workpackage 2.5: potential risks associated with strategies deliverable 2.5.6: data on how bacterial...

WORKPACKAGE 2.5: Potential risks associated with strategies

DELIVERABLE 2.5.6: Data on how bacterial interactions contribute to (i) biofilm formation ability of individual strains, and (ii) their resistance to sanitizers

PILLAR 2: Control and intervention strategies along the fork-to-farm chain to ensure beef safety

Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens

Vienna, 25 - 26 March 2010


PILLAR 2, WP2.5, D2.5.6

Biofilm formation & implications in beef industry In the meat industry, biofilms of both spoilage and pathogenic

bacteria may be related to serious problems of food contamination (lowered shelf-life of products, disease transmission)

In the majority of natural & industrial environments, monospecies biofilms are relatively rare Conversely, microorganisms are associated with surfaces in complex multispecies communities

Bacterial interactions are believed to influence the biofilm forming capacity of individual strains, as well as their antimicrobial resistance


PILLAR 2, WP2.5, D2.5.6

Main objectives / tasks

1. Investigate attachment to and biofilm forming ability on model abiotic surfaces of some food-relevant bacteria in monoculture and in mixed-culture

1. Evaluate disinfection efficiency of some commercial disinfectants against mono & mixed-culture biofilms


PILLAR 2, WP2.5, D2.5.6

39 bacterial strains* screened for biofilm formation These belonged to bacterial species which are typically

found in complex food industrial ecosystems

o Listeria monocytogenes (11 strains)o Salmonella enterica (8 strains)o Staphylococcus aureus (3 strains)

o Pseudomonas sp. (6 species/strains)

o Lactobacillus sakei (11 strains)

representatives of pathogens

representatives of spoilage bacteria

representatives of useful technological bacteria

* All tested strains had been previously identified by 16S rRNA analysis and separated by PFGE

P. fluorescens, P. fragi, P. aeruginosa, P. phsychrophilla, P. gessardii, Pseudomonas sp.


PILLAR 2, WP2.5, D2.5.6

Isolation origin of strains

All strains were provided by the microorganisms collection of Laboratory of Microbiology and Biotechnology of Foods* (Department of Food Science and Technology, AUA) & had been previously isolated from different sources

FOODS (51.3%)UNKNOWN (28.2%)

HUMANS (7.7%) FOOD

INDUSTRY SURFACE(12.8%)

* Code used FMCC_B, Food Microbiology Culture Collection_Bacteria

203

11

5


PILLAR 2, WP2.5, D2.5.6

Surfaces used

Two surfaces of different physicochemical properties were used as abiotic substrates for biofilm development:

1. Polystyrene (PS) - 96-well microplates

2. Stainless steel (SS) - rectangular coupons of 3 x 1 x 0.1 cm, type AISI-304

- material commonly used for the manufacture of food-processing equipment


PILLAR 2, WP2.5, D2.5.6

Biofilm formation assay using PS microplates Commonly applied method for easy screening biofilm formation by different strains (many

repetitions)

Stain biofilm cells with crystal violet, dissolving bound dye by ethanol/acetone & quantification with absorbance measurements (A575nm)

microplate readerPS microplate with stained biofilm cells


PILLAR 2, WP2.5, D2.5.6

Temperature: 15oC

Growth media: TSB and 1/10 dTSB

Initially, bacteria were left to adhere on PS microplates for 3 h (at 15oC). For this bacterial suspension of ca. 108cfu/ml in ¼ Ringer solution was used

Loosely attached cells were then removed by rinsing (with ¼ Ringer)

Growth media were added, followed by incubation under static conditions (except for Pseudomonas sp.) for 48 h

Growth media were renewed at 24 h

Biofilm formation assay using PS microplates


PILLAR 2, WP2.5, D2.5.6

Results: biofilm formation on PS microplates

0

0.5

1

1.5

2

2.5

3

3.5

4

FMCC

B-1

60

FMCC

B-1

25

FMCC

B-1

26

FMCC

B-1

30

FMCC

B-1

65

FMCC

B-1

29

FMCC

B-1

64

FMCC

B-1

24

FMCC

B-1

69

FMCC

B-1

66

FMCC

B-1

27

FMCC

B-1

7

FMCC

B-1

9

FMCC

B-4

2

FMCC

B-5

6

FMCC

B-6

2

FMCC

B-6

7

FMCC

B-1

37

FMCC

B-1

94

FMCC

B-9

5

FMCC

B-1

34

FMCC

B-1

35

FMCC

B-2

9

FMCC

B-3

4

FMCC

B-2

6

FMCC

B-5

5

FMCC

B-4

6

FMCC

B-4

3

FMCC

B-2

26

FMCC

B-2

37

FMCC

B-2

38

FMCC

B-2

27

FMCC

B-2

39

FMCC

B-2

36

FMCC

B-2

30

FMCC

B-2

48

FMCC

B-2

28

FMCC

B-2

25

FMCC

B-2

29

A57

5nm

0

0.5

1

1.5

2

2.5

3

3.5

4

FMCC

B-1

60

FMCC

B-1

25

FMCC

B-1

26

FMCC

B-1

30

FMCC

B-1

65

FMCC

B-1

29

FMCC

B-1

64

FMCC

B-1

24

FMCC

B-1

69

FMCC

B-1

66

FMCC

B-1

27

FMCC

B-1

7

FMCC

B-1

9

FMCC

B-4

2

FMCC

B-5

6

FMCC

B-6

2

FMCC

B-6

7

FMCC

B-1

37

FMCC

B-1

94

FMCC

B-9

5

FMCC

B-1

34

FMCC

B-1

35

FMCC

B-2

9

FMCC

B-3

4

FMCC

B-2

6

FMCC

B-5

5

FMCC

B-4

6

FMCC

B-4

3

FMCC

B-2

26

FMCC

B-2

37

FMCC

B-2

38

FMCC

B-2

27

FMCC

B-2

39

FMCC

B-2

36

FMCC

B-2

30

FMCC

B-2

48

FMCC

B-2

28

FMCC

B-2

25

FMCC

B-2

29

A57

5nm

L. monocytogenes Salm. enterica Staph.

aureus

Pseudomonas sp. Lactobacillus sakeiRic

h gr

owth

med

ium

(TSB)

Nut

rien

t lim

ited

gro

wth

med

ium

(1/1

0 T

SB)


PILLAR 2, WP2.5, D2.5.6

Results: biofilm formation on PS microplates For most L. monocytogenes strains no significant differences were

observed on biofilm formation between the 2 growth media

All S. enterica strains (except FMCC_B-62) formed more biofilm (p < 0.05) when cultured in 1/10 TSB compared to TSB

The 3 Staph. aureus and the 11 L. sakei strains were poor biofilm producers in both nutritional conditions

Pseudomonas fluorescens (FMCC_B-29), Pseudomonas aeruginosa (FMCC_B-26) and Pseudomonas gessardii (FMCC_B-46) formed high amount of biofilm in both growth media. On the contrary, the other 3 Pseudomonas species produced low biofilm


PILLAR 2, WP2.5, D2.5.6

Sterile SS coupons were fully immersed in bacterial suspensions of ca. 108 cfu/ml in ¼ Ringer solution for 3 h at 15oC (ATTACHMENT STEP)

Loosely attached cells were removed by rinsing (with ¼ Ringer)

Coupons were then incubated in TSB at 15oC for 6 days (144 h)

(BIOFILM FORMATION STEP)

Growth medium was renewed every 48 h

Biofilm formation assay using SS coupons

SS coupons in TS broth


PILLAR 2, WP2.5, D2.5.6

Quantification of biofilm formation on SS coupons

Method based on detaching attached biofilm cells by “bead vortexing” followed by quantification by “agar plating”

SS coupon in inoculated growth medium (TSB)

Removal of coupon using forceps

Rinsing with ¼Ringer Vortexing (2’) with glass beads

Agar plating


PILLAR 2, WP2.5, D2.5.6

Results: Attachment to and biofilm formation on SS coupons

0

10

20

30

40

50

60

70

80

FM

CC

B-1

60

FM

CC

B-1

25

FM

CC

B-1

26

FM

CC

B-1

30

FM

CC

B-1

65

FM

CC

B-1

29

FM

CC

B-1

64

FM

CC

B-1

24

FM

CC

B-1

69

FM

CC

B-1

66

FM

CC

B-1

27

FM

CC

B-1

7

FM

CC

B-1

9

FM

CC

B-4

2

FM

CC

B-5

6

FM

CC

B-6

2

FM

CC

B-6

7

FM

CC

B-1

37

FM

CC

B-1

94

FM

CC

B-9

5

FM

CC

B-1

34

FM

CC

B-1

35

FM

CC

B-2

9

FM

CC

B-3

4

FM

CC

B-2

6

FM

CC

B-5

5

FM

CC

B-4

6

FM

CC

B-4

3

FM

CC

B-2

26

FM

CC

B-2

37

FM

CC

B-2

38

FM

CC

B-2

27

FM

CC

B-2

39

FM

CC

B-2

36

FM

CC

B-2

30

FM

CC

B-2

48

FM

CC

B-2

28

FM

CC

B-2

25

FM

CC

B-2

29

ΠΡΟ

ΣΚΟ

ΛΛ

ΗΣ

Η (

%)

0

1

2

3

4

5

6

7

8

9

FM

CC

B-1

60

FM

CC

B-1

25

FM

CC

B-1

26

FM

CC

B-1

30

FM

CC

B-1

65

FM

CC

B-1

29

FM

CC

B-1

64

FM

CC

B-1

24

FM

CC

B-1

69

FM

CC

B-1

66

FM

CC

B-1

27

FM

CC

B-1

7

FM

CC

B-1

9

FM

CC

B-4

2

FM

CC

B-5

6

FM

CC

B-6

2

FM

CC

B-6

7

FM

CC

B-1

37

FM

CC

B-1

94

FM

CC

B-9

5

FM

CC

B-1

34

FM

CC

B-1

35

FM

CC

B-2

9

FM

CC

B-3

4

FM

CC

B-2

6

FM

CC

B-5

5

FM

CC

B-4

6

FM

CC

B-4

3

FM

CC

B-2

26

FM

CC

B-2

37

FM

CC

B-2

38

FM

CC

B-2

27

FM

CC

B-2

39

FM

CC

B-2

36

FM

CC

B-2

30

FM

CC

B-2

48

FM

CC

B-2

28

FM

CC

B-2

25

FM

CC

B-2

29

ΒΙΟ

-ΥΜ

ΕΝ

ΙΟ (

log

cfu/

cm2)

L. monocytogenes Salm. entericaStaph. aureus

Pseudomonas sp. Lactobacillus sakei

BIO

FIL

M (

log

cfu/

cm2)

AT

TA

CH

MEN

T (

%)

The attachment ability of each strain was expressed as the percentage (%) of cells being attached, compared to the total population of cells contained in bacterial suspension in which the SS coupon was immersed (for 3 h)


PILLAR 2, WP2.5, D2.5.6

Results: Attachment to and biofilm formation on SS coupons

Species Attachment (%) Biofilm (log cfu/cm2)

Listeria monocytogenes 47.1 - 63.5 4.71 - 6.27

Salmonella enterica 53.6 - 64.1 4.63 - 5.64

Staphylococcus aureus 69 - 71.6 4.71 - 5.42

Pseudomonas sp. 30.5 - 74.4 4.33 - 7.81

Lactobacillus sakei 26.8 - 61.9 3.59 - 5.65

3

4

5

6

7

8

9

20 30 40 50 60 70 80

ATTACHMENT (%)

BIO

FIL

M (

log

cfu/

cm2)

Variations at levels of attachment and biofilm formation for each species

Relationship between attachment & biofilm forming ability for the 39

bacterial strains


PILLAR 2, WP2.5, D2.5.6

Future work (…to be done the next 6 months) Select 3 strains from each species & test biofilm formation

on SS in monospecies mixed culture

Study multispecies biofilm formation on SS

- L. monocytogenes – S. enterica

- L. monocytogenes – S. aureus

- L. monocytogenes – Pseudomonas sp.

- L. monocytogenes – L. sakei

- L. monocytogenes – S. enterica - S. aureus – Pseudomonas sp. – L. sakei

Test disinfection efficiency of 3 commercial disinfectants (benzalkonium chloride, chlorine, PAA) against mono- and mixed-culture biofilms

DUAL SPECIES


PILLAR 2, WP2.5, D2.5.6

Thank you

Acknowledgments

BSc Student Elli Braxou

workpackage 2.5: potential risks associated with strategies deliverable 2.5.6: data on how bacterial...

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