAPTER 3

METHODOLOGY


______________________________________________________________________

48

3.1 Overview

Due to the aim and nature of the project, an array of investigation techniques has

been selected. The latter has been applied to different bacterial taxonomic lineages

represented by nine bacteria (described below). The adhesive behaviour and metabolic

response of selected bacteria to three chemically and structurally diverse surfaces has

been investigated.

3.2 Bacteria

Nine bacterial strains that differ not only by their source of isolation, i.e., being

pathogenic, opportunistic, soil and marine but also by surface and adhesive

characteristics were used throughout the experimental designs. They are of exceptional

academic significance, and have been a subject of intensive research over the last

decade. Amongst bacteria selected for this project, Escherichia coli K12,

Staphylococcus aureus CIP 68.5 and Pseudomonas aeruginosa ATCC 9027 are well

studied terrestrial bacteria in regards to their pathogenic effects. Cobetia marina DSM

4741T, Pseudoalteromonas issachenkonii KMM 3549

T, Salegentibacter flavus CIP

107843, Staleya guttiformis DSM 11458T, Sulfitobacter mediterraneus ATCC 700865

T

and Alivibrio fischeri DSM 507T, are marine bacteria with significant environmental

impact.

3.2.1 Non-marine bacteria

3.2.1.1 Escherichia coli K12

E. coli is a Gram negative, oxidase-negative bacterium belonging to the

Enterobacteriaceae family. This bacterium is a facultative anaerobe having both a

respiratory and a fermentative type of metabolism (Garrity, 1984, Castellani and


______________________________________________________________________

49

Chalmers, 1919). E. coli cells are elongated, 1–2 µm in length and 0.1–0.5 µm in

diameter. E. coli K12 is a noteworthy producer of EPS and LPS consisted of three

components; keto-deoxy-octulonate (KDO), core polysaccharide and O-antigen (Burks

et al., 2003).

E. coli K12 is harmless inhabitant of the human intestinal tract and has a long

history of being in the focus of intense metabolic, biochemical and genetic

investigations (Nelson and Cox, 2000). As such remains the best-studied bacterium and

the primary reference organism (Nelson and Cox, 2000).

3.2.1.2 Pseudomonas aeruginosa ATCC 9027

P.aeruginosa is a Gram-negative, aerobic rod belonging to the Pseudomonadaceae

family. Cells are 0.5-0.8µm in diameter and 1.5- 3.0µm long, motile by means of a

single polar flagellum. This bacterium has simple nutritional requirements; while grown

on nutrient agar can produce three types of colonies: small rough, smooth and mucoid,

the last two morpho-types are mostly formed by the clinical isolates. Bacteria belonging

to the Pseudomonadaceae family are ubiquitous inhabitants that are regularly isolated

from the surfaces of plants, soil and occasionally animals. P.aeruginosa is regarded as

opportunistic pathogen that breaks the host defense system and is capable of causing

urinary, respiratory, gastrointestinal, bone and joint infections and a variety of systemic

infections (Todar, 2007). These pathogenic affects are particularly prominent in

immunocompromised, hospitalized, patients with cancer, cystic fibrosis, and burns

(Todar, 2007). This bacterium caused considerable interest in the past years, mostly

because of its strong propensity towards biofilms formation on biological (e.g., on lung

tissue in cystic fibrosis) and abiotic surfaces (w.g., contact lenses, catheters, implants,

etc.) (Bruinsma et al., 2002, Donabedian, 2003, Kim et al., 2007, Ryder et al., 2007,

Wagner et al., 2006).


______________________________________________________________________

50

3.2.1.3 Staphylococcus aureus CIP 68.5

S. aureus belongs to the genus Staphylococcus of the family Staphylococcaceae. S.

aureus forms large, round golden-yellow colonies on rich medium and is β-hamolytic if

grown on blood agar. The bacterium can grow at high NaCl concentrations and wide

range of temperature variations; from 15 to 45°C. All Staphylococci are catalase-

positive, oxidase-negative facultative anaerobes. In 1884 two colony types of

staphylococci were described by Rosenbach, the yellow pigmented, S. aureus and the

white pigmented Staphylococcus albus later on named Staphylococcus epidermidis1. S.

aureus is invariable inhabitant of the nasal passages and skin surfaces in approximately

20% of healthy humans, hence should always be considered as potential pathogen

(Todar, 2007). S. aureus can cause various infections mostly due to the expression of

surface proteins that can promote attachment (Todar, 2007).

3.2.2 Marine bacteria

3.2.2.1 Cobetia marina DSM 4741T

C. marina DSM 4741T belongs to the genus Cobetia of the family

Halomonadaceae12

. There has been certain controversy in the taxonomic affiliation of

this bacterium over the years, mostly because of the heterogeneity of the genus

Halomonas (Arahal et al., 2002). The bacterium is an aerobic, straight, rod shaped,

Gram negative. Cell size ranged from 0.8-1.2µm wide and 1.6-4.0µm long. Cells are

motile by means of two-five peritrichous flagella. Can occur single or in pairs and forms

round, bright, smooth, creamy colonies. Bacteria of this species are known by their

particular metabolic activity such as production of alkaline phosphatases (APs) with

high specific activity, etc (Plisova et al., 2004).


______________________________________________________________________

51

3.2.2.2 Pseudoalteromonas issachenkonii KMM 3549T

P. issachenkonii is a Gram negative, rod-shaped, 0.7-0.9 µm in diameter and 1.0 -

1.2 µm long bacterium, motile by single polar flagella. It is also oxidase and catalase

positive, forms uniform round colonies, 2-3 mm in diameter (Silipo et al., 2004). This

bacterium derived from a symbiotrophic association of the degraded thallus of brown

algae Fucus evanescens and was identified as a novel species of the genus

Pseudoalteromonas of the Gammaprotobacteria (Ivanova et al., 2002b). The latter is a

group of abundant marine prokaryotes that carry out several critical ecological

functions, including the reduction and/or oxidation of sulphur compounds,

biodegradation of hydrocarbons and other compounds (Garrity, 1984). Members of this

family coexist in complex symbiotic associations with other microorganisms (Ivanova

et al., 2002b).

3.2.2.3 Salegentibacter flavus CIP 107843T

S. flavus is a new species in the genus Salegentibacter of the family

Flavobacteriaceae that includes a rather complex group of halophilic organisms, many

of which are psychrophilic(Ivanova et al., 2006b). The taxonomic structure of this group

has been progressively unraveled following the emended description of the family

Flavobacteriaceae (Bernardet et al., 2002). S. flavus Fg 69T is a Gram negative, aerobic,

non-motile, asporogenic bacterium. Cells are rod shaped 0.5-0.7µm wide and 2.5 - 4.0

µm long and form circular colonies 1-3 mm in diameter when grown on marine agar at

22-25ºC. This bacterium was first isolated from sediment sample collected in Chazma

Bay (Sea of Japan) that has been radioactively contaminated as a result of a nuclear

submarine accident (Ivanova et al., 2006b).

3.2.2.4 Staleya guttiformis DSM 11458T

The genus Staleya so far comprises only one species, S. guttiformis (Labrenz et al.,

2000), that was isolated from hypersaline, heliothermal and meromistic Ekho Lake


______________________________________________________________________

52

(Vestfold Hills, East Antarctica), bacteria of this taxonomic lineage, namely marine

Alpharoteobacteria, from ‘Roseobacter–Sulfitobacter–Silicibacter’ group (Sorokin,

1995, Staley, 1968, Wagner-Döbler et al., 2003, Vogler, 1998), represent the second

most abundant 16S rRNA gene clone type in marine environments (Rappé et al., 2000)

playing an important role in nutrient cycling, e.g. by oxidation or degradation of

sulphite (Sorokin, 1995),(Pukall et al., 1999), dimethyl sulfoniopropionate (González et

al., 2003), methylamine (Doronina et al., 2000), lignin (González et al., 1997), aromatic

compounds (Buchan et al., 2000), etc. This is a Gram-negative rod bacterium, motile

via flagella and forms smooth, circular pink-brownish colonies. Taking into

consideration that bacteria belonging to this group are poorly studied in the context of

their attachment capabilities, the present study is an extension of our investigation to

probe the attachment of marine Alpharoteobacteria and their biofilm formation on

polymeric surfaces (Ivanova et al., 2002a, Ivanova et al., 2006a).

3.2.2.5 Sulfitobacter mediterraneus ATCC 700865T

The genus Sulfitobacter was first established in 1995 involving only two

heterotrophic strains that were initially isolated from H2O/O2 interface in the Black

Sea(Sorokin, 1995). Few years later, bacteria of this genus were detected in samples

isolated from the hyper-saline Ekho Lake of east Antarctica and natural seawater

collected from the Mediterranean Sea (Pukall et al., 1999). At present this genus

comprises three species, Sulfitobater pontiacus, S. mediterraneus and Sulfitobacter

brevis. Recent data suggest that they are rather ubiquitous marine bacteria widely

distributed in coastal and open-sea environments at the Black Sea, Sargassao Sea, the

Mediterranean Sea the South China Sea and the Japan Sea where they most likely play

an important role in the organic sulfur process (Ivanova et al., 2002a). They all are

Gram negative, aerobic members of the α-Protobacteria closely related to the

Roseobacter genus. S. mediterraneus ATCC 700865T

is non-sporogenic, catalase and

oxidase positive strictly anaerobic bacterium. Calls were found to be 1-3µm long and

0.5-0.8µm in diameter, motile by means of 1-5 subpolar flagella(Pukall et al., 1999).

Bacteria are readily cultivated on marine agar on 25°C; in cases of cultivation on marine

agar with supplemented acetate cells tend to form rosettes. Although typically rod-


______________________________________________________________________

53

shaped it has been confirmed that under unfavourable conditions and prolonged

incubation, 24-72h, S. mediterraneus cells can undergo morphological change from

vegetative into coccoid forms (Ivanova et al., 2002a). The same study further confirmed

that these coccoid bodies can be easily resuscitated in standard nutrient media, pointing

out to their viability and cultivability.

3.2.2.6 Alivibrio fischeri DSM 507T

A. fischeri belongs to the Vibrionaceae family, a large family

of marine

Gammaproteobacteria (Ruby et al., 2005). A. fischeri is a Gram negative, facultative

anaerobe capable of both fermentative and respiratory metabolism ref. Species of the

Vibrio genus are straight or slightly curved rods, 0.5 - 0.8 µm wide and 1.4 - 2.6 µm

long. A. fischeri also possesses 2-8 polar flagella (Skerman et al., 1980). The

luminescent A. fischeri is best known as the specific symbiont in the light-emitting

organs of eukaryotic hosts (fish, squids) (Ruby, 1996), where it produces luminescence

by expressing the lux operon, a small cluster of genes found in several Vibrio species.

Luminescence is controlled by acyl-homoserine lactone quorum sensing, which was

first discovered in A. fischeri but is a common feature of host-associated bacteria in a

number of genera (Whitehead et al., 2001, Miller and Bassler, 2001).

3.2.3 Culture conditions, attachment experiments and staining protocols

3.2.3.1Culture conditions

All marine bacteria, P. issachenkonii, C. marina, S. flavus, S. guttiformis and A.

fischeri were routinely cultured on marine agar 2216 (Difco) plates and stored at –80°C

in marine broth (Difco) supplemented with 20% (v/v) glycerol as described elsewhere

(Ivanova et al., 2002a). E. coli and P.aeruginosa, and S. aureus were routinely cultured

on nutrient agar (Merck) plates and stored at -80°C in storing solution prepared of ¾

nutrient broth and ¼ glycerol. Fresh bacterial suspension was prepared prior to each


______________________________________________________________________

54

experiment with marine (Difco) or nutrient (Merck) agar, depending on the bacterial

strain.

3.2.3.2 Bacterial attachment experiments

• Bacterial adsorption on nano-structured glass surfaces (as-received and

chemically modified)

The experimental set up was designed as follows: prior to each experiment, a fresh

bacterial suspension of OD(600) between 0.2-0.3 was prepared from bacterial cells grown

in marine/nutrient broth at room temperature (ca 25°C) for 24 hours. The optical density

for all bacterial suspensions was adjusted to OD600 0.2-0.3 on GeneQuant Pro

Spectrophotometer (Amersham Biosciences) (OD600 1= 8x108

cells/ml).

A portion of 3-5 ml of bacterial suspension was poured into a sterile Petri dishes

where the glass slides (one glass slide per Petri dish) were completely immersed and left

for 12 h at room temperature (ca 25°C). All of the slides were washed with nanopure

H2O (18.2 MΩcm-1

Barnstead/Thermolyne NANOpure® Infinity water purification

system) after incubation. This approach allowed the experiments for bacterial

attachment to be performed under identical conditions for each half of each microscope

slide (Mitik-Dineva et al., 2008a, Mitik-Dineva et al., 2008b).

• Bacterial adsorption on nano-structured P(t)BMA polymer surfaces (native

and photolithographycally modified)

Sterile Petri dishes (Interpath Services, Pty Ltd) were inoculated with log-phase

culture (3-5 ml); the cell density was adjusted as previously described. Glass cover slips

(22 x 60mm, 1oz, Deckgläser) covered with thin layers of P(t)BMA, native and

photolithographycally modified, were completely immersed in cell culture and

incubated at 25°C for 12 hours. After incubation all glass cover slips were rinsed three

times with sterilized nanopure H2O (18.2 MΩcm-1

Barnstead/Thermolyne NANOpure®


______________________________________________________________________

55

Infinity water purification system) and left to dry at room temperature (ca. 22ºC, 45%

humidity) without additional fixation to prevent cell deformation. Polymer slides

containing statistically grown bacteria were imaged on the same day in order to avoid

cell deformation. Duplicate independent experiments and triplicate samples were

performed.

• Bacterial adsorption on optical fibres (as received and chemically modified)

Prior to each experiment bacterial cells were grown in marine/nutrient agar for 24

hours. On the day of the experiment 2 ml of the suspension with OD(600) adjusted to 0.2-

0.3 depending on the strain was stored in centrifuge tubes (Interpath Services, Pty Ltd).

Duplicate fibre samples were placed into each of the tubes and were incubated for 12

hours at room temperature (ca 22°C). After incubation, all fibres were rinsed three times

with sterilized nanopure H2O (18.2 MΩcm-1


Infinity water purification system) and kept under sterile condition until further

examination.

3.2.3.3 Fluorescent labelling of produced EPS and viable cells

Two dyes were used in order to simultaneously visualize viable cells and their

production of extracellular substances while attaching to any of the tested surfaces.

CFDA SE Vybrant Cell Tracer (Molecular Probes Inc.) was used to colour viable cells

and Concanavalin A 488 (Molecular Probes Inc.) was used to label EPS.

Concanavalin A Alexa Fluor® 488 Conjugate (Molecular Probes Inc.), was applied

in order to visualize EPS. This dye selectively binds to α-mannopyranosyl and α-

glucopyranosyl residues in EPS (Goldstein et al., 1964). In neutral and alkaline

solutions, this bright green dye exists primarily as a tetramer with a molecular weight of

104,000 daltons (Sumner and Howell, 1936). The Alexa Fluor 488 conjugate was

applied as it is superior to other spectrally similar conjugates such as fluorescein,

exhibiting more intense fluorescence and photostablity, allowing more time for image

capturing. Fluorescence of the Alexa Fluor 488 fluorophore is independent of pH from

4 to 10. This pH insensitivity is a major improvement over fluorescein, which emits


______________________________________________________________________

56

fluorescence that is significantly affected by pH (Invitrogen, 2006). At the same time

the wide range of pH stability allowed simultaneous use with the carboxyfluorescein

diacetate, succinimidyl ester (Vybrant CFDA SE Cell Tracer Kit) and scanning of the

same field of view for both, viable cells as well as synthesized EPS. Concanavalin A

stock solution was prepared by dissolving 5 mg in 5 ml of 0.1 M sodium bicarbonate at

pH 8.3 and stored at 20°C. Working solution was prepared by diluting stock solution to

1:20 using the same buffer to avoid changes in pH. Excitation and emission

wavelengths for Concanavalin A are 495 and 519 nm, respectively. It is important to

mention that the overall distribution of the green fluorescent signal on top of the

cell/substratum surface will very much depend on the chemical composition and the

distribution of the produced EPS. Staining cells that produce capsular-like EPS

overlaying the whole cell surface that contain α-mannopyranosyl and α-glucopyranosyl

residues as their main constituents will result in green fluorescent bacterium shaped

signal on the CLSM. Contrary to this, the fluorescent signal from cells producing lumpy

like EPS on their surface will not represent the cells couture.

The Vybrant CFDA SE Cell Tracer dye was applied in order to trace viable cells

adsorbed on each of the probed surfaces. This assay uses carboxyfluorscin diacetate

succinimidyl ester (CFDA SE) that successfully labels viable cells. The kit contains

CFDA SE (carboxyfluorescein diacetate, succinimidyl ester) that is initially colourless

and nonfluorescent. It passively diffuses into cells where the acetate groups are cleaved

by intracellular esterases to yield highly fluorescent, amine-reactive carboxyfluorescein

succinimidyl ester. The dye–protein adducts that form in labelled cells are retained by

the cells and inherited by daughter cells after division. CFDA SE stock solution

(10mM) was diluted to 20µM in PBS and was further used as working solution.

Working solutions of the dye as well as cell labelling conditions were prepared as

described elsewhere (Invitrogen, 2006). The excitation and emission wavelengths for

CFDA SE are 495 nm and 517 nm, respectively. In general glass, polymer or fibre

substrates were incubated in the bacterial suspension for 11h before an aliquot of

Concanavalin A 488 was added in ratio 1:5 (cell suspension/dye). The dye was allowed

1h to diffuse when CFDA SE dye, in the same ratio, was added to the suspension and

incubated for additional 15min at 37°C.


______________________________________________________________________

57

After incubation the samples were washed with sterilized nanopure H2O (18.2

MΩcm-1

), left to dry for several hours at room temperature without additional fixation to

prevent the deformation of the cells and further processed.

3.3 Surfaces

3.3.1 Glass

The surfaces of standard glass microscope slides (7105-PPA premium glass slides,

Livingstone International) were chemically modified (etched) by treatment with a

buffered solution of hydrofluoric acid to achieve a nanometer scale variation in surface

roughness. In particular, one half of each slide was treated by dipping it into the

buffered hydrofluoric (BHF) etching solution for 20 minutes (White and Stoddart,

2005). This resulted in different topographical characteristics on each half of the same

glass slide. The chemical composition of the buffered hydrofluoric acid was as follows:

6 parts of 40 % ammonium fluoride NH4F, 1 part of 49 % HF hydrofluoric acid and 14

parts of 36.8% HCl hydrochloric acid. All slides were thoroughly washed with sterile

nanopure H2O (18.2 MΩcm-1

Barnstead/Thermolyne NANOpure® Infinity water

purification system) and stored in 96% alcohol (Aldrich). Prior to each experiment the

slides were washed again with deionised water and placed in sterile Petri dishes

(Interpath Services Pty Ltd, AU, catalogue number 632-180).

3.3.2 Polymers

3.3.2.1 Overview

The model polymeric surface selected for probing bacterial adhesion was Poly-

(tert) butyl methacrylate P(t)BMA (Ivanova et al., 2006c). This particular polymer was


______________________________________________________________________

58

selected simply because it is a hydrophobic surface, hence supportive of bacterial

adhesion. Furthermore, its influence on bacterial adhesion was already tested and

revealed some interesting notion, that bacteria might undergo morphological changes in

order to sustain their survival on this surface (Ivanova et al., 2006c).

P(t)BMA is a photosensitive polymer, which is classed as a “photoresist”. These

polymers endure a photochemical reaction when exposed to either UV light, visible

light, electrons, ions or X-rays, where the physical properties are subject to change due

to their ability to alter the orientation of their surface functional groups (Jaeger, 2002).

Photoresists are classified into two groups; positive and negative. When exposed to

UV light, the chemical structure of a positive resist is changed so that the polymer is

weakened and becomes more soluble in the photoresist developer, meaning the resist is

washed away where the light struck it, transferring a positive image to the resist layer. A

negative photoresist reacts in an opposite manner and becomes relatively insoluble to

the photoresist developer, meaning the resist that was not exposed to UV light is washed

away and a negative image is transferred to the resist (Jaeger, 2002).

P(t)BMA was originally designed as a positive photoresist in which chemical

contrast is readily induced through exposure to deep UV irradiation (Osaki and Carsten,

2003). This polymer has been frequently employed as a positive photoresist due to its

excellent mechanical and optical properties, e.g. transparency (>90% transmission),

stiffness, low water absorption and high abrasion resistance (Ivanova et al., 2006c).

P(t)BMA in its as-received state is typically hydrophobic, this is thought to be primarily

due to the presence of methyl (CH3) groups on the polymer backbone and the tert-butyl

ester (Raczkowska et al., 2004).

3.3.2.2 Polymer film preparation

Polymeric films were prepared as described elsewhere (Ivanova et al., 2006c).

Briefly, a 4 wt% solution of P(t)BMA (MW~170,000) in propylene glycol methyl ether

acetate (PGMEA), (Sigma Aldrich Co.) 99% was used. Polymer films were prepared on

22 x 60 mm glass substrates (glass cover slips, 1oz, Deckgläser) that were previously

sonicated in isopropanol (PriOH) for 30 min, washed with copious amounts of sterile

nanopure H2O (18.2 M cm-1

, Barnstead/Thermolyne NANOpure® Infinity water


______________________________________________________________________

59

purification system), and dried under a stream of high purity nitrogen prior to priming

with hexamethyldisilazane (HMDS) (Sigma Aldrich Co.). Primer was spun at 1000 rpm

for 15 seconds and polymers at 3000 rpm for 40 seconds using a Specialty Coating

Systems spin coater (Model P6708). Finally, polymer covered slides were post-

exposure baked for 60 minutes at 950C and stored in a desiccators prior to use.

3.3.2.3 Photolithography

Photolithography was carried out as described elsewhere (Ivanova et al., 2006c).

The as-received P(t)BMA substrates, prepared as described above, were exposed to UV

light (254 nm, 760µWcm-2

) for 10 minutes. The UV-irradiated sample was post-

exposure baked at 90oC for 20 minutes to facilitate diffusion of the photo-generated acid

thereby initializing tert-butyl ester deprotection. Excess PAG was washed away with

ethanol. The sample was dried at room temperature and stored in desiccators prior to

use.

3.3.3 Optical fibres

3.3.3.1 Overview

The effects of micro-scale surface roughness on bacterial adhesion were studied by

exposing bacteria to spatially designed surfaces with fabricated micro-scale surface

topographies. Model surfaces used for this purpose were optical glass fibres FIGH-70-

1300N (Fujikura Ltd). This is a standard optical/imaging fibre, with approximately

10000 picture elements, and total outer diameter of 1.3mm. According to the

manufacturer specification, these fibres are made from silica glass cores surrounded by

fluorine-doped silica cladding. This exact chemical structure of the fibres itself and the

surrounding cladding was the base for further modification achieved by etching the fibre

surface with buffered hydrofluoric acid. The final result was occurrence of typical,

micro-scale rough honeycomb pattern on the fibre surface.


______________________________________________________________________

60

Differences in bacterial adhesive behaviour were observed by comparing cell-

surface interaction on the as-received and on the fabricated fibre surface after the

exposure to the etching solution.

3.3.3.2 Surface preparation

The fibre as purchased from the manufacturer was 100 cm long bar. Using an

automatic dicing saw (DISCO DAD 321 Automatic Dicing Saw, Equipment

Acquisition Resources Inc.) and diamond blade (NBC-ZH 2050-J-SE, 0.09mm),

workable 5 mm fibre subdivisions were produced. As the fibre through the whole

length is protected with silicone coating, 99% Aldrich acetone was used to remove this

coating. All fibres were initially washed with copious amounts of sterile nanopure H2O

(18.2 MΩcm-1

Barnstead/Thermolyne NANOpure® Infinity water purification system),

sterilized at 121ºC for 15 minutes and stored under sterile conditions until prior to use.

SEM images of the final working surface area are presented in Figure 3.1.

(a) (b)

Figure 3.1: SEM images of the as-received optic fibre surfaces, scale bar 250µm on

image (a) and 1µm on image (b)


______________________________________________________________________

61

3.3.3.3 Surface modification

In order to fabricate micro-scale surface topographies, number of the 5 mm long

fibre subdivisions were exposed to BHF (buffered hydrofluoric acid) by dipping the

fibre in the acid solution for certain period of time (White and Stoddart, 2005). The

ultimate effect after the acid exposure is actually based on the difference in the chemical

structure between the fibre components. The cladding around each picture element

etches at a slower rate than the silica core, thus resulting in a defined honeycomb pattern

on the fibre surface, with each well being approximately 2.5µm in diameter and 2.5µm

deep. The time of etching can be optimised depending on the preferred well size. Since

the requirements for this study were to attain well size same or double the bacterial size

(2.5µm x 2.5µm ± 15%), fibres were acid treated for 20 minutes. SEM images of the

fibre surface after treatment with the etching solution are presented in Figure 3.2. After

the exposure to the etching solution all samples were rinsed with sterile nanopure H2O

(18.2 MΩcm-1

Barnstead/Thermolyne NANOpure® Infinity water purification system)

number of times, sterilized on 121ºC for 15 minutes and kept under sterile conditions

just prior to inoculation.

(a) (b)

Figure 3.2: SEM images of the optic fibre after exposure to the etching solution for

20min. Scale bar equals 250µm on image (a) and 1µm on image (b)


______________________________________________________________________

62

To ensure excess BHF from the fibre surface was removed simple screening

technique was employed; after fibres were washed the dissipated solution was tested for

acidity with Phenolphthalein and Bromothymol blue indicator dyes. The deficient

change in colour was considered sufficient indicator of BHF deficiency on the fibre

surfaces.

3.4 Qualitative analyses of the abiotic and biological surfaces

3.4.1 Contact angle measurements

Bacterial and substrata surface wettability was inferred from contact angles

measurements. To determine the surface tension components of surfaces, it is necessary

to perform contact angle measurements using probe liquids with well-known surface

tension properties. Most frequently used diagnostic liquids are water (LW

= 21.8 and +

= - = 25.5 mJ m

-2) and formamide (

LW = 34.0,

+ = 3.92 mJ m

-2, and

- = 57.4 mJ m

-2)

as polar, and diiodomethane (LW

= 50.8 and + =

- = 0 mJ m

-2) as apolar(Ong et al.,

1999, Brant and Childress, 2002). For the purpose of this study water was selected as

the most suitable diagnostic liquid since it can be used for measurement of contact

angles on tested abiotic surfaces as well as on lawns of bacterial cells for determent of

the cell surface wettability (Dong et al., 2002).

3.4.1.1 Bacterial surface wettability

For the purpose of measuring cell surface wettability static contact angles were

measured using the sessile drop method. Bacterial cells suspension was prepared by

growing the cells overnight in nutrient/marine broth. Cell were than harvested by

centrifugation and resuspended in 0.1M NaCl buffer (OD470 =0.4). This suspension was

deposited on cellulose acetate membrane filters (Sartorius, pore diameter 0.2µm, filter

diameter 47mm). The wet filters were left on ambient temperature for approximately

30-40 minutes to air dry until a “plateau state” (Korenevsky and Beveridge, 2007),

(Bakker et al., 2002).


______________________________________________________________________

63

The FTA200 equipped with charge-coupled device (CCD) camera was used to

observe the droplet contact angle. After the initial deposition the drop was allowed to

settle for 2 seconds without needle contact (for static contact angle measurements).

Images were digitally saved and contact angle values obtained by processing the image

with the accompanied program (Korenevsky and Beveridge, 2007) (Bakker et al.,

2002).

3.4.1.2 Substratum surface wettability

The glass surface wettability was inferred by advancing contact angle

measurements using the embedded needle method (Öner and McCarthy, 2000, Quéré et

al., 2003). For advancing contact angle prior to measurements each glass slide was

washed with sterile nanopure H2O (18.2 MΩcm-1


Infinity water purification system) and left to air dry in sterile Petri dish (Interpath

Services, Pty Ltd). FTA200 was used again, but in this instance movies were taken, five

for each of the surfaces, as received and modified. Each of the movies delivered up to

100 images for later analysis. After magnification, images were analysed using the

instrument software. The final values represent the average of those measurements.

The sessile drop method was also used for measurement of the polymer surface

wettability for both, the as-received and the UV-modified. Images of static, water

contact angles (θ) were taken using the FTA200. The accompanying software was used

for processing the data after what observed values were averaged over six different

readings.

Surface wettability of both, the as-received and the chemically eroded fiber surface

was inferred from water contact angle measurements using the FTA200 and the sessile

drop method. FTA200 was used for liquid deposition and image capturing. Captured

images were digitally saved and contact angle data was obtained by processing the

images with the accompanied software. Prior to measurements all fibres were washed

with sterile nanopure H2O (18.2 MΩcm-1

Barnstead/Thermolyne NANOpure® Infinity

water purification system) and left to air dry. Selected fibres (as-received and modified)

were mounted onto rigid (micro-slide glass) support thus enabling/maintaining vertical

position required for deposition of 1µl water droplet onto the fibre surface. Due to the


______________________________________________________________________

64

limited fibre surface (1.33mm2) each fibre was used for only single measurements.

Experiments were repeated 10 times and the final values represent average of those

measurements.

3.4.2 Surface free energy

Apart from experimental measurement of contact angles of specific diagnostic

liquids, characterization of the surface’s wettablity can be evaluated by calculating the

surface free energy or the surface tension. To determine the surface tension it is

necessary to perform contact angle measurements using probe liquids with well-known

surface tension properties. Most frequently used diagnostic liquids are water and

formamide as polar, and diiodomethane as apolar (Ong et al., 1999, Brant and

Childress, 2002). Their surface tension parameters are presented in Table 3.1

Table 3.1: Surface tensions and its parameters (mJ/m2) of common solvent in the

measurement of contact angles.

γlv γlvLW

γlvAB

γlv+

γlv-

Water 72.8 21.8 51.0 25.5 25.5

Formamide 58 39 19 2.28 39.6

Diiodomethane 50.8 50.8 ~0

3.4.3 Surface charge measurements

Bacterial and substratum cell surface charge was inferred from the zeta potential

measurements. Zeta potentials provide an indication of the overall net surface charge

and can be obtained by measuring the electrophoretic mobility (EPM) (de Kerchove and

Elimelech, 2005, Eboigbodin et al., 2006, Pearson et al., 2004, van Merode et al., 2007),

(Sanders et al., 1995).


______________________________________________________________________

65

3.4.3.1 Bacterial surface charge

Bacterial cell surface charge was inferred from the zeta potential measurements. It

is important to always have pure cell cultures when measuring cell surface charge, as

the presence of subpopulations with different surface properties in single-strain cultures

can lead to erroneous conclusions (van der Mei and Busscher, 2001). Even then it is to

be remembered that no ζ potential value can be assigned to bacteria at the strain and

species level and that even different isolates of the same strain can express different ζ

potentials (Busscher and Norde, 2000).

The EPM was measured as a function of ionic strength by microelectrophoresis

using a zeta potential analyser (ZetaPALS, Brookhaven Instruments Corp). The data

were processed with the accompanying software, which employs the Smoluchowski

equation. Cell suspensions were prepared as follows; after 24 hours growth in

nutrient/marine broth, cells were harvested by centrifugation for 5 minutes at 5000 rpm.

Harvested cell pallets were re-suspended in 10 mM potassium chloride (KCl) and then

washed and centrifuged again. This step was repeated four times to eliminate residual

extracellular polysaccharides that may influence the surface electric potential. After the

final wash, cell pallets were re-suspended in 10 mM KCl solution to OD(600nm) = 1, as

suggested by De Kerchove and Elimelech (de Kerchove and Elimelech, 2005). This cell

solution was then diluted 1000 times in 5 ml 10mM KCl, pH 7.5, for use in the EPM

measurements. Measurements were conducted in electric field of 2.5 V cm-1

and

frequency of 2 Hz (Eboigbodin et al., 2006). All measurements were done in triplicates

and for each sample the final EPM represents the average of 5 successive ZetaPALS

readings, each of which consisted of 14 cycles per run.

3.4.3.2 Substratum surface charge

Similarly as cell surface charge, the substratum charge was also inferred by

measuring zeta potentials and electrophoretic mobility as described elsewhere (Sanders

et al., 1995). For the purpose of measuring the glass surface charge small pieces of both,

acid treated and the as received, glass surfaces were grinded using ceramic grinder in


______________________________________________________________________

66

order to prevent contamination into fine particles. Obtained samples were suspended in

5 ml of 0.01M NaCl, the suspension concentration being 0.01g/100ml. The optical

density of the obtained suspension was adjusted to OD450=0.2 and measurements were

taken(Sanders et al., 1995).

As for the polymer surface charge, polymer coated slides were prepared in the

same manner as described in Chapter 3.3.2. The few nanometres thick polymer film was

removed from the covers slips using plastic spatula in order to avoid abrasion of the

glass and contamination of the specimen. Obtained polymer fragments were

resuspended in 5ml of 0.01M NaCl (concentration 0.01g/100ml). In order to obtain

homogeneous mixture containing only micro-size particles the suspension was blended

in stainless steel blender. Same as for the glass, the suspension absorbance was

measured, optical density was adjusted to OD450=0.2 and measurements were taken.

3.4.4 AFM characterization of the surfaces

A scanning probe microscope (SPM) (Solver P7LS, NT-MDT) was used to image

the glass, polymer and fibre surface morphology and to quantitatively measure and

analyse the surface roughness. The analysis was performed in the semi-contact mode

which reduces the interaction between the tip and sample and thus allows the

destructive action of lateral forces that exist in contact mode due to be avoided. The

carbon “whisker” type silicon cantilevers (NSC05, NT-MDT) with a spring constant of

11 N/m, tip radius of curvature of 10 nm, aspect ratio of 10:1 and resonance frequency

of 150 KHz were used to obtain good topographic resolution. Scanning was performed

perpendicular to the axis of the cantilever at a typical rate of 1 Hz. Image processing of

the raw topographical data was performed with first order horizontal and vertical

levelling and the topography and surface profile of the samples were obtained

simultaneously. In this way the surface features of the samples were measured with a

resolution of a fraction of nanometer and the surface roughness of the investigated areas

could be statistically analysed using the standard instrument software (LS7-SPM

v.8.58).


______________________________________________________________________

67

3.4.5 Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIM)

The Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) uses a pulsed

primary ion beam, typically liquid metal ions such as Ga+ and Cs

+ to bombard and

ionize species from a sample surface. The resulting secondary ions that emit from the

surface are then electrostatically accelerated into a mass spectrometer, where they are

mass analysed by measuring their time-of-flight from the sample surface to the detector.

TOF-SIMS can provide mass spectroscopy for surface chemical characterization,

images to visualize the distribution of individual chemical species on the surface and

depth profiles for thin film characterization and can be used for surface analysis of

inorganic, organic materials and biological cells, applied to conductors, insulators and

semiconductors.

The primary requirement from this investigation was to enhance our understanding

of the fibre surface chemistry and thereafter determine any difference in the surface

composition between the two fibre surfaces that may have influenced the cells’

behaviour. All measurements were performed using a ToF-SIMS IV instrument (ION-

TOF GmbH, Munster, Germany) with a reflection analyser and a pulsed electron flood

source for charge neutralization. Both positive and negative spectra were acquired from

a 100 µm × 100 µm area. Samples were exposed to the atmosphere for less than 5 min

during mounting in the TOF-SIMS instrument. All experiments were performed using a

cycle time of 100 µs. A monoisotopic 69

Ga+ primary ion source was operated at 25 keV

in the “burst alignment” mode, which gives very high spatial resolution at the expense

of mass resolution and positive and negative spectra were acquired with a mass

resolution typically greater than 6000 at m/z = 27, sufficient to identify most of the

fragments. The spectra acquired were analysed using the accompanying software.

3.4.6 X-ray Photoelectron Spectroscopy (XPS)

The chemical composition of both glass surfaces and the polymer P(t)BMA was

analysed using an Axis Ultra spectrometer (Kratos Analytical Ltd., UK), equipped with

a monochromatised X-ray source (Al Kα, hν = 1486.6 eV) operating at 150 W. The


______________________________________________________________________

68

spectrometer energy scale was calibrated using the Au 4f7/2 photoelectron peak at

binding energy (EB = 83.98 eV). Photoelectrons emitted at 90° to the surface from an

area of 700 x 300 µm2 were using 160 eV survey spectra and 20 eV for high-resolution

region spectra for selected elements (O 1s, C 1s, Ca 2p, N 1s, Si 2p) at 285.0 eV. The

relative atomic concentration of elements detected by XPS was quantified from the area

of peaks in the survey spectra sensitivity factors appropriate for the Kratos instrument

(Mitik-Dineva et al., 2008a, Mitik-Dineva et al., 2008b, Ivanova et al., 2008).

3.4.7 X-ray fluorescence spectroscopy (XRF)

Since the levels of few of the elements detected on the glass and the polymer

surface by XPS were close to the sensitivity limit, the obtained differences in the

relative contributions could not be regarded as significant. In this light further detail

regarding the chemical composition of the glass and the polymer surface were inferred

through XRS. Both samples were prepared by accurately weighing approximately

500mg +/- 0.1mg of each sample into 95%Pt/Au crucibles with approximately 5g +/-

0.1mg of 12-22 lithium tetraborate/metaborate flux previously dried at 550°C. The

sample was fused into a homogeneous melt over an oxy-propane flame at a temperature

of approximately 1050°C for approximately 10 minutes.

A commercially available ammonium iodide doped cellulose tablet was added

approximately 100 seconds before the molten glass was poured into a 32 mm diameter

95%Pt/Au mould heated to a similar temperature. Air jets then cooled the mould and

melt for approximately 300 seconds. The resulting glass discs were analysed on a

Philips PW2404 Wavelength Dispersive XRF spectrometer using an in-house

calibration and algorithms developed in this laboratory and control program developed

by Philips.


______________________________________________________________________

69

3.4.8 Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) was employed to provide more in depth

understanding of bacterial adhesion on all micro-nano structured surfaces used

throughout the experiments. A FeSEM – ZEISS SUPRA 40VP was used to obtain the

high-resolution images of the substratum surface, bacterial morphology as well as the

adhesion pattern. Primary beam energies of 3 to 15 kV were used, which allowed

features on the sample surface or within a few microns of the surface to be observed.

Prior to imaging all samples were mounted on pin type aluminium SEM mounts with

double-sided conducting carbon tape and then coated in Dynavac CS300 coating unit

with carbon and gold to achieve better conductivity of the specimen surface. The

thickness of the coating was not measured but should be in the order of few nm. The

working distance (WD) varied between 6-7mm, and images were mostly captured on

500x, 1000x and 1000x magnification. Control images of all surfaces before bacterial

inoculation, with and without broth were also taken.

3.4.9 Confocal scanning laser microscope (CSLM)

The confocal scanning laser microscope (CSLM) Olympus Fluoview FV1000

Spectroscopic Confocal System which included an inverted Microscope System

OLYMPUS IX81 (20X, 40X (oil), 100X (oil) UIS objectives) that operates using

multiple Ar, He and Ne laser lines (458, 488, 515, 543, 633 nm) was used. The system

was equipped with a transmitted light differential interference contract attachment and a

CCD camera (Cool view FDI). Excitation and emission wavelengths for Concanavalin

A and CFDA SE Vybrant are 495Em/Ex519 nm and 492Em/517Ex, respectively.

Chapter 4:

The effects of nano-structured glass surfaces on bacterial attachment

______________________________________________________________________

70

CHAPTER 4

Chapter 4:


______________________________________________________________________

71

THE EFFECTS OF NANO-STRUCTURED GLASS

SURFACES ON BACTERIAL ATTACHMENT

4.1 Bacterial surface characteristics

4.1.1 Overview

Number of factors, resolving from both, bacteria and glass (as-received and

modified) are believed to be responsible for the specific bacterial adhesive behaviour.

Bacterial surface properties such as wettability, charge and production and

composition of surface EPS as well as glass wettability, surface free energy, charge and

roughness are presented herein.

4.1.2 Cell surface wettability

Cell surface wettability is by far the most studied microbial surface characteristic

due to its imperative role in microbial adsorption. It is an important bacterial

characteristic that is mainly dependant on the presence of EPS and their geometrical

microstructure. Cell surface wettability varied among bacteria studied, most likely

reflecting the different chemical composition of surface-expressed polymeric

substances (Korenevsky and Beveridge, 2007). Obtained mean contact angles are

presented in Table 4.1. The water contact angles (θ) of all strains were in the range of

33-83°. Obtained values are consistent with already reported data for E. coli (Li and

Logan, 2004, Burks et al., 2003) and P. aeruginosa (Li and Logan, 2004). As for S.

aureus the contact angle reported in here, 72.23° ± 8°, is significantly higher that the

already reported 27° ± 4° by Vermeltfoort et al. (Vermeltfoort et al., 2005) This

difference can be attributed to the fact that in their study they have used different strain,

S. aureus 835. Due to this significant difference the surface wettability of two more S.

aureus strains, S. aureus ATCC 25923 and S. aureus ATCC 12600T, was also

measured. Obtained water contact angles of 49.85° ± 6° and 27.43° ± 3°, respectively

Chapter 4:


______________________________________________________________________

72

point out to the suggest that the vast variety in the surface wettability is most likely

type specific. The hydrohobic nature of S. aureus cells might be due to highly

negatively charged and hydrophobic teichoic and lipoteichoic acids which are the main

constitutes of S. aureus cell wall (Gross et al., 2001). Cell wall teichoic acids are

composed of a linear chain of approximately 40 1,3-phosphodiester-linked ribitol

phosphate residues linked to O6 of the N-acetylmuramyl residues of peptidoglycan

(Canepari et al., 1990). Lipoteichoic acids are composed of a single, unbranched 1,3-

linked poly(glycerophosphate) chain, which units may be partly substituted with

positively charged D-alanine ester (Canepari et al., 1990). In the latter case S. aureus

strains exhibit significantly less hydrophobic characteristics; as, for example was

reported by Vermeltfoort et al.(Vermeltfoort et al., 2005).

For C. marina, P. issachenkonii, S. flavus, S. guttiformis, S. mediterraneus and A.

fischeri cell surface wettability was evaluated for the first time, therefore there is no

data available to compare obtained results. If contact angle of 60-65° can be taken as

the borderline denoting hydrophobicity, according to definition by Vogler et al (Vogler,

1998), than one can foretell that the cell surface of E. coli, P. aeruginosa, P.

isschenkonii, S. flavus, S. guttiformis and S. mediterraneus is hydrophilic (Korenevsky

and Beveridge, 2007). Accordingly, only C. marina, S. aureus and A. fischeri exhibit

hydrophobic cell-surface character.

Table 4.1: Water contact angles of bacterial cell surfaces

Strain Water (θ)*

E. coli K12 33.0 ± 4

P. aeruginosa ATCC 9027 43.27 ± 8

S. aureus CIP 68.5 72.23 ± 8

C. marina DSM 4741T 75.14 ± 9

P. issachenkonii KMM 3549T 51.90 ± 3

S. flavus CIP 107843T 47.22 ± 6

S. guttiformis DSM 11458T 55.45 ± 4

S. mediterraneus ATCC 700865T 39.09 ± 7

Alivibrio fisheri DSM 507 T

83.19 ± 5

Chapter 4:


______________________________________________________________________

73

*Presented values represent average of 10 independent measurements

As expected, the most significant difference was between strains belonging to different

taxonomic groups.

Translated into adhesive tendency the results displayed below indicate that A.

fischeri, S. aureus, C. marina and S. guttiformis will have stronger propensity in

attaching to hydrophilic surfaces (Bos et al., 1999, Bruinsma et al., 2001). Contrary to

this E. coli, P. aeruginosa, P. issachenkonii, S. flavus and S. mediterraneus will adhere

better to hydrophobic surfaces due to the thermodynamically predicted preference of

the hydrophilic cell surfaces towards hydrophilic substrata, and hydrophobic towards

hydrophobic substrata (Bruinsma et al., 2001).

4.1.3 Cell surface charge

Apart from cell surface wettability, microbial electrokinetic properties are the other

most significant bacterial surface characteristic in predicting cellular behaviour while

attaching to number of surfaces. Measurement of the cells electrophoretic mobility and

its conversion to zeta potential using Smoluchowski’s approximation were used to

evaluate bacterial surface charge.

Surface charge values of the bacterial cells of nine selected strains are presented in

Table 4.2. Obtained results for E. coli, P. aeruginosa and S. aureus are within close

proximity to already reported statistics (Li and Logan, 2004, Gottenbos et al., 2001,

Soni et al., 2007). As for the other strains no data was reported yet.

Chapter 4:


______________________________________________________________________

74

Table 4.2:. Electrophoretic mobility and calculated zeta potential values on bacterial

cell surfaces*

Species

Electrophoretic

Mobility **

(µs-1

Vcm-1

)

Zeta

Potential (ζ)

(mV)

E. coli K12 -3.1 ± 0.6 -38.41 ± 0.3

P. aeruginosa ATCC 9027 -1.1 ± 0.1 -14.36 ± 0.7

S. aureus CIP 68.5 -2.7 ± 0.8 -35.15 ±1.0

C. marina DSM 4741T -2.5 ± 0.6 -32.50 ± 0.5

P. issachenkonii KMM 3549T -2.9 ± 0.2 -35.27 ± 0.2

S. flavus CIP 107843T -1.6 ± 0.7 -21.04 ± 0.4

S. guttiformis DSM 11458T -3.3 ± 0.5 -43.18 ± 0.2

S. mediterraneus ATCC 700865T -3.0 ± 0.1 -38.65 ± 1.1

A. fisheri DSM 507 T

-2.7 ± 0.7 -34.95 ± 0.9

* calculated using Smoluchowski’s equation (Gallardo-Moreno and Calzado-Montero,

2006)

** All measurements were done in triplicates and for each sample the final EPM

represents the average of 5 successive ZetaPALS readings, each of which consisted of

14 cycles per run.

All bacterial strains used in this study were fund to bear net negative surface

charge, which is in agreement with the well accepted notion that the majority of

microbial cell surfaces are negatively charged (van der Mei and Busscher, 2001,

Busscher and Norde, 2000). As the results indicate (Table 4.2), the least

electronegative species were P. aeruginosa and S. flavus with EMP ranging from

-1.1(µs-1

)(V/cm) to -1.65(µs-1

)(V/cm), respectively, followed by C. marina, P.

issachenkoii, S. aureus and A. fisheri displaying EMP in the range of - 2.5(µs-1

)(V/cm)

– 2.9(µs-1

)(V/cm) and the most electro-negatively charged cells at our experimental

setup were S. mediterraneus, E. coli and S. guttiformis with EMP above -3(µs-1

)(V/cm).

Previous studies have indicated that cell surface charge is inversely correlated with

bacterial adhesion (Li and Logan, 2004). In this light it can be expected that S.

Chapter 4:


______________________________________________________________________

75

guttiformis would exhibit weakest and P. aerugionosa strongest attachment

preferences.

Zeta potentials different from those presented here can be measured if different

bacterial strain, or heterogeneous culture containing subpopulations is used for

measurements (Soni et al., 2007, Gottenbos et al., 2001) or if cells are resuspended in

solution with ionic strength other than 10mM; increase of the solutions ionic strength

will result in decreased zeta potential, hence increased number of cells will be able to

successfully attach (Li and Logan, 2004, Camesano and Logan, 1998, Gross and

Logan, 1995, Jewett et al., 1995).

4.2 Substratum surface characteristics

4.2.1 Overview

Substratum surface properties such as wettability, surface tension and roughness

were characterised in order to comparatively differentiate the glass surfaces before and

after modification. Summary of the physicochemical characteristics of the as-received

and the modified glass surfaces is presented herein

4.2.2 Substratum surface wettability and surface tension

The substratum surface hydrophilicity have ubiquitous role in the cell-substratum

interactions. For this reason surface hydrophilicity of both, the as-received and the

modified glass surfaces was evaluated via advancing contact angle measurements

(Figure 4.1).

Chapter 4:


______________________________________________________________________

76

(a) (b)

Figure 4.1: Advancing water contact angles measured on the as-received (a) and on

the modified (b) glass surface

The values listed in Table 4.3 represent the average of multiple measurements.

They indicate that the glass surface was slightly hydrophilic with measured average

contact angle of 44° for the as-received and 41° for the modified surfaces.

Once the mean contact angle values for each of the diagnostic liquids have been

determined, the surface free energies (total, dispersive and acid-base) could be

calculated based on the theoretical model by Van Oss (van Oss, 1994, Bayoudh et al.,

2006). The surface free energy was estimated being 50.3 mJ/m2 and 48.7 mJ/m

2 for as-

received and modified glass surfaces.

Table 4.3: Substratum surface wettability and surface free energy before and after

modification

Contact angle*, (θ) Surface free energy**, γ, (mJ/m2) Glass

Surface θW θF θD γLW

γAB

γ+

γ-

γTOT

As-received 44±5 41±2 31±5 38.9 11.4 0.9 35.6 50.3

Modified 41±4 39±2 32±5 39.9 8.8 0.5 43.3 48.7

* Contact angle of water, formamide and diidomethane (θW, θF and θD respectively);

** Lifshitz/van der Waals component (γLW

), acid/base component (γAB

), electron

acceptor (γ+) and electron donor (γ

-)

Chapter 4:


______________________________________________________________________

77

Presented results indicate minimal, insignificant decrease of the surface free energy on

the modified glass surface.

4.2.3 Substratum surface charge

Glass surface charge was measured as described in Chapter 3.4.3. Obtained values

for surface charge on the as–received and the modified glass surface are presented in

Table 4.4. Presented data shows that both surfaces exhibit negative net surface charge,

typical for soda-lime glass surfaces (Li and Logan, 2004).

Table 4.4: Glass surface charge as inferred from zeta potential measurement

Sample Electrophoretic Mobility *

(µs-1

)(V/cm)

Zeta Potential ζ

(mV)

As received glass - 5.18 ± 0.1 -66.3 ± 1.1

Modified glass -4.52 ± 0.2 -57.8 ± 2.2

* All measurements were done in triplicates and for each sample the final EPM


14 cycles per run.

As the vast majority of bacteria, including those selected for this study, carry

negative surface charge, their adhesion to the negatively charged glass surfaces is

discouraged (Gottenbos et al., 2001). Taking into account this, it can be hypothesized

that all nine bacterial strains would all exhibit low susceptibility towards both glass

substrates.

Another interesting observation is the approximately 10% lower net surface charge

of the modified glass surface when compared with the as-received. Translated into

cellular adhesive tendency this would suggest that cells are expected to attach in lesser

degree to this surface.

Chapter 4:


______________________________________________________________________

78

4.2.4 XPS analysis of the as-received and the modified glass surface

The relative atomic concentrations of the chemical elements detected on both glass

surfaces are shown in Table 4.5. XPS results indicate that the most abundant elements

on both surfaces were O, Si and C. Modest increase in the relative concentration of O,

Si, Ca and Na on the modified glass surface, consistent with the removal of superficial

carbon during the etching is evident. The levels of Al, F and Fe were close to the

sensitivity limit for these elements and therefore the differences in relative

concentration could not be regarded as significant.

Table 4.5: Relative atomic concentration of the chemical elements presented at the

glass surfaces as determined by XPS analysis

Relative Atomic Concentration (%) Element

As-received Glass Modified Glass

O 54.1 57.2

Si 21.2 22.3

C 22.3 16.9

Ca 0.4 1.1

Al 0.7 0.9

N 0.5 0.6

Na 0.1 0.6

F 0.8 0.2

Fe - 0.2

Regional and wide spectra collected from the modified and as-received glass

surfaces against O 1s, C 1s, N1s, Si 2p and Ca 2p are presented in Figure 4.2. The

similarity in the surface chemical composition of the glass substrates before and after

modification is evident from the presented spectra.

Chapter 4:


______________________________________________________________________

79

a b

c d

e f

g h

Chapter 4:


______________________________________________________________________

80

i j

k

l

Figure 4.2: Regional and wide spectra collected from the modified (a, c, e, g, i, k) and

the as-received glass surface (b, d, f, h, j, l).

Chapter 4:


______________________________________________________________________

81

Peaks in the high resolution region spectra were fitted with synthetic Gaussian-

Lorentzian components after removal of linear backgrounds (using Kratos Vision II

software). The summary of high-resolution region spectra shown in Table 4.6 indicate

that Si and O were, as expected, predominantly present as silica and Ca was present in

the 2+ oxidation state.

Table 4.6: Relative contributions of different chemical states assigned to the XPS peaks

Analysis of the C1s high resolution spectra confirmed the presence of

hydrocarbons (C-C, C-H), carbon singly bonded to oxygen or nitrogen (C-O, C-N),

carbon doubly bonded to oxygen (C=O) and carbonate species (CO3). Although less C

was detected on the surface of the etched glass compared to native glass, which may

indicate removal of organic contaminants, the relative concentration of the various

organic species measured by XPS is similar to that of the native glass. From these

results it can be concluded that the fictionalisation of the glass surfaces did not cause

significant changes in the chemical composition of the glass surface.

4.2.5 XRF analysis of the as-received and the modified glass surfaces

Results obtained by X-ray fluorescence spectroscopy (XRF) indicated that the bulk

chemical composition of the glass slides showed a typical soda-lime glass composition,

with the most abundant chemical components in both samples being SiO2, Na2O, CaO,

MgO and Al2O3 (data presented in Table 4.7). The XRF results also indicated that the

percentage of all detected components (29 in total) in both glass structures was almost

Native glass Etched glass Element /

transition Assignment Binding

energy (eV)

Relative

Contribution

Binding

energy (eV)

Relative

Contribution

N 1s C-N 400.5 100 400.3 100

Ca 2p Ca2+ 347.9 100 347.5 100

C 1s C-C,C-H 285.0 80 285.0 80

C-O,C-N 286.5 12 286.5 12

CO3 289.3 4 289.3 4

C=O 288.1 3 288.3 4

Si 2p SiO2 103.3 100 103.3 100

Chapter 4:


82

identical, with the exception of fluorine, which was found to be present at a level of

0.37 at% in the modified glass, compared to 0.24 at% in the as-received material. This

observation is consistent with the fact that HF was the main component of the etching

solution.

Table 4.1: Detection limits and percentages of all detected components in the as-

received and the modified glass surfaces

Number Sample I.D. Detection Limit (DL)

Modified

glass

As-received

glass

1 SiO2 0.02 % 68.6 68.8

2 Al2O3 0.02 % 1.68 1.68

3 Fe2O3 0.005 % 0.072 0.075

4 CaO 0.005 % 7.52 7.54

5 Cr2O3 0.003 % < DL 0.004

6 CuO 0.003 % < DL < DL

7 K2O 0.005 % 4.08 4.05

8 MgO 0.01 % 4.07 4.06

9 Mn3O4 0.001 % 0.01 0.01

10 Na2O 0.01 % 16.4 16.2

11 NiO 0.004 % < DL < DL

12 P2O5 0.005 % 0.015 0.015

13 SO3 0.005 % 0.224 0.230

14 TiO2 0.004 % 0.099 0.102

15 V2O5 0.003 % < DL < DL

16 ZnO 0.004 % 0.004 0.004

17 ZrO2 0.004 % 0.032 0.032

18 Cl 0.005 % 0.072 0.072

19 F 0.2 % 0.37 0.24

20 Co3O4 0.005 % < DL < DL

21 As2O3 0.003 % < DL < DL

22 BaO 0.01 % 0.053 0.053

23 CdO 0.003 % < DL < DL

24 MoO3 0.002 % < DL < DL

25 PbO 0.004 % < DL < DL

26 SnO2 0.005 % < DL < DL

27 WO3 0.002 % 0.005 0.006

28 SrO 0.001 % 0.007 0.009

29 Ga2O3 0.001 % < DL < DL

Total Sum % 100 99.9

Chapter 4:


______________________________________________________________________

83

4.2.6 AFM analysis of the as-received and the modified glass surfaces

The substratum surface topography of the as-received and the modified glass

surface –were completed using SPM. According to the typical topographic images

shown in Figure 4.3, the modified glass surface appears uniformly smoother and

without the relatively prominent 14-17 nm high protrusions observed on the as-received

sample.

(a)

(b)

Figure 4.3: Typical AFM images of the as-received (a) and modified (b) glass surfaces.

Imaged areas represent 5 × 5 µm2 and 5 × 6 µm

2, respectively.

Chapter 4:


______________________________________________________________________

84

Three conventional roughness parameters, the average surface roughness (Ra)

representing the mean value of the surface relative to the centre plane, the root mean

square (Rq) and the maximum roughness (Rmax) being the difference in height between

the highest and the lowest points on the surface relative to the centre plane (Arnold and

Bailey, 2000), were all approximately 70% higher on the native glass surface. The

separation between the protrusions on the as-received glass is approximately 0.5-1 µm

and the distance from the lowest point to the highest can reach up to 16 nm.

However, these parameters do not necessarily provide a satisfactory indication of

the topographical differences, given the number of relatively prominent protrusions that

are visible in Figure 4.3 (a) but not in Figure 4.3(b).

Table 4.8: Glass surfaces roughness parameters

Roughness parameters (nm)* Sample

Ra Rq Rmax Rz

As-received glass surfaces 2.1 2.8 16.4 12.2

Modified glass surfaces 1.3 1.6 14.2 4.8

* Presented values represent average of 5 independent measurements

An alternative roughness measure that has been suggested for use in the context of

biofouling is the ten point average roughness Rz. The ten point average roughness is

defined as the difference in height between the average of the five highest peaks and

the five lowest valleys along a profile (Whitehead and Verran, 2006). If this definition

is adapted to provide the difference in height between the average of the five highest

peaks and the five lowest valleys over a given surface, then the modified glass surface

appears to be approximately 60-70 % smoother then the as-received glass. Overall, all

four parameters (Ra, Rq, Rmax, Rz) suggest that the as-received glass surface was

rougher than the modified glass surface.

Chapter 4:


______________________________________________________________________

85

4.2.7 SEM of the as-received and modified glass surface

4.2.7.1 Overview

Detailed visualisation of the surface morphology before and after bacterial

cultivation as well as the bacterial attachment pattern was obtained by SEM. Details

regarding sample preparation and SEM were described in Chapter 3.2.4.

SEM images enabled quantitative as well as qualitative cell analyses. For

quantification of the number of adsorbed bacteria, cells from at least five representative

images/areas per slide (three slides per bacterium) was transformed into number of

bacteria per unit area using the Image-Pro software (Waar et al., 2002). The final

densities have estimated errors of approximately 10% due to local variability in the

coverage.

4.2.7.2 Evaluation of control glass surfaces

As already detailed in Chapter 3.2.9, control experiments were set up to evaluate

the change on the glass surfaces, if any, after incubation with sterile marine broth. The

surfaces before incubation were also examined. These experiments were aimed to

verify whether the media used modified the surface morphology or topography and

whether any culture media ingredients deposited on the surface interfered with bacterial

attachment.

(a) Bare modified glass (b) Modified glass with marine broth

Chapter 4:


______________________________________________________________________

86

(c) Bare as-received glass (d) As-received glass with marine broth

Figure 4.4: Typical SEM images of glass surfaces. The scale bar observed on all

images is equal to 1µm. (a) Modified glass surface (b) modified glass surface with

marine broth 2216 (c) as-received glass surface (d) as-received glass surface with

marine broth

The results (Figure 4.4.) indicate that neither of the glass surfaces has changed

their appearance.

As both media are approximately neutral in pH and it is fair to assume that any

effects observed by marine broth would be equal to if not greater than that of the

Nutrient broth.

4.3 Investigation of bacterial adhesion on nano-smooth glass

surfaces

4.3.1 Attachment of Escherichia coli cells on as-received and modified glass

surfaces

The below presented images (Figure 4.5) represent the particular morphology of

E. coli cells after attaching to both glass surfaces as inferred from the high-resolution

SEM images. The initial inspection of both surfaces revealed noticeable differences in

Chapter 4:


______________________________________________________________________

87

the number of attached cells to the two surface regions. Namely, the number of attached

cells on the modified glass surface, tentatively estimated to be 7.1 x 106 cells/cm

2, was

almost double the number on the as-received, 3.25 x 106 cells/cm

2.These densities have

estimated errors of approximately 10% due to local variability in the coverage.

(a) (b)

(c) (d)

Figure 4.5: Typical SEM representing the attachment pattern of E. coli cells after 12 h

incubation on the as-received glass surface (a and b), and on the modified glass

surface (c and d)

Apart from the quantitative difference in the E. coli attachment behaviour on both

surfaces after 12 h incubation, there was obvious difference in the cells appearance. As

evident from the high resolution SEM images (Figure 4.5 b and d), cells attached to the

Chapter 4:


______________________________________________________________________

88

as-received surface appeared flatter and smoother, contrary to the cells attached to the

modified glass who started forming multiyear structures coated with jelly-like

substance believed to be EPS. SEM images also revealed presence of granular-like

features on the modified glass surface.

Observed difference in the cells morphology as well as the surface topography was

confirmed by AFM imaging (Figure 4.6 (a) and (b)).

(a) (b)

Figure 4.6: Selected AFM images representing the morphology and surface

topography of E. coli cells after 12 h of incubation on the as-received glass (a), and on

the modified (b) glass surfaces

The AFM images did confirm that more cells were attached on the modified glass

surface. It was almost impossible to isolate and image single cell on the modified glass

surface contrary to the as-received. They also confirm the morphological difference in

the cells appearance on both surfaces. Namely, cells dimensions after 12 h incubation

on the as-received glass surface were 1.7 µm length, 1µm width and 200 nm height.

These values are in concordance with the original E. coli description cells, 1-2 µm long

and 0.1-0.5 µm in diameter (Burks et al., 2003), but different from observed cell

dimensions after attaching on the modified glass surface. In this instance cells were 2.1

µm long, 1.3 µm high and 250 nm high. Hence pointing out to the 20% increase in the

Chapter 4:


______________________________________________________________________

89

cells size while attaching to the modified glass surface. This morphological

transformation is most likely stimulated by the changes in the surface topography.

The AFM also detected presence of lumpy deposits on the modified glass surface

itself believed to be EPS (circled areas Figure 4.7 (b)). This assumption was confirmed

by CLSM imaging (Figure 4.7 a, b, c, d and e).

(a) (b)

(c)

Chapter 4:


______________________________________________________________________

90

(d) (e)

Figure 4.7:Typical CLSM images showing the EPS production (a, d) and the viable (b,

e) E. coli cells after 12 h of incubation on as-received (a, b, c) and modified (d, e)

glass surfaces. Scale bar on image (a),(b), (d) and (e) is 10 µm and 2 µm on image (c)

Prior to confocal imaging, cells and EPS were coloured as described in Chapter

3.2.5. As evident from image (a) and (b) cluster of attached cells was observed on the

as-received glass surface. From both images it is evident that attached cells were viable

(b) and covered with EPS (a). Apart from the cluster of cells, two single cells are

visible in the left top corner on Figure 4.7 (a), but absent on Figure 4.7 (b). This is

might be an indication of the better survival prospects of cells when gathered in

organised consortia. Superimposing of image (a) and (b) (presented in Figure 4.7 (c))

revealed that the EPS produced by E. coli attached to the as-received glass surface are

most certainly of capsular nature and coat each cell separately. There were not any gel-

like deposits found on the bear glass surface. On the other hand images (d) and (e)

(Figure 4.7) indicate that apart from the significant number of viable cells attached to

the modified glass surface extra-cellular products (EPS/LPS) were found not only in

relation to the cell surface but also on the substrate in a cloudy-like appearance (circled

area top left corner Figure 4.7 (d)).

Chapter 4:


______________________________________________________________________

91

4.3.2 Attachment of P. aeruginosa cells on as-received and modified glass

surfaces

The high-resolution SEM images presented below (Figure 4.8) demonstrate the

attachment behaviour of P. aeruginosa cells while adhering to both glass surfaces, the

as-received and the modified. As evident by image (a) and (c) Figure 4.8, substantial

number of cells attached and maintained their presence to both surfaces after 12h

incubation. When translated into number of attached cells per unit area, the number of

P. aeruginosa cells attached to the as-received glass was estimated at 10.3 x

106cells/cm

2, whereas the number of cells attached to the modified glass surface was

almost double that, 18.45 x 106 cells/cm

2. Cell densities have estimated errors of

approximately 10% due to local variability in the coverage.

In addition to the numerical differences, clear changes in the surface topography

and the production of extra-cellular polymeric material (presumably EPS) were also

observed. As evident from the SEM images cells preferred to organise in multicellular

consortia interconnected with extra-cellular products. Apart from the interconnecting

channel-like EPS, granular like deposits were also observed on the surface. These

extra-cellular products were particularly visible on the modified glass surface after 12h

incubation (Figure 4.8(e)).

Contrary to E. coli, the SEM images did not reveal any significant changes in the

appearance between cells attached to each of the surfaces.

(a) (b)

Chapter 4:


______________________________________________________________________

92

(c) (d)

(e)

Figure 4.8: Typical SEM images showing the attachment behaviour of P. aeruginosa

cells after 12 h incubation on the as-received (a) and (b), and on the modified glass

surface (c) and (d). Scale bar represents 10 µm on (a), (c) and (e) and 1 µm on (b) and

(d)

Even so, the AFM images presented in Figure 4.9 (a) and (b) revealed some

noticeable differences. For example, cells attached to the modified surface appeared

slightly longer and significantly wider (2.4µm x 1.8µm x 250nm) contrary to the cells

attached to the as-received glass (2.1µm x 1.1µm x 170nm). These differences are

presumably attributed to the diversity if the substratum surface topography as well as to

the excessive quantities of EPS synthetised by the P. aeruginosa cells while attaching

to the modified glass (Figure 4.9 (c)).

Chapter 4:


______________________________________________________________________

93

(a) (b)

(c)

Figure 4.9: Selected AFM representing the morphology and surface topography of P.

aeruginosa cells after 12h incubation on the as-received(a), and on the modified glass

surface (b and c)

Figure 4.9 (c) illustrates typical appearance of P. aeruginosa cell after 12 h

incubation on the modified glass surface. It also gives an indication of the overall cell

height (top transverse profile) and the height of the EPS deposits in the near-cell

surrounding (bottom transverse profile). Judging by the bottom transverse profile the

overall height of the EPS was 156nm.

Since significant difference in the cell dimensions was observed, the roughness

parameters of selected 0.5µm x 0.5µm areas from the cell surfaces were also evaluated.

Chapter 4:


______________________________________________________________________

94

As the results presented in Table 4.9 indicate all three parameters indicative of surface

roughness were 2-4 times higher for cells attached to the modified glass surface, thus

pointing out to the probability of excessive production of EPS by P. aeruginosa cells

wile adsorbing on the modified glass surface.

Table 4.9: P. aeruginosa cells surface parameters after attachment on the as-received

and modified glass surfaces

* Scanned areas are 0.5 x 0.5 µm

** Presented values represent average of 5 independent measurements

The elevated production of EPS on the modified surface was also confirmed after

analysis of the confocal scanning images presented in Figure 4.10. As evident from the

images significant number of viable P. aeruginosa cells managed to maintain their

existence on the glass surface before and after modification. Bacteria seamed to attach

in typical pattern on both glass surfaces with the only evident difference being the

excessive number of calls attached to the modified glass surface, as was also seen the

SEM.

It is clear that there are varieties of EPS produced by the bacteria while attaching

on both surfaces. There are EPS observed on and in close proximity to the cells as well

as EPS spreading on the glass surface in a cloudy-like manner (pointing arrows Figure

4.10 (a) and (c)). Considering the specific affinity of Concanavalin Alexa 488 towards

α-mannopyranosyl and α-glucopyranosyl, it is to be expected that they are the

constituent components in all the varieties of EPS deposits. Observed different ranges

of strength of the Alexa 488 green fluorescent signal indicated the probability of

variable representation of each of the polysaccharide components or even presence of

polysaccharide component not entirely stained by Concanavalin Alexa 488.

Parameter P.aerugionsa attached on

as-received glass*, **

P. aerugionsa attached

on modified glass*, **

Ra 5.25 nm 19.66 nm

Rq 8.02 nm 23.61 nm

Rmax 55.1 nm 94.54 nm

Chapter 4:


______________________________________________________________________

95

(a) (b)

(c) (d)

Figure 4.10: Selection of CLSM images representing the viability (viable cells are red

stained) and the EPS production (produced EPS are green stained) of P. aeruginosa

cells after 12h incubation on as-received glass surface (a and b) and the modified glass

surface

The EPS produced by this bacterium while attaching to different surfaces were

previously described as formation of capsules, sheaths or slimes depending on their

proximity to the cell wall (Beveridge and Graham, 1991). The chemical composition of

the EPS varies depends on their location as well as on their function. Namely, gel-like

EPS surrounding the cell usually have protective role, contrary to the “free-EPS”

released into the culture medium whose role is mostly related to irreversible cell

adhesion (Beech et al., 1999). The distinction between different types of EPS is

Chapter 4:


______________________________________________________________________

96

difficult and imprecise, mainly due to the limited quantities produced and the tightly

bonds between them and the cellular surface or the substratum. Beech at al. (Beech et

al., 1999) isolated three different types of EPS (capsular, free EPS from the culture

medium and EPS associated with biofilm) from Pseudomonas sp. They have

discovered structural differences between the three exopolymers and have concluded

that although the three types of EPS shared some of the chemical components, the

exopolymer chemistry depends on the cellular mode of growth.

4.3.3 Attachment of S. aureus cells on as-received and modified glass

surfaces

Attachment behaviour of the coccoid S. aureus was investigated by means of SEM,

AFM and CLSM.

Contrary to the previous two terrestrial strains, E. coli and P. aeruginosa, the

morphologic and metabolic transformation of S. aureus while attaching to the as-

received and modified glass surfaces was not as dramatic. As indicated by the SEM

images presented in Figure 4.11 cells appearance was almost identical regardless of the

surface. The only apparent difference was bacterial organisation in clusters of few

(Figure 4.11 (c) and (d)) while attaching to the modified glass surface contrary to the

majority of single cells observed on the “as-received surface” (Figure 4.11 (a) and (b)).

(a) (b)

Chapter 4:


______________________________________________________________________

97

(c) (d)

Figure 4.11: Typical SEM images showing the attachment behaviour of S. aureus cells

after 12h incubation on the as-received (a and b), and on the modified glass surface (c

and d). Scale bar indicates 10µm on image (a) and(c), and 1µm on (b) and (d)

The AFM images presented on Figure 4.12 represent the S. aureus cells

morphology and surface topography as they attach to the as-received, Figure 4.12 (a)

and to the modified, Figure 4.12 (b) glass surfaces. Image (a) presents one dividing cell

attached to the as-received surface after 12h incubation contrary to image (b) where

typical cluster of few spherical cells is presented. Appraisal of the cells dimensions on

both surfaces revealed that the average S. aureus size after 12h incubation on both

surfaces is approximately 0.9µm x 0.9µm. Contrary to the previously presented strains

where average cell height varied between 200-250nm the S. aureus height was found to

be in the range of 400nm. This considerable difference in the cells height can be

attributed either to the spherical shape of S. aureus cells contrary to E. coli and P.

aeruginosa or to the overlaying coat of EPS as later presented on the confocal images.

Chapter 4:


______________________________________________________________________

98

(a) (b)

Figure 4.12: Selected of AFM representing the morphology and surface topography of

S. aureus cells after 12h incubation on the as-received glass surface (a), and on the

modified glass surface (b)

Already revealed adhesive conduct of S. aureus by the SEM and the AFM analysis

was confirmed by CLSM images (Figure 4.13).

(a) (b)

Chapter 4:


______________________________________________________________________

99

(c) (d)

Figure 4.13: Typical CLSM images of S. aureus cells attaching to the as-received (a

and b) and to the modified (c and d) glass surface after 12h incubation. Scale bar on all

images is 10 um

The number of viable cells attached to the modified glass surface after 12h

incubation appeared to be significantly higher when compared to the number of cells

attached to the as-received surface. Even though grape-like clusters, typical for S.

aureus, (Figure 4.13 (a) and (b)) were observed on the as-received surface after 12h

incubation, they were all unilayered and significantly smaller than those detected on the

modified (Figure (c) and (d)).

4.3.4 Attachment of C. marina cells on as-received and modified glass

surfaces

Scanning electron images presenting the attachment behaviour of C. marina cells

after 12 h incubation on glass surfaces before and after treatment with buffered

hydrofluoric acid are presented in Figure 4.14. It is evident that substantial number of

cells attached to both surfaces, although when translated into number of attached cells

per unit area considerable increase in the number of attached cells to the modified glass

surface was observed. Namely, the number of C. marina cells attached to the as-

received glass was 5 660 000 cells/cm2, whereas the number of cells attached to the

Chapter 4:


______________________________________________________________________

100

modified glass surface was 9.78 x 106 cells/cm

2. According to the original description

C. marina cells are supposed to be rod shaped, 1.6-4.0µm long (Arahal et al., 2002).

Contrary to this, almost all cells attached on the modified surface observed on the SEM

and later on confirmed by the AFM (Figure 4.15) were of oval shape. Hence, pointing

out to the possibility of C. marina cells undergoing some sort of morphological

transformation triggered by the cultivation conditions during the 12h incubation period.

Apart from the different shape, the surface of cells attached to the modified glass

surface appeared to be more irregular and lumpier, when compared to the relatively

smooth surface of cells attached to the as-received glass.

(a) (b)

(c) (d)

Figure 4.14: Typical SEM images showing the attachment behaviour of C. marina cells

after 12h incubation on the as-received (a) and (b), and on the modified glass surface

(c) and (d). Scale bar on all images represents 2 µm

Chapter 4:


______________________________________________________________________

101

The AFM images presented below represent the C. marina cells morphology as

well as the surface topography after 12h incubation on the as-received (a) and modified

(b) glass surface.

(a) (b)

Figure 4.15: Typical AFM images of C. marina cells attaching to the as-received (a)

and to the modified (b) glass surface after 12h incubation. Scanned areas

approximately 3.0µm x 3.0µm and 4.5µm x 4.5µm, respectively

As already evident by the SEM images significant number of C. marina cells

attached to both glass surfaces. The slight increase of the number of attached cells to

the modified glass surface already determined by the SEM and consistent with the

already presented data for other strains was confirmed by the AFM (Figure 4.15).

Image (a) presents one dividing C. marina cell adsorbed on the as-received surface

after 12h incubation, whereas on image (b) cluster of four cells is presented. Cells

dimensions after attaching to both surfaces were as follows, 1.8µm x1.2µm x180nm on

the as-received and 1.5µm x 1.0µm x178nm on the modified glass surface. Previously

mentioned observation that cells attached to the modified surface appeared to be oval

shaped was confirmed by these measurements. This change in the cells shape is

consistent with the observed 15% decrease in cells length and width when attaching to

the modified glass which is contrary to previously reported attachment behaviour for E.

coli and P. aeruginosa where cell dimensions showed tendency to increase after 12h

incubation on the modified glass.

Chapter 4:


______________________________________________________________________

102

Cell surface topography after attachment to both glass surfaces appeared to be

similar as inferred from the almost identical parameters representative for surface

roughness, data presented in Table 4.10.

Table 4.10: C. marina cell surface roughness on selected 0.5µmx0.5µm areas on top of

the cells attached to the as-received and modified glass surface

Parameter C. marina attached on

as-received glass*

C. marina attached on

modified glass*

Ra 11.58 nm 10.29 nm

Rq 13.60 nm 12.48 nm

Rmax 67.78 nm 68.93 nm

*Presented values represent average of 5 independent measurements

Although the presence of EPS was not detected on AFM and SEM images, the

same were observed on the cell surface by CLSM. Images presented bellow (Figure

4.16) indicate that substantial amounts of EPS were synthetised by C. marina cells

whiles attaching to both glass surfaces.

(a) (b)

Chapter 4:


______________________________________________________________________

103

(c) (d)

Figure 4.16: Typical CLSM images of C. marina cells attaching to the as-received (a)

(b) and to the modified (c) and (d) glass surface after 12h incubation. Scale bar on all

images is 10 um

Images (a) and (c), Figure 4.16, indicate the EPS production of C. marina cells

while attaching to the as-received (a) and to the modified (c) glass surface, while

corresponding red fluorescent images (b) and (d) indicate the number of viable cells

after 12 h incubation on both surfaces. As indicated by the images, noteworthy number

of cells managed to attach and survive on both surfaces. Also obvious is the presence of

EPS encapsulating each cell regardless of the surface on which its being attached, or if

it exists as single cell or in cluster of cells. As concanavalin A specifically binds to α-

mannopyranosyl and α-glucopyranosyl residues, it can be assumed that these

polysaccharide components are part of the capsular EPS found on the cell surfaces.

Apart from the capsular EPS, no other extra-cellular products were observed on both

surfaces. This does not exclude the possibility of C. marina cells producing varieties of

EPS, but simply points out to the possibility of distinct chemical composition from the

already observed capsular-like EPS.

Chapter 4:


______________________________________________________________________

104

4.3.5 Attachment of P. issachenkonii cells on as-received and modified glass

surfaces

The particular morphology of the P. issachenkonii cells after adhering to both glass

surfaces was examined by means of high-resolution scanning electron microscope

(SEM), as shown by the images in Figure 4.17.

An initial inspection of the bacterial attachment revealed striking differences in the

bacterial response to the two surface regions. The number of attached cells observed in

the SEM images (1000× magnification) was transformed into a number of bacteria per

unit area and was tentatively estimated to be 3.6 x 106 cells/cm

2 on the as-received

glass surface, while the bacterial density increased by a factor of three on the modified

glass surface, reaching approximately 11 x 106 cells/cm

2. These densities have

estimated errors of approximately 10% due to local variability in the coverage.

(a) (b)

Chapter 4:


______________________________________________________________________

105

(c) (d)

Figure 4.17: Typical SEM images of P. issachenkonii cells attaching to the as-received

(a) and (b) and to the modified (c) and (d) glass surface after 12h incubation

In addition to the obvious numerical differences, clear changes were observed in

the cell morphology and the production of extra-cellular polymeric material

(presumably EPS). Morphological differences in the cells attached to both surfaces

after 12h incubation were seen on SEM images (Figure 4.17 (c) and (d)) and were latter

confirmed by AFM analysis (Figure 4.18).

(a) (b)

Figure 4.18: Selected AFM images of P. issachenkonii cells attaching to the as-

received (a) and to the modified (b) glass surface after12h incubation

Chapter 4:


______________________________________________________________________

106

According to the original description (Ivanova et al., 2002b), cells of P.

issachenkonii are 0.7-0.9µm wide and 1.0-1.2µm long. In these experiments, the

majority of the cells attached to the as-received glass were 2.0µm long, 1.0µm wide and

140 nm high (Figure 4.18(a)). However, on the modified glass surfaces, the average

width of the bacterial cells was found to be 1.3µm, the length 2.9µm and the average

height 170nm. Apart from the EPS coating the cells, additional quantities of EPS 80-

120 nm in height were also found on the modified glass surface (circled points (a) and

(b) in Figure 4.18(b)). This is indicative of the surface modification strategy utilized by

P. issachenkonii in order to better sustain their existence on this surface. The

production of extracellular substances during the process of adhesion was observed

using CSLM.

(a) (b)

(c) (d)

Figure 4.19: Selected CLSM images of P. issachenkonii cells attaching to the as-

received (a) and (b) and to the modified (c) and (d) glass surface after 12h incubation.

Scale bar on all images is 2 um

Chapter 4:


______________________________________________________________________

107

The attachment pattern, viability and the production of EPS of bacterial cells on

two regions of glass surfaces after 12 hours of incubation is presented in Figure 4.19.

The number of bacterial cells remained greater on the modified glass surfaces after

incubation similarly as E. coli and P. aeruginosa. It appears that the cells attached to

the modified glass surfaces began to form a multilayer structure and produced greater

quantities of EPS (circled area on Figure 4.19 (d)), according to the fluorescence

images. As concanavalin A specifically binds to α-mannopyranosyl and α-

glucopyranosyl residues, it can be assumed that these sugars are components of EPS

found on the cell surfaces. However, the granular EPS observed by AFM on the etched

glass surface (see points (a) and (b) in Figure 4.19(b)) was also detected in the confocal

images but with lesser intensity of the Alexa 488 signal (pointing arrows on Figure 4.20

(d)), suggesting a distinct chemical composition for this type of EPS. This is an

indication of the possible changes in the cells metabolic activity while attaching to the

modified surface, most likely triggered by changes in the nanoscale surface roughness,

as later detailed in chapter 7. However, identification of the chemical composition of

the EPS produced not only by P. issachenkonii cells on both types of glass surface

remains a challenging task due to the small amounts of material available for analysis.

4.3.6 Attachment of S. flavus cells on as-received and modified glass surfaces

High resolution SEM images showing the attachment behaviour of S. flavus cells

after 12h incubation on the as-received and the modified glass surfaces are presented in

Figure 4.20. As indicated by the images the number of attached cells was not as

generous when compared to the previously presented data for other strains. When

translated into number of cells per unit area it was revealed that the number of attached

cells after surface modification was approximately 20 times higher. Namely, calculated

number of attached cells to the as-received glass surface was 146 000cells /cm2,

contrary to the modified where it was found to be 2.75 x 106cells /cm

2. In general

terms, this is extremely low surface spread and may suggest that this strain lacks some

of the adhesion mechanisms already present in the other strains.

Chapter 4:


______________________________________________________________________

108

Granular-like deposits located in close proximity to attached cells were also

observed on the modified glass. As the CLSM will later confirm these deposits did not

contain α-mannopyranosyl and/or α-glucopyranosyl residues, thus were not labelled

with Concanavalin A. This does not exclude the possibility of them being EPS with

distinct chemical composition.

Considering this is a newly detected/described strain it is to be expected that most

of its surface and metabolic features are yet to be determined (Ivanova et al., 2006b).

(a) (b)

(c) (d)

Figure 4.20: Typical SEM images of S. flavus cells attaching to the as-received (a) and

(b) and to the modified (c) and (d) glass surface after 12h incubation. Scale bar

represents 10µm on image (a) and (c) and 1 µm on image (b) and (d)

Chapter 4:


______________________________________________________________________

109

The AFM imaging of both glass surfaces after 12h incubation in the presence of S.

flavus bacterial suspension are presented bellow (Figure 4.21). The initial scanning of

the as-received glass surface confirmed the SEM observation, only few cell were

detected (Figure 4.21(a)). Apart from the cells itself lumpy-like deposits whose height

varied between 100-450nm were observed in the near-cell surrounding (pointing arrow

Figure 4.21(b)). The cells themself were approximately 2.5µm long, 1.0µm wide and

between 150-200nm high.

Slightly higher number of cells was noted to be able to successfully colonise the

modified glass surface (Figure 4.21(c)). Although the cell appearance on both surfaces

is almost identical, S. flavus cells adsorbed on the modified surface appeared to be

approximately 10% bigger, with measured dimensions of 2.7µm x 1.2µm x 250nm.

(a) (b)

Chapter 4:


______________________________________________________________________

110

(c) (d)

Figure 4.21: Selected AFM images of S. flavus cells attaching to the as-received (a,

scanned area 50µmx50µm), (b, scanned area 4.0µmx4.0µm) and to the modified (c,

scanned area 35µmx35µm), (d, scanned area 4.5µmx4.5µm) glass surfaces after 12h

incubation

Due to the low cell density after 12 h incubation period, for the purpose of CLSM

both surfaces, the as-received and the modified were inoculated with cell suspension

for 24 h. CLSM images (Figure 4.22) indicate that after extended incubation time

substantial number of cells managed to adsorb on both surfaces. Cellular metabolic

activity was confirmed with the presence of EPS. Each green fluorescent signals

(Figure 4.22 (a) and (c)) correspond to red fluorescent signals (Figure 4.22 (b) and (d)),

thus suggesting that the extra-cellular deposits produced by the adhering cells are most

likely of capsular nature.

(a) (b)

Chapter 4:


______________________________________________________________________

111

(c) (d)

Figure 4.22: Typical CLSM images of S. flavus cells attaching to the as-received (a)

and (b) and to the modified (c) and (d) glass surface after 12 h of incubation. Scale bar

on all images is 2 um

The CLSM images also indicate that bacteria adsorbed on both surfaces in random,

not-patterned way, however they did express tendency to gather in multi-cellular

consortia (circled areas on Figure 4.22(c)) when adsorbing on the modified glass

surface.

Unusual observation was the presence of cell-shaped EPS (pointing arrows on

Figure 4.22(c)) in the absence of corresponding red fluorescent (viable cell) signal after

24 h incubation only on the modified glass surface, suggesting that not all initially

adsorbed cells were capable of maintaining their viability.

4.3.7 Attachment of S. guttiformis cells on as-received and modified glass

surfaces

Images presented on Figure 4.23 are indicative of S. guttiformis adhesive

behaviour after 12h incubation on the as-received, images (a) and (b), and on the

modified glass surface images, (c) and (d). They also give an indication of the

approximate number of cells attached to each of the surfaces after 12h incubation.

Chapter 4:


______________________________________________________________________

112

The number of S. guttiformis cells adsorbed on the as-received glass was estimated

at 3.28 x 106calls/cm

2, contrary to the 7.67 x 10

6calls/cm

2 adsorbed on the modified

surface.

(a) (b)

(c) (d)

Figure 4.23: Typical SEM images of S. guttiformis cells attaching to the “as-received’

(a) and (b) and to the modified (c) and (d) glass surface after 12 h of incubation. Scale

bar represents 10µm on image (a) and (c) and 1 µm on image (b) and (d)

High resolution SEM images revealed that cells presented in a tear-like shape,

typical for this strain, regardless of the surface with granular-like deposits on their

surface. Better insight into the cell surface characteristics was achieved by AFM.

Chapter 4:


______________________________________________________________________

113

S. guttiformis cell size and surface topography were observed by AFM (Figure

4.24). The bellow presented images confirmed previous observations. Cell morphology

appeared to be similar regardless of the type of the surface cells were attached to. Cell

exterior appeared granular, regardless of the surface of attachment.

(a) (b)

Figure 4.24: Selected AFM images of S. guttiformis cells attaching to the as-received

(a) and to the modified (b) after 12 h of incubation. Scanned areas repreent 4.0µm x

4.0µm and 7.0µm x 7.0µm, respectively.

(a) (b)

Chapter 4:


______________________________________________________________________

114

(c) (d)

Figure 4.25: Typical CLSM images of S. guttiformis cells attaching to the as-received

(a) and (b) and to the modified (c) and (d) glass surface after 12 h of incubation. Scale

bar on all images represents 10 µm

It is obvious that substantial number of cells attached to both surface after 12h

incubation. Cells not only attached but also maintained their viability (Figure 4.25 (b)

and (d)) and synthetised significant quantities of extra-cellular products. Appeared that

majority of the produced extra-cellular substances /EPS are of capsular nature, coating

each cell individually. Apart from the capsular EPS that coat each cell individually,

lawn-like deposits were also observed on the modified glass surface in between cells

(marked areas image (c)). The fact that they were presented with lighter green

fluorescent signal suggests distinct chemical composition from the capsular EPS which

contained mainly α-mannopyranosyl and/or α-glucopyranosyl residues as their main

components.

4.3.8 Attachment of S. mediterraneus cells on as-received and modified glass

surfaces

S. mediterraneus attracted significant interest over the last years due to its ability to

undergo morphological conversion from vegetative form into coccoid bodies (Ivanova

Chapter 4:


______________________________________________________________________

115

et al., 2002a). This is phenomenon that has been recently discovered and studied mainly

in spiral-shaped, freshwater and marine Gram-negative bacteria of the genera

Aquaspirillum (Terasaki, 1979), Oceanospirillum (Kreig and Holt, 1984),

Marinospirillum (Satomi et al., 1998), and some pathogenic bacteria of significant

medical impact, such as Alivibrio cholerae (Ravel et al., 1995, Sörberg et al., 1996) and

Helicobacter pylori35

. There have been different opinions if the coccoid bodies of

pathogenic bacteria are viable but non-culturable (Sörberg et al., 1996, Oliver, 1995),

or non-viable degenerate forms that might even cause diseases in laboratory animal

models (Koechlein and Krieg, 1998). Hence, their functions and physiological role as

either a degenerative or a viable resting stage, remains unclear. The transformation

capabilities of this strain were first discovered noted while studding its attachment

behaviour on P(t)BMA polymeric surfaces (Ivanova et al., 2002a).

While studding the attachment behaviour of S. mediterraneus on glass surfaces

with different physical characteristics its transformation capability was once again

confirmed. It is interesting to note though that cell transformation from vegetative into

coccoid form was only observed on cells adhering to the as-received glass surface.

As the below presented SEM images indicate (Figure 4.27) the overall number of

cells successfully adsorbed on the as-received glass (Figure 4.27(a)) was noticeably

lower when compared to the modified (Figure 4.27(c)). When translated into number of

cells per unit area, 553 000 cells/cm2 on the as-received and 891 500 cells/cm

2 on the

modified surface, it was confirmed that the overall colonisation of both surfaces was

very low in comparison to some of the other strains presented in this study.

Observation of the high resolution SEM images presented in Figure 4.26 (b) and

(d) indicate that there was significant difference in the cells appearance. Cells adsorbed

on the as-received surface initially appeared to be flat and initially elongated, 1.0-

2.0µm long, which is in concordance with their original description. It was also

obvious that they have started their transformation by shrinking (pointing arrows image

(b)) and concentrating their cellular material in the middle of the cell, resulting in

decreasing of their overall length, but increasing of their diameter. The conglomerate

formed in the middle of what appears to be a cell is 0.9-1.2µm long, which again is in

agreement with reported dimensions for S. mediterraneus coccoid bodies (Ivanova et

Chapter 4:


______________________________________________________________________

116

al., 2002a). In the study Ivanova et al. (Ivanova et al., 2002a) conducted they were able

to observe completely formed coccoid bodies but after 24h incubation. Here we present

what appears to be the initial stage of S. mediterraneus cellular morphologic

transformation after only 12 h incubation.

(a) (b)

(c) (d)

Figure 4.26: Selected SEM showing the attachment behaviour of S. mediterraneus cells

after 12 h incubation on the as-received glass surface (a) and (b), and on the modified

glass surface (c) and (d). Scale bar represents 10µm on images (a) and (c), and 1µm on

image (b) and (d).

Contrary to this cells attached to the modified glass appeared to be of typical

vegetative appearance, elongated and encapsulated in extra-cellular deposits. Judging

Chapter 4:


______________________________________________________________________

117

by the high resolution SEM images (Figure 4.26(d)) it appears that they randomly

colonise the surface, although the organized, multi-cellular clusters were formed in

surface irregularities most likely resulting from the etching. Apart from the capsular-

like EPS coating each of the cells separately, deposits of irregular size were also

observed on the glass surface itself.

Detailed insight into the surface topography and appearance of cells adsorbed on

both surfaces after 12h incubation was inferred through AFM. The images presented in

Figure 4.27 confirmed the previously observed cellular behaviour by SEM. The image

presented in Figure 4.28 (a) represents typical S. mediterraneus cell attached to the as-

received glass after 12h incubation. It is evident that the cellular transformation has

already begun with the intracellular matrix condensing towards the left pole of the cell.

Contrary to this and in alliance with previously observed changes by the SEM, S.

mediterraneus cells adsorbed to the modified glass managed to retain their original

form (Figure 4.27 (b)). The same image also indicates the presence of lumpy extra-

cellular deposits, most likely EPS, on the glass surface in the near cell surrounding.

Measured cell dimensions were also indicative of the ongoing cellular

transformation. Namely, cells adsorbed on the as-received surface were approximately

3.0µm x 1.4µm x 220nm, contrary to cells adsorbed to the modified surfaces, whose

dimensions were 2.7µm x 1.0µm x 170nm on average. Particularly indicative of the

cellular transformation was the 30% increase of the height of cells adsorbed to the as-

received glass.

(a) (b)

Chapter 4:


______________________________________________________________________

118

(c)

Figure 4.27: Selected AFM images of S. mediterraneus cells attaching to the as-

received ((a), scanned area 4.5x4.5µm) and to the modified ((b), scanned area

4.5x4.5µm) glass surface after 12 h of incubation. Image (c) represents the appearance

of S. mediterraneus cells adsorbed to the modified glass surface after 18 h incubation

(scanned area 14x14µm).

Since cellular transformation from vegetative into coccoid form was observed on

the as-received glass, additional set of AFM imaging was conducted but in this

occasion S. mediterraneus bacterial suspension was incubated for extended period of

time, 18h, on the modified glass surface. As evident by the image (Figure 4.27 (c))

prolonged incubation time resulted in cellular conversion similar to that observed on

the as-received surface after only 12h incubation. Pointing arrows on the same image

indicate the end poles of the attached cells where measured cell height was

approximately 180nm. Contrary to this the maximum height towards the right pole of

the cell (circled area) was approximately 270nm, indicating the position of condensed

cellular matrix.

The cell viability as well as the production of S. mediterraneus cells attaching to

the as-received and to the modified glass were revealed through CLSM (Figure 4.28).

As the bellow presented images indicate the overall cellular presence on both surfaces

was extremely low. Another interesting observation was the absence of EPS deposits

on the as-received surface (Figure 4.28 (a)). On the other hand image (b) indicates that

Chapter 4:


______________________________________________________________________

119

cells managed to attach and maintain their viability. Although in concordance with

previous observations (SEM and AFM) this is not a firm indication of EPS absence.

Viable cells presented on Figure 4.28 (b) confirm the beginning stage of cellular

transformation already observed on the SEM and the AFM images.

(a) (b)

(c) (d)

Figure 4.28: Selected CLSM images of S. mediterraneus cells attaching to the as-

received (a) and (b) and to the modified (c) and (d) glass surface after 12 h of

incubation. Scale bar on all images is 2 µm

Contrary to this S. mediterraneus cells attached to the modified glass after 12 h

incubation were found to be encapsulated in α-mannopyranosyl and α-glucopyranosyl,

Chapter 4:


______________________________________________________________________

120

as suggested by image Figure 4.28 (c). Extra-cellular deposits presented with pale green

fluorescent signal were also observed on the glass surface in the near cell surrounding.

The appearance of the two viable cells attached to the modified glass (Figure 4.28 (d))

confirms previous observation that during the 12h incubation S. mediterraneus cells are

capable of mentioning their natural form.

4.3.9 Attachment of A. fischeri cells on as-received and modified glass

surfaces

In a similar manner as for the other strains, the adhesive behaviour of A. fischeri on

both glass surfaces was explored by means of SEM, AFM and CLSM. Initial inspection

of the SEM images (Figure 4.29) revealed striking difference in the cell appearance on

each of the surfaces. The overall cell presentation over both surfaces appeared to be

similar, with cells organised in single-layered conglomerates on the as-received as well

as to the modified surface. Yet, closer inspection of the high resolution SEM images

revealed that A. fischeri cells attached to the modified surface have lumpier surface,

most likely as result of the presence of extra-cellular products. Similar lumpy features

with obvious compositional dissimilarity, that act as a bond sustaining attached cells in

close proximity, were also observed on the modified surface in the near-cell

surrounding.

Apart from the significant morphologic differences, considerable variation in the

quantity of attached cells was also observed. Namely, when translated into number of

cells per unit area it appeared that the number of attached cells to the modified glass

(13.2 x 106cells/mm

2) was approximately 30% higher than cells adsorbed onto the as-

received surface.

Chapter 4:


______________________________________________________________________

121

(a) (b)

(c) (d)

Figure 4.29: Selected SEM showing the attachment behaviour of A. fischeri cells after

12 h incubation on the as-received glass surface (a) and (b), and on the modified glass

surface (c) and (d). Scale bar on all images represents 2 µm

The AFM images presented on Figure 4.30 (a) and (b) represent the overall

substratum surface topography including few attached cells. Observed cellular

morphology after 12 h incubation on the as-received glass surface is very much similar

to that detected by SEM. As evident by Figure 4.30 (a) two A. fischeri cells attached to

the surface without noticeable presence of any extra-cellular products. Contrary to this,

AFM of the A. fischeri’s attachment to the modified glass surface differed from the

SEM. Namely on the SEM images presented in Figure 4.30 (c) and (d) it is obvious that

significant numbers of cells are attached to the modified glass surface but still in a

Chapter 4:


______________________________________________________________________

122

single-layered group. On the other hand the AFM imaging of the same surface

presented in Figure 4.30 (b) reveals the beginning stage of multi-layered biofilm

structure.

Comparison of the cell dimensions on the both surface revealed 10-15%

discrepancy between cells attached to the as-received and cells attached to the

modified. As with all other rod shaped bacteria presented in this study, A. fischeri cells

attached to the modified surface were slightly bigger. Namely, cells adsorbed on the as-

received glass were 1.5µm long, 0.7µm wide and 180nm high. Similarly to this cells

attached directly on the modified surface were 1.6µm long and 0.8µm wide. The height

of cells adsorbed on the modified glass was difficult to judge due to the stacks of cells

and EPS. On the other hand the top two cells were approximately 40% longer and

wider when compared to the cells being in direct contact to the glass surface. The

average height of these two cells was 0.47nm, but again this is not to be taken as

absolute as stack of cells and EPS are to be found below. The EPS height varied

between 130-170nm, as supported by the transverse profile presented on image (c).

(a) (b)

Chapter 4:


______________________________________________________________________

123

(c)

Figure 4.30: Selected AFM images of A. fischeri cells attaching to the as-received (a,

3.5x3.5µm) and to the modified (b, 7.0x7.0µm) glass surface after 12h incubation.

Image (c) presents transverse profile of the EPS deposited on the modified glass

surface

A. fischeri viability and production of EPS during 12 h incubation on the as-received

and modified glass surfaces was evaluated through CLSM (Figure 4.31).

Chapter 4:


______________________________________________________________________

124

(a) (b)

(c) (d)

Figure 4.31: Typical CLSM images of A. fischeri cells attaching to the as-received (a)

and (b) and to the modified (c) and (d) glass surface after 12 h of incubation. Scale bar

on all images is 2 µm

Presented images indicate that the number of viable cells (red fluorescent signal,

Figure 4.31 (d)) is higher on the modified glass surface as is the production EPS (green

fluorescent signal, Figure 4.31 (b)). The weaker green fluorescent signal on Figure 4.31

(a) when compared to the one on Figure 4.31 (c) suggests the probability in distinct

chemical composition of the EPS produced by the same bacterium while attaching to

two surfaces with different surface roughness.

Chapter 4:


______________________________________________________________________

125

4.4. Conclusion

Notwithstanding individual species specific pattern of attachment, consistent

bacterial preference towards the smoother, modified glass surfaces was observed.

Bacterial inclination towards the nano-smooth glass surfaces was accompanied with

significant morphologic and metabolic transformations. Cellular morphologic

transformations were in particular prominent for rod-shaped bacteria in contrast to the

spherical shaped S. aureus. All rod-shaped bacteria, excluding C. marina and S.

mediterraneus appeared to be more voluminous, longer and wider, after attaching on the

modified glass surfaces. C. marina and S. mediterraneus cells adsorbed on the modified

glass were of oval-shape compared with the predominantly elongated cells attached to

the rougher, as-received glass surfaces.

Bacterial metabolic activity, such as production of EPS during the incubation

period on the two glass surfaces, was observed by CLSM. It is evident that all of the

studied bacteria produced significant amounts of capsular-like EPS (as labeled by

concanavalin A). Since concanavalin A specifically binds to α-mannopyranosyl and α-

glucopyranosyl residues, it can be assumed that these sugars are present in EPS found

on the bacterial cell surfaces. In a few cases EPS were located not only on the cell

surface but also on the modified glass. It is suggested that these extra-cellular deposits

serve as primers that modify the substratum surface and thereby facilitate bacterial

adhesion. The average height of these depositions varied between 20-200 nm depending

on the strain. It is also noted that some of the extracellular deposits clearly shown on the

SEM and AFM images were not detected by the CLSM. This observation suggests that

cells might produce a few types of EPS. Similar observations have been reported

previously. Notably, however, S. flavus cells, while exhibiting the weakest capacity to

colonize both types of glass surfaces did not produce EPS.

Chapter5:

Effects of nano-structured P(t)BMA polymer surfaces on bacterial attachment

______________________________________________________________________

126

CHAPTER 5

THE EFFECTS OF NANO-STRUCTURED P(t)BMA

POLYMER SURFACES ON BACTERIAL

ATTACHMENT

Chapter5:


______________________________________________________________________

127

5.1 Overview

In order to further investigate and confirm the effects of nano-smooth rough

surfaces on bacterial adhesion, the attachment behavior of the same bacterial strains

(E. coli, P. aeruginosa, S. aureus, P. issachenkonii, C, marina, S. flavus, S. guttiformis,

Sulfitomacter mediteraneus and A. fischeri) was tested against P(t)BMA polymeric

surfaces. Tested P(t)BM micro-texture and topography were similar to the previously

tested glass, where as its physicochemical characteristics were notably different.

P(t)BMA was used as a base for creating thin polymer layers with roughness

parameters resembling those already displayed on the as-received glass. Polymer

substrates were selected as sample surfaces because of their exceptional mechanical and

optical characteristics (>90% transparency, stiffness, low water absorption, high

abrasion resistance, etc) for which they have been frequently used as a positive

photoresist (Ivanova et al., 2006c). The same polymer surface was letter modified by

UV exposure as in detail described in section 3.3.2.b. The photolithographycaly

modified P(t)BMA surface exerted minor changes in the surface physicochemical

characteristics but considerable in the surface topography. Details regarding the surface

transformation as well as the bacterial behavior after 12 hours incubation on each of the

polymer surfaces are herein presented.


Identical as previously described in chapter 4.1

Chapter5:


______________________________________________________________________

128

5.3 P(t)BMA surface characteristics

5.3.1 Surface wettability and tension

Contact angle measurements were applied to both the native and modified

P(t)BMA surfaces in order to accurately identify modifications in the polymer surface

hydrophobicity following photolithography. Static contact angles of water droplets

deposited on both polymers surfaces were measured as detailed in chapter 3.4.1.b.

Results presented in Table 5.1 represent the average of these measurements. Images

presented in Figure 5.1 represent typical appearance of water droplet deposited on the

native (a) and on the modified (b) P(t)BMA surface.

Table 5.1: Observed water contact angle values for native and modified P(t)BMA.


(a) (b)

Figure 5.1: Static water contact angles measured on the native (a) and on the modified

(b) polymer surfaces

According to the above presented results the modified P(t)BMA surface was

found to be moderately less hydrophobic following photolithography in comparison to

Surface Contact Angle*

Native P(t)BMA 86.50 ± 1.5

Modified P(t)BMA 63.53 ± 3.0

Chapter5:


______________________________________________________________________

129

native. Approximately 15-20% reduction in the contact angles between both surfaces

was observed. This alteration in the surface hydrophobicity following UV irradiation

and subsequent heating is an indicator of the possible transformation of the surface

chemistry and topography occurring after the UV exposure. As latter on described, the

observed decrease in the surface hyderophobicity is due to the production of acid

carboxylic groups as a result of the post-exposure baking of the polymer films. The

reaction scheme for formation of activated P(t)BMA is presented in Figure 5.2.

Figure 5.2: Reaction scheme for formation of activated P(t)BMA. Image adopted from

journal article, Ivanova et al. (Ivanova et al., 2006c)

Apart from water contact angles, the contact angles for diiodomethane and

formamide were also measured. Once the mean contact angle values for each of the

diagnostic liquids have been determined, the surface free energies (total, dispersive and

acid-base) on both polymers surfaces could be calculated based on the theoretical

model by Van Oss (van Oss, 1994, Bayoudh et al., 2006). The surface free energy was

estimated at 35.7 mJ/m2 for the native and 34.1 mJ/m

2 for the modified polymer

surfaces (Table 5.2).

Chapter5:


______________________________________________________________________

130

Table 5.2: Substratum surface wettability and surface free energy before and after

modification

Contact angle*, (θ) Surface free energy**, γ, (mJ/m2) Polymer

Surface θW θF θD γLW

γAB

γ+

γ-

γTOT

Native 86 ±5 69±2 49±5 35.7 0 0 4.8 35.7

Modified 63 ±4 71±2 49±5 34.1 0 0 22.4 34.1

* Contact angle of water, formamide and diidomethane (θW, θF and θD respectively);



), electron


-)

As indicated by the above presented results, the UV exposure resulted in insignificant,

less than 5% decrease in the surface free energy.

5.3.2 Surface charge

Overall estimate of the net surface charge of the native and the modified P(t)BMA

was obtained as described in chapter 3.4.3. Acquired average results are presented in

Table 5.3.

Table 5.3: Polymer surface charge as inferred from zeta potential measurements

Sample Electrophoretic Mobility

(µs-1

)(V/cm) *

Zeta Potential ζ

(mV)

Native P(t)BMA -3.78 ± 0.09 -48.41 ± 1.2

Modified P(t)BMA -3.54 ± 0.09 -45.26 ± 1.1

* All measurements were done in triplicates and for each sample the final EPM


14 cycles per run.

As indicated from the above presented data both surfaces exhibit negative net

surface charge. Taking into account the negative charge of the polymer surfaces as well

as the negative surface charge of all nine bacterial strains involved in this study it can be

hypothesized that they would all exhibit low attachment susceptibility towards both

Chapter5:


______________________________________________________________________

131

P(t)BMA substrates. Another interesting observation is the approximately 10% lower

net surface charge measured on the modified polymer surface following

photolithography. Translated into cellular adhesive tendency this would suggest that

fewer cells are expected to be able to successfully attach after the UV exposure.

5.3.3 XPS analysis

Elemental analysis of the polymer chemical composition before and after UV

exposure was analyzed by XPS. Details regarding the instrument and the methodology

are presented in chapter 3.4.6.

As expected, surface analysis confirmed the presence of carboxylic groups on the

UV exposed P(t)BMA surface as indicated by the elevated oxygen presence (Table 5.3).

Table 5.4: Relative contributions of different chemical states assigned to the XPS peaks.

Relative elemental contribution,

atom %

Surface

C O

Native P(t)BMA 59.43 32.25

Modified P(t)BMA 19.30 46.01

Elemental analysis of the polymeric surfaces presented in Table 5.3 indicated

carbon as the predominant element present on the surface of the native P(t)BMA, with a

calculated atomic concentration of 59%. Oxygen content was secondary with an

analysed relative atomic concentration of 32%. A large difference in chemical

composition was noted between the native and the photolithographically modified

P(t)BMA, suggesting chemical modification of the surface functional groups provoked

by UV irradiation and postexposure baking. From the above presented results it is

evident that the percentage of detected oxygen on the native polymer increased from

32% to 46%. At the same time decrease in the carbon concentration from 59% to 19%

was noted.

Chapter5:


______________________________________________________________________

132

Traces of other elements were also observed on both types of P(t)BMA surfaces,

however these were not considered to be significant due to low atomic concentrations.

The typical XPS regional and wide spectra collected from both P(t)BMA surfaces is

shown in Figure 5.3.

a b

c d

e f

Chapter5:


______________________________________________________________________

133

g h

k

l

Figure 5.3: Regional and wide spectra collected from the modified (a, c, e, g, i, k) and

the native polymer surfaces (b, d, f, h, j, l).

Chapter5:


______________________________________________________________________

134

The proposed structural changes on the polymer surface caused by the

photolithography and suggested by the above presented XPS data were reproduced

by molecular modelling using the software program “SYBYL”, version 7.2. (Tripos:

www.tripos.com). Data presented in Figure 5.4.

Native P(t)BMA UV irradiated P(t)BMA

Figure 5.4: The structural re-arrangement undertaken by the P(t)BMA monomer

through photolithographic treatment is visualized by the use of molecular modelling.

Oxygen molecules are indicated by red sections, hydrogen molecules are indicated by

blue sections and carbons are indicated by grey sections. Figure adopted from

Murphy’s honours report (Murphy, 2007)

The above presented image illustrates that the number of methyl groups from the

native polymer was significantly reduced which on the other hand is indicative for

reduction of the surface hydrophobicity. Namely, the presence of tert-butyl ester and

methyl-groups on the polymer backbone are known to increase surface wettability

(Ivanova et al., 2006c). On the other hand the UV irradiation and the subsequent

heating in order to catalyse the chemical reaction resulted in formation of surface

carboxylic acid groups. The ultimate effect resulting from the presence of carboxylic

acid groups on the polymer surface was decrease of surface wettability (data presented

in Table 5.1).

5.3.4 AFM analysis

AFM analysis of both, native and modified P(t)BMA indicated a topographical

alteration in the surface roughness on the nanometer scale following photolithographic

Chapter5:


______________________________________________________________________

135

treatment. As shown on Figure 5.4 an increase in the uniformity of P(t)BMA surface

topographical features was observed after UV exposure. From image (a) which

represents the topography of the native P(t)BMA is evident that two types of surface

irregularities are present on this surface. One, the non-uniform high peaks

(approximately 40-50 nm high) randomly spread over the surface and the second type

the more uniform lower peaks (approximately 10-15 nm high) closely concentrated

across the polymer surface.

(a)

(b)

Figure 5.5: Typical 3D AFM images of the native (a) and modified (b) P(t)BMA

surfaces Scanned areas represent 7.0µm x 7.0µm.

Chapter5:


______________________________________________________________________

136

In contrast to the native form of the polymer, the topographical image of modified

P(t)BMA (b), indicates more uniform surface characteristics. The high, 50 nm, peaks

are completely absent from the surface. Observed surface irregularities are almost

entirely of the same size, 11-20 nm high and heavily packed across the polymer surface.

Table 5.5: Surface roughness parameters of the P(t)BMA before and after exposure to

UV light as inferred from the AFM measurements

Native

P(t)BMA (nm)

Modified

P(t)BMA (nm)

Ra 5.52 1.56

Rq 8.55 2.36

Rmax 53.65 21.46

* Scanned areas were 7.0 x7.0 µm


The roughness parameters presented in Table 5.5 indicate that the native P(t)BMA

surface has an average roughness (Ra) of 5.52 nm, a root-mean-square (Rq) roughness

of 8.55 nm and maximum roughness (Rmax) of 53.65 nm. On the other hand the overall

roughness parameters calculated on the modified surface measured 1.56 nm for the

average surface roughness; (Rq) was 2.36 nm and the maximum surface roughness

(Rmax) 21.46nm. These parameters suggest that the native P(t)BMA is approximately

twice as rough (on the nano-meter scale) when compared to the modified P(t)BMA.

5.3.5 SEM analysis

5.3.5.1 Overview

Detailed visualisation of the polymer surface morphology before and after bacterial

cultivation as well as the bacterial attachment pattern was obtained through SEM.

Details regarding sample preparation and microscopy technique were presented in

chapter 3.4.8.

Chapter5:


______________________________________________________________________

137

SEM images enabled analysis of the numbers of cells attached to each of the

polymer surfaces. For quantification of the number of adsorbed bacteria, cells from at

least five representative images/areas was transformed into number of bacteria per unit

area using the Image-Pro software (Waar et al., 2002). Final densities have estimated

errors of approximately 10% due to local variability in the coverage. Despite

quantitative, SEM images also enabled qualitative analyses of the cell morphology.

5.3.5.2 Control P(t)BMA surfaces

As already detailed in section 3.2.4.b, negative control experiments on both

surfaces with growth media were carried out in order to verify or discard the potential of

the used media to modify the surface. Eventual surface modifications would affect

bacterial behaviour and interfere with cellular attachment.

(a) (b)

(c) (d)

Figure 5.6: Negative control SEM images of the P(t)BMA. Scale bar equals 2µm on all

Chapter5:


______________________________________________________________________

138

images. (a) Native P(t)BMA (b) Native P(t)BMA with marine broth (c) Modified

P(t)BMA) (d) Modified P(t)BMA with marine broth.

As indicated by the images presented in Figure 5.6 the visual appearance of both, the

polymer surfaces cultivated with and without growth medium was identical, thus

indicating that any observed differences in cellular behaviour on each of the tested

surfaces would not be influenced by the medium itself.

5.4 Investigation of bacterial adhesion on nano-smooth

P(t)BMA surface

5.4.1 Attachment of E. coli cells on native and modified P(t)BMA surfaces

High resolution SEM images presented in Figure 5.7 illustrate the attachment

behaviour of E. coli cells to both, native and modified P(t)BMA. Image (a) and (c)

indicate that the overall colonization of the modified P(t)BMA surface was greater when

compared to the native. This visually attained observation was confirmed when the

number of cells per cm2 was calculated. It was estimated that the overall number of E.

coli cells attached to the native P(t)BMA is 3.1 x 106/cm

2 ± 10% which was less than

half the number of cells attached to the modified polymer surface (7.6 x 106/mm

2 ±

10%).

Monolayered cellular clusters as well as isolated sessile cells were present on both

surfaces. Although repetitive pattern of attachment was apparent on both surfaces,

differences in the cellular behaviour were still observed. For instance the overall length

of the cells attached to the native polymer surface is in the range of 1.5-2 µm Figure 5.7

(b) contrary to the approximately 2 µm long cells attached to the modified P(t)BMA

surface (Figure 5.7 (d)). Image (b) revealed that the attachment of E. coli cells to the

native polymer surface was not accompanied with deposition of exta-cellular products.

Opposite this, image (d) clearly indicates that noteworthy amounts of EPS are present

on the modified P(t)BMA.

Chapter5:


______________________________________________________________________

139

Apart from the relatively ordered pattern of distribution over both surfaces and the

absence of EPS on the native P(t)BMA surface, the SEM did not disclose any

significant variations in the cells morphology between both surfaces.

(a) (b)

(c) (d)

Figure 5.7: Selection of SEM representing the attachment behaviour of E. coli cells

after 12h incubation on the native P(t)BMA, (a) and (b), and on the modified P(t)BMA

surface (c) and (d). Scale bar represents 10 µm on image (a) and (c), 2 µm on (b) and

(d).

Observed extra-cellular deposits on the modified P(t)BMA surfaces on SEM

images were confirmed by AFM (Figure 5.8). The height of these deposits varied

between 5-40 nm. As appears by the AFM images they were mostly located in the near

Chapter5:


______________________________________________________________________

140

cell surrounding (Figure 5.8 (c)). The presence of extra-cellular deposits are indication

of the substantial metabolic activity by the cells attached on the modified P(t)BMA .

Similarly to the SEM, the AFM did not detect noteworthy differences in the cells

length and width. Measurements revealed less than 10% increase of those two

parameters, however significant 25-34% increase in the height for cells attached to the

modified polymer after 12h incubation. These differences can be most certainly credited

to the topography diversity between both polymer surfaces.

(a) (b)

(c)

Figure 5.8: Selection of AFM representing the morphology and surface topography of

E. coli cells after 12h incubation on the: (a) native P(t)BMA surface and (b): on the

modified P(t)BMA surface. Image (c) represents transverse profile of the extra-cellular

deposits on the modified P(t)BMA

Chapter5:


______________________________________________________________________

141

Bacterial viability as well as the cellular metabolic activity was reconfirmed by

CLSM (figure 5.9). Cluster of viable E. coli cells as well as viable single cells attached

to the native P(t)BMA are presented on Figure 5.8 (b). The exact corresponding image

representing the production of extra-cellular products (Figure 5.8 (a)) indicated that the

only extra-cellular deposits observed on the native P(t)BMA surfaces were most likely

capsular like nature as were all associated with the cells.

(a) (b)

(c) (d)

Figure 5.9: Selection of CLSM images representing the EPS production (a, c) and the

viability (b, d) of E. coli cells after 12h incubation on native (a, b) and modified (c, d)

P(t)BMA surface. Scale bar represents 5µm on all images

CLSM image presented in Figure 5.9 (c) show that in contrast to native P(t)BMA a

considerable quantities of EPS containing α-mannopyranosyl and α-glucopyranosyl

Chapter5:


______________________________________________________________________

142

residues were observed not only overlaying the cells attached to the modified P(t)BMA

but were also deposited on the polymer surface (marked areas). Image (d) indicates that

majority of attached cells maintained their viability after successfully colonizing the UV

irradiated P(t)BMA surface.

5.4.2 Attachment of P. aeruginosa cells on native and modified P(t)BMA

surfaces

In a similar manner as E. coli, the attachment behaviour, bacterial morphology as

well as metabolic activity of P. aeruginosa cells was also observed by means of SEM,

AFM and CSLM.

SEM images presented in Figure 5.10 indicate that P. aeruginosa cells attached in

modest quantities on the native polymer surface. Transferred into number of cells per

unit area it appeared that approximately 4.35 x 106 ± 10% cells attached to the native

and at least three times more to the UV exposed polymer surface. Even though majority

of cells attached to the native P(t)BMA surface were organised in clusters, sessile cells

were also observed. On the other hand cells attached to the modified polymer surface

started forming multylayered colonies contrary to the unilayered clusters of cells present

on the native P(t)BMA. Another noteworthy observation are the atypically long P.

aeruginosa cells located on the native P(t)BMA surface (middle section on Figure 5.10

(b)).

Apart from the evident numerical difference, presented images also indicate

noteworthy changes in the cellular morphology. To be exact, cells adsorbed onto the

modified polymer surfaces appeared more voluminous and uneven when compared to

the relatively smooth and flattened cells adsorbed on the native P(t)BMA. These

differences in the cells morphology as well as the granular EPS deposition on the

polymer surface after the UV exposure were confirmed by AFM and CLSM.

Chapter5:


______________________________________________________________________

143

(a) (b)

(c) (d)

Figure 5.10: Selection of SEM representing the attachment behaviour of P. aeruginosa

cells after 12h incubation on the native images (a) and (b), and on the modified

P(t)BMA surface, images (c) and (d). Scale bar represents 10 µm on image (a) and (c),

2 µm on (b) and (d).

As suggested by the AFM images presented in Figure 5.11 and indicated by the

AFM surface analysis cells attached to the native P(t)BMA were 1.7µm long, 0.9µm

wide and approximately 0.17nm high, contrary to this cells attached to the modified

P(t)BMA were 2.3µm long, 1.3µm wide and 0.24µm high.

AFM scan across the modified P(t)BMA surface (Figure 5.11 (b)) incubated for

12h in the presence of P. aeruginosa cell suspension confirmed the existence of EPS

deposits on the polymer surface (marked areas) and the cell surface as well.

Chapter5:


______________________________________________________________________

144

(a) (b)


P. aeruginosa cells and produced EPS after 12h incubation on the native (a) and

modified (b) P(t)BMA surface.

]

Better visualization of the EPS produced by P. aeruginosa cells after 12h

incubation on each of the P(t)BMA surfaces was achieved by colouring with

Concanavalin Alexa 488 (Figure 5.12 (a) and (b)). All the green fluorescent signal

observed on image (a) derive from the cells indicating that all the EPS produced by the

cells attaching to the native polymer surface are most likely of capsular nature. Contrary

to this granular EPS deposits containing α-mannopyranosyl and α-glucopyranosyl

residues were observed on the photolithoraphically modified polymer surface (pointing

arrows image (c)). Cells attached to this surface were also overlayed with capsular EPS.

(a) (b)

Chapter5:


______________________________________________________________________

145

tT

(c) (d)


viability (b, d) of P. aeruginosa cells attaching to the native (a, b) and to the UV-

exposed (c, d), P(t)BMA polymer surface after 12h incubation. Scale bar on all images

represents 2µm

Apart from the production of EPS, the CLSM images on Figure 5.12 (b) and (d)

also represent the cellular viability after 12h incubation on both polymer surfaces. It is

evident that significant number of cells attached and managed to maintain their viability

on each of the surfaces.

5.4.3 Attachment of S. aureus cells on native and modified P(t)BMA surfaces

The attachment behaviour of S. aureus cells on the native and modified P(t)BMA

surface was also observed by means of SEM, AFM and CLSM. As the SEM images

presented in Figure 5.13 indicate sufficient number of cells organised in grape-like

clusters, typical for this strain, were observed on both surfaces. Estimated number of

cells per unit area was 13.26 106 ± 10% to the native and 18.7 x 10

6 ± 10% to the

modified P(t)BMA surface. These values demonstrate severe variations when compared

with previously observed for E. coli and P. aeruginosa. Namely, the ratio between cells

adsorbed on the native and modified P(t)BMA surface for E. coli was 1:2, where as for

Chapter5:


______________________________________________________________________

146

P. aeruginosa was 1:3. It appeared that even the native polymers surface attracted

significant number of S. aureus cells. However cells adsorbed on the modified P(t)BMA

surface was double that on the native, where as three times more for the P. aeruginosa.

One of the possible reasons for such adhesive behaviour might be the fact that all

species belonging to the S. genus are coccoid contrary to E. coli and P. aeruginosa who

are rod shaped. The shape and morphology appeared to be identical for cells attached to

the native as well as to the modified P(t)BMA.

.

(a) (b)

(c) (d)

Figure 5.13: Selection of SEM representing the attachment behaviour of S. aureus cells


surface (c) and (d). Scale bar represents 10 µm on (a) and (c), 1 µm on (b) and (d).

Images presented in Figure 5.13 also indicate that that regardless of the type of the

surface, after 12h incubation cells managed to form only monolayered multicellular

Chapter5:


______________________________________________________________________

147

colonies. It appeared that jelly-like material, most likely EPS was secreted by S. aureus

cells attached on both surfaces most likely to keep/glue cells in close proximity to eash

other and provide some sort of support. Apart from the EPS observed in between cells,

the SEM did not detect other extra-cellular deposits on each of the surfaces. The same

observation was confirmed by AFM images presented in Figure 5.14.

AFM statistical analysis of the cells adsorbed on both polymer surfaces confirmed

what appeared to be obvious even on the SEM images. Namely, cells appeared to be of

approximately the same size regardless of the surface they were attached to. Inferred

dimensions indicated approximately 1.0x1.0 µm in length and width and close to 500

nm height.

(a) (b)


S. aureus cells after 12h incubation on the native (a) and modified (b) P(t)BMA surface.

The CSLM images presented in Figure 5.15 established the existence of α-

mannopyranosyl and α-glucopyranosyl deposits on the cellular surface after attachment

to both surfaces. This is a strong indication that the EPS produced over the 12h

incubation period are most likely to be of capsular origin.

Chapter5:


______________________________________________________________________

148

(a) (b)

(c) (d)


viability (b, d) of S. aureus cells attaching to the native (a and b) and to the UV-exposed

(c and d) P(t)BMA polymer surface after 12h incubation. Scale bar on all images is

2um

The pointing arrows on Figure 5.15 (d) suggest that extra-cellular deposits might be

present not only on the cell surface but also between cells. The existence of such jelly-

like deposits over-coating bacterial clusters was already pointed on the SEM images

(Figure 5.13).

Chapter5:


______________________________________________________________________

149

5.4.4 Attachment of C. marina cells on native and modified P(t)BMA

surfaces

Similarly as with non-marine bacteria, the attachment behaviour and subsequent

growth on both polymer surfaces for six marine bacteria was also tested.

High resolution SEM images presented in Figure 5.16 (a) and (b) revealed that

single cells randomly colonised native P(t)BMA surface. Contrary to this, C. marina

cells attaching to the modified polymer surface were organised in multicellular, even

multilayered clusters. Images presented in Figure 5.16 (c) and (d) indicate that the outer

surface of cells attached to the modified P(t)BMA was not as smooth and flattered as

that of cells attached to the native polymer. The presence of granular like deposits, most

likely EPS, noted on the surface of cells attached to the modified P(t)BMA was also

noted on the polymer surface as well. It is noteworthy mentioning that various cell

dimensions and forms were observed on this surface in contrast to the native where cells

appeared to be of more uniform morphology.

The difference in the cells appearance as well as the presence of extra-cellular

deposits was also detected on the AFM images presented in Figure 5.17 and on the

CLSM images presented in Figure 5.18.

(a) (b)

Chapter5:


______________________________________________________________________

150

(c) (d)

Figure 5.16: Selection of SEM representing the attachment behaviour of C. marina

cells after 12h incubation on the native P(t)BMA, (a) and (b), and on the modified

P(t)BMA surface (c) and (d). Scale bar represents 10 µm on image (a) and (c), 2 µm on

image (b) and (d).

Bellow presented AFM images verified the observed higher cellular density on the

modified surface. They also confirm the existence of primarily elongated cells on the

native polymer surface, contrary to the predominantly spherical and elliptical cells

attached on the UV-exposed polymer. In contrast to the SEM where C. marina cells

attached to the native P(t)BMA surface appeared smooth, the AFM revealed that the

outside of these cells was also irregular and roughened, identical as with cells adsorbed

on the UV-irradiated P(t)BMA.

Table 5.6: Roughness parameters taken from the surface of C. marina cells attached to

the native and modified P(t)BMA surface

*Scanned area 0.5µm x 0.5µm


C. marina cells attached

to native P(t)BMA

C. marina cells attached to

modified P(t)BMA

Ra 13.66 nm 17.18 nm

Rq 16.54 nm 20.16 nm

Rmax 76.08 nm 89.81 nm

Chapter5:


______________________________________________________________________

151

At first observed cell surface irregularities appeared to be similar regardless of the

type of surface cells were adsorbed to. Detailed analysis of roughness parameters taken

from 0.5x0.5µm areas from the surface of cells adsorbed onto each of the P(t)BMA

surfaces revealed significant variations. Namely all roughness parameter measured on

the surface of C. marina cells adsorbed on the UV-exposed polymer appeared to be by

approximately 20% higher when compared to those taken from the surface of cells

adsorbed onto the native P(t)BMA. Data presented in Table 5.6.

Contrary to previously observed morphologic changes (increase of cell dimensions)

for E. coli and P. aeruginosa cells, surface modification did not induce such behaviour

for C. marina cells. Namely, cells adsorbed on the native P(t)BMA were found to be

2.4µm long, 1.6µm wide and 210nm high, opposite cells adsorbed on the UV-exposed

surface whose observed dimensions were as follows; 1.3µm length and 1.0µm width. It

was difficult to estimate the height of C. marina cells attached to the modified polymer

surface as it is suspected that cells adsorbed onto this surface were embedded in layer of

EPS. As image (c) indicates the overall height of the extra-cellular products was in the

range of 180nm.

(a) (b)

Chapter5:


______________________________________________________________________

152

(c)


C. marina cells and produced EPS after 12h incubation on the native (a) and modified

(b) P(t)BMA surfaces. Image (c) represents transverse profile of the overall height of

cells and EPS adsorbed on the modified P(t)BMA

In order to better visualize produced EPS, Concanavalin A, fluorescent dye was

used. At the same time red fluorescent dye, staining only the viable cells attached to

both surfaces was also applied.

As the CLSM images presented bellow indicate, C. marina cells attached and

sustained their existence on both polymer surfaces. It is also evident that almost all EPS

produced by the bacteria during the attachment is in close association with the cells. It

can be speculated that lesser quantities of EPS are produced by bacteria while attaching

to the native polymer surfaces as few viable cells without EPS coating were observed

(arrows on Figure 5.18 (a)).

Contrary to this, all red florescent signals detected from viable cells attached to the

UV-exposed P(t)BMA (Figure 5.18 (c)) corresponded to identical green fluorescent

signal (Figure 5.18 (d)); thus indicating that every single cell adsorbed on the modified

surface is enclosed in EPS of a capsular nature.

Chapter5:


______________________________________________________________________

153

(a) (b)

(c) (d)

Figure 5.18: Selection of CLSM images representing the EPS production (b, d) and the

viability (a, c) of C. marina cells attaching to the native (a, b) and to the UV-exposed

(c,d)) P(t)BMA polymer surface after 12h incubation. Scale bar on all images is 2um

Apart from the capsular like EPS there were no isolated polysaccharide deposits

evident on the polymer surface itself. Excess of EPS were noted on few locations

(pointing arrows on Figure 5.18 (d)) but even those were in close proximity and

associated with the cells.

It is worth mentioning that detected green fluorescent signals arising from the

capsular like EPS deposited on cells adsorbed on the UV-exposed P(t)BMA surface

(Figure 5.18 (d)) are presented in discontinuity, in a granular-like manner, contrary to

those presented in image (b) where viable cells seemed to be completely encapsulated in

a continuous layer of extra-cellular deposits. This might be an indication of distinct

Chapter5:


______________________________________________________________________

154

chemical composition between EPS produced by the cells attached to each of the

surfaces.

5.4.5 Attachment of P. issachenkonii cells on native and modified P(t)BMA

surfaces

High resolution SEM images presented in Figure 5.19 represent the attachment

behaviour of P. issachenkonii cells on the native and modified P(t)BMA surface. They

clearly point to the differences in the quantity of adsorbed cells, as well as the cells’

morphology. Calculated numbers of cells per unit area confirmed that the number of P.

issachenkonii cells attached to the modified P(t)BMA is 9.25 x 106cells/cm

2, which is

three times higher than the that calculated on the native polymer surface. Also, cells

adsorbed on the native P(t)BMA appeared to be flatter (Figure 5.19 (a) and (b)) when

compared to those adsorbed on the modified (Figure 5.19 (c) and (d)).

(a) (b)

Chapter5:


______________________________________________________________________

155

(c) (d)

Figure 5.19: Selection of SEM representing the attachment behaviour of P.

issachenkonii cells after 12h incubation on the native P(t)BMA, (a) and (b), and on the

modified P(t)BMA surface, (c) and (d). Scale bar represents 10 µm on images (a) and

(c), 1 µm on images (b) and (d).

SEM imaging of the modified polymer surface under higher magnification (Figure

5.19 (d)) detected the presence of granular like deposits on the surface believed to be

EPS. Their existence was also confirmed by AFM.

AFM scanning of both polymer surfaces after 12h incubation in bacterial

suspension supported already observed cellular behaviour by SEM. It was evident that

significant number of cells successfully attached to each of the surfaces. The AFM also

revealed that granular like deposits were present on both polymer surfaces, native and

modified, as well as on the P. issachenkonii cell surface (Figure 5.20).

Chapter5:


______________________________________________________________________

156

(a) (b)


P. issachenkonii cells and produced EPS after 12h incubation on the native (a) and

modified (b) P(t)BMA surfaces

Statistical analysis of the cellular surface roughness was also considered.

Table 5.7: Roughness parameters taken from the surface of P. issachenkonii cells

attached to the native ad modified P(t)BMA surface

P. issachenkonii cells attached on

the native P(t)BMA

P. issachenkonii cells attached

on the modified P(t)BMA

Ra 6.3nm 10.5nm

Rq 7.8nm 11.9nm

Rmax 40.9nm 50.6nm

*Scanned areas represent 0.5x0.5µm


As the data in Table 5.7 indicates, cells adsorbed onto the modified polymer surface

are rougher, thus suggesting they might carry more EPS deposits on their surface.

Inferred cell dimension revealed that P. issachenkonii cells attached to the modified

P(t)BMA were slightly bigger when compared to those adsorbed onto the native

polymer surface (data presented in Table 5.8).

Chapter5:


______________________________________________________________________

157

Table 5.8: Dimensions of P. issachenkonii cells attached to the native ad modified

P(t)BMA surface


on the native P(t)BMA


on the modified P(t)BMA

Length 1.5 µm 1.8 µm

Width 800 nm 900 nm

Height 80 nm 100 nm


This observation is consistent with previously reported trend for other rod shaped

bacteria. On the other hand, the average height of cells attached to either of the surfaces

was extremely low, 80 nm for cells adsorbed on the native and 100 nm for cells

adsorbed on the modified polymer surface. Coming back to the previous observation

that both surfaces appeared to be completely covered in granular EPS, it can be

speculated that the low cellular height might be due to their partial embedment of the

cells in the EPS deposits.

The EPS nature of the extra-cellular surface deposits was confirmed by CLSM. As

the images presented in Figure 5.21 indicate each cell regardless of the surface it was

adsorbed to was surrounded with EPS. Images (a) and (b) show that majority of cells

adsorbed to the native P(t)BMA maintained their viability during the proposed

incubation interval. They also illustrate the mono-layered pattern of colonization after

12h incubation. Contrary to this cells adsorbed on the modified polymer surface during

the same incubation period started to form multilayered cellular structures (bottom right

corner on Figure 5.21 (c) and (d)).

(a) (b)

Chapter5:


______________________________________________________________________

158

(c) (d)


viability (b, d) of P. issachenkonii cells attaching to the native (a, b) and to the UV-

exposed (c, d) P(t)BMA polymer surface after 12h incubation. Scale bar on all images

is 10µm

The existence of such multilayered colonies as well as increased cellular density

can be attributed to the amplified inclination of Pseudoalterononas issachnkonii cells

towards the UV exposed P(t)BMA surface when compared to the native.

5.4.6 Attachment of S. flavus cells on native and modified P(t)BMA surfaces

In a similar manner as for all the other strains used in this study, the adhesive

characteristics of S. flavus were tested against native and modified P(t)BMA. Initial

inspection of images obtained by SEM (Figure 5.22), AFM (Figure 5.23) and CLSM

(Figure 5.24) indicated that S. flavus cell presented different adhesive behaviour

compared to the other tested strains. The most striking observation was the extremely

low adhesive propensity towards each of the surfaces. Estimated numbers of

approximately 1 x 106cells/cm

2 on the native and close to 1.2 x 10

6cells/cm

2 on the

UVexposed polymer support this observation.

SEM image presented in Figure 5.22 (a) indicate that there is extremely low cellular

density on the native polymer. Higher magnification image of the same field of view

Chapter5:


______________________________________________________________________

159

(Figure 5.22 (b)) revealed the presence of sufficient quantities of granular extra-cellular

products in the near-cell surrounding. It appeared that few of the cells that managed to

successfully attach are completely embedded in these EPS deposits. This on the other

hand is an indication of the somewhat “complicated” adhesive preferences of this strain

which eventually will lead to significant structural transformation of the polymer

surface topography.

(a) (b)

(c) (d)

Figure 5.22: Selection of SEM representing the attachment behaviour of S. flavus cells


surface (c) and (d). Scale bar represents 2 µm on all images.

Chapter5:


______________________________________________________________________

160

Similar conclusion can be drawn by observing images (c) and (d) on Figure 5.22

which represent overview of the modified P(t)BMA surface topography and the cellular

appearance after 12 h incubation. It is obvious that more cells managed to secure their

existence on this surface; however it appears that the near-cell surface modification

plays imperative role in the process.

The existence of the surface deposits was confirmed by AFM. As image (a) and (b)

Figure 5.23 indicate cellular absence was obvious, however granular-like surface

deposit believed to be EPS were detected on the native P(t)BMA surface. The average

diameter of these EPS varied between 200 and 400 nm whereas the average height was

in the range of approximately 10-20 nm.

Almost identical surface features were observed on the UV exposed P(t)BMA

surface; images presented in Figure 5.23 (c) and (d). The absence of cells was again

noted and the existence of granular EPS confirmed. The only difference in comparison

to the native polymer surface is the relatively larger size of the EPS. Namely their

height remained the same, 15-20 nm, but their diameter could reach up to 900 nm.

(a) (b)

Chapter5:


______________________________________________________________________

161

(c) (d)

Figure 5.23: Selection of AFM representing the native and modified P(t)BMAS surface

topography after 12h incubation in S. flavus culture medium.

The fact that no cells were detected on the AFM images on either of the surfaces,

does not exclude their existence nighter contradicts the SEM conclusions, it might

simply be result of the more complex nature of this microscopy technique when

compared to the SEM.

(a) (b)

Figure 5.24: Selection of CLSM images representing the EPS production (a, b) of S.

flavus cells attaching to the native (a) and to the UV-exposed (b) P(t)BMA polymer

surface after 12h incubation. Scale bar on all images is 2µm

As the CSLM images indicate (Figure 5.24) extra-cellular deposits containing α-

mannopyranosyl and α-glucopyranosyl were detected on both polymer surfaces, native

Chapter5:


______________________________________________________________________

162

and modified, however the same field of view did not indicate presence of viable cells

capable to produce fluorescent, amine-reactive carboxyfluorescein succinimidyl ester.

5.4.7 Attachment of S. guttiformis cells on native and modified P(t)BMA

surfaces

The attachment behaviour of this bacterium on native and exposed P(t)BMA

surfaces aroused particular interest, mostly because this investigation was a continuation

of previous work designed to probe the attachment of marine α-Proteobacteria and their

biofilm formation on polymeric surfaces (Ivanova et al., 2002a).

It was previously found that S. brevis, S. mediterraneus, and S. pontiacus, all

belonging to the same taxonomic lineage as S., expressed different attachment

behaviour on P(t)BMA. In particular, vegetative cells of S. mediterraneus underwent

morphological conversion into coccoid forms, while S. pontiacus and S. brevis failed to

attach onto poly(tert-butyl methacrylate) polymeric surfaces. In contrast, S. guttiformis

was able to successfully sustain its existence on this surface and form a biofilm

(Ivanova et al., 2002a, Ivanova et al., in press). In light of this finding, it was of interest

to study in more detail the attachment pattern of S. guttiformis cells on native and

modified P(t)BMA surfaces. Same as for the other strains whose adhesive

characteristics onto the two types of polymer surface were tested, the attachment pattern

of S. guttiformis was visualised using SEM and AFM imaging and the cell viability and

production of EPS was confirmed by confocal microscopy.

Analysis of a series of SEM images taken at different magnifications (as shown on

Figure 5.25 (a) and (b)) revealed that a particular biopolymer network had formed by

the cells while they had been attaching on the native polymeric surface. The

biopolymer, apparently EPS, appears to have served as a primer to support bacterial cell

adhesion and formed some sort of bridge to facilitate connection between the cells and

the substrate surface. It was also noted that the bacterial cell surface was not smooth,

but somewhat rough with lumpy features, observation that was latter confirmed by

AFM (Figure 5.26 (a) and (b)).

Chapter5:


______________________________________________________________________

163

Contrary to this observed cellular behaviour on the modified P(t)BMA surface was

noticeably different (Figure 5.25 (c) and (d)). Greater number of cells with variable

dimensions and forms appeared to be able to successfully colonise this surface. It was

also noted that the EPS produced by the cells during the 12 h incubation period

appeared different when compared to those already observed on the native P(t)BMA

surface. Each cell appeared to be enclosed in jelly-like matrix that stretches in-between

cells ensuring better inter-cellular connection.

Granular like extra-cellular deposits were not only observed on the cell surface as

but also in the near cell surrounding. The existence of these deposits on the native as

well as on the modified polymer surfaces was confirmed on the AFM and CLSM

images. Even though their exact chemical structure is yet to be revealed it is more than

clear that they are of a different composition judging by the different appearance under

SEM.

(a) (b)

Chapter5:


______________________________________________________________________

164

(c) (d)

Figure 5.25: Selection of SEM representing the attachment behaviour of S. guttiformis

cells after 12h incubation on the native P(t)BMA, (a) and (b), and on the modified

P(t)BMA surface (c) and (d). Scale bar represents 10 µm on images (a) and (c), 2 µm

on images (b) and (d).

The AFM image in Figure 5.26 (a) represents 65µm x 65µm scanned area of the

native P(t)BMA surface colonised by S. guttiformis cells after 12 h incubation. It is

evident that cells were gathered in clusters of variable size. Closer view (image (b)

enabled measurements of the cell dimensions. It revealed that majority of attached cells

were 1.5µm long, 0.7µm wide and approximately 200nm high.

(a) (b)

Figure 5.26: Selection of AFM images representing the morphology and surface

topography of S. guttiformis cells after 12h incubation on the native P(t)BMA surface

Chapter5:


______________________________________________________________________

165

Even though extra-cellular deposits were not observed on closer view (Figure 5.26

(b)) further analysis of the cell surface topography on a nano-meter scale (Figure 5.27)

as well as the polymer surface (Figure 5.28) using a non-contact AFM was undertaken.

This allowed further insight into the fine structure and distribution of bacterial extra-

cellular surface materials.

(a) (b)

Figure 5.27: Typical high-resolution AFM topographical images (non-contact mode) of

S. guttiformis cells; (a) cell attached to the native P(t)BMA surface and a lose granular

EPS surrounding the cell; (b) zoomed area on the surface of the cell showing cell

surface topography.

(a) (b)

Figure 5.28: A typical AFM topographical image of the loose granular EPS on the

native P(t)BMA surface; (a) high resolution image obtained in the non-contact mode;

(b) a transverse profile of granular EPS in a nano-meter scale. Similar images were

obtained in different regions of at least two different samples.

Chapter5:


______________________________________________________________________

166

Roughness analysis of the cell surface as well as on the polymer surface was also

performed (marked areas, Figure 5.26 (b)). Data presented in Table 5.9.

Table 5.92: Roughness parameters taken from the surface of S. guttiformis cells

attached to the native P(t)BMA surface and from the polymer surface itself.

As the roughness parameters presented in Table 5.9 and the images presented in

Figure 5.28 indicate, certain quantities of EPS materials encapsulating the entire cell

and spreading to the surrounding substrate surface/neighbouring cell are present. The

thickness of the EPS layer, as measured against the substrate surface, ranged between 5-

35nm.

Further on, S. guttiformis cells attached to the modified P(t)BMA surfaces were

also observed. As the images presented in Figure 5.29 (a) and (b) indicate significant

number of cell gathered in clusters of few. Thick layer of extra-cellular products

surrounding and embedding the cells was also evident. Cellular and EPS height was

measured (transverse profile presented on image (c), Figure 5.29).

(a) (b)

S. guttiformis cell surface

roughness on native P(t)BMA

Substratum surface roughness

(native P(t)BMA)

Ra 2.33 nm 4.67 nm

Rq 3.03 nm 6.35 nm

Rmax 13.37 nm 35.59 nm

Chapter5:


______________________________________________________________________

167

(c)

Figure 5.29: Selection of AFM images representing the morphology and surface

topography of S. guttiformis cells after 12h incubation on the modified P(t)BMA

surface; image (c) represents transverse profile of the overall height of EPS deposited

on the surface

Roughness analysis of the cell surface as well as the modified polymer surface was

also taken. As the data presented in Table 5.10 indicate and when compared with the

data presented in Table 5.9 it is evident that overall all substratum roughness parameters

are at least double those measured on the native P(t)BMA.

The roughness on top of the cells themselves was also increased by factor of two

when compared to the cells attached to the native P(t)BMA, thus suggesting distinct

chemical/structural composition of the synthetised EPS encapsulating the cell.

At the same time, cells attached to the modified polymer surface appeared to be

approximately 25% bigger, thus supporting the already suggested theory that the

polymer modification stimulates some-sort of metabolic/morphologic cellular change.

Chapter5:


______________________________________________________________________

168

Table 5.10: Roughness parameters taken from the surface of S. guttiformis cells

attached to the modified P(t)BMA surface and from the polymer surface itself.


Fluorescent conjugate of a carbohydrate-binding protein, concanavalin A, was used

in attempt to clarify the nature of the extracellular polymeric substances produced by S.

guttiformis cells during the proposed incubation period on both polymer surfaces.

(a) (b)

(c) (d)

S. guttiformis cells attached to the

modified P(t)BMA

Substratum surface

roughness (modified

P(t)BMA)

Ra 4.52 nm 16.85 nm

Rq 5.42 nm 19.78 nm

Rmax 24.23 nm 72.68 nm

Chapter5:


______________________________________________________________________

169

(e) (f)


viability (b, d) of S. guttiformis cells after 12h incubation on native (a, b) and modified

(c, d) P(t)BMA surface. Scale bar indicates 10µm on image a, b, c and d. Face contrast

images of S. guttiformis cells attached to the native (e) and to the modified (f) P(t)BMA

surface representing the overall cell distribution and the presence od EPS on the cell

surface

As can be seen from the images presented on Figure 5.30, significant number of viable

cells colonized both surfaces, the native (b) and the modified (d) P(t)BMA. However,

images (a) and (c) indicate that almost all cells attached to either of the surfaces were

encapsulated in EPS containing α-mannopyranosyl and α-glucopyranosyl residues. As

concanavalin A specifically binds to these saccharides, it can be assumed that they are

components of S. guttiformis surface EPS. However, lumpy-granular EPS deposited on

the substratum observed on the SEM and AFM images were not detected on the

confocal images, suggesting the distinct chemical nature of this type of EPS.

5.4.8 Attachment of S. mediteraneus cells on native and modified P(t)BMA

surfaces

The attachment behaviour of S. mediteraneus on native P(t)BMA surface has

already been studied by Ivanova et al. (Ivanova et al., 2002a). As concluded in their

Chapter5:


______________________________________________________________________

170

study, and already reported here in Chapter 4.3.8; vegetative cells of S. mediteraneus

are capable of transforming into coccoid bodies when cultivated for 24h on P(t)BMA

surface.

In a similar manner as for all other strains, the adhesive characteristics of this

particular bacterium were tested against native and UV-exposed P(t)BMA during 12h

incubation timeframe. As the SEM images presented in Figure 5.31 and later on

confirmed by the AFM (Figure 5.32) and CLSM (Figure 5.33) indicate, significant

difference in the cellular adhesive behaviour onto each of the surfaces was observed. In

both cases it appeared that adsorbed cell were elongated, tear-like shaped. The only

clear difference between cells adsorbed on the native and on the modified P(t)BMA

observed on the SEM images is the existence of granular-like deposits on the cells

adsorbed on the modified polymer. It is also evident that the number of cells attached

this surface is considerably higher in comparison to the number of cells attached to the

native polymer surface. When translated into number of cells per unit area, it was

confirmed that 5.2 x 106cells/cm

2 were adsorbed on the native and 8.1 x 10

6cells/cm

2 on

the UV-exposed.

SEM imaging also revealed that significant amounts of extra-cellular deposits,

believed to be EPS were located in the near-cell surrounding on the modified P(t)BMA.

(a) (b)

Chapter5:


______________________________________________________________________

171

(c) (d)

Figure 5.31: Selection of SEM images representing the attachment behaviour of S.

mediteraneus cells after 12h incubation on the native P(t)BMA, (a) and (b), and on the

modified P(t)BMA surface (c) and (d). Scale bar represents 10 µm on images (a) and

(c), 2 µm on images (b) and (d.)

Differences in individual cell dimensions were not as obvious as for other reported

strains. It appeared that cells attached to the native polymer surface were 1.2µm x

0.9µm x 0.3µm, whereas cell attached on the UV-exposed polymer measured 1.7µm x

0.8µm x 0.35µm. It is noteworthy that clear determination of cells dimensions was

challenging due to two main causes. First, cells appeared to be surrounded by expressed

EPS in the near-cell environment (Figure 5.31 (b)), and second their form varied from

tear-like to more spherical, which again is indication of their capability to transform into

coccoid bodies (Figure 5.32).

Chapter5:


______________________________________________________________________

172

(a) (b)

Figure 5.32: Selection of AFM images representing the attachment behaviour of S.

mediteraneus cells after 12h incubation on the native P(t)BMA, (a) and on the

modified(b) P(t)BMA surface.

CLSM (Figure 5.33) revealed that S. mediteraneus cells successfully attached and

maintained their viability on both surfaces. Concanavalin Alexa 488, effectively

coloured extra-cellular products deposited on the cell surface but did not those deposited

on the substratum surface already noted by the AFM. As already stated, this dye only

labels α-mannopyranosyl and α-glucopyranosyl residues which indicates that the EPS

deposited on the surface are of distinct chemical composition when compared to those

adjacent to the cell surface.

(a) (b)

Chapter5:


______________________________________________________________________

173

(c) (d)


viability (b, d) of S. mediteraneus cells after 12h incubation on native (a, b) and

modified (c, d) P(t)BMA surface. Scale bar represents 1um

The CLSM images presented in Figure 5.32 also supported previous observation

that the overall shape of the S. mediteraneus cells attached to each of the surfaces is

unstable and inconsistent with the original description for this strain (Pukall et al.,

1999).

5.4.9 Attachment of A. fischeri cells on native and modified P(t)BMA

surfaces

SEM images presented in Figure 5.34 illustrate the attachment behaviour of A.

fischeri cells after 12h incubation on native and modified P(t)BMA surfaces. From the

below presented images it is evident that substantial amount of A. fischeri cells were

able to successfully colonize the native P(t)BMA surface in an ordered manner. Cells

observed in Figure 5.34 (a) appear to follow growth ‘channels’ across the surface.

Higher magnification images (Figure 5.34 (b)) indicated that sessile cells are able to

branch from these channels, probably to further colonize the surface. It is obvious that

cells are rod shaped and between 1.5-2µm in length typically for this species (Krieg and

Chapter5:


______________________________________________________________________

174

Holt, 1984). The number of attached cells per unit area was approximately 8.7 x 106

cells/cm2.

SEM images presented in Figure 5.34 (c) and (d) indicate the attachment pattern of

A. fischeri to the surface of modified P(t)BMA. A greater amount of cell appear to have

been able to successfully attach to the modified polymer and proliferate in comparison

to native P(t)BMA (12 880 000cells/cm2). Surface colonization however appears non-

directional with no clear growth patterns. The distribution of suspected EPS materials

(white substance overlaying the cell surface) produced by A. fischeri is shown in Figure

in Figure 5.34 (d). The greatest quantities of EPS are present in areas where cells are

within close proximity of one another (probably indicating some sort of beginning stage

of biofilm formation). Although cells are characteristically rod shaped and

approximately 2µm in length, cells morphology is difficult to distinguish. Nevertheless,

they are clearly different than the cells attached to the native P(t)BMA. Namely cell

surface appears to be granular and cells them self seemed to be more voluminous, most

likely due to the generous EPS production.

(a) (b)

Chapter5:


______________________________________________________________________

175

(c) (d)

Figure 5.34:: Selection of SEM images representing the attachment behaviour of A.

fischeri cells after 12h incubation on the native P(tBMA), (a) and (b), and on the

modified P(t)BMA surface (c) and (d). Scale bar represents 10 µm on images (a) and

(c), 2 µm on images (b) and (d.)

The granular-like cell surface appearance was confirmed by AFM. As the images

presented in Figure 5.35 (a) and (b) indicate A. fischeri cells attached to either of the

polymer surfaces appeared to carry granular EPS deposits on their surface.

(a) (b)

Figure 5. 35: AFM images of A. fischeri cells attaching to the native (a) and to the

modified (b) P(t)BMA surface after 12h incubation

Chapter5:


______________________________________________________________________

176

Small, 0.5µm x 0.5µm areas, located on the surface of cells attached on each of

the surfaces were scanned. Collected roughness parameters (date presented in Table

5.11) revealed no significant difference from the cell surface. This observation and the

fact that cells attached to the UV-exposed polymer were slightly higher (data presented

in Table 5.12) indicates that secreted EPS are most likely of identical chemical

composition but the quantities of secreted EPS varied depending on the probed surface.

This on the other hand is an indication of the cellular metabolic transformation

stimulated by the diversity of the substratum characteristics.

Table 5.11: Roughness parameters taken from the surface of A. fischeri cells attached

to the native ad modified P(t)BMA surface

A. fischeri cells attached on

the native P(t)BMA

A. fischeri cells attached on

the modified P(t)BMA

Ra 7.33nm 7.31nm

Rq 10.32nm 10.23nm

Rmax 58.23nm 59.46nm

*Scanned area represents 0.5µm x 0.5µm


Table 5.3: Dimensions of A. fischeri cells attached to the native ad modified P(t)BMA

surface

A. fischeri cells attached to

the native P(t)BMA

A. fischeri cells attached to

the modified P(t)BMA

Length 1.5µm 1.8µm

Width 0.9µm 0.9µm

Height 100nm 140nm


Cell viability and production of EPS during 12h incubation period on each of the

polymer surfaces was observed by CLSM, using two dyes simultaneously, as already

detailed in Chapter 3.2.5.

Chapter5:


______________________________________________________________________

177

(a) (b)

(c) (d)


viability (b, d) of A. fischeri cells after 12h incubation on native (a, b) and modified (c,

d) P(t)BMA surface. Scale bar represents 10um on all images.

The attachment of viable A. fischeri cells on modified P(t)BMA is observed in

Figure 5.36 (d). When compared to the number of viable cells attached to the native

polymer surface (Figure 5.36 (b)) it appeared that greater number of cells were able

successfully attach and proliferate on the modified P(t)BMA surface. It is also evident

that almost every cell is enveloped in capsular like extra-cellular product containing

residues of α-mannopyranosyl and α-glucopyranosyl, as evidenced by images (a) and

(c). It also noteworthy that cells attached to the modified P(t)BMA surface appeared to

gather in multi-cellular even multilayered groups. Contrary to this, cells attached to the

native polymer surfaces appeared to spread and colonise evenly across the surface,

which eventually contributed to overall higher cell density on the UVexposed P(t)BMA.

Chapter5:


______________________________________________________________________

178

5.5 Conclusion

A decrease in the overall P(t)BMA roughness upon UV exposure provided nano-

smooth surfaces with increased uniformity. SEM and AFM analysis in combination

with CSLM revealed that studied bacteria showed consistent adsorption preference

towards the photolithographycally modified P(t)BMA surfaces. Approximately 30-50%

increase in the number of rod-shaped bacteria attached to the modified polymer surfaces

was observed. Contrary to this the coccoid, S. aureus cells maintained higher presence

on the native polymer surfaces.

Increased bacterial presence on the UV-irradiated polymer surfaces was

accompanied with evident morphologic cellular transformations. Namely, all rod-

shaped bacteria studied herein appeared to be longer after 12 h incubation on the

modified polymer surfaces. The only exclusion was observed for C. marina cells whose

overall length decreased after attaching to the smoother, UV-irradiated polymer

surfaces.

In addition to the obvious numerical and morphologic transformation, changes in

bacterial metabolic activity were also observed. CSLM findings highlighted a large

quantity of EPS in the near-cell surrounding on the modified polymer surface. Bacteria

observed to the most successful colonizers of both surfaces were also observed to be

excessive producers of EPS. This is indicative of the surfaces modification strategy

utilized by studied strains to better sustain their existence on this surface. Apart from the

cell microenvironment, extracellular deposits were also observed on the cell surfaces.

Considering they were all labelled with concanavalin A, which specifically binds to

mannose and glucose it can be assumed that these sugars are the main constituents of

detected EPS. Therefore, these EPS can be considered as positive contributors to the

attachment patterns of studied bacteria.

Chapter 6:

Bacterial cells interactions with the surface of micro-nano-structured surfaces

______________________________________________________________________

179

CHAPTER 6

BACTERIAL CELLS INTERACTIONS WITH THE

SURFACE OF MICRO-NANO-STRUCTURED

OPTIC FIBRES

Chapter 6:


______________________________________________________________________

180


Identical as already presented in Chapter 4.

6.2 Substratum surface characteristics

6.2.1 Overview

Several studies have already investigated the influence of substratum surface

characteristics such as topography, charge, wettability and chemistry on cell-surface

interactions. Most of these investigations have focused on planar surfaces of glass,

different polymers or metal, whilst optical fibre surfaces have not yet been investigated.

In this study, a series of experiments were designed to investigate the attachment pattern

of three commonly studied medically important marine bacteria of different taxonomic

affiliations during interaction with the surfaces of optical fibres.

6.2.2 Substratum surface wettability and tension

Fibre surface analyses involved quantification of the surface wettability and tension

before and after exposure to the etching solution. Three diagnostic fluids, water,

formamide and diodomethane, were selected for the purpose. Measured contact angles

on both the ‘as received’ and the modified fibre surface are presented in Figure 6.1 are,

whereas the surface tensions (and their parameters (mJ/m2)) of selected diagnostic fluids

are presented in Table Table 6.2 (Van Oss et al., 1988).

Chapter 6:


______________________________________________________________________

181

(a) (b)

Figure 6.1: Images representing measured water contact angles on the as-received (a)

and on the eroded (b) fibre surface.

Table 6.1: Surface wettability and surface tensions of the as-received and the modified

fibre surfaces.

* Contact angle of water, formamide and diiodomethane (θW, θF and θD respectively);



), electron


-)

Incorporating inferred parameters into the Young equation as detailed in chapter 3.2.5.2

enabled calculation of the surface tension on the as-received and the modified fibre

(data presented in Table 6.3).

6.2.3 ToF-SIMS analysis

The chemical composition of both fibre surfaces was analysed using ToF-SIMS.

The results obtained showed a similarity between both surfaces. As evident from the

Fibre

surface Contact angle*, θ (°)

Surface tensions**, γ (mJ/m2)

Water Diiodomethane Formamide γLW

γAB

γ+ γ

-

As-received 107.2 129.6 102.6 1.67 7.51 3.34 4.22

Modified 106.9 102.3 101.3 7.89 1.77 0.18 4.38

Chapter 6:


______________________________________________________________________

182

images presented in Figure 6.3 (a) and (b) and collected positive and negative spectra

presented in Figure 6.4, the most abundant component on both surfaces was Si,

followed by SiC3H9, SiCH3, CH3 and Na, representing 70% of the elemental composition

for both surfaces. The only difference was the lesser representation of Ge on the

modified surface. This observation was expected since the precise effect of the etching

solution is to removal of some of the germanium and fluorine ions from the fibre

surface. Higher concentrations of F were found on the modified sample, indicating that

residual F remained on the surface from the etching solution. Both surfaces showed an

appreciable presence of carbon, most likely due to surface air contamination.

(a)

(b)

Figure 6.2: ToF-SIMS scans from the (a) as-received and (b) eroded fiber surface

Chapter 6:


______________________________________________________________________

183

(a)

(b)

(c)

Chapter 6:


______________________________________________________________________

184

(d)

Figure 6.1: Positive (a, b) and negative (c, d) spectra collected from the as-received (a,

c) and the eroded (b, d) fibre surface

6.2.4 AFM analysis

Since the ToF-SIMS data did not reveal substantial differences in the chemical

composition between the two surfaces, AFM analyses were conducted to characterize

the surface topography. The typical AFM images presented in Figure 6.5 show a

topographical profile of the two surfaces.

(a) (b)

Figure 6.2: Surface topography of the as-received and the eroded fibre as inferred from

AFM

Chapter 6:


______________________________________________________________________

185

The observed differences in the surface topography were confirmed by a statistical

analysis of the surface height, leading to the roughness parameters presented in Table

6.2.

Table 6.1: Roughness parameters from the as-received and the eroded fibre surface as

inferred from AFM

Fibre surface* Ra (nm) Rq (nm) Rmax (nm)

‘As received’ fibre surface 180 ± 9 235 ± 9 1740 ± 84

Modified fibre surface 1563 ± 77 3428 ± 173 2400 ± 96

*Scanned area is approximately 40 µm x 40 µm


The results indicate that the fibre surface containing the micro-structured honeycomb

pattern of wells as a result of exposure to the etching solution is significantly rougher

than the as-received fibre. The modified fibre is more than 10 times rougher that the ‘as

received’ surface according to the Rq measure.

6.2.5 SEM analysis

6.2.5.1 Overview

A detailed visualization of the substratum surface morphology before and after

bacterial cultivation, together with the bacterial attachment pattern, was obtained via

SEM analysis. An analysis of the images indicated that the presence of growth medium

did not modify either of the fibre surfaces in a way that might influence bacterial

adhesive behaviour.

Details regarding sample preparation and SEM were described in chapter 3.2.4. a.

SEM images enabled quantitative as well as qualitative cell and substratum surface

analyses. For quantification of the number of adsorbed bacteria, cells number from at

least ten representative images/areas was transformed into number of bacteria per unit

Chapter 6:


______________________________________________________________________

186

area using the Image-Pro software (Waar et al., 2002). Final densities have estimated

errors of approximately 10-15% due to local variability in the coverage.

6.2.5.2 Control fibre surfaces

As already detailed in section 3.2.4.a, negative control experiments of the both fibre

surface (as-received and modified) with and without sterile marine broth were

performed in order to verify that the media used does not have the ability to leave any

deposits that might interfere the attachment progression (Figure 6.5).

(a) (b)

(b) (d)

Figure 6.5: Control SEM images of the as-received fibre surfaces without (a) and with

marine broth (b) and the chemically eroded fibre surface without (c) and with marine

broth (d). Scale bar on all images is 10µm.

An analysis of the images indicated that the presence of growth medium did not

modify either of the fibre surfaces in a way that might influence bacterial adhesive

behaviour.

Chapter 6:


______________________________________________________________________

187

6.3 Observed bacterial adhesive behaviour on micro-nano-

structured fibre surfaces

High resolution SEM images representing the attachment behaviour of the six

bacterial strains on the “as-received” fibre surfaces are given in Figure 6.6. As evident

in the images, the fibre surface has a different appearance as a result of the presence of

the extra-cellular products (EPS) secreted by the cells. Granular deposits of variable size

can be seen, particularly in the case of E. coli, P. aeruginosa, P. issachenkonii and S.

guttiformis attachment. It is likely that these deposits serve as primers, facilitating the

modification of the fibre surfaces to assist in the bacterial adhesion process. The

secretion of EPS by these strains during colonization of other surfaces has already been

reported (Mitik-Dineva et al., 2008a, Lam et al., 1992, Ivanova et al., in press). In

contrast, C. marina and S. aureus cells, while also being successful colonizers of the

“as-received” fibre surface, did not produce EPS to the same extent as E. coli, P.

aeruginosa, P. issachenkonii or S. guttiformis.

The difference between the observed bacterial attachment patterns was seen via the

number of bacteria attached to the fibre surfaces. The data presented in Table 6.3 clearly

indicates that C. marina and P. issachenkonii were the two most successful colonizers.

Apart from observed differences in the cellular metabolic activity, quantitative

differences in the cellular response to both surfaces were also detected. The data

presented Table 6.3 clearly indicate that C. marina and P. issachenkonii were the two

most successful colonizers of the surface, with 5.5 x 106 and 5.3 x 10

6 cells attached per

square cm respectively.

These two strains are also the only belonging to the γ-proteobacteria, hence

suggesting strain subordinate adhesive behaviour.

Chapter 6:


______________________________________________________________________

188

Table 6.3: Numbers of attached cells per surface area (mm2) on the as-received

fibre.

Strain Number of attached cells/ cm2

E. coli 2.98 x 105

P. aeruginosa 1.15 x 105

S. aureus 1.38 x 105

C. marina 55.46 x 105

P. issachenkonii 53.3 x 105

S. guttiformis 3.6 x 105

A remarkably different bacterial response was observed on the modified fibre

surfaces. None of the nine studied bacterial strains managed to attach to the modified

fibre surface. Despite varying quantities of granular EPS detected on the modified fibre

surface (around and inside the wells), no bacterial cells were observed to adhere to this

surface.

Apparent cellular absence may be an indication that the surface modifications

occurring as result of the 20 minute exposure to the etching solution have the potential

to resist bacterial “adoptive” mechanisms, thus serve as “model method” in creating

potentially “cell-resistant surface”.

E. coli cells on as-received fibre surface

P. aeruginosa cells on as-received

surface

Chapter 6:


______________________________________________________________________

189

S. aureus cells on as-received fibre surface

C. marina cells on as-received fibre

surface

P. issachenkonii cells on as-received fibre

surface

S. guttiformis on as-received fibre

surface

Figure 6.6: SEM images of the attachment pattern of E. coli, P. aeruginosa, S. aureus,

C. marina, P. issachenkonii and S. guttiformis on the as-received fibre surface

Visualization of the extra-cellular products produced by the cells while interacting

with both surfaces was achieved by labelling α-mannopyranosyl and α-glucopyranosyl

residues commonly found in bacterial EPS (Goldstein et al., 1964) with the fluorescent

dye Concanavalin A. CLSM was subsequently used to image the EPS distribution. The

images presented in Figure 6.7 confirmed the presence of EPS produced by E. coli, P.

aeruginosa, P. issachenkonii and S. guttiformis after 12 h incubation on the as-received

fibre surface. Granular EPS deposits produced by E. coli (a), P. aeruginosa (b) and P.

issachenkonii (d) were mostly located on and around fiber cores, whilst granular EPS of

different size produced by S. guttiformis (c) were randomly deposited over the fiber

Chapter 6:


______________________________________________________________________

190

cores and the surrounding cladding. No EPS produced by C. marina and S. aureus were

observed using CLSM, confirming the data obtained using SEM.

It is noteworthy that despite appreciable amounts of EPS being produced, no viable

cells could be detected on these surfaces. Figures 7.7 (a) – (c) highlight that the bacterial

cells can be clearly seen on the surfaces, however, cell viability was not confirmed by

the fluorescent labelling process.

(a) (b)

(c) (d)

Figure 6.7: CLSM image representing the EPS production of E. coli (a), P. aeruginosa

(b), S. guttiformis (c) and P. issachenkonii (d) after 12h incubation on the as-received

fibre surfaces

Chapter 6:


______________________________________________________________________

191

(a) (b) (c)

(d) (e) (f)

Figure 6.8: CLSM image representing the EPS production of E. coli (a), P. aeruginosa

(b), S. aureus (c), C. marina (d), P. issachenkonii (e) and S. guttiformis (f) after 12h

incubation on the modified fibre surface

The EPS produced by bacteria whilst interacting with the modified fibre surfaces

was clearly detectable (Figure 6.8). However, the amount, size and area of the EPS

deposition varied, depending on the strain. For instance, EPS synthesized by S.

guttiformis (f) were deposited inside and around the wells i.e. on the fibre cores and the

surrounding cladding, whereas EPS produced by E. coli (a), P. aeruginosa (b) and P.

issachenkonii (e) were predominantly located inside the wells (on the fibre cores). As

expected S. aureus (c) and C. marina (d) produced minimal amounts of EPS.

6.4 Conclusion

Although a limited number of bacterial strains representing different phylogenetic

lineages were able to sustain their existence on the as-received fibre surfaces, it is clear

Chapter 6:


______________________________________________________________________

192

that their different adhesive behaviour is strongly dependant on their outer surface

characteristics; production of EPS in particular. It also appeared that as important

bacterial surface characteristics are, substratum surface roughness is of equal, if not

more significant importance. Obtained results indicated that although belonging to

different taxonomic linage bacteria noted to be EPS overproducers (P. aeruginosa, P.

issachenkonii, C. marina, E. coli and S. guttiformis) expressed similar attachment

preference towards the nano-smooth, as-received fibre surfaces. S. aureus cells are the

only exclusion from this pattern, but then again it is the only coccoid bacterium studied

herein, thus pointing out to the importance of bacterial shape on cellular attachment

behaviour. On the other hand all tested strains, regardless of the origin and their shape

expressed identical repulsion towards the chemically modified fibre surfaces, thus

suggesting that substratum surface topography might be extremely influential surface

characteristic in establishing bacterial adhesive response.

Chapter 7:

Discussion

______________________________________________________________________

193

CHAPTER 7

DISCUSSION

Chapter 7:

Discussion

______________________________________________________________________

194

7.1 Overview

The interactions between bacteria and surfaces and their attachment patterns have

been intensively studied, but the results to date are somewhat contradictory and do not

allow formulation of a reliable correlation (Busscher and van der Mei, 1997, Bos et al.,

2000, Bos et al., 1999, Pereira et al., 2000, Teixeira and Oliveira, 1999). Current

theoretical predictions regarding the propensity for bacterial attachment to different

surfaces are based on the physicochemical characteristics of bacterial and substratum

surfaces. The bacterial surface properties believed to be most influential are cell surface

wettability and charge, as well as the presence and composition of surface EPS (Bell et

al., 2005, Pham et al., 2003, Beech et al., 2005, de Rezende et al., 2005, Danese et al.,

2000), whereas substratum surface characteristics that positively influence bacterial

adhesion for some species include high surface hydrophobicity (Hogt et al., 1985,

Tegoulia and Cooper, 2002, Marshall et al., 1971), surface functionality (Tegoulia and

Cooper, 2002) and topographical variation on the micro-scale (Characklis, 1973, An et

al., 1995).

The physicochemical characteristics of nine taxonomically diverse bacteria, and

three different surfaces (glass, P(t)BMA polymer and optical fibres) and their modified

counterparts, as well as peculiarities of bacterial adhesive behaviour after 12 hours

incubation on each of the surfaces were presented in previous sections. As the initial

inspection of bacteria attached on all tested surfaces and their modified equivalents

revealed considerable differences, the aim of this section is to correlate bacterial and

substratum surface characteristics in an attempt to integrate them all into a model that

may accurately predict bacterial adhesive patterns.

Although a species-specific pattern of attachment was evident, a consistent

inclination was observed for all tested strains towards the smoother surfaces (Table 7.1).

In particular, the modified glass and polymer surfaces appeared to attract more cells

than the as-received glass and native P(t)BMA surfaces, respectively. In a similar

manner, the smoother, as-received fibre surface was colonized by some of the strains,

whereas the modified, rougher fibre surface appeared to have a cyto-repellent potential.

It is of particular interest that the same adhesion tendency was observed for all tested

Chapter 7:

Discussion

195

strains, regardless of their different taxonomic affiliation and their cell surface

characteristics.

As the results presented in Table 7.1 indicate, the number of cells attached to the

modified glass and polymer surfaces was greater than the numbers attached to the as

received surfaces by 40% to 90% depending on the bacterial strain. Increased cellular

presence on the modified glass and polymer surfaces as well as on the as-received fibre

surface was also evident in the images presented in Chapters 4, 5 and 6.

Table 7.1: Number of bacteria/cm2 attached to all tested surfaces and their modified

equivalents*

Glass P(t)BMA Fibre*

Strain

Phylogenetic lineage

As-received

Modified

% of cells

on as-

received

As-received

Modified

% of cells on

the as-

received

As-received

E. coli K12 γ-proteobacteria 3.25x 106 7.1 x106 46 3.1 x106 7.6 x106 41 2.98x105

P. aeruginosa γ-proteobacteria 10.3x106 18.4 x106 56 4.3x106 13 x106 33 1.15x105

S. aureus Firmicutes-Bacilli 4.95x 106 6.68 x106 73 13.2x106 18.7 x106 72 1.38x105

C. marina γ-proteobacteria 5.66x106 9.78 x106 58 7.23x106 10.3 x106 70 5.5x106

P. issachenkonii γ-proteobacteria 3.6 x106 11 x106 33 3.1x106 9.25 x106 33 5.3x106

S. flavus Bacteroidetes

(Flavobacteriaceae

1.5 x 105 2.74 x106 5 1x105 1.2 x106 8 0

S. guttiformis α-proteobacteria 3.28x106 7.67 x106 43 3.41x106 8.12 x106 37 3.6x105

S. mediterraneus α-proteobacteria 5.5 x105 8.9 x105 62 5.2x105 8.15 x105 63 0

A. fischeri γ-proteobacteria 9.23x106 13.2 x106 70 8.7x106 13 x106 67 0

* E. coli, P. aeruginosa, S. aureus, C. marina, P. issachenkonii and S. guttiformis did

not attach to the modified fibre surface, whereas S. flavus, S. mediterraneus and

A. fischeri did not attach to either of the fibre surfaces.

Chapter 7:

Discussion

______________________________________________________________________

196

In addition to the remarkable quantitative differences in the cells’ viability, changes

in the metabolic activity (production of EPS) and morphological changes were also

detected for all strains.

In order to address its aim, this chapter is divided into three sections. The subject of

the first two is the correlation between bacterial adhesion and surface wettability and

charge (bacterial and surface), with respect to both theoretical considerations and

previously-reported data. The remainder of the chapter is devoted to interpolating

substratum surface topography and roughness and bacterial adhesion. This section will

also illuminate the importance of surface topography as a factor of significant interest in

understanding cell-surface interactions.

7.2 Bacterial attachment and surface wettability

7.2.1 Overview

Previously reported data on the interrelations between cell surface wettability and

bacterial adhesion are somewhat contradictory. For instance, data published by van

Loosdrecht et al. (van Loosdrecht et al., 1990) suggest strong linear dependence

between bacterial adhesion and bacterial surface wettability, whilst others provided

evidence suggesting bacterial adhesion is inversely correlated with bacterial surface

contact angle (Li and Logan, 2004).

According to the thermodynamic theory, hydrophilic cells are expected to exhibit

greater propensity to adhere to hydrophilic glass surfaces and hydrophobic cells to

hydrophobic substrata (Bruinsma et al., 2001, Howell and Behrends, 2006, Bos et al.,

1999). Nevertheless, several studies have indicated that this might not always be the

case, because molecules located on the outer-cell membrane are not inert but at least

partially active components that can respond to various environmental stimuli

(Korenevsky and Beveridge, 2007). The latter statement - which is in agreement with

the results of the current study - favours the hypothesis that bacterial survival strategies

include an attachment dependent on the presence, chemical composition and structure of

surface exo-cellular properties Sutherland (Sutherland, 2001a, Sutherland, 2001b,

Chapter 7:

Discussion

______________________________________________________________________

197

Wright et al., 1990, Yildiz and Schoolnik, 1999, Wozniak et al., 2003, Watnick and

Kolter, 1999).

7.2.2 The effects of cell surface wettability on bacterial adhesion to glass,

polymer and fibre surfaces

Preliminary results indicated that bacteria might exhibit different surface

characteristics regardless of the taxonomic linage. For instance, E. coli, P. aeruginosa,

C. marina and P. issachenkonii all belong to the Gammaproteobacteria, however, E.

coli and P. aeruginosa had hydrophilic surfaces, P. issachenkonii was on the borderline

with a measured water contact angle of 52°, whereas C. marina displayed typical

hydrophobic surface characteristics. Results for the cell surface wettability of all of the

studied strains were presented in Table 4.1. 106

The relationship between bacterial surface wettability and their attachment

propensity towards the hydrophilic glass surfaces is presented in Figure 7.1.

Chapter 7:

Discussion

______________________________________________________________________

198

Figure 7.1: Evaluation of the attachment patterns of E. coli (ec), S. mediterranues

(sm), P. aeruginosa (pa), S. flavus (sf), P. issachenkonii (pi), S. guttiformis (sg), S.

aureus (sa), C. marina (cm) and A. fischeri (af) on the as-received and modified glass

surfaces: number of attached cells compared to the bacterial surface wettability

The data presented herein suggest that Pseudomonas aerugionsa, A. fischeri and P.

issachenkonii were the most successful colonisers of the hydrophilic glass surface. Two

of the three - P. aeruginosa and P. issachenkonii - displayed hydrophilic surface

characteristics according to the contact angle measurements (43° and 52°, respectively),

hence complied with the thermodynamically predicted preference of hydrophilic cells to

hydrophilic substrata. Contrary to this, A. fischeri was found to have hydrophobic cell

surface characteristics with a measured contact angle (θ) of 83°; this was the highest

measured contact angle off all tested strains, thus suggesting that A. fischeri would

exhibit the lowest attachment propensity. Nevertheless, A. fischerii was the second most

successful coloniser of both the as-received and the modified glass surfaces. The

attachment inclination of E. coli cells also contradicted theoretical expectations; as the

strain with lowest measured water contact angle (33°), they were expected to display

intense affinity towards the hydrophilic glass surfaces, however results presented in

Chapter 7:

Discussion

______________________________________________________________________

199

Figure 7.1 indicate that E. coli was a moderate coloniser of both as-received and

modified glass surfaces.

A continuous trend of inconsistent correlation between cell surface wettability and

bacterial adhesion was observed on the native and modified P(t)BMA surfaces (Figure

7.2). Both polymer surfaces were found to be hydrophobic, with water contact angles of

86° and 63° respectively; accordingly (and in line with the thermodynamic theory) A.

fischeri, C. marina and S. aureus were expected to be the most prominent colonizers on

both P(t)BMA surfaces. To some extent obtained results did comply with the theoretical

expectations, as S. aureus cells maintained the highest cellular presence, whereas A.

fischeri and C. marina were outnumbered by P. aeruginosa cells which were noted to

be excessive producers of EPS. Nonetheless, as contact angles decreased and cell

surface wettability changed from hydrophobic to hydrophilic, deviations from this trend

were observed. S. flavus and S. mediterraneus were expected to inhabit both surfaces in

greater extent than E. coli due to being less hydrophilic (47° and 39°, respectively), yet

E. coli cells appeared to be more prominent colonisers than either. In the same line, P.

aeruginosa cells - instead of being amongst the weakest colonisers - expressed strong

adhesive tendency, in particular to the modified P(t)BMA surface.

Figure 7.2: Evaluation of the attachment patterns of E. coli (ec), S. mediterranues

Chapter 7:

Discussion

______________________________________________________________________

200

(sm), P. aeruginosa (pa), S. flavus (sf), P. issachenkonii (pi), S. guttiformis (sg), S.

aureus (sa), C. marina (cm) and A. fischeri (af) on the as-received and modified

P(t)BMA surfaces compared to the number of attached cells versus bacterial surface

wettability

Nevertheless, a certain correlation was detected between cell surface wettability

and bacterial adhesion. In compliance with van Loosdrecht’s theory that there is a

strong dependence between bacterial adhesion and bacterial surface wettability (van

Loosdrecht et al., 1990), S. mediterraneus, S. flavus, Staleya guttigormis, C. marina and

A. fischeri all demonstrated approximately linear increase of cellular presence with

increase of cell surface hydrophobicity on both glass and polymer modified surfaces.

(Figure 7.1 and Figure 7.2). This result also agrees with the previously reported

observation that decrease of cell surface wettability can lead to depleted cellular

presence (van Loosdrecht et al., 1990, Bruinsma et al., 2001) .

Bacterial attachment on the optical fibre surfaces was also probed in the same way

as glass and polymer surfaces,. Of the nine tested strains, only six attached to the as-

received nano-smooth fibre surface, while the modified, micro-rough surface remained

uncolonised (Figure 7.4). Two strains - C. marina and S. aureus - were found to be

highly hydrophobic; they complied with the theoretical expectations and maintained

their presence on the hydrophobic, as-received fibre surface.

Chapter 7:

Discussion

______________________________________________________________________

201

Figure 7.3: Evaluation of the attachment patterns of E. coli (ec), P. aeruginosa (pa), P.

issachenkonii (pi), S. guttiformis (sg) and C. marina (cm) on the as-received fibre

surfaces: number of the attached cells compared to the bacterial surface wettability.

Nevertheless, despite having somewhat similar surface wettability, C. marina and

S. aureus displayed fairly divergent attachment tendencies. C. marina cells were

prominent colonisers of the as-received fibre surface, whiles S. aureus cells attached in

minimal numbers, displaying similar attachment tendencies to the hydrophilic P.

aeruginosa (Figure 7.4). E. coli cells, on the other hand - even though hydrophilic and

expected to be the poorest colonisers - managed to outnumber the more hydrophobic S.

aureus.

Chapter 7:

Discussion

______________________________________________________________________

202

7.2.3 The effects of substratum surface wettability on bacterial adhesion

In addition to cell surface wettability, the effects of substratum surface wettability

on bacterial interactions with all tested surfaces were considered. Previous

investigations of bacterial attachment processes have concluded that in general, bacteria

favour hydrophobic substrata over hydrophilic (Busscher et al., 1990, Doyle and

Rosenberg, 1990). In this respect both polymer surfaces, with their hydrophobic surface

characteristics, were expected to attract more cells than the hydrophilic glass; however,

the experimental results clearly indicate this was not the case (Figure 7.4).

Figure 7.4: Evaluation of the attachment patterns of E. coli (ec), S. mediterranues (sm),

P. aeruginosa (pa), S. flavus (sf), P. issachenkonii (pi), S. guttiformis (sg), S. aureus

(sa), C. marina (cm) and A. fischeri (af)on both glass and polymer surfaces: number of

attached cells compared to the substratum surface wettability

Data presented in Table 7.1 and Figure 7.4 suggest that only S. aureus cells

conformed to the expected pattern of attraction; they were able to colonise the

Chapter 7:

Discussion

______________________________________________________________________

203

hydrophobic polymer surfaces (native and modified) in significantly higher numbers

than the glass surfaces. E. coli cells were also present in higher numbers on the

modified polymer surface, however the number of cells detected on the modified,

hydrophilic, glass surface was almost double that on the native P(t)BMA. Considering

S. aureus was the only spherical bacterium included in the study, it can be hypothesised

that cell shape is of considerable influence in cell-surface interactions. The influence of

cell shape and size on bacterial adhesion has already been suggested (Dworkin, 2007).

It is interesting to notice that both optical fibre surfaces displayed somewhat similar

surface hydrophobicity. However the observed bacterial behaviour on the as-received

and on the modified fibre surfaces was severely different; therefore suggesting that

factors other than substratum surface wettability might play a more important role in

controlling bacterial adhesive behaviour.

The results presented in Figure 7.4 indicate that irrespective of the substratum

surface wettability (hydrophilic or hydrophobic), bacteria employ different attachment

strategies. They also suggest that no clear correlation can be drawn between bacterial

adhesion and substratum or cell surface wettability. It seams probable that this is due to

the unpredictable structure of the cellular surface and the possibility of dynamic and

conformational transformations on the bacterial surface as a response to various

environmental stimuli. The ultimate effect of this is an alteration in the cell surface

structure, resulting in modification of the cell’s hydrophobic/hydrophilic potential and

its attachment inclination (Korenevsky and Beveridge, 2007). For this reason,

identification of a correlation between the substratum or cell surface wettability and the

extent of bacterial adhesion has proven elusive. Note that the present results confirm

that a significant change in cell morphology occur together with a change in number if

attached bacteria on the as-received and modified surfaces.

Chapter 7:

Discussion

______________________________________________________________________

204

7.3 Bacterial attachment and surface charge

7.3.1 Overview

In terms of cell surface charge, all bacteria had negative surface charge as expected

(van der Mei and Busscher, 2001), mainly in the range -3 2mV to -38 mV (data

presented in Table 4.2). The highest surface charge of -43 mV was measured on S.

guttiformis cell surfaces, whereas P. aeruginosa cells had the lowest measured surface

charge values of -14mV.

From an electrostatic perspective, cells having greater negative charge would be

expected to have the weakest propensity for attachment to negatively charged surfaces

and vice versa. Yet studies that suggest negative, positive or no correlation between

bacterial electrokinetic properties and surface attachment have been reported (Pearson et

al., 2004, Rozhok and Holz, 2005, van Merode et al., 2007, Soni et al., 2007). These

observations are supported by data from earlier studies which reported that due to the

presence of outer surface EPS, their constant dynamic motion and the presence or

absence of areas with variable molecular polarity or charge, bacterial cells can exhibit

variable surface adhesion characteristics (Li and Logan, 2004, Vadillo-Rodriguez et al.,

2004).

7.3.2 The effects of cell surface charge on bacterial adhesion to glass,

polymer and fibre surfaces

Taking into account the suggested inverse correlation between cell surface charge

and bacterial adhesion (Li and Logan, 2004) and the electrostatic repulsion between two

negatively charged areas (bacteria and surface) (Jucker et al., 1996), it can be expected

that P. aeruginosa and S. flavus cells - having the lowest measured surface charge -

would exhibit the strongest and S. guttiformis the weakest attachment preferences.

However, S. flavus cells showed relatively weak attachment, particularly to the “as-

received” surface. The most successful colonisers on both glass surfaces was P.

aeruginosa, followed by A. fischeri, P. Issachenkonii and C. marina. Conforming to the

Chapter 7:

Discussion

______________________________________________________________________

205

theoretical predictions S. mediterraneus cells exhibited the weakest attachment, contrary

to S. guttiformis which although expected to be weak colonizers managed to attach in

similar numbers as E. coli and S. aureus (Figure 7.5).

Figure 7.5: Evaluation of the attachment patterns of P. aeruginosa (pa), S. flavus (sf),

C. marina (cm), S. aureus (sa), P. issachenkonii (pi), A. fischeri (af), E. coli (ec), S.

mediterraneus (sm) and S. guttiformis (sg)_to the as-received and modified glass

surfaces: number of the attached cells compared to the bacterial surface charge

Similar cellular behaviour was observed when bacteria were cultivated on the

P(t)BMA polymer surfaces. Despite the theoretical considerations and regardless of

having the highest charge, S. guttiformis cells were again better colonisers than S.

mediterraneus and S. flavus (Figure 7.6).

Chapter 7:

Discussion

______________________________________________________________________

206

Figure 7.6: Evaluation of the attachment patterns of P. aeruginosa (pa), S. flavus (sf),

C. marina (cm), S. aureus (sa), P. issachenkonii (pi), A. fischeri (af), E. coli (ec), S.

mediterraneus (sm) and S. guttiformis (sg) to the as-received and modified P(t)BMA

surfaces: number of the attached cells compared to the bacterial surface charge

To some extent, the experimental results obtained did comply with the theoretical

predictions. For instance P. aerugionsa cells, which bear lower charge than C. marina

were more successful in colonizing both surfaces (glass and polymer). In contrast, S.

aureus - although bearing identical surface charge (-35mV) to A. fischeri and P.

issachenkonii - did not display similar behaviour. It is noteworthy that this bacterium is

of spherical shape, in contrast to the previous two which are rod-shaped, and does not

belong to the Gammaproteobacteria, thus conforming to previous observations that

bacterial shape may affect cellular behaviour (Dworkin, 2007). Overall, the most

proficient colonisers on both surfaces were A. fischeri and P. aeruginosa, which were

also noted to be the most excessive producers of EPS, thus pointing out the importance

of EPS in bacterial attachment.

The correlation between bacterial zeta potentials and their attachment patterns on

the fibre surfaces are presented in Figure 7.7.

Chapter 7:

Discussion

______________________________________________________________________

207

Figure 7.7: Evaluation of bacterial attachment pattern to the as-received and modified

fibre surfaces: number of the attached cells versus bacterial surface charge

Beside the fact that only six of the nine tested strains managed to maintain their

presence on the as-received surface and none on the modified, a consistent linear

increase in the number of attached cells with decrease of cell surface charge was

observed for P. aeruginosa, S. aureus, E. coli and S. guttiformis cells (Figure 7.7);

however, C. marina and P. issachenkonii cells did not follow the same pattern.

Chapter 7:

Discussion

______________________________________________________________________

208

7.3.3 The effects of substratum surface charge on bacterial adhesion

The as-received glass, being the least negatively charged surface, was expected to

attract the highest number of negatively charged cells. Nevertheless the obtained results

indicate that this was not the case for any of the tested strains, thus supporting Li and

Logan’s theory that no correlation can be drawn between bacterial adhesion and

substratum surface charge (Li and Logan, 2004).

7.4 Bacterial attachment and surface roughness

Notwithstanding the different taxonomic affiliations and individual species-

specific patterns of attachment of the bacteria studied, the data obtained in this study

indicated amplified bacterial attachment on the modified glass and polymer surfaces and

as-received fibre surface (Table 7.1). Noticeably low adhesive tendencies, in particular

on the as-received glass and P(t)BMA surfaces, were observed for S. flavus and S.

mediterraneus, although a consistent trend of increased cellular presence on the

modified surfaces was still evident.

Considering that the chemical treatment and the UV exposure altered some of the

glass, polymer and fibre surface characteristics, it was of interest to further investigate

whether a particular parameter or a combination of parameters might have influenced

the observed consistent trends of increased bacterial attachment to smoother surfaces.

Several additional investigative techniques - such as, XPS, XRF, SEM and AFM - were

considered in order to compare and differentiate surface characteristics before and after

modification.

Physicochemical analysis of both the as-received and modified glass and fibre

surfaces reported herein suggested that almost all of the surface modifications occurring

as a result of exposure to the etching solution have insignificant effects on the surface

properties. For example, water contact angles and surface tension on both surfaces were

almost identical. Analysis of the surface chemistry by XPS and XRF for the glass and

ToF-SIMS for the fibre surfaces also did not reveal appreciable differences between as-

Chapter 7:

Discussion

______________________________________________________________________

209

received and modified equivalents. On the other hand, a significant increase in all

roughness parameters was found on the chemically modified surfaces - approximately

50% on the modified glass surface and a more than ten-fold increase on the modified

fibre surface was found for all roughness parameters. Thus, detailed analysis of the

modified glass and fibre surfaces indicated a nano-scale change in the surface

topography caused by the etching process, and an insignificant change in surface

chemical and physicochemical characteristics.

The photolithographic treatment of P(t)BMA also resulted in the modification of

several polymer surface characteristics such as hydrophobicity, surface chemistry and

nanotopography. An observed water contact angle of 85° on the native polymer surface

is consistent with previously reported values (Pham et al., 2003). Modified P(t)BMA,

on the other hand, was found to be moderately less hydrophobic (63o), which is likely to

be due to the loss of methyl groups and the tertbutyl ester on the polymer backbone and

the formation of carboxylic acid groups (Ivanova et al., 2006c, Michielsen and Lee,

2007). XPS analysis revealed conclusive evidence to support the premise that surface

carboxylic acid groups were present on the modified P(t)BMA polymer surface. AFM

analysis of both P(t)BMA surfaces indicated a topographical alteration of surface

roughness caused by the UV-irradiation. In contrast to the native polymer, the modified

surface displayed more uniform characteristics; an overall decrease of approximately

60% was detected for all roughness parameters.

The results presented in section 5.3 suggest that the surface charge, tension and

chemistry were not significantly altered by UV exposure, therefore they cannot be

considered influential enough to affect bacterial attachment; however, a 25% decrease

in surface wettability and a 60% decrease in surface roughness were observed. Although

the decrease in surface wettability is appreciable, the analysis presented in Chapter 7.1.3

shows that there is no significant correlation between substrate contact angles and

attachment of the studied bacteria. This observation conforms to previously reported

data and already existing theoretical considerations (Busscher et al., 1990, Doyle and

Rosenberg, 1990).

Existing knowledge of the effects of surface roughness on bacterial adhesion

suggests that bacteria prefer surface irregularities as the starting point for their

attachment as these provide shelter from unfavourable environmental influences

Chapter 7:

Discussion

______________________________________________________________________

210

(Dworkin, 2007, Shellenberger and Logan, 2002, Jones and Velegol, 2006, Whitehead

and Verran, 2006). The work presented in this thesis suggests that this might not always

be the case. In particular, this research has proven that nano-scale surface roughness

may also have stimulating effects on bacterial adhesion. Moreover, differences in the

surface roughness of only a few nanometres may exhibit a strong influence on the

cellular response towards certain surfaces (Ivanova et al., in press, Mitik-Dineva et al.,

2008a). Regardless of their taxonomic origin or surface characteristics such as

wettability, charge or shape, all of the tested strains displayed a considerable preference

for attaching to the modified, nano-rough glass and polymer surfaces and the nano-scale

rough as received fibre surface. It is our understanding that the smoother surfaces have a

generally stimulating effect on bacterial metabolism. Bacterial preference towards these

surfaces appeared to be accompanied with changes in the cell’s morphology and its

metabolic activity.

The stimulating effects of the nano-smooth surfaces on bacterial metabolic activity

were suggested by elevated amounts of EPS produced by cells while attaching to the

modified surfaces. The presence of extra-cellular deposits was particularly noticeable on

the modified glass and polymer surfaces when compared to their native equivalents.

Although bacterial presence was not noted on the modified fibre surfaces, extra-cellular

deposits containing α-mannopyranosyl and α-glucopyranosyl were still detected.

Another interesting observation is the elevated EPS production for all of the tested

strains, suggesting that the investigated bacteria employed somewhat similar strategies

for attachment to nano-smooth surfaces by producing increased amounts of EPS. As

indicated by the CLSM images presented in Chapters 4, 5 and 6, all of the studied

bacteria were able to synthesise capsular-like EPS (as labelled by concanavalin A).

Nevertheless, extra-cellular deposits were also located on the tested surfaces, modified

surfaces in particular; the average height of these depositions varied between 20-450

nm. It is suggested that the EPS may serve as primers that modifies the substratum

surface and thereby facilitates bacterial adhesion; as it was observed when cells were

incubated on modified glass and polymer surfaces. However the existence of extra-

cellular deposits on the modified fibre surfaces (Figure 6.8) without bacterial presence

indicates that there may be other contributing factors. Extra-cellular products secreted

by E. coli, P. aeruginosa, Pseudolateromonas issachenkonii and A. fischeri detected by

Chapter 7:

Discussion

______________________________________________________________________

211

the SEM or AFM were not always observed on the CLSM images, thus suggesting

differing composition of the extracellular materials produced. This observation implies

that cells might produce a few different types of EPS that can vary widely in

composition, structure and physical properties. Similar observation have been reported

previously (Dong et al., 2002, Sutherland, 2001a, Sutherland, 2001b, Wright et al.,

1990, Yildiz and Schoolnik, 1999, Wozniak et al., 2003, Watnick and Kolter, 1999).

It is noteworthy that P. aeruginosa, P. issachenkonii and A. fischeri were the most

successful colonisers on all tested surfaces regardless of their own or the substratum

surface characteristics. On the other hand, CLSM images presented in Chapter 4, 5 and

6 indicate that P. aeruginosa, P. issachenkonii and A. fischeri were also the most

excessive producers of EPS. In the same way, S. flavus and S. mediterraneus cells -

while exhibiting the weakest attachment capacity - appeared to be the poorest producers

of EPS. This observation suggests that of all cell surface characteristics, production and

composition of surface EPS is most likely the most influential factor in initial bacterial

attachment. A positive correlation between the presence of EPS and surface adhesion

has been shown previously (Kreft and Wimpenny, 2001, Flemming and Wingender,

2001, Sutherland, 2001a, Sutherland, 2001b, Ryu et al., 2004, Gross et al., 2001, Beech

et al., 2005), however, not in the context of surface nano-topography. Nevertheless, due

to the small quantities of EPS produced by all bacteria throughout this study, as well as

the limitations of the methods used for in situ EPS characterization, only tentative

characterisation of EPS is presented as inferred from the application of Concanavalin A.

Further work concerning the nature and chemical composition of the EPS is necessary

in order to reasonably confirm the predictions discussed above.

A detailed inspection of cell morphology after attachment to each of the tested

surfaces and their modified counterparts revealed striking differences in the cell

morphology. Bacteria attached to the modified glass and polymer surfaces appeared

approximately 5-25% longer and 15-40% wider and higher than those attached to the

as-received (Figure 7.8).

Chapter 7:

Discussion

______________________________________________________________________

212

Figure 7.8: Variations in the length of E. coli (ec), P. aeruginosa (pa), S. aureus (sa),

Pseudoalteromons issachenkonii (pi), C. marina (cm), S. flavus (sf), S. guttiformis (sg),

S. mediterraneus (sm) and A. fischeri (sf) cells after attaching to the as-received

surfaces and their modified equivalents

*Variations in the length of S. flavus cells adsorbed on the as-received and modified

P(t)BMA surfaces could not be estimated

Nevertheless the nominated bacteria did exhibit some individual, species-specific

attachment patterns. It was observed that all rod shaped bacteria - E. coli, P. aeruginosa,

P. issachenkonii, S. flavus, S. guttiformis, S. mediterraneus and A. fischeri - expressed

extreme susceptibility to cellular transformation (enlargement) on the modified surfaces.

On the other hand, C. marina cells attached to the modified surfaces appeared to be

smaller and of an oval shape when compared with the predominantly elongated cells

detected on the as-received surfaces that conform to the original species description

(Arahal et al., 2002).

S. aureus did not undergo morphological transformations. The variation in the

number of S. aureus cells attached to the as-received and the modified glass was also

not as pronounced as occurred for the rod-shaped bacteria. This observation is in

agreement with the “attachment point” theory which states that small sphere-shaped

Chapter 7:

Discussion

______________________________________________________________________

213

micro-organisms (such as S. aureus) exhibit different attachment patterns compared to

large or elongated cells because of the fewer available access points (Advincula et al.,

2007).

Of particular interest was the attachment pattern and cellular transformation of S.

mediterraneus. Cells of this bacterium, while exhibiting the weakest adhesion

propensity, showed an extraordinary tendency to modify their morphology. As can be

seen from the high magnification SEM and AFM images in Figures 4.26 and 4.27

(Chapter 4) and Figures 5.31 and 5.32 (Chapter 5), the cells are transformed from

typically elongated into more spherical shapes. The remarkable attachment behaviour of

S. mediterraneus on P(t)BMA surfaces has previously been reported (Ivanova et al.,

2002a). Ivanova et al. showed that S. mediterraneus vegetative cells transformed into

coccoid forms after 24-48 h of incubation while in contact with a P(t)BMA polymer

surface (Figure 7.9).

Figure 7.9: Conversion of vegetative cells of S. mediterraneus ATCC 700856T into

coccoid forms after attachment to Pt BMA, 24 h. Left: Vegetative cells with subpolar

flagella; middle: initial step towards coccoid body formation; right: coccoid form of S.

mediterraneus ATCC 700856T(Ivanova et al., 2002a).

In the current study the period of incubation was 12 hours, so it is possible that the time

of incubation was insufficient for the cells to fully undertake a morphological change.

Nevertheless, morphological transformations believed to be the beginning stages of

cellular transformation into coccoid bodies were observed.

Both bacterial morphological transformations and production of EPS are indicative

of the modification strategy utilized by bacteria to better sustain their existence on the

Chapter 7:

Discussion

______________________________________________________________________

214

smoother surfaces. This observation implies that the nano-smooth surfaces may induce

cellular transformations as well as EPS production.

7.5 General discussion

Although bacteria deriving from different taxa were selected for investigation, it

has been shown that they displayed similar preference for the nano-smooth surfaces;

notwithstanding individual species-specific patterns of adhesion, a consistent tendency

for increased bacterial adsorption onto the smoother surfaces was observed. Increases of

20%-60% (depending on the strain) were observed in the number of Gram-negative

bacteria attached to the smoother modified glass and modified polymer and as-received

fibre surfaces in contrast to the rougher as-received glass, native polymer and modified

fibre. Increased cellular metabolic activity as indicated by the elevated EPS presence

was also observed on the smoother surfaces. Therefore, it is suggested that the

modification of surface roughness on the nanometre scale might trigger an elevated

production of EPS and a subsequent increase in the number of attached bacterial cells.

This also suggests that the tested strains employ similar strategies for attachment by

producing variable amounts of EPS. Nevertheless, regardless of their origin, all of the

tested bacteria expressed a similar repulsion towards the chemically modified fibre

surface, suggesting that not only the surface roughness but also the substratum’s surface

topography might be influential in determining bacterial adhesive response.

Alterations in the bacterial morphology were also observed; specifically, it was

observed that cells attaching to the smoother surfaces were enlarged overall. These

changes were particularly consistent for rod-shaped bacteria and may reflect an

attachment strategy common to different bacterial taxa. Contrary to this finding, more

spherical strains - such as C. marina and the typically coccoid S. aureus - followed the

pattern of amplified cellular presence on the smoother surfaces, but did not suffer any

morphological modifications. This suggests that bacterial shape may also be a

contributing factor in cell-surface interactions.

Chapter 7:

Discussion

______________________________________________________________________

215

In summary, the results obtained in this study lead to the conclusion that variations

in surface roughness have a potentially significant effect on cell-surface interactions.

The data also suggest that bacterial attachment is consistently promoted on nano-smooth

surfaces and reflect an attachment strategy common to different bacterial taxa. The

finding that bacteria are susceptible to nanoscale surface roughness has significant

implications for biomedical and industrial applications, e.g. design of orthopaedic

prosthesis, contact lenses, artificial valves, drainage tubes, laboratory glassware, water

filtration systems, food packaging materials, ect.

Chapter 8:

Conclusion and future directions

______________________________________________________________________

216

CHAPTER 8

CONCLUSION AND FUTURE DIRECTIONS

Chapter 8:


______________________________________________________________________

217

8.1 Summary

The attachment pattern of nine bacterial strains was tested on a series of chemically

and structurally diverse surfaces; glass, P(t)BMA polymeric surfaces and optical fibre.

Selected microorganisms comprised a group of strains from the following different

taxonomic lineages; Gammaproteobacteria: E. coli, P. aeruginosa, C. marina P.

issachenkonii and A. fischeri; Alphaproteobacteria: S. guttiformis and S. mediterraneus;

Bacteriodetes: S. flavus and Firmicutes-Bacilli : S. aureus, all of which possess

characteristics of significant medical and environmental impact. Surface modification

techniques such chemical treatment by exposure to BHF etching and UV irradiation

allowed surfaces exhibiting transformation of the roughness and topography to be

obtained whilst maintaining all other substratum characteristics at a near constant level.

A number of microscopic as well as physicochemical investigative techniques assessing

cell and substratum surface characteristics were employed.

The results obtained in the course of this study have confirmed that bacterial

adhesive behaviour is a complex phenomenon that cannot be explained solely by

physicochemical parameters, as there are numbers of factors affecting bacterial

interactions with surfaces. The results obtained have displayed a consistent inclination

for bacteria to adhere preferentially to nano-smooth surfaces. The result also indicate

that no clear correlation exists between bacterial wettability or surface charge and the

level and extent of bacterial attachment. Therefore, none of the cell surface

characteristics surveyed herein could provide a reasonable explanation for the different

bacterial response towards each of the tested surfaces. Nevertheless, it is clear that

bacterial adhesion is significantly influenced by nanometre scale changes in the surface

topography.

The degree of roughness of the substratum is known to play a significant role in

bacterial attachment to different surfaces, yet it was not previously considered a factor

of primary interest; most research attention was directed towards the effects of surface

wettability, charge and surface free energy on the adhesion process. Existing knowledge

about the effects of surface roughness on cell-substratum interactions is also lacking in

consistency, controversial and an overall understanding of the interrelationship is yet to

be obtained. Results suggesting bacterial adhesion is encouraged on rougher surfaces do

exist, the hypothesis being that surface pits, cracks, grooves and abrasion defects can

Chapter 8:


______________________________________________________________________

218

provide shelter for attached bacteria from unfavourable environmental factors (Verran

and Boyd, 2001, Verran et al., 1980, Whitehead and Verran, 2006, Taylor et al., 1998,

Messing and Oppermann, 1979). It has also been proposed that surface imperfections

also allow time for cells to establish stable, irreversible attachment following the initial

reversible physicochemical attachment (Taylor et al., 1998). There is still disagreement

over whether there is a threshold below and above which surface roughness can

promote or inhibit bacterial adhesion. It is believed that surface irregularities of an order

of magnitude that is comparable to the size of bacteria (1-1.5 µm in diameter) are

capable of retaining more cells than smoother surfaces. Adhesion would increase on

such surfaces due to an increased contact area between the cell and its surrounding

environment. The latter consideration is reflected in the conventional wisdom that

smoother surfaces represent a more repellent environment to bacteria.

Current study suggests that nano-smooth glass, polymer and optical fibre surfaces

have stimulating effects on bacterial adhesion. The consistent increase in the numbers of

attached cells was in particularly pronounced for the rod-shaped bacteria. Combination

of SEM, AFM and CLSM analysis revealed that under similar conditions these bacteria

showed consistent preference towards the smoother, modified glass and polymers

surfaces in contrast to the as-received surfaces. A decrease in the surface roughness

after exposure to BHF and UV irradiation provided an increase in the surface

uniformity, which on the other hand appeared to have stimulating effect on the

attachment preferences of rod-shaped bacteria. However, the variation in the number of

sphere-shaped, S. aureus, cells attached to the as-received/native and modified surfaces

were not as pronounced. This observation is in agreement with the ‘attachment point’

theory which states that small sphere-shaped organisms exhibit a different attachment

pattern compared to elongated, rod-shaped bacteria due to the different number of

available access points (Scardino et al., 2006).

Although both fibre surfaces appeared not to favour bacterial adhesion, some

bacterial species managed to successfully attach and maintain their existence on the as-

received fibre surface. On the other hand, modification of the fibre surface as described

herein, with exposure to BHF etching solution, appears to have cito-repellant potential

resulting in a decreased affinity between the bacteria and the fibre surface. The as-

received, nano-rough fibre surface was found to be substantially smoother than that of

Chapter 8:


______________________________________________________________________

219

the modified surface, again suggesting that the nano-smooth surface may in fact result

in an increased propensity for attachment.

The surface of optical fibres with a structure similar or equal to that presented

herein can serve as a base for development of SERS substrates, chemical or

environmental sensors. The cyto-repellent characteristics of the modified optical fibre

surfaces would provide a definite advantage in designing chemical sensors, SERS

probes or optical instruments. However the same fibre characteristic can be considered a

disadvantage when creating whole-cell biosensors when accurate data acquisition is

dependant on the cellular presence on the fibre surface.

Apart from the change in the number of cells undergoing attachment on the

smoother nano-structured surfaces, a remarkable change in cellular metabolic activity

was observed during the attachment process, as demonstrated by the characteristic cell

morphologies and the production of EPS. P. aeruginosa, P. issachenkonii, C. marina

and A. fischeri, which appeared to be the most successful colonisers of all tested

surfaces, were also noted to be excessive producers of EPS. S. flavus and S.

mediterraneus on the other hand showed the weakest attachment propensity towards

both glass and polymer surfaces, and failed to show any tendency to attach to the optical

fibre surfaces. It is of interest that they also did not produce any EPS. This observation

implies that bacteria employ somewhat ‘simple’ strategies for attaching to nano-smooth

surfaces by producing elevated quantities pf EPS. However, it is possible that the nano-

smooth surface itself has a stimulating effect on bacterial EPS production. A positive

correlation between presence of EPS and surface bacterial surface adhesion has been

previously suggested, yet not in the context of surface nano-topography (Kreft and

Wimpenny, 2001, Sutherland, 2001a, Sutherland, 2001b, Flemming and Wingender,

2001).

8.2 Future directions

Elucidation of the mechanisms responsible for the surface-induced changes in

bacterial behaviour has considerable potential to impact on both our fundamental

understanding of bacterial attachment and biofilm formation and on industrial and

Chapter 8:


______________________________________________________________________

220

medical applications. For this reason, further work is needed to determine if there is a

particular range of surface roughness that controls, or strongly influences bacterial

response to solid surfaces. A broader study incorporating larger numbers of bacterial

strains with differing taxonomic affiliations is needed in order to determine whether the

observed cellular response to nano-smooth surfaces is a fundamental, generic

mechanism. Investigations on the molecular level and extensive sequencing data should

also be considered in attempts to unravel the genetic mechanisms behind bacterial

attachment.

In order to systematically address the influence of surface roughness on bacterial

adhesion, studies incorporating more surfaces with variations not only in surface

roughness but also in surface topography should be considered. The potential to gain

absolute control of surface chemistry through techniques such as atom layer deposition

(Elam et al., 2002) is also of significant interest in confirming the effects described here.

Although recent advances have transformed our understanding of bacterial

interactions, we are still only just beginning to appreciate the chemical and physical

basis of cell-surface interactions. The application of nano-technological tools to create

surfaces with controlled roughness and topography, thereby controlling the chemical

and physical microenvironment surrounding the bacteria, will assist subsequent

researchers to enhance our understanding of bacterial attachment behaviour.

8.3 Close

Enhanced attachment of selected bacteria on smoother surfaces is a novel

discovery. The results of this study are of particular interest as they suggest that bacteria

may be far more susceptible to nano-scale surface roughness than to micron scale

irregularities during the process of attachment and biofilm formation. This observation

casts serious doubt on the conventional wisdom that smoother surfaces represent a more

repellent environment to bacteria than rougher surfaces. Based on the data presented in

this thesis, nano-scale surface roughness is hypothesised to have the potential to exert a

greater influence on bacterial adhesion than previously believed, and should therefore

Chapter 8:


______________________________________________________________________

221

be considered as a parameter of primary interest alongside other well-recognized factors

that control initial bacterial attachment.

The results of this study are of particular commercial interest. Although recent

advances have transformed our understanding of bacterial interactions with number of

substrates, we are only beginning to appreciate the chemical and physical basis behind

this complex phenomenon. Application of nanotechnological tools to control the

microenvironments surrounding cells will significantly aid in enhancing our

understanding of bacterial attachment behaviour. In this respect, better understanding of

the effect of nano-scale surface roughness on bacterial adhesion will have far-reaching

implications in designing surfaces for use in surgical implants, the food industry and

sterile environments such as hospitals and pharmaceutical laboratories. The suggestion

that nano-scale manipulation of surface roughness and topography might represent a

novel method to control the extent of bacterial adhesion provides a useful signpost for

further work.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 222

LIST OF REFERENCES

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 223

ABDELSALAM, M. E., BARTLETT, P. N., KELF, T. & BAUMBERG, J. (2005)

Wetting of regularly structured gold surfaces. Langmuir, 21, 1753-1757. ADVINCULA, M. A., PETERSEN, D., RAHEMTULLA, F., ADVINCULA, R. &

LEMONS, J. E. (2007) Surface analysis and biocorrosion properties of nanostructured surface sol-gel coatings on Ti6Al4V titanium alloy implants. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 80B, 107-120.

AIZAWA, S. I., HARWOOD, S. C. & KADNER, J. R. (2000) Signaling components in bacterial locomotion and sensory reception. Journal of Bacteriology, 182, 1459-1471.

AN, Y. H. & FRIEDMAN, R. J. (1998) Concise review of mechanisms of bacterial adhesion to biomaterial surfaces. Journal of Biomedical Materials Research 43, 338-348.

AN, Y. H., FRIEDMAN, R. J., DRAUGHN, R. A., SMITH, E. A., NICHOLSON, J. & JOHN, J. F. (1995) Rapid Quantification of Staphylococci adhered to titanium surfaces using image analyzed epifluorescence microscopy. Journal of Microbiological Methods, 24, 29-40.

ARAHAL, D. R., CASTILLO, A. M., LUDWIG, W., SCHLEIFER, K. H. & VENTOSA, A. (2002) Proposal of Cobetia marina gen. nov., comb. nov.,within the family of Halomonadaceae to include the species Halomonas marina. Systematic and Applied Microbiology, 25, 207-211.

ARNOLD, J. W. & BAILEY, G. W. (2000) Surface finishes on stainless steel reduce bacterial attachment and early biofilm formation: scanning electron and atomic force microscopy study. Pollution science, 79, 1839-1845.

BAKKER, D. P., BUSSCHER, H. J. & VAN DER MEI, H. C. (2002) Bacterial deposition in a parallel plate and a stagnation point flow chamber: microbial adhesion mechanisms depend on the mass transport conditions. Microbiology, 148, 597-603.

BAKKER, D. P., HUIJS, F. M., DE VRIES, J., KLIJNSTRA, J. W., BUSSCHER, H. J. & VAN DER MEI, C. H. (2003) Bacterial deposition to fluoridated and non-fluoridated polyurethane coatings with different elastic modulus and surface tension in a parallel plate and a stagnation point flow chamber Colloids and Surfaces B: Biointerfaces, 32, 179-190.

BASSON, A., FLEEMING, L. A. & CHENIA, H. Y. (2007) Evaluation of adherence, hydrophobicity, aggregation and biofilm development of Flavobacterium johnsoniae-like isolates. Microbial Ecology, 55, 1-14.

BATES, R. J. (2001) Optical switching and networking handbook, New York, McGraw-Hill.

BAYLES, K. W. (2007) The biological role of death and lysis in biofilm development. Nature Reviews Microbiology, 5, 721-726.

BAYOUDH, S., OTHMANE, A., BETTAIED, F., BAKHROUF, A., OUADA, B. H. & PONSONNET, L. (2006) Quantification of the adhesion free energy between bacteria and hydrophobic and hydrophilic substrata. Materials science and engineering: C, 26, 300-305.

BEECH, B. I., HANJAGSIT, L., KALAJI, M., NEAL, A. L. & ZINKEVICH, V. (1999) Chemical and structural characterization of exopolymers produced by

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 224

Pseudomonas sp. NCIMB 2021 in continuous culture. Microbiology, 145, 1491-1497.

BEECH, B. I., SUNNER, A. J. & HIRAOKA, K. (2005) Microbe-surface interactions in biofoulding and biocorrosion processes. International Microbiology, 8, 157-168.

BEECH, B. I. & SUNNER, J. (2004) Biocorrosion: towards understanding interactions between biofilms and metals. Current Opinion in Biotechnology, 15, 181-186.

BEECH, I. B., SMITHA, R. J., STEELEB, A. A., PENEGARA, I. & CAMPBELLA, A., S. (2002) The use of atomic force microscopy for studying interactions of bacterial biofilms with surfaces. Colloids and Surfaces B: Biointerfaces, 23, 231-247.

BELL, C. H., ARORA, B. S. & CAMESANO, T. A. (2005) Adhesion of Pseudomonas putida KT2442 Is mediated by surface polymers at the nano- and microscale. Environmental Engineering Science, 22, 629-641.

BENITO, Y., PIN, C., MARTIN, L. M., GARCIA, L. M., SELGAS, D. M. & CASAS, C. (1997) Cell surface hydrophobicity and attachment of pathogenic and spoilage bacteria to meat surfaces. Meat Science, 45, 419-425.

BERNARDET, J. F., NAKAGAWA, Y. & HOLMES, B. (2002) Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. International Journal of Systematic and Evolutionary Microbiology, 52, 1049-1070.

BERNTSSON, K. M., JONSSON, P. R., LEJHALL, M. & GATENHOLM, P. (2000) Analysis of behavioural rejection of micro-textured surfaces and implications for recruitment by the barnacle Balanus improvisus. Journal of Experimental Marine Biology and Ecology, 251, 59-83.

BEVERIDGE, T. J. & GRAHAM, L. L. (1991) Surface layers of bacteria. Microbiological Reviews, 55, 684-705.

BHATIA, S. K., TEIXEIRA, J. L., ANDERSON, M., SHRIVER-LAKE, L. C., CALVERT, J. M., GEORGER, J. H., HICKMAN, J. J., DULCEY, C. S., SCHOEN, P. E. & LIGLER, F. S. (1993) Fabrication of surfaces resistant to protein adsorption and application to two-dimensional protein patterning. Analytical Biochemistry, 208, 197-205.

BHOSLE, N., SUCI, P. A., BATY, A. M., WEINER, R. M. & GEESEY, G. G. (1998) Influence of divalent cations and pH on adsorption of a bacterial polysaccharide adhesin. Journal of Colloid and Interface Science, 205, 89.

BIRAN, I., RISSIN, D. M., RON, E. Z. & WALT, D. R. (2003) Optical imaging fiber-based live bacterial cell array biosensor. Analytical Biochemistry, 315, 106-113.

BORMASHENKO, E., BORMASHENKO, Y., WHYMAN, G., POGREB, R. & STANEVSKY, O. (2006a) Micrometrically scaled textured matallic hydrophobic interfaces validate Cassie-Baxter wetting hypothesis. Journal of Colloid and Interface Science, 302, 308-311.

BORMASHENKO, E., POGREB, R., WHYMAN, G. & ERLICH, M. (2007) Resonance Cassie-Wenzel wetting transition for horizontally vibrated drops deposited on a rough surface. Langmuir, 23, 12217-12221.

BORMASHENKO, E., STEIN, T., WHYMAN, G., BORMASHENKO, Y. & POGREB, R. (2006b) Wetting properties of the multiscaled nanostructured polymer and metallic superhydrophobic surfaces. Langmuir, 22, 9982-9985.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 225

BOS, R., VAN DER MEI, C. H., GOLD, J. & BUSSCHER, H. J. (2000) Retention of bacteria on a substratum surface with micro-patterned hydrophobicity. FEMS Microbiology Letters, 189, 311-315.

BOS, R., VAN DER MEI, H. C. & BUSSCHER, H. J. (1999) Physico-chemistry of initial microbial adhesive interactions - its mechanisms and methods for study. FEMS Microbiology Reviews, 23, 179-230.

BRADING, M., JASS, J. & LAPPIN-SCOTT, H. M. (1995) Dynamics of bacterial biofilm formation. IN COSTERTON, J. W. & LAPPIN-SCOTT, H. M. (Eds.) Microbial biofilms. Cambridge, Cambridge University Press.

BRANT, A. J. & CHILDRESS, E. A. (2002) Assessing short-range membrane–colloid interactions using surface energetics. Journal of membrane science, 203, 257.

BRUHN, J. B., GRAM, L. & BELAS, R. (2007) Production of antibacterial compounds and biofilm formation by Roseobacter species are influenced by culture conditions. Applied and Environmental Microbiology, 73, 442-450.

BRUINSMA, G. M., RUSTEMA-ABBING, M., DE VRIES, J., STEGENGA, B., VAN DER MEI, H. C., VAN DER LINDEN, M. L., HOOYMANS, J. M. M. & BUSSCHER, H. J. (2002) Influence of wear and overwear on surface properties of etafilcon a contact lenses and adhesion of Pseudomonas aeruginosa. Investigative Ophthalmology & Visual Science 43, 3646-3653.

BRUINSMA, G. M., RUSTEMA-ABBING, M., VAN DER MEI, H. C. & BUSSCHER, H. J. (2001) Effects of cell surface damage on surface properties and adhesion of Pseudomonas aeruginosa. Journal of Microbiological Methods, 45, 95-101.

BUCHAN, A., COLLIER, L. S., NEIDLE, E. L. & MORAN, M. A. (2000) Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine Roseobacter lineage. Applied and Environmental Microbiology, 66, 4662-4672.

BURKS, G. A., VELEGOL, S. B., PARAMONOVA, E., LINDENMUTH, B. E., FEICK, J. D. & LOGAN, B. E. (2003) Macroscopic and nanoscale measurements of the adhesion of bacteria with varying outer layer surface composition. Langmuir, 19, 2366-2371.

BUSSCHER, H. J., HANDLEY, P. S., BOS, R. & VAN DER MEI, H. C. (1999) Physico-chemistry of microbial adhesion – from an overall approach to the limits, New York, Marcel Dekker.

BUSSCHER, H. J. & NORDE, W. (2000) Limiting values for bacterial ζ potentials. Journal of Biomedical Materials Research, 50, 463-464.

BUSSCHER, H. J., SJOLLEMA, J. & VAN DER MEI, C. H. (1990) Relative importance of surface free energy as a measure of hydrophobicity in bacterial adhesion to solid surfaces, Washington, American Sociaety for Microbiology.

BUSSCHER, H. J. & VAN DER MEI, H. C. (1997) Physico-chemical interactions in initial microbial adhesion and relevance for biofilm formation. Advances in Dental Research, 11, 24-32.

CALDWELL, D. E., KORBER, D. R. & LAWRENCE, J. R. (1992) Cofocal laser microscopy and digital image analysis in microbial ecology. Advances in Microbial Ecology, 12, 1-67.

CALLOW, M. E., JENNINGS, A. R., BRENNAN, A. B., SEEGERT, C. E., GIBSON, A., WILSON, L., FEINBERG, A., BANEY, R. & CALLOW, J. A. (2002) Microtopographic cues for settlement of zoospores of the green fouling alga Enteromorpha. Biofouling, 18, 229-236.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 226

CAMESANO, T. A. & LOGAN, B. E. (1998) Influence of fluid velocity and cell concentration on the transport of motile and nonmotile bacteria in porous media. Environmental Science and Technology, 32, 1699-1708.

CANEPARI, P., BOARETTI, M., LLEÓ, M. M. & SATTA, G. (1990) Lipoteichoic acid as a new target for activity of antibiotics: mode of action of daptomycin (LY146032). Antimicrobial Agents and Chemotherapy, 34, 1220–1226.

CAO, T., TANG, H., LIANG, X., WANG, A., AUNER, G. W., SALLEY, S. O. & NG, S. K. Y. (2006) Nanoscale investigation on adhesion of Escherichia coli to surface modified silicone using atomic force microscopy. Biotechnology and Bioengineering, 94, 167-176.

CAREY, P. R. (1999) Raman spectroscopy, the sleeping giant in structural biology, awakes. Journal of Biological Chemistry, 274, 26625-26628.

CARMAN, M. L., ESTES, T. G., FEINBERG, A. W., SCHUMACHER, J. F., WILKERSON, W., WILSON, L. H., CALLOW, M. E., CALLOW, J. A. & BRENNAN, A. B. (2006) Engineered antifouling microtopographies - correlating wettability with cell attachment. Biofouling, 22, 11-21.

CASTELLANI, A. & CHALMERS, A. J. (1919) Manual of topical medicine 3rd ed., Type genus: Bacteroides, New York.

CASTELLANOS, T., ASCENCIO, F. & BASHAN, Y. (1997) Cell-surface hydrophobicity and cell-surface charge of Azospirillum spp. FEMS Microbiology, Ecology, 24, 159-172.

CHA, C., GAO, P., CHEN, Y.-C., SHAW, D. P. & FERRAND, K. S. (1998) Production of acyl-homoserine lactone quorum-sensing signals by Gram-negative plant-associated bacteria. MPMI, 11, 119-1129.

CHAE, M. S., SCHRAFT, H., TRUELSTRUP HANSEN, L. & MACKERETH, R. (2006) Effects of physicochemical surface characteristics of Listeria monocytogenes strains on attachment to glass. Food Microbiology, 23, 250-259.

CHARACKLIS, W. G. (1973) Attached microbial growths-II. Friction resistance due to microbial slimes. Water Research, 7, 1249-128.

CHEN, Y. & PEPIN, A. (2001) Nanofabrication: conventional and nonconventional methods. Electrophoresis 22, 187-207.

CHMIELEWSKI, R. A. N. & FRANK, J. F. (2003) Biofilm formation and control in food processing facilities. Comprehensive Reviews in Food Science and Food Safety, 2, 22-32.

COSTERTON, J. W. & LAPPIN-SCOTT, H. M. (1995) Microbial biofilms, Cambridge, Cambridge University Press.

COSTERTON, J. W. & LAPPIN-SCOTT, H. M. (1995a) Introduction to microbial biofilms. IN COSTERTON, J. W. & LAPPIN-SCOTT, H. M. (Eds.) Microbial biofilms. Cambridge, Cambridge University Press.

D'SOUZA, S. F. (2001) Microbial biosensors. Biosensors and Bioelectronics, 16, 337-353.

DANESE, P. N., PRATT, L. A. & KOLTER, R. (2000) Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. Journal of Bacteriology, 182, 3593-3596.

DAVEY, M. E. & O'TOOLE, G. A. (2000) Microbial biofilms: from ecology to molecular genetics. Microbiology and Molecular Biology Reviews, 64, 847-867.

DE KERCHOVE, A. J. & ELIMELECH, M. (2005) Relevance of electrokinetic theory for "soft" particles to bacterial cells: implication for bacterial adhesion. Langmuir, 21, 6462-6472.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 227

DE REZENDE, C. E., ANRIANY, Y., CARR, L. E., JOSEPH, S. W. & WEINER, R. M. (2005) Capsular polysaccharide surrounds smooth and rugose types of Salmonella enterica serovar typhimurium DT104. Applied and Environmental Microbiology, 71, 7345-7351.

DECKERT, V., ZEISEL, D., ZENOBI, R. & VO-DINH, T. (1998) Near-field surface enhanced Raman imaging of dye-labeled DNA with 100-nm resolution. Analytical Chemistry, 70, 2646-2650.

DONABEDIAN, H. (2003) Quorum sensing and its relevance to infectious diseases. Journal of Infection, 46, 207-214.

DONG, H., ONSTOTTA, T. C., KOB, C.-H. A., HOLLINGSWORTH, A. D., BROWN, D. G. & MAILLOUX, B. J. (2002) Theoretical prediction of collision efficiency between adhesion-deficient bacteria and sediment grain surface. Colloids and Surfaces B: Biointerfaces, 24, 229.

DONLAN, R. M. (2002) Biofilms: microbial life on surfaces. Emerging Infectious Diseases 8, 881-890.

DORONINA, N. V., TROTSENKO, Y. A. & TOUROVA, T. P. (2000) Methylarcula marina gen. nov., sp. nov. and Methylarcula terricola sp. nov.: novel aerobic, moderately halophilic, facultatively methylotrophic bacteria from coastal saline environments. International Journal of Systematic and Evolutionary Microbiology, 50, 1849-1859.

DOYLE, R. J. & ROSENBERG, M. (1990) Microbial cell surface hydrophobicity Washington, American Sociaety for Microbiology.

DUBAJ, V., MAZZOLINI, A., WOOD, A. & HARRIS, M. (2002) Optic fibre bundle contact imaging probe employing a laser scanning confocal microscope. Journal of Microscopy, 207, 108-117.

DULCEY, C. S., GEORGER, J. J. H., KRAUTHAMER, V., STENGER, D. A., FARE, T. L. & CALVERT, J. M. (1991) Deep UV photochemistry of chemisorbed monolayers: patterned coplanar molecular assemblies. Science and technology of advanced materials, 252, 551-554.

DUPRES, V., MENOZZI, D. F., LOCHT, C., CLARE, H. B., ABBOTT, L. N., CUENOT, S., BOMPARD, C., RAZE, D. & DUFRENE, Y. F. (2005) Nanoscale mapping and functional analysis of individual adhesins on living bacteria. Nature Methods, 2, 515-520.

DWORKIN, M. (2007) Shape, polarity, and multicellular morphogenesis. Current Opinion in Microbiology, 10, 588-590.

EBOIGBODIN, E. K., NEWTON, A. R. J., ROUTH, F. A. & BIGGS, A. C. (2005) Role of non-adsorbing polymers in bacterial aggregation. Langmuir, 21, 12315-12319.

EBOIGBODIN, E. K., NEWTON, A. R. J., ROUTH, F. A. & BIGGS, A. C. (2006) Bacterial quorum sensing and cell surface electrokinetic properties. Applied Microbiology and Biotechnology, 73, 669-675.

ELAM, J. W., GRONER, M. D. & GEORGE, S. M. (2002) Viscous flow reactor with quartz crystal microbalance for thin film growth by atomic layer deposition. Review of Scientific Instruments, 73, 2981-2987.

EMERSON, R. J., BERGSTROM, T. S., LIU, Y., SOTO, E. R., BROWN, C. A., MCGIMPSEY, W. G. & CAMESANO, T. A. (2006) Microscale correlation between surface chemistry, texture, and the adhesive strength of Staphylococcus epidermidis. Langmuir, 22, 11311-11321.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 228

ENGINEERING, T. C. F. B. (2008) A friendly guide to biofolm basics and the CBE. Montana State University.

ETZLER, F. M. (2006) Surface free energy of solids: A comparison of models. IN MITTAL, K. L. (Ed.) Contact angle, Wettability and Adhesion. 1st ed., Brill Academic.

FERNÁNDEZ, N., DÍAZ, E., AMILS, R. & SANZ, J. (2007) Analysis of microbial community during biofilm development in an anaerobic wastewater treatment reactor. Microbial Ecology.

FILLOUX, A. & VALLET, I. (2003) Biofilms: set-up and organization of a bacterial community. Medical Sciences (Paris), 19, 77-83.

FLEISCHMAN, M., HENDRA, P. J. & MCQUILLAN, A. J. (1974) Raman spectra of pyridine adsorbed at a silver electrode. Chemical Physics Letters, 26, 163-166.

FLEMMING, H. C. & WINGENDER, J. (2001) Relevance of microbial extracellular polymeric substances (EPSs) - Part I: Structural and ecological aspects. Water Science and Technology, 43, 1-8.

FLETCHER, M. (1996) Bacterial attachment in aquatic environments: a diversity of surface and adhesion strategies, New York, Wiley-Liss.

FLETCHER, M. & FLOODGATE, G. D. (1973) An electron-microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. Journal of General Microbiology, 74, 325-334.

FLETCHER, M. & MARSHAL, K. C. (1982) Bubble contact angle method for evaluation of substratum interfacial characteristics and its relevance to bacterial attachment. Applied and Environmental Microbiology, 44, 184-192.

GALLARDO-MORENO, A. M. & CALZADO-MONTERO, R. (2006) Zeta potential aspects of dispersed solvents involved in the determination of microbial cell surface hydrophobicity. Journal of Dispersion Science and Technology, 27, 23.

GALLARDO-MORENO, A. M. & GONZALEZ-MARTIN, M. L. (2002) Serum as a factor influencing adhesion of Enterococcus faecalis to glass and silicone. Applied and Environmental Microbiology, 68, 5784-5787.

GANNOT, I. & BEN-DAVID, M. (2003) Optical fibers and waveguides for medical applications IN VO-DINH, T. (Ed.) Biomedical Photonics Handbook. CRC Press, Boca Raton.

GARRITY, G. M. (1984) Bergey's manual of systematic bacteriology, Williams & Wilkins.

GESSNER, R., ROSCH, P., KIEFER, W. & POPP, J. (2002) Raman spectroscopy investigation of biological materials by use of etched and silver coated glass fiber tips. Biopolymers, 67, 327-330.

GOERTZ, P. M., HOUSTON, J. E. & Y., Z. X. (2007) Hydrophilicity and the viscosity of interfacial water. Langmuir, 23.

GOLDSTEIN, I. J., HOLLERMAN, C. E. & SMITH, E. E. (1964) Protein-carbohydrate interaction. II. Inhibition studies on the interaction on concanavalin A with polysaccharides. Biochemistry 4, 876-883.

GONZÁLEZ, J. M., COVERT, J. S., WHITMAN, W. B., HENRIKSEN, J. R., MAYER, F., SCHARF, B., SCHMITT, R., BUCHAN, A., FUHRMAN, J. A., KIENE, R. P. & MORAN, M. A. (2003) Silicibacter pomeroyi sp. nov. and Roseovarius nubinhibens sp. nov., dimethylsulfoniopropionate-demethylating bacteria from marine environments. International Journal of Systematic and Evolutionary Microbiology, 53, 1261-1269.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 229

GONZÁLEZ, J. M., MAYER, F., MORAN, M. A., HODSON, R. E. & WHITMAN, W. B. (1997) Sagittula stellata gen. nov., sp. nov., a lignin-transforming bacterium from a coastal environment. International Journal of Systematic Bacteriology, 47, 773-780.

GOOD, R. J. (1952) A thermodynamic derivation of Wenzel's modification of Young's equation for contact angles; together with a theory of hysteresis. Journal of the American Chemical Society, 74, 5041.

GOTTENBOS, B., GRIJPMA, D. W., VAN DER MEI, C. H., FEIJEN, J. & BUSSCHER, H. J. (2001) Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria. Journal of Antimicrobial Chemotherapy, 48, 7-13.

GOTTENBOS, B., VAN DER MEI, H. C. & BUSSCHER, H. J. (2000) Initial adhesion and surface growth of Staphylococcus epidermidis and Pseudomonas aeruginosa on biomedical polymers. Journal of Biomedical Materials Research, 50, 208-214.

GROSS, M., CRAMTON, S. E., GOTZ, F. & PESCHEL, A. (2001) Key role of teichoic acid net charge in Staphylococcus aureus colonization of artificial surfaces.

GROSS, M. J. & LOGAN, B. E. (1995) Influence of different chemical treatments on transport of Alcaligenes paradoxus in porous media. Applied and Environmental Microbiology, 61, 1750.

HALL-STOODLEY, L., COSTERTON, J. W. & STOODLEY, P. (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nature Reviews Microbiology, 2, 95-108

HAYNES, C. L., YONZON, C. R., ZHANG, X. & VAN DUYNE, R. P. (2005) Surface-enhanced Raman sensors: early history and the development of sensors for quantitative biowarfare agent and glucose detection. Journal of Raman Spectroscopy, 36, 471.

HICKMAN, J. J., BHATIA, S. K., QUONG, J. N., SHOEN, P., STENGER, D. A., PIKE, C. J. & COTMAN, C. W. (1994) Rational pattern design for in vitro cellular networks using surface photochemistry. Journal of Vacuum Science & Technology B: Microelectronics and Nanometer Structures, 12, 607-616.

HOGT, A. H., DANKERT, J. & FEIJEN, J. (1985) Adhesion of Staphylococcus epidermidis and Staphylococcus saprophyticus to a hydrophobic material. Journal of General Microbiology, 131, 2485-2491.

HOIPKEMEIER-WILSON, L., SCHUMACHER, J. F., CARMAN, M. L., GIBSON, A. L., FEINBERG, A. W., CALLOW, M. E., FINLAY, J. A., CALLOW, J. A. & BRENNAN, A. B. (2004) Antifouling potential of lubricious, micro-engineered, PDMS elastomers against zoospores of the green fouling alga Ulva (Enteromorpha). Biofouling, 20, 53-63.

HOWELL, D. & BEHRENDS, B. (2006) A review of surface roughness in antifouling coatings illustrating the importance of cutoff length. Biofouling, 22, 401-410.

INVITROGEN (2006) Vybrant CFDA SE Cell Tracer Kit. www.probes.invitrogen.com. IVANOVA, E. P., ALEXEEVA, Y. V., PHAM, D. K., WRIGHT, P. J. & NICOLAU,

D. V. (2006a) ATP level variations in heterotrophic bacteria during attachment on hydrophilic and hydrophobic surfaces. International Microbiology, 9, 37-46.

IVANOVA, E. P., BOWMAN, P. J., CHRISTEN, R., ZHUKOVA, V. N., LYSENKO, M. A., GORSHKOVA, M. N., MITIK-DINEVA, N., SERGEEV, A. &

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 230

MIKHAILOV, V. V. (2006b) Salegentibacter flavus sp. nov. International Journal of Systematic and Evolutionary Microbiology, 583-586.

IVANOVA, E. P., PHAM, D. K., WRIGHT, P. J. & NICOLAU, D. V. (2002a) Detection of coccoid forms of Sulfitobacter mediterraneus using atomic force microscopy. FEMS Microbiology Letters, 214, 177.

IVANOVA, E. P., SAWABE, T., ALEXEEVA, Y. V., LYSENKO, A. M., GORSHKOVA, N. M., HAYASHI, K., ZUKOVA, N. V., CHRISTEN, R. & MIKHAILOV, V. V. (2002b) Pseudoalteromonas issachenkonii sp. nov., a bacterium that degrades the thallus of the brown alga Fucus evanescens. International Journal of Systematic and Evolutionary Microbiology, 52, 229-234.

IVANOVA, E. P., WRIGHT, J. P., PHAM, D. K., BRACK, N., PIGRAM, P., ALEKSEEVA, Y. V., DEMYASHEV, G. M. & NICOLAU, D. V. (2006c) A comparative study between the adsorption and covalent binding of human immunoglobulin and lysozyme on surface-modified poly(tert-butyl methacrylate). Biomedical materials, 1, 24-32.

IVANOVA, P. E., MITIK-DINEVA, N., MOCANASU, C. R., MURPHY, S., WANG, J., VAN REISSEN & CRAWFORD, R. J. (2008) Vibrio fischeri and Escherichia coli tendencies towards photolithographically modified nanosmooth poly (tert-butyl methacrylate) polymer surfaces. Nanotechnology, Science and Applications, in press.

IVANOVA, P. E., MITIK-DINEVA, N., WANG, J., PHAM, K. D., WRIGHT, P. J., NICOLAU, D. V., MOCANASU, C. R. & CRAWFORD, R. J. (in press) Staleya guttiformis attachment on poly(tert-butylmethacrylate) polymeric surfaces. Micron.

JAEGER, R. C. (2002) Introduction to Microelectronic Fabrication, New York, Upper Saddle River.

JEWETT, D. G., HILBERT, T. A., LOGAN, B. E., ARNOLD, R. G. & BALES, R. C. (1995) Bacterial transport in laboratory columns and filters: Influence of ionic strength and pH on collision efficiency. Water Research, 29, 1673.

JONES, J. F. & VELEGOL, D. (2006) Laser trap studies of end-on E. coli adhesion to glass. Colloids and Surfaces B: Biointerfaces, 50, 66-71.

JOSEPH, L. A. & WRIGHT, A. C. (2004) Expression of Vibrio vulnificus capsular polysaccharide inhibits biofilm formation. Journal of Bacteriology, 186, 889-893.

JUCKER, B. A., HARMS, H. & ZEHNDER, A. J. (1996) Adhesion of the positively charged bacterium Stenotrophomonas (Xanthomonas) maltophilia 70401 to glass and Teflon. Journal of Bacteriology, 178, 5472-5479.

KELLER, M. D., KANTER, E. M. & MAHADEVAN-JANSEN, A. (2006) Raman spectroscopy for cancer diagnosis. Spectroscopy, 21, 33-41.

KIM, B. H., CHANG, I. S. & GADD, G. M. (2007) Challenges in microbial fuel cell development and operation. Applied Microbiology and Biotechnology, 76, 485-494.

KOECHLEIN, D. J. & KRIEG, N. R. (1998) Viable but nonculturable coccoid forms of Prolinoborus fasciculus (Aquaspirillum fasciculus). Canadian Journal of Microbiology, 44, 910-912.

KORENEVSKY, A. & BEVERIDGE, T. J. (2007) The surface physicochemistry and adhesiveness of Shewanella are affected by their surface polysaccharides. Microbiology, 153, 1872-1883.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 231

KREFT, J. U. & WIMPENNY, J. W. (2001) Effect of EPS on biofilm structure and function as revealed by an individual-based model of biofilm growth. Water Science and Technology, 43, 135-141.

KREIG, N. R. & HOLT, J. G. (1984) Bergey's manual of systematic bacteriology, Baltimore, Wiliams and Wilkins.

KRIEG, N. R. & HOLT, J. G. (1984) Bergey’s manual of bacteriology Wiliams and Wilkins.

LAARMANN, S. & SCHMIDT, M. A. (2003) The Escherichia coli AIDA autotransporter adhesin recognizes an integral membrane glycoprotein as receptor. Microbiology 149, 1871-1882.

LABRENZ, M., TINDALL, B. J., LAWSON, P. A., COLLINS, M. D., SCHUMANN, P. & HIRSCH, P. (2000) Staleya guttiformis gen. nov., sp. nov. and Sulfitobacter brevis sp. nov., Alpha-3-Proteobacteria from hypersaline, heliothermal and meromictic antarctic Ekho lake. International Journal of Systematic and Evolutionary Microbiology, 50, 303-313.

LAM, J. S., GRAHAM, L. L., LIGHTFOOT, J., DASGUPTA, T. & BEVERIDGE, T. J. (1992) Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution. Journal of Bacteriology, 174, 7159-7167.

LI, B. & LOGAN, B. E. (2004) Bacterial adhesion to glass and metal-oxide surfaces. Colloids and Surfaces B: Biointerfaces, 36, 81-90.

LI, B. & LOGAN, B. E. (2005) The impact of ultraviolet light on bacterial adhesion to glass and metal oxide-coated surface. Colloids and Surfaces B: Biointerfaces, 41, 153-161.

LI, Y., LI, C., CHO, S. O., DUAN, G. & CAI, W. (2007) Silver hierarchical bowl-like array:synthesis, superhydrophobicity and optical properties. Langmuir, 23, 9802-9807.

LIU, C., ZHAO, Q., LIU, Y., WANG, S. & ABEL, E. W. (2008) Reduction of bacterial adhesion on modified DLC coatings. Colloids and Surfaces B: Biointerfaces, 61, 182-187.

LIU, Y. & ZHAO, Q. (2005) Influence of surface energy of modified surfaces on bacterial adhesion. Biophysical Chemistry, 117, 39-45.

LLAMAS, I., QUESADA, E. & MARTÍNEZ-CÁNOVAS, M. J. (2005) Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas. Extremophiles, 9, 333-341.

LYNCH, A. S. & ROBERTSON, G. T. (2008) Bacterial and fungal biofilm infections. Annual Review of Medicine, 59, 415-428.

MANDLIK, A., SWIERCZYNSKI, A., DAS, A. & TON-THAT, H. (2008) Pili in Gram-positive bacteria: assembly, involvement in colonization and biofilm development. Trends in Microbiology, 16, 33-40.

MARAZUELA, M. D. & MORENO-BONDI, M. C. (2002) Fiber-optic biosensors – an overview. Analytical and bioanalytical chemistry, 372, 664–682.

MARSHALL, K. C. (1992) Biofilms: an over-view of bacterial adhesion, activity and control at surfaces. ASM News, 58, 202-207.

MARSHALL, K. C., STOUT, R. & MITCHELL, R. (1971) Mechanisms of the initial events in the sorption of marine bacteria to surfaces. Journal of General Microbiology, 68, 337-348.

MEI, Y., WU, T., XU, C., LANGENBACH, J. K., ELLIOT, T. J., VOGT, D. B., BEERS, L. K., AMIS, J. E. & WASHBURN, R. N. (2005) Tuning cell adhesion

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 232

on gradient poly(2-hydroxyethil methacrylate)-grafted surface. Langmuir, 21, 12309-12314.

MESSING, R. A. & OPPERMANN, R. A. (1979) Pore dimensions for accumulating biomass. I. Microbes that reproduce by fission or by budding. Biotechnology and Bioengineering, 21, 49-58.

MICHIELSEN, S. & LEE, J. H. (2007) Design of superhydrophobic surface usinh woven structures. Langmuir, 23, 6004-6010.

MILLER, M. B. & BASSLER, B. L. (2001) Quorum sensing in bacteria. Annual Reviews in Microbiology, 55, 165-99.

MITIK-DINEVA, N., STODDART R., P., CRAWFORD, R. J. & IVANOVA, E. P. (2006 ) Adhesion of bacteria. IN AKAY, S. M. (Ed.) Encyclopedia of Biomedical Engineering. New York, John Wiley & Sons.

MITIK-DINEVA, N. & STODDART, R. P. (2006) Applications of atomic force microscopy in topographic imaging. IN IVANOVA, E. P. (Ed.) Nanoscale structure and properties of microbial cell surfaces. New York, Nova Science Publishers, Inc.

MITIK-DINEVA, N., WANG, J., MOCANASU, C. R., STODDART, R. P., CRAWFORD, R. J. & IVANOVA, E. P. (2008a) Impact of nano-topography on bacterial attachment. Biotechnology Journal, 3, 536-544.

MITIK-DINEVA, N., WANG, J., STODDART, R. P., CRAWFORD, R. J. & IVANOVA, P. E. (2008b) Nano-structured surfaces control bacterial attachment

ICONN. Melbourne, Australia. MITTELMAN, M. W. (1996) Adhesion to biomaterials. Bacterial adhesion: Molecular

and ecological diversity. New York, Willey. MOONEY, J. F., HUNTDAGGER, A. J., MCINTOSHDAGGER, J. R.,

LIBERKODAGGER, C. A., WALBADAGGER, D. M. & ROGERS, C. T. (1996) Patterning of functional antibodies and other proteins by photolithography of silane monolayers. Proceedings of the National Academy of Sciences of the United States of America, 93, 12287-12291.

MURPHY, S. (2007) Employment of polymer UV photolithography to control bacterial attachment. Faculty of Life and Social Sciences. Melbourne, Swinburne University of Technology.

MURPHY, T., LUCHT, S., SCHMIDT, H. & KRONFELDT, H. D. (2000) Surface-enhanced Raman scattering (SERS) system for continuous measurements of chemicals in sea-water. Journal of Raman Spectroscopy, 31, 943-948.

NELSON, D. L. & COX, M. M. (2000) Lehninger principles of biochemistry: third edition, Worth Publishers.

NISHINO, T., IKEMOTO, E. & KOGURE, K. (2004) Application of atomic force microscopy to observation of marine bacteria. Journal of Oceanography, 60, 219-225.

O'TOOLE, G. A., KAPLAN, H. B. & KOLTER, R. (2000) Biofilm formation as microbial development. Annual Reviews in Microbiology, 54, 49-79.

O'TOOLE, G. A. & KOLTER, R. (1998a) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Molecular Microbiology, 30, 295-304.

O'TOOLE, G. A. & KOLTER, R. (1998b) Initiation of biofilm formation in Pseudomonas fluorescens. Molecular Microbiology, 28, 449-461.

OLIVER, J. (1995) The viable but nonculturable state in the human pathogen Vibrio vulnificus. FEMS Microbiology Letters, 133, 203-208.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 233

ÖNER, D. & MCCARTHY, T. J. (2000) Ultrahydrophobic surfaces. Effects of topography length scales on wettability. Langmuir, 16, 7777-7782.

ONG, Y., RAZATOS, A., GEORGIOU, G. & SHARMA, M. M. (1999) Adhesion forces between E. coli bacteria and biomaterial surfaces. Langmur, 15, 2719-2725.

OSAKI, T. & CARSTEN, W. (2003) Ionization Characteristics and Structural Transitions of Alternating Maleic Acid Copolymer Films. Langmuir, 19, 5787-5793.

PALMER, J., FLINT, S. & BROOKS, J. (2007) Bacterial cell attachment, the beginning of a biofilm. Journal of Industrial Microbiology and Biotechnology, 34, 577-588.

PARSEK, M. R. & SINGH, P. K. (2003) Bacterial biofilms: an emerging link to disease pathogenesis. Annual Review of Microbiology, 57, 677-701.

PASMORE, M. & COSTERTON, J. W. (2003) Biofilms, bacterial signaling, and their ties to marine biology. Journal of Industrial Microbiology and Biotechnology 30, 407-413.

PEARSON, C. R., HENG, M., GEBERT, M. & GLATZ, C. E. (2004) Zeta potential as a measure of polyelectrolyte flocculation and the effect of polymer dosing conditions on cell removal from fermentation broth. Biotechnology and Bioengineering, 87, 54-60.

PEREIRA, M. A., ALVES, M. M., AZEREDO, J., MOTA, M. & OLIVEIRA, R. (2000) Influence of physico-chemical properties of porous microcarriers on the adhesion of an anaerobic consortium. Journal of Industrial Microbiology and Biotechnology, V24, 181-186.

PETRONIS, S., BERNTSSON, K., GOLD, J. & GATENHOLM, P. (2000) Design and microstructuring of PDMS surfaces for improved marine biofouling resistance. Journal of Biomaterials Science, Polymer Edition, 11, 1051-1072.

PETRY, R., SCHMITT, M. & POPP, J. (2003) Raman Spectroscopy - a prospective tool in the life sciences. ChemPhysChem 4, 14-30.

PHAM, D. K., IVANOVA, E. P., WRIGHT, J. P. & NICOLAU, D. V. (2003) AFM analysis of the extracellular polymeric substances (EPS) released during bacterial attachment on polymeric surfaces. Manipulation and Analysis of Biomoleucles, Cells and Tissues, 4962, 151-159.

PLISOVA, E. Y., BALABANOVA, L. A., IVANOVA P., E., KOZHEMYAKO, V. B., MIKHAILOV, V. V., AGAFANOVA, E. V. & RASSKAZOV, V. A. (2004) A highly active alkaline phosphates from the marine bacterium Cobetia. Marine Biotechnology, 7, 173-178.

POLWART, E., KEIR, R. L., DAVIDSON, C. M., SMITH, W. E. & SADLER, D. A. A. S. (2000) Novel SERS-active optical fibers prepared by the immobilization of silver colloidal particles. Applied spectroscopy, 54, 522.

POTYRAILO, R. A., HOBBS, S. E. & HIEFTJE, G. M. (1998) Optical waveguide sensors in analytical chemistry: today’s instrumentation, applications and trends for future development. Journal of Analytical Chemistry, 362, 349–373.

POWER, L., ITIER, S., HAWTON, M. & SCHRAFT, H. (2007) Time Lapse Confocal Microscopy Studies of Bacterial Adhesion to Self-Assembled Monolayers and Confirmation of a Novel Approach to the Thermodynamic Model. Langmuir, 23, 5622-5629.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 234

PRATT, L. A. & KOLTER, R. (1998) Genetic analysis of E. coli biofilm formation: roles of flagella motility, chemotaxis and type I pili. Molecular Microbiology, 30, 285-293.

PRATT, L. A. & KOLTER, R. (1999) Genetic analyses of bacterial biofilm formation. Current Opinion in Microbiology, 2, 598-603.

PRINGLE, J. H. & FLETCHER, M. (1986) Influence of substratum hydration and adsorbed macromolecules on bacterial attachment to surfaces. Applied and Environmental Microbiology, 51, 1321-1325.

PUKALL, R., BUNTEFU, D., FRUHLING, A., ROHDE, M., KROPPENSTEDT, R. M., BURGHARDT, J., LEBARON, P., BERNARD, L. & STACKEBRANDT, E. (1999) Sulfitobacter mediterraneus sp. nov., a new sulfite-oxidizing member of the alpha-Proteobacteria. International Journal of Systematic Bacteriology, 49, 513-519.

QIAN, P. Y., LAU, S. C., DAHMS, H. U., DOBRETSOV, S. & HARDER, T. (2007) Marine biofilms as mediators of colonization by marine macroorganisms: Implications for antifouling and aquaculture Marine Biotechnology, 9, 399-410.

QUÉRÉ, D., LAFUMA, A. & BICO, J. (2003) Slippy and sticky microtextured solids. Nanotechnology, 14, 1109-1112.

RACZKOWSKA, J., BERNASIK, A., BUDKOWSKI, A., SAJEWICZ, K., PENC, B., LEKKI, J., LEKKA, M., RYSZ, J., KOWALSKI, K. & CZUBA, P. (2004) Structures formed in spin-cast films of polystyrene blends with poly(butyl methacrylate) isomers. Macromolecules, 37, 7308-7315.

RAPPÉ, M. S., VERGIN, K. & GIOVANNONI, S. J. (2000) Phylogenetic comparisons of a coastal bacterioplankton community with its counterparts in open ocean and freshwater systems. FEMS Microbiology, Ecology, 33, 219-232.

RAVEL, J., KNIGHT, I., MONAHAN, C. & HILL, R. (1995) Temperature-induced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation? Microbiology, 141, 377-383.

RAWLINGS, D. E. & JOHNSON, D. B. (2007) The microbiology of biomining: development and optimization of mineral-oxidizing microbial consortia. Microbiology 153, 315-324.

RAZATOS, A. (2001) Application of atomic force microscopy to study initial events of bacterial adhesion. Methods Enzymol 337, 276-285.

RAZATOS, A., ONG, Y., SHARMA, M. M. & GEORGIOU, G. (1998) Molecular determinations of bacterial adhesion monitored by atomic force microscopy. Proceedings of the National Academy of Sciences, 95, 11059-11064.

RIEDEWALD, F. (2006) Bacterial adhesion to surfaces: The influence of surface roughness. PDA Journal of Pharmaceutical Science and Technology, 60, 164-171.

RIJNAARTS, H. H. M., NORDE, W., LYKLEMA, J. & ZEHNDER, A. J. B. (1999) DLVO and steric contributions to bacterial deposition in media of different ionic strengths. Colloid Surf. B: Biointerfaces, 14, 179–195.

ROOSJEN, A., BUSSCHER, H. J., NORDE, W. & VAN DER MEI, H. C. (2006) Bacterial factors influencing adhesion of Pseudomonas aeruginosa strains to a poly(ethylene oxide) brush. Microbiology, 152, 2673-2682.

ROSCH, P., HARZ, M., SCHMITT, M., PESCHKE, K. D., RONNEBERGER, O. & BURKHARDT, H. (2005) Chemotaxonomic identification of single bacteria by micro-Raman spectroscopy: Application to clean-room-relevant biological contaminations. Applied and Environmental Microbiology, 71, 1626-1637.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 235

ROZHOK, S. & HOLZ, R. (2005) Electrochemical attachment of motile bacterial cells to gold. Talanta, 67, 538-542.

RUBY, E. G. (1996) Lessions from a cooperative, bacterial-animal association: The Vibrio fischeri-Euprymna scolopes Light Organ Symbiosis. Annual Review of Microbiology, 50, 591-624.

RUBY, E. G., URBANOWSKI, M., CAMPBELL, J., DUNN, A., FAINI, M., GUNSALUS, R., LOSTROH, P., LUPP, C., MCCANN, J., MILLIKAN, D., SCHAEFER, A., STABB, E., STEVENS, A., VISICK, K., WHISTLER, C. & GREENBERG, E. P. (2005) Complete genome sequence of Vibrio fischeri: A symbiotic bacterium with pathogenic congeners. Procedings of the National Academy of Sciences USA, 102, 3004-3009.

RYDER, C., BYRD, M. & WOZNIAK, D. J. (2007) Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Current Opinion in Microbiology, 10, 644-648.

RYU, J. H., KIM, H. J. & BEUCHAT, L. R. (2004) Attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel as influenced by exopolysaccharide production, nutrient availability, and temperature. Journal of Food Protection, 64, 2123-2131.

SANDERS, R. S., CHOW, R. S. & MASLIYAH, J. H. (1995) Deposition of bitumen and asphaltene-stabilized emulsions in an impinging jet cell Journal of Colloid and Interface Science 174, 230-245.

SATOMI, M., KIMURA, B., HAYASHI, M., SHOUZEN, Y., OKUZUMI, M. & FUJII, T. (1998) Marinospirillum gen. nov., with description of Marinospirillum megaterium sp. nov., isolated from kusaya gravy and transfer of Oceanospirillum minutulum to Marinospirillum minutulum comb. nov. International Journal of Systematic Bacteriology, 48, 1341-1348.

SAUER, K. & CAMPER, A. K. (2001) Characterization of phyenotypic changes in Pseudomonas putida in response to surface associated growth. Journal of Bacteriology, 183, 6579-6589.

SCARDINO, A. J., HARVEY, E. & DE NYS, R. (2006) Testing attachment point theory: diatom attachment on microtextured polyimide biomimics. Biofouling, 22, 55-60.

SCHEUERMAN, T. R., CAMPER, A. K. & HAMILTON, M. A. (1998) Effects of substratum topography on bacterial adhesion. Journal of Colloid and Interface Science, 208, 23-33.

SCHUSTER, K. C., REESE, I., URLAUB, E., GAPES, J. R. & LENDL, B. (2000) Multidimensional information on the chemical composition of single bacterial cells by confocal Raman microspectroscopy. Analytical Chemistry, 72, 5529-5534.

SENARATNE, W., ANDRUZZI, L. & OBER, C. K. (2005) Self-assembled monolayers and polymer brushes in biotechnology: current applications and future perspectives. Biomacromolecules, 6, 2427 - 2448

SHARON, N. (2006) Carbohydrates as future anti-adhesion drugs for infectious diseases. Biochimica et Biophysica Acta (BBA) - General Subjects, 1760, 527-537.

SHELLENBERGER, K. & LOGAN, B. E. (2002) Effect of molecular scale roughness of glass beads on colloidal and bacterial deposition. Environmental Science and Technology, 36, 184-189.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 236

SILIPO, A., LEONE, S., LANZETTA, R., PARRILLI, M., STURIALE, L., GAROZZO, D., NAZARENKO, L. E., GORSHKOVA, P. R., IVANOVA, E. P., GORSHKOVAC, M. N. & MOLINARO, A. (2004) The complete structure of the lipooligosaccharide from the halophilic bacterium Pseudoalteromonas issachenkonii KMM 3549T. Carbohydrate Research, 339, 1985-1993.

SIMONI, S. F., BOSMA, T. N. P., HARMS, H. & ZEHNDER, A. J. B. (2000) Bivalent cations increase both the subpopulation of adhering bacteria and their adhesion efficiency in sand columns. Environmental Science and Technology, 34, 1011-1017.

SINGH, R., PAUL, D. & JAIN, R. K. (2006) Biofilms: implications in bioremediation. Trends in Microbiology, 14, 389-397.

SKERMAN, V. B. D., MCGOWAN, V. & SNEATH, P. H. A. (1980) Approved lists of bacterial names. International Journal of Systematic Bacteriology, 30, 225-420.

SONI, K. A., BALASUBRAMANIAN, A. K., BESKOK, A. & PILLAI, S. D. (2007) Zeta potential of selected bacteria in drinking water when dead, starved, or exposed to minimal and rich culture media. Current Microbiology.

SÖRBERG, M., NILSSON, M., HANBERGER, H. & NILSSON, L. (1996) Morphological conversion of Helicobacter pylori from bacillary to coccoid form. European Journal of Clinical Microbiology and Infectious Diseases 15, 216-219.

SOROKIN, D. Y. (1995) Sulfitobacter pontiacus gen. nov., sp. nov. - a new heterotrophic bacterium from the Black Sea, specialized on sulfite oxidation. Mikrobiologiya, 64, 354-365

SOTO, G. E. & HULTGREN, S. J. (1999) Bacterial adhesins; common themes and variations in architecture and assembly Journal of Bacteriology, 181, 1059-1071.

SPERANZA, G., GOTTARDI, G., PEDERZOLLI, C., LUNELLI, L., CANTERI, R., PASQUARDINI, L., CARLI, E., MANIGLIO, D., BRUGNARA, M. & ANDERLE, M. (2004) Role of cehmical interactions in bacterial adhesion. Biomaterials, 25, 2029-2037.

STALEY, J. T. (1968) Prosthecomicrobium and Ancalomicrobium: New Prosthecate Freshwater Bacteria. Journal of Bacteriology, 95, 1921-1942.

STENGER, D. A., GEORGER, J. H., DULCEY, C. S., HICKMAN, J. J., RUDOLPHT, A. S., NIELSEN, T. B., MCCORT, S. M. & CALVED, J. M. (1992) Coplanar molecular assemblies of amino- and perfluorinated alkylsilanes: characterization and geometric definition of mammalian cell adhesion and growth. Journal of the American Chemical Society 114, 8435-8442.

STODDART, P. R. & BRACK, N. (2007) Physical techniques for cell surface probing and manipulation. IN IVANOVA, E. P. (Ed.) Nanoscale Structure and Properties of Microbial Cell Surfaces. Nova Science.

STOKES, D. L., CHI, Z. H. & VO-DINH, T. (2004) Surface enhanced Raman scattering inducing nanoprobe for spectrochemical analysis. Applied Spectroscopy, 58, 292-298.

STUART, D. A., YUEN, J. M., SHAH, N., LYANDRES, O., YONZON, C. R., GLUCKSBERG, M. R., WALSH, J. T. & VAN DUYNE, R. P. (2006) In vivo glucose measurement by surface-enhanced Raman spectroscopy. Analytical Chemistry, 78, 7211-7215.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 237

SUMNER, J. B. & HOWELL, S. F. (1936) The role of divalent metals in the reversible inactivation of jack bean hemagglutinin. Journal of Biological Chemistry 115, 583.

SUTHERLAND, I. W. (2001a) Biofilm exppolysaccharides: a strong and sticky framework. Microbiology and Molecular Biology Reviews, 147, 3-9.

SUTHERLAND, I. W. (2001b) Exopolysaccharides in biofilms, flocs and related structures. Water Science and Technology, 43, 77 -86.

SUZUKI, S., NAKAJIMA, A., YOSHIDA, N., SAKAI, M., HOSHIMOTO, A., KAMESHIMA, Y. & OKADA, K. (2007) Hydrophobicity and freezing of water droplet on fluoroalkylsilane coatings with different roughnesses. Langmuir, 23, 8674-8677.

TAYLOR, R. L., VERRAN, J., LEES, G. C. & WARD, A. J. P. (1998) The influence of substratum topography on bacterial adhesion to polymethyl methacrylate. Journal of Material Science: materials in medicine, 9, 17-22.

TEGOULIA, V. A. & COOPER, S. L. (2002) Staphylococcus aureus adhesion to self-assembled monolayers: effect of surface chemistry and fibrinogen presence. Colloids and Surfaces B: Biointerfaces, 24, 217-228.

TEIXEIRA, P. & OLIVEIRA, R. (1999) Influence of surface characteristics on the adhesion of Alcaligenes denitrificans to polymeric substrates. Journal of Adhesion Science and Technology, 13, 1243-1362.

TERASAKI, Y. (1979) Transfer of five species and two subspecies of Spirillum to other genera (Aquaspirillum and Oceanospirillum), with emended descriptions of the species and subspecies. International journal of systematic bacteriology, 29, 130-144.

TODAR, K. (2007) Todar's online book of bacteriology. IN TODAR, K. (Ed.), University of Wisconsin, department of Bacteriology.

TON-THAT, H. & SCHNEEWIND, O. (2003) Assembly of pili on the surface of Corynebacterium diphtheriae. Molecular Microbiology, 50, 1429-1438.

TRIANDAFILLU, K., BALAZS, D. J., ARONSSON, B. O., DESCOUTS, P., TU QUOC, P., VAN DELDEN, C., MATHIEU, H. J. & HARMS, H. (2003) Adhesion of Pseudomonas aeruginosa strains to untreated and oxygen-plasma treated poly(vinyl chloride) (PVC) from endotracheal intubation devices. Biomaterials, 24, 1507-1518.

UBBINK, J. & SCHÄR-ZAMMARETTI, P. (2005) Probing bacterial interactions: integrated approaches combining atomic force microscopy, electron microscopy and biophysical techniques. Micron, 36, 293.

UDD, E. (1995) Fiber optic smart structures, New York, Wiley. VADILLO-RODRIGUEZ, V., BUSSCHER, H. J., NORDE, W., VRIES, J. & VAN

DER MEI, H. C. (2004) Atomic force microscopy corroboration of bond aging for adhesion of Streptococcus thermophilus to solid substrata. Journal of Colloid and Interface Science, 278, 251-254.

VADILLO-RODRÍGUEZ, V. & LOGAN, B. E. (2006) Localized attraction correlates with bacterial adhesion to glass and metal oxide substrata. Environmental Science and Technology, 40, 2983-2988.

VAN DER MEI, H. C., BOS, R. & BUSSCHER, H. J. (1998) A refernce guide to microbial cell surface hydrophobicity based on contact angles. Colloids and Surfaces B: Biointerfaces, 11, 213-221.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 238

VAN DER MEI, H. C. & BUSSCHER, H. J. (2001) Electrophoretic mobility distributions of single-strain microbial populations. Applied and Environmental Microbiology, 67, 491-494.

VAN DER MEI, H. C., DE VRIES, J. & BUSSCHER, H. J. (2000) X-Ray photoelectron spectroscopy for the study of microbial cell surfaces. Surface Science Reports, 39, 1-24.

VAN LOOSDRECHT, M. C., LYKLEMA, J., NORDE, W., SCHRAA, G. & ZEHNDER, A. J. (1987a) Electrophoretic mobility and hydrophobicity as a measure to predict the initial steps of bacterial adhesion. Applied and Environmental Microbiology, 53, 1898-1901.

VAN LOOSDRECHT, M. C., LYKLEMA, J., NORDE, W., SCRAA, G. & ZEHNDER, A. J. (1987b) The role of bacterial cell wall hydrophobicity in adhesion. Applied and Environmental Microbiology, 53, 1893-1897.

VAN LOOSDRECHT, M. C. M., NORDE, W., LYKLEMA, J. & ZEHNDER, A. J. B. (1990) Hydrophobic and electrostatic parameters in bacterial adhesion. Aquatic Sciences - Research Across Boundaries, 52, 103-114.

VAN MERODE, E. J. A., POTHOVEN, D. C., VAN DER MEI, C. H., BUSSCHER, H. J. & KRORN, B. P. (2007) Surface charge influences enterococcal prevalence in mixed-species biofilms. Journal of Applied Microbiology, 102, 1254-1260.

VAN OSS, C. J. (1994) Interfacial forces in aqueous media, New York, Marcel Dekker. VAN OSS, C. J., GOOD, R. J. & CHAUDHURY, M. K. (1988) Additive and

nonadditive surface tension components and the interpretation of contact angles. Langmuir, 4, 884-891.

VEISEH, M., ZAREIE, M. & HADI., Z. M. (2002) Highly Selective Protein Patterning on Gold-Silicon Substrates for Biosensor Applications. Langmuir, 18, 6671-6678.

VERMELTFOORT, P. B. J., VAN KOOTEN, T. G., BRUINSMA, G. M., HOOYMANS, A. M. M., VAN DER MEI, H. C. & BUSSCHER, H. J. (2005) Bacterial transmission from contact lenses to porcine corneas: An ex vivo study. Investigative Ophthalmology & Visual Science, 46, 2042-2046.

VERRAN, J. & BOYD, R. D. (2001) The relationship between substratum surface roughness and microbiological and organic soiling: A review. Biofoulding, 17, 59-71.

VERRAN, J., DRUCKER, D. B. & TAYLOR, C. J. (1980) Feasibility of using automatic image-analysis for measuring deposition of Streptococcus mutans on glass, in terms of percentage coverage and mean clump size. Microbios, 29, 161.

VO-DINH, T. (1998) Surface-enhanced Raman spectroscopy using metallic nanostructures. Trends in Analytical Chemistry, 17, 557.

VO-DINH, T., ALLAIN, L. R. & STOKES, D. L. (2002) Cancer gene detection using surface-enhanced Raman scattering (SERS). Journal of Raman Spectroscopy, 33, 511-516.

VO-DINH, T. & KASILI, P. (2005) Fiber-optic nanosensors for single-cell monitoring. Analytical & Bioanalytical Chemistry, 382, 918-925.

VO-DINH, T. & STOKES, D. L. (2002) IN CHALMERS, J. M. & GRIFFITHS, P. R. (Eds.) Handbook of Vibrational Spectroscopy. New York, Wiley.

VOGLER, E. A. (1998) Structure and reactivity of water at biomaterial surfaces. Advances in Colloid and Interface Science, 74, 69-117.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 239

WAAR, K., VAN DER MEI, H. C., HARMSEN, H. J. M., DEGENER, J. E. & BUSSCHER, H. J. (2002) Enterococcus faecalis surface proteins determine its adhesion mechanism to bile drain materials. Microbiology, 148, 1863-1870.

WAGNER-DÖBLER, I., RHEIMS, H., FELSKE, A., PUKALL, R. & TINDALL, B. J. (2003) Jannaschia helgolandensis gen. nov., sp. nov., a novel abundant member of the marine Roseobacter clade from the North Sea. International Journal of Systematic and Evolutionary Microbiology, 53, 731–738.

WAGNER, E. V., FRELINGER, G. J., BARTH, K. R. & IGLEWSKI, H. B. (2006) Quorum sensing: dynamic response of Pseudomonas aeruginosa to external signals. Trends in Microbiology, 14, 55-58.

WATNICK, P. I. & KOLTER, R. (1999) Steps in the development of a Vibrio cholerae biofilm. Molecular Microbiology, 34, 586-595.

WEIBEL, D. B., DILUZIO, W. R. & WHITESIDES, G. M. (2007) Microfabrication meets microbiology. Nature Reviews Microbiology, 5, 209-218.

WENZEL, N. R. (1936) Resistance of solid surfaces to wetting by water. Industrial and Engineering Chemistry, 28, 988-994.

WHITE, D. J., MAZZOLINI, A. P. & STODDART, P. R. (2007) Fabrication of a range of SERS substrates on nanostructured multicore optical fibres. Journal of Raman Spectroscopy, 38, 377-382.

WHITE, J. D. & STODDART, R. P. (2005) Nanostructured optical fiber with surface-enhanced Raman scattering functionality. Optics Letters, 30, 598-600.

WHITEHEAD, A. K. & VERRAN, J. (2006) The effect of surface topography on the retention of microorganisms. Food and Bioproducts Processing, 84, 253-259.

WHITEHEAD, A. N., BERNATRD, M. L. A., SLATER, H., SIMPSON, J. L. N. & SALMOND, P. C. G. (2001) Quorum-sensing in Gram-negative bacteria. FEMS Microbiology Letters, 25, 365-404.

WHITTAKER, C. J., KLIER, C. M. & KOLENBRANDER, P. E. (1996) Mechanisms of adhesion by oral bacteria. Annual Review of Microbiology, 50, 513-552.

WONG, H.-C., CHUNG, Y.-C. & YU, J.-A. (2002) Attachment and inactivation of Vibrio parahaemolyticus on stainless steel and glass surface. Food Microbiology, 19, 341-350.

WOZNIAK, D. J., WYCKOFF, T. J., STARKEY, M., KEYSER, R., AZADI, P., O'TOOLE, G. A. & PARSEK, M. R. (2003) Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms. Proc Natl Acad Sci U. S. A, 100, 7907-7912

WRIGHT, A. C., SIMPSON, L. M., OLIVER, J. D. & MORRIS JR, J. G. (1990) Phenotypic evaluation of a capsular transposon mutants of Vibrio vulnificus. Infection and Immunity, 58, 1769-1773.

XIE, C., MACE, J., DINNO, M. A., LI, Y. Q., TANG, W. & NEWTON, R. J. (2005) Identification of single bacterial cells in aqueous solution using conflocal laser tweezers Raman spectroscopy. Analytical Chemistry, 77, 4390-4397.

YILDIZ, F. H. & SCHOOLNIK, G. K. (1999) Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Procedings of the National Aacademy of Sciences USA, 96, 4028-4033.

ZEIRI, L. & EFRIMA, S. (2005) Surface-enhanced Raman spectroscopy of bacteria: the effect of excitation wavelength and chemical modification of the colloidal milieu. Journal of Raman Spectroscopy, 36, 667-675.

____________________________________ ___________ _ Bibliography

______________________________________________________________________ 240

ZUO, R. (2007) Biofilms: strategies for metal corrosion inhibition employing microorganisms. Applied Microbiology and Biotechnology, 76, 1245-53.

bacterial attachment to micro- and nano- structured surfaces - swinburne research … ·...

Documents