biofilms 2015: multi-disciplinary approaches shed light into
TRANSCRIPT
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Biofilms 2015: Multi-disciplinary approaches shed light into 1
microbial life on surfaces 2
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Karen L. VISICK*1; Mark A. SCHEMBRI2; Fitnat YILDIZ3 and Jean-Marc GHIGO*4 5 6 1 Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL 7
USA 8 2 Australian Infectious Diseases Research Centre, School of Chemistry and Molecular 9
Biosciences, The University of Queensland, Brisbane, Australia 10 3 Department of Microbiology and Environmental Toxicology, University of California, 11
Santa Cruz, Santa Cruz, CA 95064, USA. 12 4 Institut Pasteur, Unité de Génétique des Biofilms, Département de Microbiologie, 25-28 13
rue �du Dr. Roux F-75015 Paris, France. � 14
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* Corresponding authors 16
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E-mail: [email protected]; [email protected]; 18
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JB Accepted Manuscript Posted Online 14 March 2016J. Bacteriol. doi:10.1128/JB.00156-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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SUMMARY 23
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The 7th ASM Conference on Biofilms was held in Chicago, Illinois, from October 24th to 25
October 29th, 2015. The conference provided an international forum for biofilm 26
researchers across academic and industry platforms, and from different scientific 27
disciplines, to present and discuss new findings and ideas. The meeting covered a wide 28
range of topics, spanning environmental sciences, applied biology, evolution, ecology, 29
physiology and molecular biology of the biofilm lifestyle. This report summarizes the 30
presentations with regard to emerging biofilm-related themes. 31
32
The 7th American Society for Microbiology Conference on Biofilms was held in downtown 33
Chicago, Illinois, Saturday, October 24 – Thursday, October 29, 2015. The meeting covered 34
an exciting range of topics across the scope of biofilm research and comprised 4 keynote 35
lectures and 72 talks in 13 thematically organized sessions. The meeting also included two 36
extensive poster sessions that featured 304 posters. In this review, we attempt to convey the 37
depth and breadth of the topics that were presented during Biofilms 2015, and to provide a 38
synopsis of recent developments and emerging trends in the field. 39
40
Biofilms are matrix-enclosed single or multispecies microbial communities that can form on 41
virtually any surface. Biofilms form in most natural or engineered systems, with both 42
positive and negative impacts. The composition and physical structure of biofilms reflect 43
a multitude of complex interactions that take place at different levels between the biofilm 44
constituents and their environment, thus the study of many intrinsic functions and attributes 45
of biofilms now encompasses multiple research fields. The 2015 ASM Biofilm conference 46
highlighted the need to employ multidisciplinary approaches to advance fundamental and 47
applied research on clinical, industrial and environmental biofilm systems. 48
49
The conference was preceded by three parallel and highly attended hands-on workshops on 50
models and approaches to study, image and quantify biofilms and biofilm infections in vitro 51
and in vivo. For many, these workshops provided an excellent introduction to the four-day 52
conference, which compr i sed a comprehens ive and wide- rang ing scientific 53
program organized by a committee composed of 10 international biofilm experts. Overall, 54
the program was built around 13 thematic sessions, each comprising a series of 25 minute 55
talks given by a mix of invited speakers and speakers selected from abstracts submitted to the 56
scientific committee. 57
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BIOFILM COMMUNITIES IN NATURE 59
60
The conference was launched by the keynote lecture delivered by Dianne Newmann 61
(California Institute of Technology, California, USA), who gave an overview of the complex 62
biological and physico-chemical parameters that define different biofilm lifestyles. Dianne 63
discussed the importance of using environmentally-informed reductionist approaches to 64
study biofilms using examples drawn from her work on bacterial growth and metabolism in 65
the microenvironment of cystic fibrosis sputum. These studies revealed that bacterial growth 66
rates, on average, are far slower than typically studied in the laboratory (1). By performing a 67
proteomic study of Pseudomonas aeruginosa to determine which proteins are actively being 68
made under anaerobic survival conditions, she described the discovery of a small, acidic 69
protein, SutA (survival under transitions), that is post-transcriptionally up-regulated during 70
slow growth. SutA associates with RNA polymerase and regulates the expression of genes 71
required for ribosome biogenesis and others involved in biofilm development, secondary 72
metabolite production, and fitness in fluctuating conditions (2). With this insight 73
underscoring the importance of studying biofilm properties beyond the lab, the first session 74
provided a basis for understanding processes involving the formation of biofilms and the 75
dynamics of different naturally occurring biofilm communities. Matthew Powers (Univ. Of 76
North Carolina, USA) explored the interactions between a mixed consortia of 29 bacteria 77
isolated from roots of the plant Arabidopsis thaliana. His work identified combinations of 78
different species that exhibit both cooperative and competitive interactions. Combined with 79
MALDI-TOF mass spectrometry to generate metabolic profiles of each of these cultures, this 80
approach was highlighted as a method that can be used to better understand chemical 81
signaling between mixed species communities. 82
83
In a thought-provoking presentation, Roman Stocker (ETH Zurich, Switzerland) showed 84
that co-existence of marine Vibrios on the surface of marine particles depends on a trade-off 85
between the opposing phenotypes of adhesion and dispersion in nutrient-variable oceanic 86
environments (Fig. 1). Under high nutrient conditions, population specialization favoring 87
attachment and growth is promoted, while under limiting nutrient conditions, there is a 88
switch to a dispersal mode of growth (3). Deborah Hogan (Dartmouth, Hanover, NH, USA) 89
explored Candida albicans - P. aeruginosa interactions in the context of cystic fibrosis lung 90
infection. Using a powerful machine learning approach 91
(http://msystems.asm.org/content/1/1/e00025-15) to explore large-scale analysis of P. 92
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aeruginosa gene expression patterns, Deborah described how ethanol production by C. 93
albicans stimulates c-di-GMP synthesis and the formation of P. aeruginosa biofilms, which 94
in turn produce phenazines that enhance ethanol production. This positive feedback loop 95
provides insight into why co-infection with both P. aeruginosa and C. albicans is associated 96
with poor outcomes in cystic fibrosis (4). Mark Mandel (Northwestern Univ., Chicago, Il, 97
USA) described how a functional genomics approach led to the identification of novel 98
positive and negative regulators of biofilm development, including chaperone protein DnaJ 99
and the histidine kinase BinK, which are required for robust in vivo colonization of the light 100
organ of Euprymna scolopes squid by Vibrio fischeri bacteria (5). Stephen 101
Lindemann (Pacific Northwest National Laboratory, Richland, WA, USA) concluded this 102
session dedicated to naturally occurring environmental biofilms by presenting a study of 103
nitrogen flux into phototrophic microbial mats, showing that nitrogen limitation is species-104
specific and that the spatial organization and partitioning of carbon between autotrophs and 105
heterotrophs in cyanobacterial biofilms depends upon the form of nitrogen species 106
being assimilated. 107
108
Together, these talks provided a deeper understanding of the roles and activities of 109
organisms within single and multi-species biofilms in the natural environment, as well as 110
approaches to effectively study biofilms despite the complexity of environments in which 111
they are found. 112
113
BACTERIAL ADHESION FACTORS AND THE PLANKTONIC TO BIOFILM 114
LIFESTYLE SWITCH 115
116
Understanding how free-living planktonic bacteria switch to a sessile mode of growth is still 117
a topic of intense scrutiny and a traditional staple of all recent ASM biofilm conferences. 118
Two sessions were dedicated to this topic, largely dominated by the question of how 119
regulation of adhesion factors and signaling networks involving cyclic diguanosine 120
monophosphate (c-di-GMP) and other external signals, contribute to this switch. 121
122
In the first session dedicated to the transition from planktonic to biofilm lifestyle, Clay 123
Fuqua (Indiana Univ., Bloomington, IN, USA) described a novel signaling pathway 124
controlling Agrobacterium tumefaciens biofilm formation involving small metabolites called 125
pterins, the first report of their regulatory activity in bacteria. He showed that pterin 126
production by PruA controls surface colonization through the dual-function diguanylate 127
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cyclase-phosphodiesterase protein DcpA. The resulting c-di-GMP modulation regulates the 128
production of a unipolar polysaccharide (UPP) adhesion, required for A. 129
tumefaciens attachment and biofilm formation (6). 130
131
Daniel Kearns (Indiana Univ., Bloomington, IN, USA) presented data on a new surface 132
contact-dependent cellular differentiation mechanism in Bacillus subtilis, in which flagellar 133
density is controlled by regulatory proteolysis of the master flagellar activator protein SwrA, 134
the master regulator of flagellar biosynthesis, by LonA. It was further shown that LonA-135
mediated degradation of SwrA happens only in the presence of swarming motility inhibitor 136
A (SmiA). Mutants of SwrA that were resistant to proteolysis and caused hyper-swarming 137
were identified; it was speculated that these mutated residues were required for SmiA 138
interaction (7). Gerard Wong (UCLA, Los Angeles, CA, USA) presented his collaboration 139
with George O’Toole with a surprising finding on surface sensing in Pseudomonas 140
aeruginosa PA14. By tracking the first 20 generations of cells on a surface with single cell 141
resolution, they showed that surface sensing is an inherently multi-generational 142
phenomenon: Pseudomonas uses the second messenger cAMP as a kind of accumulated 143
memory to signal across generations, such that mechano-sensing of the surface in one 144
generation of cells can lead to flagellum shutdown in cells many generations later (8). 145
146
A new aspect of control for the transition between planktonic and biofilm behaviors was 147
presented by Benoît-Joseph Laventie (Biozentrum, Basel, SWITZERLAND), who 148
described a new c-di-GMP effector in P. aeruginosa, identified using capture-compound 149
mass spectrometry (9). This effector, FimA, mediates pilus-mediated attachment and biofilm 150
formation. He showed that in response to surface, FimA rapidly localizes to the new cell pole 151
in a cdG dependent manner to facilitate T4P assembly and function. 152
Aretha Fiebig (Univ. of Chicago, Chicago, IL, USA) described how fine-tuning of the 153
Caulobacter crescentus lifestyle depends on the HfiA protein inhibitor, which targets a 154
conserved glycolipid glycosyltransferase required for holdfast synthesis. HfiA is regulated 155
by a complex pathway involving kinases that act on the response regulator LirA, possibly in 156
combination with nutritional sensing in different environments (10). Another mechanism 157
involved in the fine tuning of planktonic and biofilm lifestyles was presented by 158
Alain Filloux (Imperial College London, London, UNITED KINGDOM). The HptB pathway 159
is part of the P. aeruginosa GacA/Rsm lifestyle switch that controls biofilm growth. Here it 160
was shown that the HptB control of biofilm formation and motility can be rewired into an 161
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original c-di-GMP-dependent network involving a newly identified diguanylate cyclase and 162
effector protein. 163
164
In her talk, Sonja Albers (Univ. of Freiburg, Freiburg, GERMANY) presented how she used 165
genetic, proteomic and transcriptomic approaches to study regulation of biofilm formation in 166
Archaea. She identified an archaea-specific group of regulators, the Lrs14 regulators, which 167
are involved in major cell fate decisions (11). In the crenarchaeon Sulfolobus acidocaldarius 168
the Lrs14 regulator AbfR1 antagonistically coordinates motility and EPS production during 169
biofilm development. Future work will focus on understanding the entire regulatory network 170
involved in biofilm formation in S. acidocaldarius. Christopher Jones (Univ. of California 171
Santa Cruz, Santa Cruz, CA, USA) described identification of a new c-di-GMP receptor, 172
MshE, in Vibrio cholerae. MshE is a polymerizing ATPase is required for biosynthesis of 173
MshA pili which is essential for transition from the motile to sessile lifestyle. MshE c-di-174
GMP binding activity is dependent on the MshE N-terminal domain and c-di-GMP affects 175
MshA pilus assembly and function through direct interactions with the MshE (12). 176
Maria Hadjifranjiskou (Vanderbilt Univ. School of Medicine, Nashville, TN, USA) 177
described the application of MALDI-TOF imaging mass spectrometry (IMS) to study 178
uropathogenic E. coli biofilms. Maria discussed how oxygen concentration influences the 179
expression of type 1 fimbriae. She showed that the phase-variable fim promoter favored the 180
‘on’ orientation in the presence of oxygen, while the ‘off’ orientation was favored in low 181
oxygen conditions. This illustrates how sensing natural oxygen gradients within biofilms 182
shapes localization of adhesive factors and contributes to the stratification of extracellular 183
matrix components within the biofilm (13). Using atomic force microscopy (AFM), Cécile 184
Formosa-Dague (Catholic Univ. of Louvain, Louvain- la-Neuve, BELGIUM) explored the 185
relationship between nanomechanics and adhesion and presented how multiparametric 186
imaging combined with single-cell force spectroscopy can unravel the zinc-dependent 187
adhesive and mechanical properties of the SasG adhesion protein from Staphylococcus 188
aureus. Cécile showed that zinc plays a dual role in S. aureus SasG-mediated biofilm 189
formation: it alters the surface properties of the cell to enable the projection of adhesive 190
SasG fibrils beyond other surface components that can in turn mediate specific cell-cell 191
adhesion through the formation of Zn2+-dependent homophilic bonds between β-sheet-rich 192
SasG multi-domains on neighboring cells (14). Finally, Inigo Lasa (Public Univ. of Navarra, 193
Pamplona, SPAIN) described the identification and characterization of a short amyloid stretch 194
in the S. aureus Bap protein. When processed and released, Bap beta-amyloid domains self-195
assemble into amyloid fibrils and induce bacterial aggregation in response to a decrease in 196
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pH during stationary phase growth. This amyloid behavior is inhibited in presence of 197
calcium, which is thought to induce compaction, limiting access to proteases that modulate 198
its activity (15). 199
200
Taken together, the speakers in this session presented an impressively diverse array of 201
approaches to explore the complex factors and signals involved in surface sensing and early 202
attachment events. 203
204
ASSEMBLY AND MODULATION OF THE BIOFILM MATRIX 205
206
The biofilm matrix is the glue that holds the cells together. Diverse organisms have evolved 207
a wealth of different strategies for adhering to each other and to surfaces, including the 208
production of amyloid fibers, protein adhesins and polysaccharides, as well as mechanisms 209
for modulating biofilm matrix production or interactions in response to environmental 210
signals such as oxygen or calcium. 211
212
For example, Matt Parsek (Univ.of Washington,Seattle, WA, USA) presented new data on 213
the composition of the PEL polysaccharide, a component of the P. aeruginosa biofilm 214
matrix. Matt showed that PEL matrix polymer is a cationic exopolysaccharide rich in N-215
acetylgalactosamine and N-acetylglucosamine. PEL interacts with extracellular DNA via 216
ionic interactions and could provide a rigid, yet extensible EPS shell that can accommodate 217
biofilm growth, like the envelope of an inflating balloon (16). This contribution of the 218
extracellular scaffold to the properties of biofilms was also illustrated by Nicola Stanley-219
Wall (Univ. of Dundee, Dundee, UNITED KINGDOM). Nicola described how BslA, a 220
surface-active amphiphilic extracellular protein that self-assembles and changes shape upon 221
interaction with an interface, forms a water-resistant hydrophobic coat around the Bacillus 222
subtilis biofilm and contributes to shielding of the bacterial community by fine-tuning its 223
solvent and interfacial interactions (Fig. 2) (17). 224
225
Daniel Wozniak (Ohio State Univ., Columbus, OH, USA) discussed the respective contribution 226
of P. aeruginosa exopolysaccharides involved in the mucoid (alginate) or small colony 227
variant (SCV) morphotypes (PEL, PSL) to biofilm biology. Dan presented evidence for SCV 228
fitness advantage via increased tolerance to antimicrobial and host defenses due to c-di-229
GMP-dependent aggregation, which then prevents uptake by phagocytic cells and 230
persistence in a porcine chronic wound infection model. 231
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232
Alexandra Paharik (Univ. of Iowa, Iowa City, IA, USA) discussed how, even in strains of 233
Staphylococcus epidermidis unable to produce surface polysaccharides, secreted 234
metalloprotease SepA promotes biofilm via proteolytic processing of a cell wall-anchored 235
adhesin called Aap (accumulation-associated protein). Jin Hwan Park (Seoul National 236
Univ., Seoul, KOREA) reported the characterization of the cabABC operon essential for 237
biofilm development in Vibrio vulnificus. CabA is a calcium-binding protein that is induced 238
by elevated levels of c-di-GMP and secreted in a CabBC-dependent manner. CabA is 239
localized to the biofilm matrix, multimerizes in presence of calcium and contributes to the V. 240
vulnificus robust biofilm structure and rugose colony phenotype (18). 241
242
Boo Shan Tseng (Univ. of Washington, Seattle, WA, USA - currently at UNLV, Las Vegas, 243
NV, USA) further illustrated that the biofilm matrix can be much more than a structural 244
scaffold. Boo used a proteomic approach to investigate the role of biofilm matrix proteins 245
and showed that ecotin, a serine protease inhibitor, is selectively maintained by PSL in the P. 246
aeruginosa biofilm matrix. Ecotin could protect biofilms from proteolytic attack, potentially 247
inhibiting neutrophil elastase, an enzyme produced by the host immune system during 248
respiratory infections. 249
250
Fungal biofilms are linked to many human infections were also represented in the meeting. 251
The adherence of organisms such as Candida albicans to implanted medical devices results 252
in biofilms that withstand extraordinarily high antifungal concentrations. Thus, there is a 253
strong need to understand the physiology and molecular dynamics of fungal biofilms. Aaron 254
Mitchell (Carnegie Mellon Univ., Pittsburgh, PA. USA) introduced us to fungal biofilms 255
formed by C. albicans. He described how Candida biofilm formation can result from either 256
the positive regulation of GPI-linked ALS1, ALS3 and HWP1 (through regulators such as 257
Bcr1), or from the inhibition of yeast formation via a negative regulatory cascade that leads 258
to filamentation and transition to the hyphal stage (19). David Andes (Univ. of Wisconsin-259
Madison, Madison, WI, USA) described how Candida adherence to implanted medical 260
devices can withstand extraordinarily high antifungal concentrations. He showed that the 261
Candida biofilm matrix contains mannan-glucan structures that are distinct from Candida 262
cell-wall glucans and can sequester antifungal drugs, therefore contributing to multidrug 263
resistance (19). 264
265
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In his keynote address, Yves Brun (Indiana Univ., Bloomington, IN, USA) brought together 266
the topics addressed by the 4 first sessions by discussing mechanisms of bacterial surface 267
attachment at the single cell level (Fig. 3). Drawing from his work on Caulobacter 268
crescentus and other α-proteobacteria producing holdfast adhesin structures, Yves discussed 269
the notion of surface sensing, the transition between reversible and irreversible attachment, 270
and the mechanical forces involved in holdfast-surface interaction and in anchoring the 271
holdfast to the cell envelope. These results shed new light on holdfast biophysics and 272
regulation, and revisited the role of flagella in surface mechano-sensing, while also 273
providing inspiration for the development of new bio-inspired materials with different 274
adhesion properties (20). 275
276
NEW INSIGHTS INTO ANTIMICROBIAL TOLERANCE AND NOVEL TARGETS 277
AND STRATEGIES TO FIGHT BIOFILM INFECTIONS 278
279
The remarkable resistance properties of biofilms to antimicrobials and host defenses are 280
well-known, and are likely one factor contributing to the current crisis in the availability of 281
effective antibiotics in the clinic. Understanding the mechanisms that permit biofilm cells to 282
resist or tolerate antibiotics and natural host defenses, as well as the corresponding response 283
of hosts to biofilm infections, are therefore important areas of investigation. Not surprisingly, 284
the development of novel treatments for infections, based on the properties of biofilms, was 285
a prominent theme of the conference. 286
287
Recently, it has become apparent that the ability of a subset of cells to become antibiotic-288
tolerant “persisters” is a key factor affecting antibiotic effectiveness, and thus an active area 289
of research lies in understanding the mechanisms involved in the production of persisters. 290
Kim Lewis (Northeastern Univ., Boston, MA, USA) covered a decade of work on persister 291
biology and biofilm eradication, including a discussion on how a decrease in the level of ATP 292
- but not toxin-antitoxin (TA) systems - leads to persistence in S. aureus. Kim then presented 293
his recent work on the clinical significance of persister enrichment in clinical isolates and the 294
mechanistic basis of heritable, clinically relevant antibiotic tolerance (21). He concluded by 295
presenting his recent work on the discovery of the new antibiotic Teixobactin, a cell wall 296
synthesis inhibitor with bactericidal activity against multiple pathogens including S. aureus 297
and Mycobacterium tuberculosis. Continuing the theme of antibiotic tolerance, Olga Petrova 298
(Binghamton Univ., Binghamton, NY, USA) presented results suggesting that P. 299
aeruginosa biofilm formation and biofilm tolerance to antibiotics are regulated by distinct 300
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signaling cascades, both involving the transcriptional regulator FleQ and the sensor-regulator 301
hybrid SagS, but with discrete c-di-GMP requirements and protein interaction partners. 302
Separating the factors involved in biofilm formation and biofilm tolerance will allow biofilm 303
control strategies by targeting of the two distinct pathways (22). 304
305
Luanne Hall-Stoodley (The Ohio State Univ. College of Medicine, Columbus, OH, USA) 306
introduced us to Streptococcus pneumoniae biofilms that colonize adenoid tissues and 307
develop biofilms on middle ear mucosal epithelia contributing to the severity of respiratory 308
infections and chronic otitis. Luanne showed that low doses of nitric oxide affects 309
metabolism and decreases antibiotic tolerance. This suggests that adjunctive treatment with 310
low doses of NO, which do not trigger biofilm dispersal, could reduce antibiotic tolerance in 311
pneumococcal biofilms and improve antibiotic efficacy (23). Another treatment strategy was 312
championed by Bob Hancock (Univ. of British Columbia, Vancouver, BC, CANADA), who 313
made a strong case for the use of broad-spectrum cationic antibiofilm peptides against 314
biofilm infections. Bob described the characteristics of lead peptides optimized on 315
exploratory robotized platforms that target the intracellular stringent response signal ppGpp 316
in biofilms. These peptides show synergy with existing antibiotics, work in animal models 317
and represent promising alternatives to combat resistant biofilm infections (24). 318
Lori Burrows (McMasterUniv., Hamilton, ON, CANADA) gave a thought-provoking 319
presentation that described a new approach to identify novel antibiotic activities. Lori 320
reported the identification of several molecules (including Thiostrepton) that stimulate P. 321
aeruginosa biofilm formation at sub-inhibitory concentrations. She used the biofilm inducer 322
phenotype, which likely induce defense responses to sub-lethal damage, to screen for new 323
antibiotics and new targets in complex Streptomyces extracts (25). 324
325
Suzanne Walker (Harvard Medical School, Boston, MA, USA) described a synthetic lethal 326
approach to map interactions between cell envelope pathways in S. aureus and then used a 327
synthetic lethal chemical screen to identify inhibitors of proteins in an interaction network. 328
Network mapping involved screening a transposon mutant library in presence of a molecule 329
that specifically targets a step involved in envelope biosynthesis and using Tn-Seq to identify 330
the genes that become essential when this step is inhibited. The synthetic lethal chemical 331
screen led to an inhibitor of DltB, involved in teichoic acid D-alanylation. This approach, 332
which can be generalized to other bacteria, can be used to identify new small molecules that 333
could serve as novel drugs or as probes to elucidate biological functions (26). In a 334
continuation of this theme, Hans Steenackers (KU Leuven Leuven, BELGIUM) discussed 335
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the use of approaches based on compound screening and synthetic chemistry to 336
identify biofilm inhibitors with broad spectrum activity. He elaborated on the molecular 337
mode of action of 2-aminoimidazole-based biofilm inhibitors, their low potential for 338
resistance development and their application in anti-biofilm coatings for orthopedic implants 339
(27). 340
341
Targeting enzymes involved production and degradation of exopolysaccharides involved in 342
matrix production may lead to new biofilm control strategies. Jennifer L. Dale (Univ. of 343
Minnesota, Minneapolis, MN, USA) showed that the Enterococcus faecalis 344
glycosyltransferases (GTFs) EpaI and EpaOX are involved in synthesis of a cell wall-345
associated Epa polysaccharide. A defect in GTFs, and associated Epa synthesis, negatively 346
impacts biofilm formation and leads to decreased structural integrity and susceptibility to 347
antibiotics and bile salts, suggesting that GTFs could be new targets for antimicrobial 348
design. Lynne Howell (Hospital for Sick Children/Univ. of Toronto, Toronto, ON, CANADA) 349
presented evidence that combinations of glycosyl hydrolases can degrade matrix 350
polysaccharides such as P. aeruginosa PEL, PSL or fungal galactosaminogalactan. These 351
enzymes could be used for prevention or disruption of biofilms in the treatment of chronic 352
microbial infections (28). Nicholas Jakubovics (Newcastle Univ., Newcastle upon Tyne, 353
UNITED KINGDOM) presented work demonstrating the effect of L-arginine on 354
intermicrobial interactions driving structure and stratification, including co-aggregation and 355
interspecies signaling, in dental biofilms. Interfering with these mechanisms could represent 356
a novel approach to control oral Streptococcal biofilms (29). John Gunn (The Ohio State 357
Univ., Columbus, OH, USA) showed how Salmonella Typhi biofilm formation on gallstones 358
enhances gallbladder carriage. This was demonstrated in a human study and new mouse 359
model of carriage. In vitro and in vivo studies also demonstrate involvement of the 360
gallbladder epithelium in carriage. Potential antigenic targets present in gallstone biofilms 361
or the use of biofilm-inhibiting compounds may provide potential new avenues for 362
therapeutic intervention against Salmonella biofilm formation and chronic gallbladder 363
infection (30). Finally, using an HPLC-HRAM MS untargeted lipidomic based approach, 364
Skander Hathroubi (Université de Montréal, St-Hyacinthe, QC, CANADA.) discussed how 365
planktonic and biofilm Actinobacillus pleuropneumoniae cells differed significantly in lipid 366
A structure and quantity, with larger lipid A molecular entities observed in biofilm cells. 367
This would explain at least in part the weaker ability observed in A. pleuropneumoniae 368
biofilm cells to stimulate porcine alveolar macrophages (PAMs) (31). 369
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370
Targeting therapeutics to bacterial amyloids was another important topic that was covered. 371
Fredrik Almqvist (Umea Univ., Umea, SWEDEN) discussed non-biocidal approaches targeting 372
pili fibers and curli amyloids involved in biofilm formation. Fred described how the 373
discovery and improvement of anti-ß-amyloid lead compounds could not only inhibit 374
bacterial biofilm formation, but may also inform studies on human amyloid-related diseases 375
(32). Cagla Tukel (Temple Univ., Philadelphia, PA, USA), showed surprising results suggesting 376
that amyloid-containing biofilms trigger autoimmunity. At least 40% of bacterial species 377
produce amyloid-like proteins that share a quaternary structure, as well as physical and 378
immunological properties, with human amyloids associated with complex diseases such as 379
Alzheimer’s disease, Prion Diseases and Type II diabetes. Hence, bacterial amyloids present 380
in biofilm extracellular matrix, where they can bind eDNA, could induce inflammation and 381
production anti-dsDNA as well as anti-chromatin antibodies, potentially contributing to the 382
progression of autoimmunity (33). In his work, Steven Goodman (Nationwide Children’s 383
Hospital, Columbus, OH, USA) has found that targeting the DNABII family of proteins 384
required for the maintenance eDNA structure can prevent biofilm formation by non-typeable 385
Haemophilus influenzae, P. aeruginosa, S. aureus and Burkholderia cenocepacia. Steve 386
proposed that eDNA-DNABII could be essential for EPS integrity in many bacteria and 387
represent a universal target for biofilm prevention. 388
389
EMERGING TECHNOLOGIES AND BIOFILM APPLICATIONS 390
391
One of the highlights of the biofilm meeting was the impressive application of new and high-392
powered technologies to study biofilms and increase our understanding of different aspects 393
of biofilm biology, including the role and/or identity of small molecules such as oxygen, 394
phenazines and peptides, the spatial composition of the matrix, and intercellular 395
communication. Lars Dietrich (Columbia Univ., New York, NY, USA) showed that the wrinkly 396
colony phenotype of P. aeruginosa PA14 correlates with oxygen limitation, regulation of 397
matrix production by PAS-domain-containing proteins, and defects in the synthesis of 398
endogenous redox-active antibiotics called phenazines. Measurements of cellular 399
NADH/NAD+ ratios in biofilms support redox-driven regulation of microbial community 400
morphogenesis. His group's results implicate specific P. aeruginosa terminal oxidase 401
complexes in biofilm physiology. Lars described his group's work developing miniaturized 402
redox sensor chips that can be used to map metabolite release by colony biofilms. Mapping 403
of phenazines released from intact colonies revealed unexpected influences of biofilm 404
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position on phenazine production (34). Elizabeth Shank (Univ. Of North Carolina, Chapel 405
Hill, NC, USA) described her research using imaging mass spectrometry (IMS) 406
and fluorescent reporter strains to identify metabolites from complex soil communities 407
that stimulate and repress biofilm formation in Bacillus subtilis. In particular, Elizabeth 408
described how thiocillin (35) and 2,4-diacetylphloroglucinol (36) can impact matrix-409
producing B. subtilis populations and therefore modulate different microbial cellular 410
phenotypes. Nydia Morales-Soto (Univ. of Notre Dame, Notre Dame, IN, USA) used confocal 411
Raman microscopy and secondary ion mass spectrometry imaging to fingerprint quinoline 412
quorum-sensing molecules during swarming motility and biofilm formation. This approach 413
provided a spatio-temporal map of quorum-sensing-based bacterial communication, 414
revealing the importance of quorum sensing signals in the early stages of P. aeruginosa 415
biofilm development (37). Vanessa Phelan (Univ. of California, San Diego, La Jolla, CA, USA) 416
discussed how to identify chemical communication signals in complex microbial 417
communities. Vanessa showed how studying bacterial association in S. aureus and E. coli co-418
cultures revealed new competition phenotypes against P. aeruginosa. A number of lead 419
compounds potentially involved in this growth inhibition were analyzed by tandem mass 420
spectrometry and the Global Natural Product Social Molecular Networking (GNPS) 421
platform, a collaborative crowd sourced knowledge base and analysis platform. 422
423
Lynette Cegelski (Stanford Univ., Stanford, CA, USA) presented a quantitative approach to to 424
define the molecular composition of bacterial biofilms using solid state NMR in two 425
different model systems E. coli and V. cholerae revealing power of this approach in 426
determining differences in matrix constituents (38). Florian Blauert (Karlsruher Institute of 427
Technology (KIT), Karlsruhe, GERMANY) discussed how non-invasive optical coherence 428
tomography (OCT) allows fast, quantitative and in situ three-dimensional analysis of biofilm 429
deformation, and how to access material properties of biofilms using OCT (39). 430
431
BIOFILMS IN ENGINEERED SYSTEMS 432
In engineered systems, biofilm formation could be beneficial, resulting in optimal 433
functioning engineered bioreactors and bioremediation of toxic compounds, or detrimental, 434
causing biofouling and biocorrosion. Bruce Logan (Pennsylvania State Univ., Univ. Park, 435
PA, USA) talked about hydrogen and biocatalyzed methane production from the cathode in 436
biofilm-based bio-electrochemical systems. Bruce discussed the functional consequence of 437
inactive or dead cells accumulating over time in anode Geobacter anodireducens biofilms. 438
This accumulation results in a two-layer structure with a live outer-layer responsible for 439
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current generation, covering an inactive inner-core layer that functions as an electrically 440
conductive matrix (40). Howard Stone (Princeton Univ., Princeton, NJ, USA) showed how 441
particular flow and surface structure influence bacterial biofilm dynamics. In one vignette, 442
Howard documented how the interplay of flow and twitching motility of P. aeruginosa lead 443
to upstream migration of the bacteria, which has consequences for how the bacteria spread in 444
flow networks. Second, using experiments performed in a microfluidic device, Howard 445
showed that S. aureus and P. aeruginosa form flow-induced, filamentous 3D biofilm 446
streamers that, over time, bridge the spaces between obstacles. Interestingly, while the 447
presence of surface-attached biofilms exerts a limited impact on flow rates, streamers cause 448
rapid clogging. This suggests that the formation of biofilm streamers, rather that surface 449
biofilms, may be the primary cause of flow reduction in environmental, industrial and 450
medical systems. In a final vignette, Howard indicated ways in which flow interacts with 451
quorum sensing (QS) to produce space and time dependence of the QS response (41). Allon 452
Hochbaum (Univ. of California, Irvine, Irvine, CA, USA) presented approaches to engineer 453
structure-property relationships in E. coli and P. aeruginosa co-culture biofilms through the 454
systematic variation of micro-fabricated growth substrate topography. Allon showed that 455
periodically patterned microstructures of growth substrate induce morphological changes in 456
E. coli biofilms and a differential accumulation of indole, thus altering competition dynamics 457
between E. coli and P. aeruginosa. An application of this technology was presented by 458
Ethan Mann (Sharklet Technologies, Inc, Aurora,CO, USA), who discussed how surface 459
characteristics impact biological responses and presented the Sharklet microtopography, a 460
non-biocidal anti-biofilm surface for medical devices (42). Kuang He (ExxonMobil, 461
Annandale, NJ, USA) presented a study on the role of indole signaling in anaerobic biofilm 462
formation using a model sulfate reducing bacterium Desulfovibrio vulgaris (23). Danielle 463
France (NIST, Boulder, CO, USA) presented data on the anticorrosive influence of Acetobacter 464
aceti biofilms on carbon steel surfaces, suggesting that corrosion inhibition by an acid-465
producing bacterium could be used as an inexpensive solution to industrial problems. Caitlin 466
Howell (Harvard Univ., Cambridge, MA, USA, currently at Univ. Of Maine, Orono, ME, 467
USA) presented an overview on the use of immobilized liquid layers as a non-toxic method 468
of controlling biomolecular and microbial attachment on a wide variety of different 469
substrates. Caitlin discussed numerous potential applications of the technology, inspired by 470
the slippery surface of the carnivorous pitcher plant, including the prevention of bacterial 471
biofilm adhesion to catheters, the mitigation of stable algal biofilm formation on glass 472
substrates, and the reduction of thrombosis in vivo (43). 473
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These presentations collectively showed that better understanding of biofilm formation in 474
industrial settings will allow improved utilization and control of biofilms. 475
476
477
EVOLUTION IN BIOFILMS AND THE IMPACT OF THE ENVIRONMENT ON 478
BACTERIAL LIFESTYLES 479
480
Jintao Liu (Univ. of California San Diego, La Jolla, CA, USA) discussed cooperation and 481
competition in B. subtilis biofilms: cells at the biofilm periphery protect cells at the biofilm 482
interior from external attack but also starve them through nutrient consumption. Jintao et al. 483
showed that this conflict was resolved by the emergence of long-range metabolic co-484
dependence between the two groups of cells. Consequently, the biofilm periphery halted 485
growth periodically, increasing availability of nutrients to the sheltered interior cells and 486
promoting the resilience of biofilms against external attack (44). From the same laboratory, 487
on a related topic, Gurol Suel (Univ. of California San Diego, San Diego, CA, USA) reported 488
that bacterial potassium ion channels conduct long-range electrical signals within bacterial 489
biofilm communities via spatially propagated waves of depolarization. This coordinates 490
metabolic states among cells in the interior and periphery of the biofilm. The report of a 491
community function for potassium ion channels demonstrates the existence of long-range 492
electrical signaling in biofilms (45). Vaughn Cooper (Univ. of Pittsburgh School of 493
Medicine, Pittsburgh, PA, USA) shed some light on the evolution of wrinkly small colony 494
variants during Burkholderia cenocepacia chronic infection. By following the evolution of 495
wrinkly colonies from a smooth B. cenocepacia ancestor, a phenomenon associated with 496
biofilm infections, Vaughn showed that selection favored mutations clustered in the 497
wsp operon (46). Despite phenotypic differences among wrinkly mutants, they shared similar 498
fitness properties in mixed biofilms and acted as early surface colonists, suggesting strong 499
selective forces drive the colonization of this common niche (47). Daniel López (Institute for 500
Molecular Infectious Biology, Wuerzburg, GERMANY) described the evolution of antibiotic 501
resistance in Staphylococcus biofilms using intraclonal competition. In the presence of 502
magnesium, the parental strain gave rise sequentially to two physiologically distinct sub-503
populations, a non-pigmented, quorum-sensing overproducer that overproduces the antibiotic 504
Bacteriocin, represses biofilms, but spreads better due to increased levels of surfactants, and 505
a pigmented strain with increased resistance to vancomycin due to a thicker cell wall. 506
Evolution of the strains in vivo also occurred in organs with higher magnesium levels, and 507
resulted in increased virulence (48). 508
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Joe Harrison (Univ. of Calgary, Calgary, AB, CANADA) described the identification of a 509
potentially widespread transposon that encodes a thermosensing diguanylate cyclase, TdcA, 510
which confers thermal control of P. aeruginosa biofilm formation by mediating temperature-511
dependent changes by the production of c-di-GMP at higher temperatures (37 degrees) but 512
not at lower temperatures (25 degrees). TdcA is conserved in other organisms, indicating that 513
temperature-controlled production of c-di-GMP may represent an evolutionary advantage. 514
Ákos Kovács (Univ. of Jena, Jena, GERMANY) discussed how B. subtilis populations 515
producing costly matrix components as common goods can avoid being out-competed by 516
cheaters in spatially structured environments of colony biofilms (49). Interestingly, cheaters 517
are also excluded from pellicles at the air-liquid interface, but they regain their biofilm 518
incorporation ability after prolonged repeated co-cultivation in the presence of the producer 519
population. This illustrates how general adaption to certain growth conditions can benefit a 520
cheater population at the expense of the cooperator population rather than by specific 521
adjustment. Kasper Kragh (Univ. of Copenhagen, Copenhagen, DENMARK) showed that 522
the relative fitness of aggregates depends markedly on the density of surrounding single 523
cells. When competition between aggregates and single cells is low, the aggregate is at a 524
growth disadvantage because of reduced nutrient availability in the aggregate interior. 525
However, when there are many single cells on the surface, and competition is high, 526
extending vertically above the surface gives the top of the aggregate a better access to 527
nutrients. These findings suggest that aggregates and their interaction with single cells may 528
play a previously unrecognized role during biofilm initiation and development. 529
530
George O’Toole (Geisel School of Medicine at Dartmouth, Hanover, NH, USA) delivered 531
the third keynote lecture. George discussed the topic of diguanylate cyclases, and in 532
particular why so many diguanylate cyclases are required for P. fluorescens biofilm 533
formation. He made a compelling case that the investment of a cell to switch to a biofilm 534
lifestyle requires the existence of multistep regulation checkpoints to control the early events 535
associated with surface interaction. Work by Kurt Dahlstrom, a graduate student in the lab, 536
showed one mechanism of control for c-di-GMP signaling is via protein-protein interactions 537
(50). Current studies, using Biolog to explore 192 different conditions for each of 53 538
different c-di-GMP-related mutants, together with bacterial two hybrid assays, are beginning 539
to explore the broader c-di-GMP network in this microbe. 540
541
542
543
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SOCIAL AND ASOCIAL INTERACTIONS IN BIOFILMS 544
545
The 2015 Biofilm Conference presentations also reflected an increasing focus on multi-546
species biofilms and microbe-microbe interactions. Numerous studies were directed at 547
understanding the competition and cooperation that occurs between organisms of the same or 548
different species in the context of a shared environment. 549
550
Joseph Mougous (Univ. of Washington, Seattle, WA, USA) described a new cytoplasmic Type 551
VI secretion effector, Tse6, which slows growth of target cells by degrading the universally 552
essential dinucleotides NAD(+) and NADP(+). Entry of Tse6 into target cells requires its 553
binding to an essential housekeeping protein, the translation elongation factor Tu (EF-Tu). 554
Understanding these bacterial cell-cell interactions will provide insights into interactions that 555
may be occurring within biofilms (51). Peggy Cotter (Univ. of North Carolina-Chapel Hill, 556
Chapel Hill, NC, USA) discussed the mechanism of interbacterial signal transduction 557
mediated by contact-dependent growth inhibition (CDI) system proteins. Peggy described 558
how the Burkholderia thailandensis BcpA protein not only inhibits the growth of ‘non-self’ 559
bacteria by mediating CDI (interbacterial killing) but also contributes to community 560
behaviors in ‘self’ bacteria (those producing the same BcpAIOB proteins) by inducing 561
changes in the expression of genes required for the production of pili, EPS and biofilm 562
formation (52). These results suggest that CDI system proteins control both cooperative and 563
competitive behaviors to build microbial communities composed of only closely-related 564
bacteria (53). 565
566
Marvin Whiteley (Univ. of Texas, Austin, TX, USA) discussed the importance of spatial 567
organization and the use of methods to reproduce the structural biofilm integrity in laboratory 568
settings. Using S. aureus - P. aeruginosa and Aggregatibacter actinomycetemcomitans - 569
Streptococcus gordonii interactions as examples, Marvin showed that there is an optimal 570
distance at which cells can grow adjacent to each other in an infection site (54). This underlines 571
the importance of spatial positioning in polymicrobial infections and suggests that targeting 572
biogeography and spatial location could constitute a valid therapeutic strategy (Fig. 4) (55). 573
John Kirby (Univ. of Iowa, Iowa City, IA, USA) similarly discussed spatial aspects of bacteria-574
bacteria interactions in his work investigating predator-prey dynamics within a biofilm. John 575
described how unknown metabolites including myxoprincomide produced by Myxococcus 576
xanthus induce the formation of novel megastructures by B. subtilis that are raised above the 577
surface and filled with viable endospores embedded within a dense matrix. Genetically 578
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distinguishable from colony biofilm formation, megastructures provide a mechanism for 579
survival of B. subtilis against predation, permitting them to escape into dormancy via 580
sporulation. Bacillus subtilis also produces the metabolite bacillaene to fend off predatory M. 581
xanthus. Antibiotic production at the interface between layers of M. xanthus and B. subtilis 582
spores may result in a “stand-off” between the two organisms (56). 583
584
Interactions between more than two partners were also explored. Mette Burmølle (Univ. of 585
Copenhagen, Copenhagen, DENMARK) presented her work on interactions within a mixed 586
biofilm comprising four bacterial soil isolates. Mette observed that all four strains benefitted 587
from joining the multispecies biofilm, strongly indicative of cooperative forces that shape 588
multispecies biofilm communities (57). Staffan Kjelleberg (SCELSE, Nanyang 589
Technological Univ., SINGAPORE and Centre for Marine BioInnovation, Univ. of New 590
South Wales, AUSTRALIA) discussed how defined, simple multispecies biofilms as well as 591
highly species-rich wastewater biofilm granules can be experimentally designed to explore 592
biofilm community traits and ecological theories (58). Staffan described how increased 593
species richness replaces intraspecific variants to offer community rather than population 594
stress protection, and how quorum sensing signaling is a true community trait with signal 595
production and quenching assigned to phylogenetically different organisms. These 596
approaches suggest that complex microbial biofilms can be designed to understand biofilm 597
biology also at the community level, reflecting natural biofilm systems (59). 598
599
The host environment provides additional factors that may influence microbial interactions. 600
Catherine R. Armbruster (Univ. Of Washington, Seattle, WA, USA) showed that the S. 601
aureus extracellular virulence factor SpA plays a previously undescribed role in 602
polymicrobial interactions within biofilms. SpA influences the course of P. 603
aeruginosa infection by binding type IV pili and the exopolysaccharide Psl, leading to 604
inhibition of not only biofilm formation but also phagocytosis by neutrophils. Because S. 605
aureus frequently precedes P. aeruginosa in chronic infections, Catherine proposed that SpA 606
can impact P. aeruginosa persistence and host interactions during co-infection (60). These 607
results provide an indication of the complex and potentially unexpected interactions that 608
occur in polymicrobial infections via secreted extracellular virulence factors with multiple 609
functions. Katharina Ribbeck (MIT, Cambridge, MA, USA) discussed the role of mucins, gel-610
forming components secreted by goblet cells, in interactions between different microbes. 611
Katharina described how mucin can reduce bacterial adhesion by blocking attachment, 612
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promoting dispersion or affecting inter-species communication and suppression of virulence 613
factors (61). 614
615
CONCLUSIONS 616
617
The biofilm field is rapidly growing and the 7th ASM Biofilm conference provided a 618
platform for researchers from different scientific disciplines to discuss and exchange ideas in 619
all aspects of biofilm research, including fundamentals of biofilm formation and biofilm 620
control, and encompassing biofilms in medicine, in the natural environment, and in industry. 621
In search of answers to fundamental scientific questions regarding the molecular 622
underpinnings of surface attachment, production and composition of the biofilm matrix, 623
physiological consequences of biofilm formation, and regulation of biofilm formation, 624
biofilm researchers are using interdisciplinary approaches leading to unprecedented 625
molecular detail. For example, the use of electrochemical camera chips or MALDI-TOF 626
imaging mass spectrometry for simultaneous imaging of multiple metabolites in biofilms is 627
leading to a better understanding of the biochemical processes that occur during biofilm 628
development and in biofilms formed in the natural environment. Application of non-invasive 629
imaging methods in living biofilms at high spatial and temporal resolution combined with 630
big data analysis is revealing fundamental principles of biofilm formation. It was exciting to 631
see translational work capitalizing on the advancement of our understanding of biofilm 632
formation. The increasing focus on translational work was revealed through a plethora of 633
examples, including the use of antibodies and engineered enzymes to target biofilm matrix 634
components, novel biomaterials engineered to prevent adherence of biofilm bacteria, and 635
compounds designed to target major structures and regulatory circuits to control biofilm 636
formation. Finally, the importance of understanding mechanisms of multi-species biofilm 637
formation, the diversity and functions of microbes in the community in which they live, and 638
the necessity for the development of techniques to answer these questions were highlighted. 639
The objectives of this meeting were to bring together scientists from across the world to 640
present and discuss the best and most up to date research on biofilms, to better understand 641
and control biofilms, and to foster interdisciplinary collaborations. 642
643
During the closing remarks, Jean-Marc Ghigo announced that the next and 8th edition of the 644
ASM biofilm conference would be held in 2018 and encouraged biofilm lovers to disperse 645
and recruit new talent to the biofilm field. 646
647
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648
ACKNOWLEDGMENTS 649
650
Fitnat Yildiz (chair) and Jean-Marc Ghigo (Co-chair) gratefully acknowledge Paul Stoodley, 651
Thomas Bjarnsholt, Claus Moser, Darla Goeres, Alex Rickhardt and all their colleagues for 652
their dedication and involvement in the organization of the workshops. They also thank the 653
conference program committee members Clay Fuqua, Tom Battin, Susanne Haußler, George 654
O’Toole, Phil Stewart, Mark Schembri and Pradeep Singh for their contributions, and Lisa 655
Nalker of the American Society for Microbiology for her outstanding support at all stages of 656
meeting. 657
658
We thank the following sponsors for their support of the 7th ASM conference on Biofilms: 659
Burroughs welcome Fund (Platinum supporter); Biofilm Control; Bitplane; Center for 660
Biofilm Engineering at MSU; EMD Millipore; Gordon and Betty Moore Foundation; 661
Recombina; Thorlabs; Vertex Pharmaceuticals (Gold supporters); Leica Microsystems; 662
Sharklet Technologies (Silver supporters); Biosurface technologies; Cook Medical (Bronze 663
supporters). 664
665
666
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FIGURE LEGENDS. 1
2
3
Figure 1. Biofilm formation and cell dispersal on marine particles. This cartoon depicts 4
different strategies of marine bacteria for the utilization of marine particles. Many bacteria in 5
the ocean are motile and chemotactic (red and blue cells), but only some populations can attach 6
to and form biofilms on marine particles (red cells), whereas others hover in the vicinity of 7
particles, obtain less nutrients, but in return can rapidly disperse to colonize new particles (blue 8
cells). Artwork by Yutaka Yawata, Glynn Gorick and Roman Stocker. 9
10
Figure 2. Hydrophobic nature of wrinkled colonies of Bacillus subtilis. A. Colony of B. 11
subtilis with a red-colored water droplet. B. Cartoon depicting a cross-section of the colony and 12
the factors that influence formation of the wrinkles. Figure from Cairns et al., Mol Microbiol., 13
93:587-98, 2014 (62). 14
15
Figure 3. Automated image analysis of surface contact stimulated holdfast synthesis in C. 16
crescentus. Phase contrast (Phase) images of cells arriving on a glass surface and fluorescence 17
(Fluorescence) images of lectin staining of holdfast were taken every 20 minutes and were 18
analyzed with MicrobeJ (http://www.indiana.edu/~microbej/) (63, 64). MicrobeJ detects (green 19
outline at t0) and tracks (pale blue) the cell pole and the holdfast (green hexagon at th). User-20
defined criteria automatically record two temporal events, cell and holdfast detection, 21
respectively, and these are used to automatically compute the time delay between cell arrival on 22
the surface and holdfast synthesis. 23
24
Figure 4. Confocal micrograph of a skin abscess co-infected with A. 25
actinomycetemcomitans (red) and S. gordonii (green). Shown is a confocal micrograph of a 3 26
day old murine skin abscess co-infected with the oral cavity pathogen Aggregatibacter 27
actinomycetemcomitans (Aa, red) and the oral cavity commensal Streptococcus gordonii (Sg, 28
green). Spatial analysis of abscesses revealed that Aa and Sg persist in vivo as biofilm-like 29
aggregates that are about 0.5 pL in volume and that Aa maintains a >4 µm distance away from 30
Sg. Mutation of the enzyme Dispersin B in Aa, which degrades and allows it to disperse from 31
biofilms, disrupts the ability of Aa to achieve its optimal spacing from Sg and as a result 32
mitigates its virulence in the abscess. Scale bar, 25 µm. Credit: Jake Everett and Dr. Kendra 33
Rumbaugh of Texas Tech University Health Sciences Center. 34
35
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