To support the cost of attending the conference, ASM has awarded travel grants of $500 to each of the ..... G. O'Toole; Dartmouth Medical School, Hanover, NH.
Biofilms 2009
© 2009, American Society for Microbiology 1752 N Street, N.W. Washington, DC 20036-2904 Phone: 202-737-3600 World Wide Web: www.asm.org All Rights Reserved Printed in the United States of America ISBN: 978-1-55581-5707
TABLE OF CONTENTS
ASM Conference Information General Information
4
Scientific Program
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5
Abstracts for Keynotes
15
Abstracts for Speakers
16
Abstracts for Posters
35
Index
153
ASM CONFERENCES COMMITTEE Fred C. Tenover, Ph.D., Chair Cepheid Lance R. Peterson, M.D., Vice Chair Northwestern University William Goldman, Ph.D. University of North Carolina, Chapel Hill Darren Higgins, Ph.D.* Harvard Medical School Lora Hooper, Ph.D. University of Texas Southwestern Medical Center Christine Jacobs-Wagner, Ph.D. Yale University Scott Weese, DVM University of Guelph Sean Whelan, Ph.D. Harvard Medical School *Indicates Committee Liaison for this Conference
ASM CONFERENCES MISSION To identify emerging or underrepresented topics of broad scientific significance. To facilitate interactive exchange in meetings of 100 to 700 people. To encourage student and postdoctoral participation. To recruit individuals in disciplines not already involved in ASM to ASM membership. To foster interdisciplinary and international exchange and collaboration with other scientific organizations.
ASM Conferences
GENERAL INFORMATION SCIENTIFIC PROGRAM ORGANIZERS Michael Givskov, PhD University of Copenhagen Susanne Häußler, PhD Helmholtz Centre for Infection Hilary M. Lappin-Scott, PhD Bangor University Robert J. Palmer Jr., PhD National Institute Dental and Craniofacial Research Matt Parsek, PhD University of Washington Paul Stoodley, PhD University of Southampton Fitnat Yildiz, PhD University of California Ex officio: J. William Costerton, DSc Center for Genomic Sciences, Allegheny-Singer Research Institute
5th ASM Conference on Biofilms
ACKNOWLEDGMENTS The Conference Organizers and the American Society for Microbiology would like to acknowledge the following for their contributions to this conference:
Innovotech Inc. 3M Bridge PTS Fluxion Biosciences Center for Biofilm Engineering Leica Microsystems, Inc. Biosurface Technologies Taylor & Francis Philips Oral Healthcare National Institutes of Health
GENERAL INFORMATION GENERAL SESSIONS All general sessions will be held in the Caribe Ballroom at the Hilton Cancun. A name badge is required for entry into all sessions. In consideration of other participants, no children are permitted in the sessions. POSTER SESSIONS Poster boards are located in the Miramar Ballroom. Presenters should mount posters on Sunday evening and should leave posters up until Thursday afternoon. Each poster is allotted one half a board face. Please check your assigned number in the abstract index and mount your poster on the board space bearing that number. Official poster sessions will be held on Monday, Tuesday and Wednesday as noted within the program. Please stand at your poster during the session (A, B or C) noted next to your poster number. Posters are grouped by topic area as follows: 1 – 67 Biofilm Development 68 – 160 Biofilms in Medicine 164 – 182 Biofilms in the Environment 183 – 216 Communication and Signaling 217 – 221 Evolutionary Aspects 222 – 232 Population Biology and Spatiotemporal Interactions 233 – 252 The Biofilm Matrix 253 – 296 Industrial and Applied Biofilm Research SOCIAL EVENTS Welcome Reception, Sunday, November 15, 8:00 pm - 9:30 pm, Poolside, Hilton Cancun Golf & Spa Resort There is much to explore and enjoy in Cancun when your not in a session. The hotel is ideally situated between the azure Nichupté Lagoon and the sparkling turquoise waters of the Caribbean Sea. It combines the allure of Mexico with the modern features of a world-class resort facility, set in a 150 acre private park with a magnificent infinity pool, and a beach with sand as fine as sifted sugar. Be sure to take advantage of your surroundings each day in Cancun! Conference Networking Reception, Banquet and Poster Prizes Presentation, Thursday, November 19, 7:00 pm - 11:30 pm, At the Beach, Hilton Cancun Golf & Spa Resort GUESTS Tickets for guests that wish to attend the welcome reception and Conference dinner may be purchased at the registration desk. STUDENT TRAVEL GRANTS ASM encourages the participation of graduate students and new postdocs at ASM Conferences. To support the cost of attending the conference, ASM has awarded travel grants of $500 to each of the following individuals: Elanna Bester Chelsea Boyd Louise Christensen Lindsay Dutton Jaakko Ekman Dennis Enning Mustafa Fazli
Maxsim Gibiansky Vernita Gordon Kim Hermans Masahito Ishikawa June Javens-Wolfe Megan Kiedrowski Charalampia Korea
Joung-Hyun Lee Florencia Malamud Dominick Motto Lisa Nelson Brian Peters Joseph Peterson Olga Petrova
Ranjani Prabhakara Amity Roberts Maria Rowe Cynthia Ryder Maria Salta Hans Steenackers Katarzyna Stevens
Timothy Sullivan Jason Tuckerman Maria van Gennip Emma Weir Jerry Woo Nan Yang ASM Conferences
GENERAL INFORMATION SUNDAY, NOVEMBER 15 7:00 am – 8:00 am Miramar Foyer
Workshop Registration
8:00 am – 4:00 pm
Concurrent Workshops
Concurrent Workshop 1 – Imaging and Quantifying ex vivo Biofilms and Flow Cell Operations Faculty: Rob Palmer, Annette Moter, Claus Sternberg, Haluk Beyenal, Paul Stoodley
Concurrent Workshop 2 – Standardized Biofilms Methods - Grow, Treat, Sample and Analyze: Standardizing Biofilm Methods for Routine Use Faculty: Darla Goeres, Diane Walker, Lindsey Lorenz, Merle Olsen, Nick Allan, Al Parker
Concurrent Workshop 3 – Animal Models for Studying Biofilms Faculty: Claus Moser, Peter Ostrup Jensen, Thomas Bjarnsholt, Lars Christophersen, Mark Shirtliff
2:00 pm – 6:00 pm Miramar Foyer 5:00 pm – 5:05 pm
Conference Registration
5:05 pm – 6:05 pm
Keynote Address 1 Time, Space and Microbial Diversity in the Human Body David Relman, V.A. Palo Alto Healthcare System and Stanford University (USA)
6:05 pm – 7:45 pm
Session 1 – Biofilms and the Human Microbiome Session Chair: J. William Costerton
6:05 pm – 6:15 pm
Introduction from Session Chair – Mapping of Commensal Biofilms on Human Tissues J. William Costerton, Allegheny General Hospital (USA)
6:15 pm – 6:45 pm 6:45 pm – 7:15 pm 7:15 pm – 7:45 pm 8:00 pm – 9:30 pm Poolside
Ecology of the Human Vaginal Microbiome Larry Forney, University of Idaho (USA)
Opening Remarks
The Oral Microbial “Canary in the Coal Mine” of Human Disease Bruce Paster, The Forsyth Institute (USA) Commensal-host Interactions at the Intestinal Mucosal Surface Lora Hooper, University of Texas Southwestern Medical Center (USA) Opening Reception
5th ASM Conference on Biofilms
SCIENTIFIC PROGRAM MONDAY, NOVEMBER 16
Session 2 – Biofilm Development Session Chairs: Joshua Shrout and Alison Kraigsley
8:00 am – 12:00 pm 8:00 am – 8:30 am 8:30 am – 9:00 am 9:00 am – 9:20 am
Heterologous Expression of Candida albicans Adhesins ALS3, HWP1 and EAP1 in Saccharomyces cerevisiae Reveals Differential Roles in Adherence and Biofilm Formation Angela H. Nobbs, University of Bristol (UK)
9:20 am – 9:40 am
In Search for Genes Important for Salmonella Biofilm Formation: Where Single Cell Approach and Global Integration Meet Kim Hermans, Centre of Microbial and Plant Genetics, K.U. Leuven (Belgium)
9:40 am – 10:00 am
CdrA- A Matrix Reinforcing Adhesin Important for P. aeruginosa Biofilm Development Matt Parsek, University of Washington (USA)
10:00 am – 10:30 am
Coffee Break
10:30 am – 11:00 am 11:00 am – 11:30 am 11:30 am – 12:00 pm 12:00 pm – 1:30 pm 1:30 pm – 3:30 pm 1:30 pm – 2:00 pm 2:00 pm – 2:30 pm
A Novel Signaling Network Essential for Regulating Pseudomonas aeruginosa Biofilm Development Karin Sauer, Binghamton University (USA)
Unmasking Cryptic Cell-surface Adhesin in Escherichia coli Jean-Marc Ghigo, Institut Pasteur (France) To Build a Biofilm George O’Toole, Dartmouth University (USA)
The Transition to Biofilm Formation in Bacillus subtilis Requires the Bifunctional Clutch Protein EpsE Dan Kearns, Indiana University (USA) The Matrix of Psuedomonas aeruginosa Biofilms Dan Wozniak, Ohio State University Medical Center (USA) Lunch break (on your own) Session 3 – The Biofilm EPS Matrix Session Chairs: Rajendra Deora and Hans Curt Flemming The Role of Extracellular Polysaccharides Matrix in the Establishment and Pathogenicity of Streptococcus mutans in Biofilms Michel Koo, University of Rochester Medical Center (USA) Life and Death During Staphylococcus aureus Biofilm Development Ken Bayles, University of Nebraska Medical Center (USA) ASM Conferences
SCIENTIFIC PROGRAM 2:30 pm – 2:50 pm 2:50 pm – 3:10 pm 3:10 pm – 3:30 pm 4:00 pm – 6:00 pm Miramar Ballroom
Extracellular DNA Induces Biofilm-specific Genes that Promote Antibiotic Resistance and Aggregation in Pseudomonas aeruginosa Shawn Lewenza, University of Calgary (Canada)
6:30 pm – 7:30 pm
Keynote Address 2 Electrochemically Active Biofilms: Insights into Long Range Electron Transfer Mechanisms from Adaptive Evolution and Some Novel Applications Derek Lovley, University of Massachusetts (USA)
Localization of Polysaccharide Export Proteins is Involved in the Coordinated Appearance of Holdfast in Caulobacter crescentus June Javens, Indiana University (USA) The Regulation and Role of Extracellular Nuclease Activity in Staphylococcus aureus Biofilms Alexander Horswill, University of Iowa (USA) Poster Session A
TUESDAY, NOVEMBER 17 8:00 am – 10:10 am
Session 4 – Intercellular-Signaling in Biofilm Communities Session Chairs: Alex Rickard and Bob McLean
8.00 am – 8:30 am 8:30 am – 9:00 am 9:00 am – 9:30 am 9:30 am – 9:50 am 9:50 am – 10:10 am 10:10 am – 10:40 am
Darwin, Death and Dispersal in the Biofilm Life-Cycle Jeremy Webb, Southampton University (UK) Biofilm Dinner Conversations: Tales of Evading Host Immunity and Bacterial Lobster Traps Marvin Whiteley, University of Texas at Austin (USA) Inter-kingdom chemical signaling in bacterial host associations Vanessa Sperandio, University of Texas Southwestern Medical Center (USA) Characterization of Induction of Biofilm Dispersion by cis-2-decenoic Acid David G. Davies, Binghamton University (USA) Biofilm Growth Alters the Population Dynamics of Sex Pheromone Signaling in Enterococcus faecalis Resulting in a Bistable Switch Laura C. Case, University of Minnesota (USA) Coffee Break
5th ASM Conference on Biofilms
SCIENTIFIC PROGRAM 10:40 am – 12:10 pm
Session 5 – Bioresources Session Chairs: Hilary Lappin-Scott and Paul Stoodley
10:30 am – 11:00 am 11:00 am – 11:30 am
Unexpected Succession from Compact to Filamentous Biofilm Surface Structure – Impact on Mass Transport Harald Horn, Technical University of Munich (Germany)
11:30 am – 11:50 am 11:50 am – 12:10 pm
Electrogenic Activity of Cyanobacterial Biofilms Ilia V. Baskakov, University of Maryland Biotechnology Institute (USA)
12:10 pm – 1:30 pm
Lunch break (on your own)
1:30 pm – 3:40 pm 1:30 pm – 2:00 pm 2:00 pm – 2:30 pm 2:30 pm – 3:00 pm 3:00 pm – 3:20 pm 3:20 pm – 3:40 pm
Session 6 – Biofilms in Medical and Dental Infections I Session Chairs: Thomas Bjarnsholt and Luanne Hall-Stoodley
4:00 pm – 6:00 pm Miramar Ballroom
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Nitrification in Drinking Water Systems Ann Camper, Center for Biofilm Engineering, Montana State University (USA)
Adhesive behavior of Coagulase Negative Staphylococci isolated from Italian Cheese Laura Selan, Sapienza University (Italy)
Biofilm Based Wound Care Randy Wolcott, Southwest Regional Wound Care (USA) Targeting a Haemophilus influenzae Protein Expressed During Biofilm Growth in vivo for Development of a Vaccine for Otitis media Lauren Bakaletz, The Research Institute at Nationwide Children’s Hospital (USA) E. coli Biofilms, Bottlenecks, and Host Responses in Urinary Tract Infection Scott J. Hultgren, Washington University Medical School (USA) Gallbladder Carriage of Salmonella in Mouse and Man is Enhanced by Biofilm Formation on Gallstone Surfaces John S. Gunn, University of Texas Health Sciences Center at San Antonio (USA) Candida albicans-Staphylococcus aureus Dual-species Biofilms Mediate Invasive Staphylococcal Infection Brian M. Peters, University of Maryland (USA) Poster Session B
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SCIENTIFIC PROGRAM 7:00 pm – 9:00 pm
Parallel Evening Sessions (with refreshments) Evening Session 1 – Diagnosing Biofilm Infections Moderators: Mike Givskov, Annette Moter, Paul Stoodley
Evening Session 2 – Environmental Biofilms as Pathogen Reservoirs Moderators: Hans-Curt Flemming, Jeremy Webb
Evening Session 3 – Evolution and Biofilms Moderator: Matt Parsek
Evening Session 4 – Challenges in Biofilms Studies Moderators: Staffan Kjelleberg, Fitnat Yildiz
WEDNESDAY, NOVEMBER 18 8:00 am – 10:10 am 8:00 am – 8:30 am 8:30 am – 9:00 am 9:00 am – 9:30 am 9:30 am – 9:50 am 9:50 am – 10:10 am 10:10 am – 10:30 am 10:30 am – 12:00 pm 10:30 am – 11:00 am
Session 7 – Biofilms in Medical and Dental Infections II Session Chairs: Jeff Leid and Mark Shirtliff Immune System versus Biofilms: How Chemistry may Give “An Upper Hand” Mike Givskov, University of Copenhagen (Denmark) Bacterial Biofilms on Implanted Surgical Materials in the Abdominal Wall Sandeep Kathju, Allegheny General Hospital (USA) Human Oral Microbial Biofilms are Multi-species Communities Paul Kolenbrander, National Institutes of Health (USA) In situ Measurement of Biofilm Activity by FISH: Implications for Diagnosis in Medical Biofilms Annette Moter, Charite Institute for Microbiology and Hygiene (Germany) Antibiofilm Activity of Nanosized Magnesium Fluoride Jonathan Lellouche, Bar Ilan University (Israel) Coffee Break Session 8 – Biofilm Resistance Mechanisms and Control Strategies Session Chairs: Phil Bremer and John Thomas Altering Biofilm Cohesion as a Control Strategy Phil Stewart, Center for Biofilm Engineering, Montana State University (USA)
5th ASM Conference on Biofilms
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SCIENTIFIC PROGRAM 11:00 am – 11:30 pm 11:30 am – 12:00 pm 12:00 pm – 1:30 pm 1:30 pm – 3:40 pm
Matrix-degrading Enzymes: Research Tools and Potential Therapeutic Agents Jeff Kaplan, New Jersey Dental School (USA)
1:30 pm – 2:00 pm
C-di-GMP Signaling and Biofilm Formation in vibrio cholerae Fitnat Yildiz, University of California Santa Cruz (USA)
2:00 pm – 2:30 pm 2:30 pm – 3:00 pm 3:00 pm – 3:20 pm
Signal Transduction of c-di-GMP in Pseudomonas aeruginosa Susanne Haeussler, Helmholtz Centre for Infection (Germany)
The Future Tool Box for Biofilm Control from Nanosciences to Transcriptomics Hilary Lappin-Scott, Bangor University (UK) Lunch break (on your own) Session 9 – Cyclic-di-GMP Signaling Session Chairs: Susanne Haeussler and Dan Wozniak
How Does c-di-GMP Control Biofilms in Pseudomonas aeruginosa? Carrie Harwood, University of Washington (USA) Nitric Oxide-induced Dispersal in Pseudomonas aeruginosa Biofilms Involves Cyclic di-GMP Staffan Kjelleberg, University of New South Wales (Australia)
3:20 pm – 3:40 pm 4:00 pm – 6:00 pm Miramar Ballroom 7:00 pm – 11:30 pm At the Beach
Regulation of C-di-GMP Signaling by O2 in E. coli and B. pertussis Jason Tuckerman, University of Texas Southwestern Medical Center at Dallas (USA)
*New* Poster Prizes, supported by a grant from Innovotech, will be presented in the following categories:
Poster Session C
Conference Networking Reception, Banquet and Poster Prizes Presentation
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The "Terry Beveridge Poster Award for Excellence in Biofilm Microscopy"
The "Peter Gilbert Poster Award for Excellence in Innovation and Biofilm Control"
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The "Bill Characklis Poster Award for Excellence in Engineering in Biofilm Research"
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Two awards for "Outstanding Biofilm Poster" in the Open Category ASM Conferences
SCIENTIFIC PROGRAM THURSDAY, NOVEMBER 19 8:00 am – 10:00 am 8:00 am – 8:30 am 8:30 am – 9:00 am 9:00 am – 9:20 am 9:20 am – 9:40 am 9:40 am – 10:00 am
Session 10 – Environmental Biofilms Session Chairs: Maria Pereira and Diane McDougald Roles for Redox-shuttles in Facilitating Fe Acquisition and Promoting Biofilm Formation Lars Dietrich, Massachusetts Institute of Technology (USA) Composition, Structure and Function of Hot Spring Cyanobacterial Mat Communities David Ward, Montana State University (USA) Defining the Role of Y. pestis Biofilm in Fleas Sara Vetter, CDC/DVBID (USA) Biogenic Alteration of Limestone by Fungi Isolated from Epilithic Biofilms Associated with Mayan Monuments at Chichen Itza Otto Ortega-Morales, Universidad Autónoma de Campeche (Mexico) The Effect of Carbon Limitation on Biofilm Activity, Structure and Planktonic Cell Yield Elanna Bester, University of Toronto (Canada)
10:00 am – 10:30 am
Coffee Break
10:30 am – 12:30 pm 10:30 am – 11:00 am 11:00 am – 11:30 am 11:30 am – 12:00 pm
Session 11 – Emerging Technologies for Studying Structured Systems Session Chairs: Sarah Codd and Howard Ceri
12:00 pm – 12:20 pm 12:20 pm – 2:00 pm 2:00 pm – 3:00 pm
In Vivo RNA Labeling and Separation - a Novel Tool for Biofilm Research Nikolai A. F. Stankiewicz, Institute of Functional Interfaces (Germany)
Holger Daims, University of Wien (Austria) Use of Laser Capture Microdissection Microscopy and Quantitative RT-PCR to Characterize Localized Gene Expression in Biofilms Mike Franklin, Montana State University (USA) Physiology of Shewanella oneidensis Biofilm Cells Alfred Spormann, Stanford University (USA)
Lunch break (on your own) Keynote Address 3 Extracellular Signals Dictate Cell Fate in Biofilms Roberto Kolter, Harvard Medical School (USA)
5th ASM Conference on Biofilms
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SCIENTIFIC PROGRAM
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3:00 pm – 5:10 pm 3:00 pm – 3:30 pm 3:30 pm – 4:00 pm 4:00 pm – 4:30 pm
Session 12 – Genetic Diversification and Division of Labor Session Chairs: Dao Nguyen and Garth Ehrlich
4:30 pm – 4:50 pm
High-throughput Biometric Characterization of Two Distinct Pili-driven Surface Motility Modes in Pseudomonas aeruginosa Maxsim Gibiansky, University of Illinois (USA)
4:50 pm – 5:10 pm
Diversifying the Bacterial Portfolio: Identification of Genetic Pathways Linked to the Rugose Small Colony Variant (RSCV) Phenotype of Pseudomonas aeruginosa Joe J. Harrison, University of Calgary (Canada)
5:10 pm – 5:30 pm
Closing Remarks
TBD Subpopulation Interactions during Pseudomonas aeruginosa Biofilm Development Tim Tolker-Nielsen, University of Copenhagen (Denmark) Natural Transformation of Vibrio cholerae on Chitin Surfaces Melanie Blokesch, Swiss Federal Institute of Technology (Switzerland)
ASM Conferences
SPEAKER ABSTRACTS
KEYNOTE SPEAKERS KS:1
Time, Space and Microbial Diversity in the Human Body D. Relman; Stanford University, Palo Alto, CA
Complex microbial ecosystems occupy the cutaneous and mucosal surfaces of humans. Recent advances have highlighted both the tremendous diversity of these communities and their importance to host physiology, but questions remain about the ecological processes that establish and maintain the human microbiota throughout life. We have been exploring spatial patterning and the effects of perturbation on human microbial diversity. In the subgingival crevice, we have found evidence for island-like biogeography, where every sampled tooth reveals a non-random subset of overall bacterial diversity in that mouth at that point in time. Mouths from different healthy individuals display distinct overall profiles of diversity. We are currently examining the temporal dynamics of perturbation at subgingival sites, as well as in the distal intestinal tract. The rapid return to the pretreatment community composition is indicative of factors promoting community resilience. Approaches that combine ecological theory and statistics, sequence-based and other molecular assessments of community structure, and standardized clinical measurements may improve our understanding of health and disease within the communal human organism. By recognizing the early signs of impending disturbance, we may be able to predict and avoid disease.
KS:2
Electrochemically Active Biofilms: Insights into Long Range Electron Transfer Mechanisms from Adaptive Evolution and Some Novel Applications D. Lovley; University of Massachusetts, Amherst, MA
A diversity of microorganisms are capable of using electrodes either as an electron acceptor or an electron donor for metabolism. Proposed applications for microbe-electrode interactions include: harvesting electricity from organic wastes, renewable biomass, or the environment; enhancing the bioremediation of subsurface environments; powering mobile electronics; environmental and in-body sensors; and supplying electrons for fuel and/or chemical production. Optimization of these applications requires a better understanding of the electrochemical interactions between microorganisms and electrodes. Geobacter sulfurreducens is the most effective current-producing microorganism available in pure culture. It produces thick (ca. 70 µm) biofilms on the anodes of microbial fuel cells. Depthresolved microarray analysis of gene expression within anode biofilms, as well as real-time imaging of gene expression with unstable fluorescent proteins, revealed that cells throughout the biofilm were metabolically active and likely to contribute to current production. This is despite the fact that pH-sensitive fluorescent dyes and confocal microscopy documented substantial decreases in pH near the anode surface and within the interior of biofilm pillars. Furthermore, most of the cells were 5th ASM Conference on Biofilms
not in direct contact with the anode, raising the question of how electrons are transferred over such great distances. A novel dual electrode system was devised that permitted measurements of biofilm conductance. The results demonstrated that long-range electron transfer through the biofilm is possible because the biofilm is highly conductive, with conductivities that rival those of synthetic conductive polymers. This contrasts with the biofilms of other organisms, which act as insulators. Conductance in a diversity of mutant strains of G. sulfurreducens, as well as a strain selected in adaptive evolution studies, was correlated with the abundance of electrically conductive pili, which are hypothesized to form a conductive grid of ‘microbial nanowires’ through the biofilm. The outer-surface c-type cytochrome, OmcZ, was also required for high current densities. Immunogold-labeling studies of biofilm thin sections revealed that OmcZ accumulated at the biofilm-electrode interface. This finding and electrochemical analyses suggest that OmcZ functions as an electrochemical gate promoting the flux of electrons onto the electrode. Microarray analysis and gene deletion studies suggested that electron flow in the opposite direction, i.e. from electrodes into cells, that takes place when electrodes are poised at low potentials, proceeds via a different electron transport system than for current production. A novel strategy for feeding electrode biofilms with energy derived from solar panels shows substantial promise for environmental and bioenergy applications.
KS:3
Extracellular Signals Dictate Cell Fate in Biofilms R. Kolter; Harvard Med. Sch., Boston, MA
During biofilm formation, the soil-dwelling bacterium Bacillus subtilis differentiates into distinct subpopulations of specialized cells that coexist. The coordination and interplay between these cell types requires extensive extracellular communication driven mostly by sensing self-secreted signals. These extracellular signals activate a set of specific sensor kinases, which respond by phosphorylating three major response regulators. Each phosphorylated regulator triggers a specific differentiation program while at the same time repressing other differentiation programs. This allows a particular cell fate to differentiate in response to a specific cue, even in presence of other, possibly conflicting, signals. The sensor kinases respond to the presence of an eclectic group of extracellular signals, such as quorum-sensing molecules, natural products, temperature, pH or scarcity of nutrients. In this lecture I will present the cascades of cell differentiation that are triggered by sensing extracellular signals and a working developmental model in which the diverse cell types sequentially differentiate for the proper development of the biofilm.
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SPEAKER ABSTRACTS
S1:2
Differences in vaginal bacterial communities of women in North America: implications for disease diagnosis and prevention L. J. Forney1, P. Gajer2, C. J. Williams1, Z. Abdo1, G. M. Schneider1, S. L. McCulle2, S. Karlebach3, A. Dormer3, R. Gorle2, J. Russell2, C. C. Davis4, C. Tackett2, L. Peralta5, L. Peralta2, K. A. Ault3, J. Ravel2; 1University of Idaho, Moscow, ID, 2University of Maryland, Baltimore, MD, 3Emory University, Atlanta, GA, 4Procter and Gamble, Inc., Cincinnati, OH, 5University of Maryland, Moscow, ID
Each area of the human body has a unique collection of microorganisms. Though previous studies have tended to focus on understanding etiological agents of infectious disease, there has recently been an increased emphasis on the role of indigenous microbiota in the maintenance of human health. An example is the human vagina, a dynamic and complex microbial ecosystem in which the symbiotic vaginal microbiota play an important protective role in maintaining the health of women. We hypothesize that differences in the species composition of vaginal bacterial communities in healthy women may affect the risk of developing bacterial vaginosis or acquiring communicable diseases. Characterizing differences in vaginal community composition in women within and between racial groups was a first step toward testing this. In a cross sectional study, a total of 418 North American healthy women from four ethnic groups (White, Black, Hispanic and Asian) self-collected two vaginal swabs that were used to determine Nugent scores, and characterize the composition and structure of the resident bacterial communities. Vaginal pH was measured using the Inverness VpH glove. Community analysis was done by pyrosequencing the V1-V2 region of 16S rRNA genes that were amplified from genomic DNA isolated from each sample. Cluster analysis of the data showed there were seven types of communities that differed in terms of species composition and structure that account for all those commonly found in these racial groups, and their relative frequencies significantly varied between racial groups (Pearson chi-square = 61.5, 24 df, Monte Carlo P < .0001). Although lactic acid bacteria dominated all communities, there were significant differences in the median Nugent score and vaginal pH among these communities. For example, while the overall median vaginal pH was 4.4±0.6, the median vaginal pH of Hispanic women was 5.0±0.7. These results show that fundamental differences exist in the microbial diversity of vaginal communities present in reproductive age women of different racial groups. These inherent differences have important consequences that should be taken into account in risk assessment and disease diagnosis.
S1:4
Commensal-host Interactions at the Intestinal Mucosal Surface S. Vaishnava, C. L. Behrendt, L. V. Hooper; The University of Texas Southwestern Medical Center at Dallas, Dallas, TX
The mammalian gut harbors a co-evolved microbial consortium that makes critical contributions to host health. Despite the enormous numbers of bacteria in the luminal compartment, microbial penetration of mucosal surfaces is relatively rare. We have shown that microbial colonization of germ-free mice triggers epithelial expression of RegIIIγ, a secreted C-type lectin. RegIIIγ binds intestinal bacteria but lacks the complement recruitment domains present in other microbe-binding mammalian C-type lectins. We have demonstrated that RegIIIγ and its human counterpart, HIP/PAP, are directly antimicrobial proteins that bind their bacterial targets via interactions with peptidoglycan carbohydrate. We propose that these proteins represent an evolutionarily primitive form of lectin-mediated innate immunity, and that they play a critical role in maintaining symbiotic host-microbial relationships by controlling colonization of intestinal epithelial surfaces. Analysis of the host factors that regulate RegIIIγ expression has uncovered a mechanism of epithelial cell-intrinsic sensing of intestinal bacteria in vivo. We have shown that Paneth cells, specialized secretory epithelia of the small intestine, detect bacteria via cell-intrinsic MyD88-dependent Toll-like receptor (TLR) signaling, triggering expression of multiple antimicrobial factors including RegIIIγ. Paneth cell-intrinsic MyD88 signaling limited commensal penetration of host tissues, and controlled systemic dissemination of pathogenic bacteria. These findings suggest that MyD88-dependent antimicrobial factors including RegIIIγ are essential for maintaining host-microbial homeostasis at the mucosal interface.
S1:3
S2:1
B. J. Paster; The Forsyth Institute, Boston, MA
J. Ghigo; Institut Pasteur, Paris, FRANCE
Based on culture-dependent and culture-independent molecular methods using sequence analysis of 16S rRNA genes, there are about 700 predominant oral bacterial species, of which about 35% have not yet been cultivated in vitro. The Human
Commensal and pathogenic Escherichia coli adhesion to host and environmental surfaces is mediated by a variety of adhesins. Although extensively studied as a model bacterium as well as a pathogen, E. coli genome still contains 34% of genes of
The Oral Microbial “Canary in the Coal Mine” of Human Disease
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Oral Microbe Identification Microarray, or HOMIM, is a high sample-throughput technology which allows the simultaneous detection over 300 of these bacterial species, including the “uncultivables.” HOMIMs and other molecular methods have been used to identify the potential role of specific bacterial species or bacterial complexes in oral health and infectious diseases, such as periodontitis and caries. However, HOMIM has utility beyond determining bacterial associations with oral health status. Oral microbial profiles may also serve as potential biomarkers for systemic diseases, such as pancreatic cancer, Crohn’s disease and heart disease. Consequently, specific oral bacterial profiles may be useful to determine those people at risk for disease, e.g., an “unhealthy” profile may be an early indicator of disease, the proverbial “canary in the coal mine” of human disease.
Unmasking cryptic cell-surface adhesin in Escherichia coli
ASM Conferences
SPEAKER ABSTRACTS unknown function. We hypothesized that some of them could correspond to uncharacterized adhesins, not or poorly expressed under laboratory conditions. My talk will present recent data demonstrating that E. coli adhesion arsenal includes at least 7 cryptic but functional surface structures that display homologies to type 1 fimbriae exported by the chaperone/usher pathway. These new adhesins have distinctive binding specificities and are expressed upon environmental modifications affecting H-NS and CRP activity. We propose that these fimbriae contribute to E. coli’s ability to colonize a wide diversity of host and environmental surfaces in various ecological niches.
S2:2
To Build a Biofilm G. O’Toole; Dartmouth Medical School, Hanover, NH
The intracellular signaling molecule cdiGMP has been implicated in controlling the transition between sessile and motile lifestyles in bacteria. My laboratory has been interested in the signals that modulate cdiGMP levels, the proteins that synthesize/degrade/bind this molecule and the outputs controlled by this signaling molecule. In particular, our efforts have focused on the role of cdiGMP in controlling the transition from growth as planktonic bacteria to life on a surface. I will present the most recent studies from my lab exploring the role of cdiGMP in modulating surface behaviors by pseudomonads.
S2:3
Heterologous expression of Candida albicans adhesins ALS3, HWP1 and EAP1 in Saccharomyces cerevisiae reveals differential roles in adherence and biofilm formation A. H. Nobbs1, M. M. Vickerman2, H. F. Jenkinson1; 1University of Bristol, Bristol, UNITED KINGDOM, 2SUNY Buffalo, Buffalo, NY
Candida albicans is an opportunistic pathogen found on mucosal surfaces of the mouth, urogenital and gastrointestinal tracts, and on the skin, and is able to attach to clinically relevant materials such as catheters and denture acrylic resin. C. albicans can also interact with a number of different bacteria, and is associated with >90% of oral fungal infections derived from polymicrobial biofilms. The adhesive capabilities of C. albicans are mediated, at least in part, by an array of surface-expressed proteins. C. albicans proteins ALS3 and HWP1 have been implicated in biofilm formation on catheters. We hypothesized, therefore, that these adhesins might also contribute to biofilm formation within the oral cavity, and so facilitate oral carriage and persistence of C. albicans. To test this hypothesis, ALS3 and HWP1, together with RBT1 (43% identity to HWP1) and EAP1 (implicated in biofilm formation on polystyrene) were expressed on the surface of surrogate host Saccharomyces cerevisiae. Biofilm formation (24 h) by each clone on salivacoated or polystyrene surfaces was then assessed visually by light microscopy, and biomass was measured using a crystal violet assay. Unlike wild-type S. cerevisiae cells, which failed to form biofilms on either saliva-coated coverslips or polystyrene, cells expressing ALS3 and HWP1 exhibited significant biofilm formation on both surfaces. Cells expressing RBT1 exhibited a similar biofilm profile to HWP1, but overall biomass levels 5th ASM Conference on Biofilms
were approximately 75% lower. No biofilm formation was seen for S. cerevisiae cells expressing EAP1 in the presence of saliva, but did occur on polystyrene, with twice the total biomass compared to cells with ALS3. The contribution of each adhesin to facilitating interactions with oral commensal bacterium Streptococcus gordonii was also investigated by fluorescence microscopy. S. gordonii bound to S. cerevisiae cells expressing ALS3, EAP1 and, to a lesser extent, HWP1, often forming large aggregates. No interactions, however, were seen with wild-type S. cerevisiae or cells expressing RBT1. Taken together, these data imply that a complex network of adhesins is required to enable C. albicans attachment to both synthetic and biological surfaces. Furthermore, complementary adhesive functions across adhesins ALS3, HWP1 and EAP1 are likely to play a significant role in promoting C. albicans colonization of oral surfaces and the formation of polymicrobial biofilms with other members of the oral microflora.
S2:4
In search for genes important for Salmonella biofilm formation: Where single cell approach and global integration meet K. Hermans, R. De Smet, T. Nguyen, T. Verhoeven, D. De Coster, K. Marchal, J. Vanderleyden, S. De Keersmaecker; Centre of Microbial and Plant Genetics, K.U.Leuven, Leuven, BELGIUM
Salmonella enterica serovar Typhimurium is worldwide still one of the main causes of bacterial food-borne diseases. Some of these diseases are related to the characteristic that Salmonella is capable of forming biofilms on biotic and abiotic surfaces. Global gene regulation and expression profiles are different in biofilms as compared to planktonic cells. These differential patterns, together with changes in metabolism, occurring at the transition from free-living to sessile lifestyle, are the causes of specific, species-dependent and biofilm-associated phenotypes such as an increased resistance to antibiotics. Insight in gene expression and regulation under this alternative physiological state of biofilm formation is highly needed to develop novel anti-Salmonella therapeutics, also effective against biofilms. We applied the Differential Fluorescence Induction (DFI) strategy to identify genes specifically induced under biofilm conditions. DFI, as previously developed by the Falkow group, is basically an enrichment strategy which uses small random fragments of S. Typhimurium genomic DNA, cloned upstream of a promoterless GFP gene to monitor promoter activity with a fluorescence-activated cell sorter (FACS) under specific environmental conditions, in our case conditions under which Salmonella forms biofilms. Therefore, sequence determination of the genomic inserts in the enriched pool leads to identification of specific biofilm induced DNA-fragments. We found some interesting, new elements involved in Salmonella biofilm formation, such as genes involved in polyamine metabolism, genes encoding putative cytoplasmic proteins and some nonannotated putative regulatory elements. Their role in Salmonella biofilm formation is currently being further investigated using complementary low- and high-throughput methods. The major advantage of this approach to perform a genomewide screening is the generation of data on single cell level in stead of a mean value for the whole population, as is the case with microarray studies. As such, the heterogeneity, which 17
SPEAKER ABSTRACTS is inherent to biofilms, can be taken into account. Another important advantage is the monitoring of gradations in gene expression, and this independent of prior knowledge of gene annotation. A disadvantage, however, is that DFI does not give a global view on the different pathways the identified genes are involved in. By combining the DFI identified gene information with Salmonella expression compendia using a query-driven biclustering approach, we investigated whether the identified targets show condition-dependent coexpression with genes of known pathways and how these identified pathways exhibit cross talk with physiological functions other than biofilm formation.
S2:6
A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development K. Sauer; Binghamton University, Binghamton, NY
Biofilms are complex communities of microorganisms encased in a matrix and attached to surfaces. It is well recognized that biofilm cells differ from their free swimming counterparts with respect to gene expression, protein production, and resistance to antibiotics and the human immune system. However, little is know about the underlying regulatory events that lead to the formation of biofilms, the primary cause of many chronic and persistent human infections. Here, we demonstrate that biofilm formation following the transition to the surface attached lifestyle is regulated by three previously undescribed twocomponent systems: BfiSR harboring an RpoD-like domain, an OmpR-like BfmSR, and MifSR belonging to the family of NtrC-like transcriptional regulators. These two-component systems become sequentially phosphorylated during biofilm formation. Inactivation of bfiS, bfmR, and mifR arrested biofilm formation at the transition to the irreversible attachment, maturation-1 and -2 stages, respectively, as indicated by analyses of biofilm architecture, and protein and phosphoprotein patterns. Moreover, discontinuation of bfiS, bfmR, and mifR expression in established biofilms resulted in the collapse of biofilms to an earlier developmental stage indicating a requirement for these regulatory systems for the development and maintenance of normal biofilm architecture. Interestingly, inactivation did not affect planktonic growth, motility, polysaccharide production, or initial attachment. However, virulence was affected. This novel regulatory cascade appears to be linked via BfiSdependent GacS-phosphorylation and BfmR to the previously identified LadS/RetS/GacAS/RsmA network that reciprocally regulates virulence and surface attachment. Our data thus indicate the existence of a previously unidentified transduction network regulating committed biofilm developmental steps once P. aeruginosa cells have committed to a surface associated lifestyle, in which phosphorelays appear to be key components of the regulatory machinery that coordinates gene expression during P. aeruginosa biofilm development in response to environmental cues.
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S2:7
The transition to biofilm formation in Bacillus subtilis requires the bifunctional clutch protein EpsE D. Kearns; Indiana University, Bloomington, IN
Many bacteria alternate between growth as planktonic motile individuals and non-motile aggegates called biofilms. During the transition to biofilm formation, motility is inhibited and an extracellular polysaccharide (EPS) matrix is synthesized. In the Gram positive bacterium Bacillus subtilis, the transition to biofilm formation requires the bifunctional glycosyltransferase EpsE. EpsE promotes biofilms as both an enzyme to synthesize the EPS and as a flagellar clutch to inhibit motility. EpsE inhibits flagellar rotation by interacting with the flagellar rotor protein FliG and disconnects FliG from force generating elements. EpsE is the first clutch protein identified for bacterial flagella and we conducted a genetic screen to identify residues, which when mutated, specifically abolished clutch function but left EPS synthesis. Two separate regions of the protein were found to be required for clutch activity. By identifying residues important for motility inhibition we might be able to predict clutch proteins in other bacteria. Mutation of the EpsE glycosyltransferase active site abolished EPS biosynthesis but did not abolish motility inhibition. A genetic screen was conducted to identify other residues specifically required for enzymatic activity. The screen identified residues of the conserved active site and residues possibly involved in sugar discrimination. Enzymatic and clutching activity was genetically separable indicating the EpsE is a bifunctional protein. Active site residues were adjacent to residues required for the flagellar clutch suggesting that the two functions of EpsE may be related. Both activities of EpsE promote biofilm formation. Mutations that abolish EPS biosynthesis impaired biofilm formation whereas mutations that abolish clutch function had little to no effect on biofilm structure. We conclude that the effect of EPS synthesis is dominant to that of flagellar inhibition. When EPS synthesis mutations and clutch mutations were combined however, the resulting biofilm defect was more severe than either mutation alone. We conclude that the two functions synergize to promote biofilm formation and that clutch activity stabilizes biofilms in adverse conditions.
S2:8
The Matrix of Psuedomonas aeruginosa Biofilms D. Wozniak; The Ohio State University, Columbus, OH
Pseudomonas aeruginosa is a Gram-negative pathogen responsible for a variety of opportunistic infections, including chronic airway infections in patients with cystic fibrosis (CF). During the course of chronic infection, P. aeruginosa forms matrix-encased, surface-associated communities called biofilms. Biofilms are thought to contribute to persistence in the CF airway by contributing to evasion of the host immune response and antimicrobial therapy. The exopolysaccharide (EPS) of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids and proteins. One component of the EPS is the polysaccharide Psl, which is synthesized by the polysaccharide synthesis locus (psl). Psl is required for biofilm formation in nonmucoid strains ASM Conferences
SPEAKER ABSTRACTS that do not rely on alginate as the principal biofilm polysaccharide. In this study, we directly visualized Psl EPS at different stages of biofilm development. Cell associated Psl polymerizes into a high molecular weight structure that is arranged in a helical pattern on the surface of P. aeruginosa. A model is proposed to describe how the unique organization of Psl on the cell surface may promote cell-cell interactions within biofilms formed by P. aeruginosa. We also present the first structural analysis of the psl-dependent polysaccharide, which consists of a repeating pentasaccharide containing D-mannose, D-glucose, and L-rhamnose. The structural and genetic data presented here provide a basis for further investigation of the Psl proteins and potential roles for Psl in the biology and pathogenesis of P. aeruginosa.
S3:1
The role of extracellular polysaccharides matrix in the establishment and pathogenicity of Streptococcus mutans in biofilms H. Koo; University of Rochester Medical Center, Rochester, NY
Oral diseases related to dental biofilms continue to afflict the majority of the World’s population. Among them, dental caries is the most prevalent and costly oral infectious disease. This ubiquitous disease results from the interaction of specific bacteria with constituents of the diet within biofilms formed on tooth surfaces. Streptococcus mutans is a key contributor to the formation of dental biofilms associated with caries disease because this bacterium effectively (i) utilizes dietary sucrose to rapidly synthesize large amounts of extracellular polysaccharides (EPS), (ii) adheres tenaciously to glucan-coated surfaces, and (iii) is also highly acidogenic and acid-tolerant. The majority of biofilm matrices are rich in exopolysaccharides, and dental biofilms are no exception. The polysaccharides, mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), are largely insoluble and complex in structure. The Gtfs secreted by S. mutans, particularly the products of gtfB and gtfC genes, bind avidly to the tooth and bacterial surfaces and are highly active, and when exposed to sucrose; glucans are formed rapidly on the surfaces. The polysaccharides synthesized in situ provide binding sites for colonization and accumulation of microorganisms on the apatitic surface and to each other, and contribute to the bulk, physical integrity and stability of the matrix allowing S. mutans cells to form tightly bound microcolonies. Furthermore, EPS also protect the bacteria from inimical influences of antimicrobials and other environmental assaults, and may affect the diffusion properties of the matrix influencing the microenvironment of the biofilm. Thus, EPS are essential for the establishment of S. mutans in biofilms, and play a critical role in the pathogenesis of dental caries disease. This presentation will provide an overview of the role of EPS matrix in the development of biofilms by Streptococcus mutans, and discuss the potential of biofilm-control strategies based on disruption of the synthesis of these polymeric substances.
5th ASM Conference on Biofilms
S3:2
Life and death during Staphylococcus aureus biofilm development K. Bayles; University of Nebraska Medical Center, Omaha, NE
The Staphylococcus aureus cid and lrg operons encode a putative holin-antiholin system that plays important roles in the control of bacterial cell death and lysis. The cid operon encodes the holin component of this system, having a positive effect on murein hydrolase activity and cell lysis, while the lrg operon encodes the antiholin, having a negative effect on these processes. Recently, these operons were shown to be important in biofilm development causing defects in biofilm morphogenesis and adherence. The latter was shown to be a function of the differential autolysis rates inherent to these strains and, therefore, the differing amounts of extracellular DNA (eDNA) released into the biofilm matrix. Combined, these results led to the model that a careful balance between these proteins and, thus, cell death and lysis is critical for the development of normal biofilm structures. Investigations of the regulatory control of cid and lrg expression during planktonic growth demonstrate that they are both induced by the presence of excess glucose in a CidR-dependent manner, and that lrg expression is induced by agents known to dissipate membrane potential in a process dependent on the LytSR two-component regulatory system. Recent studies on the regulatory control of the cid promoter have revealed that it is also strongly induced by growth under microaerobic conditions. To determine the relevance of these regulators and environmental stimuli on cid and lrg expression during biofilm growth, we have generated fluorescent reporterpromoter fusion constructs in which the expression of cid and lrg can be monitored in individual cells during biofilm development. The results of these experiments support a model in which the metabolic heterogeneity present in the staphylococcal biofilm plays a dramatic role in the differential control of cid and lrg expression within biofilm subpopulations and, in turn, the death and lysis of these cells.
S3:3
Extracellular DNA induces biofilm-specific genes that promote antibiotic resistance and aggregation in Pseudomonas aeruginosa H. Mulcahy, L. Johnson, S. Horsman, L. Charron-Mazenod, S. Lewenza; University of Calgary, Calgary, AB, CANADA
Microbial biofilms are surface-associated, aggregative communities encased in an extracellular matrix that is composed primarily of exopolysaccharides (EPS) and extracellular DNA (eDNA). Biofilms display a complex three-dimensional structure, increased resistance to antimicrobial compounds, environmental stresses and the host immune response. The molecular mechanisms underpinning these features of P. aeruginosa biofilms are not completely understood. DNA was initially shown to play an adhesive role in maintaining biofilm structure but we have recently identified a novel function of extracellular DNA as a divalent metal cation chelator. Cells in a DNA-rich biofilm behave as if they are cation limited and activate the 19
SPEAKER ABSTRACTS cation-sensing PhoPQ and PmrAB two-component regulatory systems. Extracellular DNA induces the expression of PhoP/ PmrA-regulated genes, including the arnBCADTEF-ugd LPS modification operon that is required for increased resistance to aminoglycosides and antimicrobial peptides. DNA-rich biofilms are up to 2500 times more tolerant to antimicrobial peptides than planktonic cultures grown in Mg2+-replete conditions. Extracellular DNA also induces expression of the PmrA-regulated operon PA4773-PA4775, which we have shown to be required for biosynthesis of the polyamine putrescine. Using HPLC, we identified putrescine in the cytoplasm, the outer surface and supernatants of wild-type cultures grown in the presence of eDNA, but not in the PA4773 mutant. We have shown a novel role for putrescine in antibiotic resistance and biofilm formation. Lastly, the addition of eDNA also strongly induces the pel and psl EPS gene expression. Mg2+ limitation is imposed on cells in the presence of eDNA and promotes biofilm formation through the transcriptional repression of retS and activation of the gacAS pathway, leading ultimately to increased Pel/Psl production. These observations highlight a novel signaling role for eDNA in activating multple two-component regulatory sytems and inducing the expression of genes required for the hallmark features of biofilms. DNA accumulation in biofilms and the CF lung is likely very important in promoting chronic infections that cannot be cleared by the immune response or antibiotic therapy.
stalked cell and a motile swarmer cell, which will, in turn, differentiate into a stalked cell. The holdfast is first observed in the late swarmer cell stage. HfsD was fused to mCherry fluorescent protein to determine if its localization during the cell cycle has a role in this coordinated appearance. A fluorescent focus first appears at the flagellar pole in late predivisional cells and remains at this pole after cell division and as the flagellum and pili are replaced by a holdfast and stalk during cell differentiation. This indicates that HfsD localizes to the swarmer pole in advance of holdfast export. HfsD is evenly distributed throughout the cell at all stages in holdfast-deficient mutants of the pleC and podJ developmental regulator genes. In a pleD mutant that exhibits delayed holdfast export, HfsD localizes to the poles, although at a lower frequency than in wild-type cells. Preliminary evidence indicates that HfsA co-localizes with the holdfast at the tip of the stalk.
S3:4
Staphylococcus aureus is a human pathogen capable of two distinct lifestyles, invasive and biofilm-forming. Recent studies have suggested that extracellular DNA (eDNA) is a major component of the biofilm matrix. Exogenous addition of DNAse I both prevented attachment and dispersed established S. aureus biofilms, confirming the important role of eDNA in maintaining biofilm integrity. Interestingly, S. aureus possesses a nuc gene that encodes a secreted nuclease enzyme (called Nuc), and our studies have demonstrated that a nuc mutant displays enhanced biofilm forming capacity. These findings lead us to hypothesize that S. aureus may modulate biofilm maturation and dispersal by controlling the available level of eDNA using the secreted Nuc enzyme. To begin investigating this question, we analyzed the regulation of nuc gene expression using transcriptional fusions and performed biochemical assays for extracellular nuclease activity. For the transcriptional analysis, a nuc-GFP promoter fusion was constructed, and the activity of this promoter was tested in agr, sigB and sarA global regulatory mutants in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. In time course experiments, expression from the nuc promoter increased dramatically in the sarA and sigB mutants, but conversely was repressed in the agr mutant. A new fluorescence-based biochemical assay for Nuc activity was developed, and similar trends in nuclease activity were measured in cell-free supernatants of agr, sigB and sarA mutants. Currently, we are extending these findings to an assessment of the role of nuclease activity in biofilm development. The nuc gene was cloned into a tetracycline-inducible expression vector and dose-response flow cell biofilm studies are being performed. These ongoing studies will allow varying levels of extracellular Nuc activity to be tested during biofilm attachment and detachment. Altogether, these findings confirm the emerging, important role for eDNA in the S. aureus biofilm matrix and will provide further insight on the contribution of nuclease activity to biofilm development.
Localization of polysaccharide export proteins is involved in the coordinated appearance of holdfast in Caulobacter crescentus J. Javens-Wolfe, Y. Brun; Indiana University, Bloomington, IN
Caulobacter crescentus is a Gram-negative alpha proteobacterium that spends the replicative part of its life cycle firmly attached to a surface via an adhesive polysaccharide holdfast structure at the end of its stalk. The genes responsible for holdfast synthesis and export are homologous to those of group 1 capsular polysaccharide biogenesis in E. coli. Caulobacter HfsD (homologous to E. coli Wza) is predicted to be an outer-membrane channel protein through which the polysaccharide polymer is secreted. HfsA and HfsB are homologous to the N-terminal and C-terminal domain (respectively) of Wzc, a polysaccharide export regulator with autokinase activity. Holdfast is neither observed on the surface of ΔhfsDAB whole cells, nor in whole cell lysates, indicating that without export machinery, upstream synthesis steps are abolished. This is likely due to the requirement of synthesis and export proteins to interact in a multi-enzyme complex for optimal biogenesis. Crystal violet staining shows that ΔhfsA, and ΔhfsD mutants do not adhere to polystyrene, while a small percentage of ΔhfsB mutant cells do adhere. ΔhfsB cells exhibit a decreased level of HfsA. The adhesion deficiency of ΔhfsB cells can be partially restored by moderate overexpression of HfsA. High level overexpression of HfsA does not restore ΔhfsB or ΔhfsA adhesion, indicating that a large excess of HfsA might upset the stoichiometry of the multiprotein complex. Overexpression of HfsB in a ΔhfsA mutant does not restore cell adhesion. These results indicate that HfsA functions downstream of HfsB. Each cell division in C. crescentus produces a sessile 20
S3:5
The regulation and role of extracellular nuclease activity in Staphylococcus aureus biofilms M. R. Kiedrowski1, J. S. Kavanaugh1, C. L. Malone1, M. S. Smeltzer2, K. W. Bayles3, A. R. Horswill1; 1University of Iowa, Iowa City, IA, 2The University of Arkansas for Medical Sciences, Little Rock, AR, 3University of Nebraska Medical Center, Omaha, NE
ASM Conferences
SPEAKER ABSTRACTS
S4:1
Darwin, Death and Dispersal in the Biofilm Life-Cycle J. S. Webb; School of Biological Sciences, University of Southampton, UNITED KINGDOM
The survival of bacteria in nature is greatly enhanced by their ability to i) develop multicellular and highly recalcitrant 3-dimensional structures within biofilms, ii) rapidly generate extensive, biofilm-specific genetic diversity and iii) disperse to and colonize new environments via nitric oxide and c-diGMP-dependant signalling pathways. We have examined the interrelationship of these processes by investigating the role of mutation in-situ in the development and dispersal of 3 dimensional biofilm structures. We transformed Pseudomonas aeruginosa cells with a plasmid harbouring the green fluorescent protein gene containing a +1 frameshift mutation. These cells were non-fluorescent until a mutation causing reversion to the wild-type sequence occurred. We observed microcolonyspecific increases in mutation frequency of at least 100-fold compared to planktonically grown cultures. Moreover, mutator phenotypes can enhance microcolony-based growth of P. aeruginosa. For a range of P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was correlated with the mutation frequency of the organism. We have also observed that characteristics of cells that disperse from biofilms include enhanced phenotypic and genetic variability compared to pre-dispersal biofilm cells. Accumulation of reactive oxygen and nitrogen species (RONS) in the interior of microcolonies triggers lysis, dispersal and phenotypic variation among dispersal cells. The predominant RONS in P. aeruginosa microcolonies is peroxynitrite. Addback of nitric oxide (NO, the precursor of stable peroxynitrite) to P. aeruginosa biofilms induces a shift from biofilm to planktonic cell physiology and greatly increases the sensitivity to antimicrobials. These phenotypic changes appear to occur through NO-mediated interference with cyclic-di-GMP signalling pathways, which are known to regulate biofilm formation and dispersal in diverse bacteria. Thus processes of mutation and diversification within biofilms can play an important role in microcolony initiation, growth and dispersal.
S4:2
Biofilm Dinner Conversations: Tales of Evading Host Immunity and Bacterial Lobster Traps M. Whiteley; The University of Texas at Austin, Austin, TX
Antibiotic resistance is a fundamental problem in clinical microbiology and numerous mechanisms for resistance have been uncovered over the last 40 years. From a mechanistic perspective, some processes, such as transfer of resistance determinants through DNA exchange, are relatively straightforward and their understanding has provided great insight into antibiotic resistance. Exacting the mechanism of antibiotic resistance in bacterial biofilms has not been straightforward. Several hypotheses have been proposed including slow growth of biofilm bacteria, reduced diffusion of antibiotics into the biofilm, sequestration of antibiotics by the biofilm EPS, the presence of persistor cells within the biofilm, and alterations 5th ASM Conference on Biofilms
in gene expression of biofilm bacteria. While these hypotheses are not mutually exclusive, the literature is equally filled with studies supporting one or more of these hypotheses. One important unifying aspect of virtually all of these studies is their use of extremely large populations of bacteria (107-1010). While the use of large populations has direct clinical significance in many infections where large biofilms have been formed (such as cystic fibrosis and urinary tract infections), nothing is currently known about antibiotic resistance in smaller aggregate populations that are likely important for seeding many infections. Here, we present a new methodology that allows for the monitoring of antibiotic resistance in low number/high density populations. Dynamic masking multiphoton lithography techniques allow us to rapidly prototype various porous protein microstructures with measurable volumes on the sub-picoliter range. Upon inoculation, bacteria enter through a small opening of a protein microstructure. Once inside, the entrance to the microstructure is closed preventing exit of the bacteria. With a constant heated flow of nutrients, bacteria enclosed within these bacterial “lobster traps” remained motile and consistently doubled, increasing cell density in a confined space. Interestingly, bacterial populations of Pseudomonas aeruginosa growing within these lobster traps display high resistance (>90%) to the antibiotic gentamicin, despite the fact that the cell number is low (approximately 1,000 - 4,000 cells) and the antibiotic readily penetrates the trap. I will discuss the utility of these traps for assessing antibiotic resistance as well as probe the molecular mechanism of resistance.
S4:3
Inter-kingdom chemical signaling in bacterial host associations V. Sperandio; UT Southwestern Medical Center, Dallas, TX
We reported that enterohemorrhagic E. coli (EHEC) serotype O157:H7, exploit cell-to-cell signaling between the microbial flora and the host as a means to gage and recognize the host environment. This inter-kingdom signaling is predicated upon hormonal communication, and utilizes the host epinephrine and/or norepinephrine (NE) stress hormones and a bacterial aromatic hormone-like signal named autoinducer-3 (AI-3). This communication relies on a conserved membrane sensor, QseC, to sense and respond to these signals to initiate a complex signaling cascade in EHEC to promote expression of virulence factors. We demonstrated that the AI-3/epinephrine/NE signaling cascade activates expression of the flagella regulon, the LEE-encoded type three secretion system and numerous effectors. Of note this signaling cascade is widespread in bacteria. Consequently, interference with this recently identified interkingdom cell-to-cell signaling constitutes a novel strategy for development of novel therapeutics to treat bacterial infections. One of the biggest challenges to medicine in the foreseeable future is the emergence of microbial antibiotic resistance. The rapid mutation rate of bacteria allows them to develop resistance to virtually all known antibiotics. This threat to humans and their food sources is immediate, global and potentially catastrophic. The appearance of multi-antibiotic resistance has been exacerbated by the emergence of new infectious diseases, and/or the spread of existing pathogens to non-traditional regions of the globe due to population or climate shifts. The increasing understanding of bacterial pathogenesis and inter21
SPEAKER ABSTRACTS cellular communication, when combined with contemporary drug discovery tools and technologies, provides a powerful platform for translating basic science into therapeutic applications to combat bacterial infections. Here we show that small molecule inhibitors of QseC-mediated signaling markedly inhibit EHEC virulence in vitro. Using a high throughput screen, we identified a potent small molecule, LED209, which inhibits binding of signals to QseC, preventing QseC’s autophosphorylation, and consequently inhibiting QseC-mediated activation of virulence gene expression in EHEC. LED209 also prevented formation of attaching and effacing (AE) lesions on epithelial cells by EHEC. LED209 selectively blocks this signaling cascade in EHEC, but does not interfere with host signaling. We have shown proof of principal that inhibition of inter-kingdom inter-cellular signaling constitutes a novel and effective strategy for the development of a new generation of antimicrobial agents. To study the structure analyses relationship of LED209, 150 analogs were synthezed and tested for their activity. Through these analyses, we aim to determine the pharmacophore of LED209.
S4:4
Characterization of induction of biofilm dispersion by cis-2-decenoic acid D. G. Davies, C. N. Marques, D. T. Amari, B. Y. Hong, J. L. Mavor; Binghamton University, Binghamton, NY
Cis-2-decenoic acid has been shown to act as a cell-to-cell signaling molecule responsible for inducing biofilm dispersion in Gram-negative bacteria, Gram-positive bacteria and fungi. Recently, we have shown that this signaling system is responsible for a number of effects that together lead to the transition from a sessile mode of existence to an active disseminating lifestyle. Induction of dispersion of P. aeruginosa biofilms was demonstrated to result in the release of degradative enzymes involved in hydrolysis of protein, nucleic acids and polysaccharides. Dispersed bacteria were observed to become motile and alter expression of virulence determinants, including elevated expression of pyochelin and exotoxin T. In addition, cis-2-decenoic acid led to increased dissemination of infection in a lettuce virulence model. The altered phenotype induced by cis-2decenoic acid has also been demonstrated to result in enhanced susceptibility of bacteria to antimicrobial agents. Against P. aeruginosa, in the presence of cis-2-decenoic acid, triclosan showed an average 3.4 Log increase in cidal activity, tobramycin and ciprofloxacin showed an average 1.8 Log increase, and polymyxin B showed a 0.9 Log increase compared to the cidal activity of the antimicrobial agents alone. It has also been determined that cis-2-decenoic acid was able to resuscitate dormant bacteria and that it enhanced the recovery of viablenon-culturable bacteria on solid medium, yielding higher cell numbers and shorter lag periods, compared to bacteria cultured on medium not containing cis-2-decenoic acid. Taken together, our results demonstrate that cis-2-decenoic acid acts to induce bacteria to transition from a biofilm phenotype typically associated with chronic infections (in which bacteria show a reduction in metabolic activity, growth and motility, and an enhanced resistance to antimicrobial agents), to bacteria with a phenotype more typically associated with acute phase infections (as characterized by enhanced growth, activity and susceptibility to 22
antimicrobial agents). These observations suggest that treatment with cis-2-decenoic acid alone or in combination with antibacterial agents, should have a significant enhancing effect on the killing of bacteria associated with chronic infections.
S4:5
Biofilm growth alters the population dynamics of sex pheromone signaling in Enterococcus faecalis resulting in a bistable switch L. C. Case, A. Barnes, A. Chatterjee, W. Hu, G. M. Dunny; University of Minnesota - TC, Minneapolis, MN
Conjugal transfer of the pCF10 plasmid of Enterococcus faecalis is induced by the chromosomally encoded peptide pheromone cCF10. PrgX, a pCF10-encoded protein acts as the molecular switch controlling the prgQ operon that leads to conjugation. Pheromone binding to PrgX changes its oligomerization state and abolishes PrgX-mediated repression. Although the regulation of this system have been studied extensively using molecular techniques such as lacZ reporter gene fusions and gene knockout strains, neither pheromone signaling nor the effects of biofilm versus planktonic growth conditions on signaling have been studied at a single cell level. To analyze the population dynamics of pheromone signaling using flow cytometric techniques, a translational fusion of the gfp gene downstream of the pheromone-inducible prgB gene was created in pCF10. PrgB mediates donor:recipient aggregation and allows for efficient conjugation. Using this construct, planktonic cell populations were induced with various levels of pheromone and examined via flow cytometry to measure the induction state of the cells. Planktonic populations appeared as single peaks with the average GFP expression increasing with increased pheromone levels over time. When cells were grown in a biofilm, however, GFP expression levels were regulated differently. Induction of cells in a biofilm or recently dispersed from a biofilm demonstrated a population bifurcation following induction. A small portion of the cells became highly induced at low pheromone levels and increasing inducer concentration led to a higher proportion of cells which were induced. The differences between the regulation of planktonic cells and biofilm cells were seen as early as four hours after inoculation of the biofilms as well as in older biofilms. Cells that were dispersed from biofilms immediately prior to induction also demonstrated a population bifurcation following induction but these dispersed cells were induced at significantly lower levels of cCF10 inducer. This indicates that although the matrix likely inhibits diffusion of pheromone in intact biofilms, this is not responsible for the altered regulation observed in biofilm cells. These data indicate that the cellular changes that occur when a cell switches from planktonic to surface growth causes them to exhibit bistability in the regulation of conjugation. Enterococcus faecalis are known to grow in biofilms during infection. Understanding the appearance of a bistable switch in peptide response that only occurs during biofilm growth could have a significant impact on the treatment and prevention of the transfer of antibiotic resistance genes in biofilms.
ASM Conferences
SPEAKER ABSTRACTS
S5:1
Unexpected succession from compact to filamentous biofilms and its impact on mass transport H. Horn1, D. Taherzadeh1, T. R. Neu2, K. Garny2; 1Institute of water quality control, TU München, GERMANY, 2Helmholtz Centre for Environmental Research – UFZ, Magdeburg, GERMANY
In a study on biofilm development and detachment a heterotrophic biofilm derived from activated sludge was cultivated in a continuous once flow-through tube reactor. The system was exposed to constant substrate and laminar flow conditions. Confocal laser scanning microscopy (CLSM) and chemical analysis was used to study the impact of detachment and sloughing on the remaining but further developing biofilm structure. An unexpected succession from a compact to a filamentous biofilm surface structure was observed directly after heavy sloughing events. This was surprising as both hydrodynamic and substrate conditions were not changed and no specific re-inoculation was applied. It is speculated that the development of filamentous microorganisms may have two reasons: Firstly, filamentous microorganisms which may have been dormant at the base biofilm adapted quicker to the conditions after sloughing. Secondly, other bacteria attached after sloughing to the remaining base biofilm quickly adapted and grew into a filamentous biofilm. Although CLSM images showed a completely different biofilm structure before and after sloughing the overall biofilm performance in terms of substrate conversion rates remained constant. A one dimensional model approach revealed that key parameters for mass transfer and diffusion have to be changed by half an order of magnitude after the start of filamentous growth to match the experimental results. The combination of experimental and simulation results are significant for applied aspects of biofilm growth and detachment under real world situations as demonstrated in this laboratory once flow through system. A further consequence of the changing biofilm structure was a change in physicochemical processes (i.e. substrate transport). The adapted model can only be applied as diagnostic tool. Nevertheless, it helps to understand the interaction of hydrodynamics, structures and processes in microbial biofilms. So far, such an unsteady behaviour of cannot be simulated with a generalized biofilm model.
S5:2
Nitrification in Drinking Water Systems A. Camper; Montana State University, Bozeman, MT
Nitrification episodes in drinking water distribution systems (DWDS) and premise plumbing are becoming increasingly problematic as water utilities switch from chlorine to chloramine to avoid the production of disinfection by-products. The degradation of chloramine leaves residual ammonia which is the substrate for microbial nitrification. A nitrification episode in the DWDS is typically defined as a decrease in the chloramine residual concomitant with an increase in nitrite concentration and heterotrophic plate counts (HPC). Unfortunately, nitrification in the DWDS is nearly impossible to stop once it has begun, and there are currently no effective methods 5th ASM Conference on Biofilms
available, short of shock chlorination, to reverse a nitrification event. Research has focused primarily on the contribution of autotrophic ammonia oxidizing bacteria and nitrite oxidizing bacteria to nitrification episodes seen in drinking water. Recent studies have shown that in environments such as soils and oceans, other groups of microorganisms contribute to nitrification more than what was previously known. Thus, the contribution of other nitrifying organisms to nitrification in drinking water systems should be evaluated as well. Research done at the CBE is investigating the importance of autotrophic bacteria, heterotrophic bacteria, and archaea in biofilms of simulated drinking water distribution systems. These reactors are actively nitrifying have been found to contain heterotrophic nitrifying bacteria and nitrifying archaea. Culturing and molecular methods have been developed and are being used to isolate and enumerate the organisms in the reactors. Other characterizations include determining rates of nitrification to assess whether the activity of these alternative organisms can account for the extent of nitrification in full scale systems. These data will be crucial in redefining how we view the ecology and possible control of nitrification in drinking water and other low organic carbon environments.
S5:3
Electrogenic activity of cyanobacterial biofilms I. V. Baskakov, J. M. Pisciotta, Y. Zou; University of Maryland Biotechnology Institute, Baltimore, MD
Cyanobacteria account for 20-30% of the Earth’s primary photosynthetic productivity and are found all over the world, from high temperate geysers to Sahara desert or Antarctic glaciers. Photosynthesis in cyanobacteria converts roughly 400 TW of solar energy into biomass-stored chemical energy, an amount that exceeds the energy demand of human society more than 25 times. In the current study, we tested electrogenic activity in 10 genera of cyanobacteria (9 biofilm-forming and one planktonic). We found that diverse genera of cyanobacteria display a conserved light-dependent electrogenic activity, i.e. transport of electrons to the extracellular environment in response to illumination. Biofilm-forming genera had a much higher electrogenic capability than a planktonic strain, however electrogenic activity was common to all ten genera tested. A naturally-growing photosynthetic biofilm collected from a fresh-water pond also displayed electrogenic activity indicating that this phenomenon is not limited to the individual genera cultivated in laboratory conditions. 16s and 23s DGGE analyses identified several genera of cyanpbacteria (Phormidium, Leptolyngbya, Pseudoanabaena and Cyanothecea) and a green algae (Scenedesmus) in the mixed biofilm consortia. Treatment with site-specific inhibitors revealed that electrons are derived from the photosynthetic electron transfer chain and that plastoquinone plays a central role in extracellular electron transfer. The yield of electron discharge from cyanobacteria to the environment was calculated as a percentage of electrons derived from the biophotolysis of water by photosystem II. While the yield of electron discharge appeared to be low under standard conditions (ranged from 0.68% for Synechocystis 6803 to 3.1% for Calothrix), it can be increased several fold by changing environmental factors or conditions for electron harvesting. Globally, cyanobacteria could convert sufficient amounts of 23
SPEAKER ABSTRACTS solar energy into electrical power to satisfy the human demand for energy. We postulate that the electrogenic activity represents a conserved mechanism evolved in cyanobacteria for adapting to unfavorable light conditions.
S5:4
Adhesive behavior of Coagulase Negative Staphylococci isolated from Italian Cheese G. Scoarughi1, R. Papa1, C. Avanzolini1, A. Cellini1, E. Mileto1, T. Bertuccio2, V. Cafiso2, S. Stefani2, P. Cocconcelli3, L. Selan1, M. Artini1; 1Sapienza University, Rome, ITALY, 2University, Catania, ITALY, 3Catholic University, Piacenza, ITALY
Background: Coagulase-negative staphylococci (CNS) are saprophytic flora on the skin and mucous membranes of warm-blooded animals and humans, but are also isolated from a wide range of foodstuffs such as meat, cheese and milk (1). All strains recognized for their technological value and involved in desirable reactions (flavor and aroma formation) during the ripening of cheeses, are CNS species. In other circumstances, some CNS can act as pathogens (2). For example S. saprophyticus, an ubiquitous species in the enviroment, can cause acute urinary tract infections. In this study various CNS isolates were analyzed, in order to determine their adhesive behavior at different temperatures, corresponding to ripening and maturation temperatures of cheese and mammalian host temperature. Methods: 112 CNS cheese isolates from five different types of Italian cheese and 4 reference strains were used. Planktonic growth and biofilm formation was assessed at 8°C, 20°C and 37°C. Proteins were fractionated by SDS-PAGE and autolytic patterns were assessed by zymogram analysis. A proteomic study was designed to identify surface proteins affected by different temperatures. Results: Among 112 CNS isolates four main species were prevalent: S. saprophyticus, S. vitulinus, S. equorum and S. caprae. All strains were analyzed for their ability to grow and form biofilm at 20°C and 37°C. Among these, 8 strains were selected for further analysis. They formed biofilm also at 8°C. Surface protein pattern at the defined temperatures was also analyzed. Furthermore, in order to find an homology between the best characterized CNS S. epidermidis a genomic analysis for icaADBC locus and atlE was carried out. Conclusions: CNS strains belonging to the same species showed different adhesive properties. Moreover, the planktonic optimal growth temperature was not necessarily the optimal sessile condition. Surface protein and autolytic patterns do not display dramatic changes among different temperatures. Furthermore, these patterns do not correlate either with the species or with the source of the isolate. References 1Irlinger. Int J Food Microbiol 2008;126:302. 2Piette, et al., Vet Microbiol 2009;134:45.
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Biofilm Based Wound Care R. Wolcott; Southwest Regional Wound Care Center, Lubbock, TX
The literature suggests that over 16 million people each year in the United States contract a chronic infection. The majority of these chronic infections have been directly linked to biofilm phenotype bacteria. More importantly, over 500,000 people die 24
from chronic infections each year (ventilator associated pneumonia 67,000, chronic wounds 50,000, COPD 30,000, etc.). Chronic infections produced by biofilm phenotype bacteria have several common characteristics regardless of the tissue involved, including a fairly stable attachment site and a host hyper inflammatory state including excessive neutrophils. Wounds are a chronic infection of the skin and are the most accessible biofilm disease to study. Acute wounds have very little biofilm on their surface, while chronic wounds have a preponderance of biofilm across their surface. It is difficult, but possible utilizing current molecular methods, to demonstrate direct cause and effect between biofilm phenotype bacteria (wound biofilm) and the hyper inflammatory milieu of the wound along with impairment of host healing mechanisms. However this knowledge is incomplete. To establish wound biofilm’s pivotal role in producing a chronic infection which subverts wound healing, a more indirect approach was taken. A definable subgroup of patients with chronic wounds that have extensive wound healing literature (that is patients with critical limb ischemia) were identified in our practice. These 190 patients were subjected to the same standard of care as described in the literature. However, our patients with critical limb ischemia also underwent a treatment regimen focusing on suppressing wound biofilm. This regimen included frequent (weekly) debridement, use of selective biocides which were rotated each month, use of targeted antibiotics at high doses for long durations and use of anti-biofilm agents. Anti-biofilm agents are a difficult to define eclectic group of agents. These agents are generally considered not cidal to bacteria but are efficacious by breaking down biofilm defenses such as attachment, EPS formation, breaking down EPS components, quorum sensing inhibitors and others. By utilizing this four-pronged approach of concurrent strategies which were rotated frequently (dynamic), clinically there was less biofilm identified in the wounds. More importantly, a statistically significant improvement in wound healing was obtained by utilizing biofilm based wound management than was obtained by using state of the art standard of care management. This clearly demonstrates biofilm’s role in the impairment of healing in chronic wounds. These therapeutic principles should translate to other biofilm disease.
S6:2
Targeting a Haemophilus influenzae protein expressed during biofilm growth in vivo for development of a vaccine for otitis media J. A. Jurcisek1, L. A. Novotny1, C. Armbruster2, W. E. Swords2, L. O. Bakaletz1; 1The Research Institute at Nationwide Children’s Hospital, The Ohio State University College of Medicine, Columbus, OH, 2 Wake Forest University School of Medicine, Dept. of Microbiology and Immunology, Winston-Salem, NC
Otitis media (OM), or middle ear infection, is one of the most highly prevalent pediatric diseases worldwide. In addition to acute disease, OM is often recurrent and/or chronic. In this latter regard, it is estimated that globally between 65 and 330 million children suffer from chronic secretory OM. Contributing to the chronic and recurrent nature of OM is the ability of the bacterial pathogens involved to form and ASM Conferences
SPEAKER ABSTRACTS reside within a biofilm in the human upper respiratory tract. During the disease course of OM, nontypeable Haemophilus influenzae (NTHI), the predominant pathogen of chronic and recurrent OM, induce the formation of a biofilm community within the middle ear cavity. One bacterial protein expressed within this biofilm is PilA, the majority subunit of the type IV pilus (Tfp). NTHI Tfp are involved in multiple key biological functions including adherence, twitching motility, uptake of foreign DNA, long- term colonization of a mucosal surface and biofilm formation. We hypothesized that due to its surface location within assembled Tfp, significant deduced amino acid conservancy among strains and the essential roles of Tfp in NTHI biology and pathogenesis, PilA might represent an ideal target for the design of a vaccine candidate to prevent OM due to NTHI. We thus tested recombinant soluble PilA (rsPilA) using a traditional (parenteral) delivery route for its ability to prevent the development of experimental NTHI-induced OM in a chinchilla model. We also assayed rsPilA for its ability to mediate eradication of a biofilm which was already established within the chinchilla middle ear space when delivered via a non-invasive (intranasal) immunization route, in order to determine its potential value as a ‘therapeutic’ vaccine. Regardless of route of delivery used, immunization with rsPilA induced the formation of antibodies that inhibited or abrogated multiple biological functions of NTHI Tfp in vitro. Moreover, rsPilAimmunized animals demonstrated either significant protection against the development and severity of experimental OM, or were able to rapidly resolve existing disease including the presence of a bacterial biofilm within the middle ear space. Further evaluation of the ability of intranasally delivered rsPilA to induce the eradication of biofilms formed by two heterologous NTHI strains yielded similar results. In both model systems, the demonstrated protective efficacy was statistically significant when compared to animals immunized with adjuvant only. Targeting bacterial proteins expressed within a biofilm for vaccine candidate design may represent an approach with broad utility for both other NTHI-induced diseases of the human airway, as well as additional diseases wherein pathogenesis includes a biofilm component to the disease course. Work was supported by NIH/NIDCD R01 DC003915 to L.O.B. and NIH/NIDCD R01 DC10051 to W.E.S.
S6:3
E. coli Biofilms, Bottlenecks, and Host Responses in Urinary Tract Infection S. Hultgren, H. L. Stoever; Washington University Medical School, St. Louis, MO
Antibiotic resistant pathogens and the emergence of new diseases, and involvement of bacterial pathogens in diseases formerly thought to be due to non-infectious agents are on the rise. In addition, the ability of bacterial pathogens to reside in intracellular niches and form biofilm-like communities, further complicates the treatment of infectious disease. All of these factors have rekindled the need to understand the “molecular logic” of virulent bacteria so that we may better develop new therapies and preventions. One of the first steps of an infectious event is the specific binding of the pathogen via an adhesin to the host epithelium, allowing the pathogen to gain a foothold. We have uncovered the fine details of a molecular 5th ASM Conference on Biofilms
machine, called the chaperone/usher pathway, used by diverse pathogenic bacteria to assemble adhesive fibers called pili on their surfaces. Pili initiate host-pathogen interactions critical in the pathogenic processes of a wide range of bacteria. We discovered that periplasmic chaperones serve as folding templates for pilus subunits, actively shaping the final structure of pilus subunits in a mechanism that represents a surprising twist of the classic Anfinsen postulate. Using uropathogenic Escherichia coli (UPEC) as a model system, we discovered that the type 1 pilus adhesin, FimH, mediates UPEC specific binding and entry into superficial umbrella cells lining the bladder lumen. This event is critical for disease progression and activates a complex genetic cascade within the bacteria leading to a pathogenic cycle involving the formation of clonal intracellular bacterial communities (IBCs) that undergo a defined maturation and differentiation program requiring the intracellular expression of type 1 pili. Both the intracellular and biofilm nature of these communities has implications for the effectiveness of antibiotic treatment. Our understanding of the critical role that pili play in the pathogenic cycle of UPEC and the molecular details of how pili are assembled and the adhesive interactions mediated by FimH, have allowed us to develop small molecule inhibitors of pilus assembly and function. Further, knowledge gained from our studies of the host response to UPEC infection has important implications for disease outcome, epithelila renewal and bladder cancers
S6:4
Gallbladder carriage of Salmonella in mouse and man is enhanced by biofilm formation on gallstone surfaces R. W. Crawford1, R. Rosales-Reyes2, M. de la Luz Ramírez-Aguilar2, O. Chapa Azuela3, C. Alpuche-Aranda4, J. S. Gunn1; 1The Ohio State University, Columbus, OH, 2Universidad Nacional Autónoma de México, Mexico City, MEXICO, 3Hospital General de México, Mexico City, MEXICO, 4Instituto Nacional de Referencia Epidemiológica, Mexico City, MEXICO
A percentage of those infected with S. enterica serovar Typhi (S. Typhi) will develop into asymptomatic, chronic carriers of the bacteria, with the main site of carriage as the gallbladder. It is not well understood why certain individuals become carriers, but it has been shown that those with gallbladder abnormalities, such as gallstones, are at a much greater risk for Salmonella carriage. We have previously shown that salmonellae form bilemediated biofilms on human gallstones and cholesterol-coated surfaces in vitro and thus hypothesize that biofilm formation on cholesterol gallbladder stones facilitates typhoid carriage. Naturally resistant 129X1/SvJ mice fed a lithogenic diet developed cholesterol gallstones and demonstrated enhanced colonization of gallbladder tissue and bile. These mice also showed pronounced fecal shedding during persistent S. enterica serovar Typhimurium infection. Furthermore, cholesterol gallstones were shown to harbor significant numbers of salmonellae that increased with time post infection. The diet and the presence of gallstones did not generally increase Salmonella colonization, as there were no significant differences in bacteria recovered from the spleen. In a human study conducted in typhoid endemic Mexico City, individuals seeking cholecystectomy due to gallstones were analyzed for S. Typhi carriage. Results 25
SPEAKER ABSTRACTS indicate that 5% of patients with cholelithiasis carried S. Typhi and that bacterial biofilms could be visualized on gallstones from 3 of 4 of these carriers. Gallstone biofilm formation was not observed in patients positive for other bacteria, suggesting that gallstone biofilms are a typhoid specific phenomenon. Histological examination of gallbladder tissue from mouse and human carriers demonstrate a lack of immune cell infiltration. These findings offer direct evidence that gallstone biofilms occur in humans and mice carrying salmonellae asymptomatically in their gallbladders, and that gallstones facilitate gallbladder colonization and shedding. An increased understanding of S. Typhi chronic infection of the gallbladder may lead to new therapeutic approaches for elimination of the carrier state.
S6:5
Candida albicans-Staphylococcus aureus dual-species biofilms mediate invasive staphylococcal infection B. M. Peters1, M. A. Jabra-Rizk1, J. G. Leid2, J. W. Costerton3, M. E. Shirtliff1; 1University of Maryland - Baltimore, Baltimore, MD, 2 Northern Arizona Univ., Flagstaff, AZ, 3University of Southern California, Los Angeles, CA
In nature, microbes rarely grow as mono-species planktonic forms. Rather, most are associated as polymicrobial biofilms attached to host and environmental surfaces. The polymorphic fungus Candida albicans and bacterium Staphylococcus aureus are both capable of forming biofilms, can be co-isolated from virtually all human mucosal sites, and are responsible for diverse localized and deep-seated infections. Despite causing a large number of mono-species infections, they have been implicated as co-infecting organisms in a variety of human diseases. Therefore, various approaches were used to understand the intricacies of this medically relevant dual-species biofilm. Confocal scanning laser microscopy and species specific PNAFISH analysis revealed that S. aureus possesses an affinity for the pathogenic hyphal form of C. albicans in vitro more so than the commensally associated yeast form. C. albicans hyphae are directly invasive and have been demonstrated previously to penetrate host epithelial tissue. Therefore, as a consequence of their interaction, we hypothesized that S. aureus may use the hyphae of C. albicans to become invasive. An in vitro co-infection of human keratinized epithelial cells demonstrated the ability of S. aureus to attach to C. albicans hyphae and penetrate epithelial cells, while cells infected with S. aureus alone showed no such breach of the epithelial surface. A similar invasive pattern was also demonstrated during infection of explanted murine tongue tissue. An in vivo mouse model of oral co-infection established the ability of S. aureus to translocate to the kidneys only in the presence of C. albicans while mono-species infections were either cleared or remained localized. Co-culture DIGE proteomic analyses were subsequently used to unravel the complexities of this inter-Kingdom relationship and revealed several virulence-related proteins to be up-regulated, including staphylococcal Ldh1 and CodY. Therefore, we present a novel mechanism of invasive S. aureus infection facilitated via interactions with C. albicans in an oral polymicrobial biofilm setting. These findings have significant impact on the consideration of treatment options for those afflicted with complicated biofilm mediated infections. 26
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Immune System versus Biofilms:How chemistry may give “an upper hand” M. Givskov; Dept. of International Health, Immunology and Microbiology, University of Copenhagen, DENMARK
The biofilm lifestyle plays a key role in many chronic bacterial infections. Pseudomonas aeruginosa is known as a notorious biofilm former. During establishment, bacteria emerge in a “harmless” state (no expression of virulence factors) as they increase in numbers and build biofilms. In the biofilm mode, P. aeruginosa uses “quorum sensing or QS” communication to inform its fellow bacteria about progress in the infectious process. When the QS system signals, the biofilm launches a cocktail of destructive virulence factors which in turn attract phagocytic white blood cells (PMN’s), a major cellular component of the innate immune system. To offer protection, a rhamnolipid based shield is also launched which on contact kill attaching PMN’s. Work in progress indicates that cross communication between bacteria and immune system are important in these Eukaryote-Prokaryote interactions. Small molecule signal blockers (QS inhibitors) can be introduced which do not kill bacteria but block QS and cross communication. The shield is not formed and the PMNs can actively break down the bacterial biofilm. QS inhibitory chemistry is available in a variety of ecological niches from coral reefs to herbs.
S7:2
Bacterial Biofilms on Implanted Surgical Materials in the Abdominal Wall S. Kathju; Center for Genomic Sciences, Pittsburgh, PA
Bacterial biofilms on implanted foreign bodies have been implicated in numerous infectious clinical scenarios, including central access catheters, heart valves, and orthopedic hardware, but have been little studied on implanted materials in the abdominal wall. We report on the clinical and microbiological characteristics of a series of patients with chronic surgical site infections after gastric bypass surgery. In each case the infection was found to derive from retained permanent suture material used for musculofascial closure. Confocal microscopy demonstrated abundant viable bacteria in biofilm configuration on the suture surfaces, and fluorescent in situ hybridization was able to speciate resident bacteria. In one case the localized suture-associated infection had progressed to frank gastrocutaneous fistula. Complete removal of all foreign suture material was required for resolution of the chronic infections, which had previously failed numerous courses of antibiotics and inadequate local wound care, consistent with the biofilm paradigm. We have also examined implanted herniorrhaphy meshes for bacterial biofilms after explantation surgery. Patients were operated for a variety of indications, including chronic nonhealing wounds, persistent pain, recurrent hernia, enterocutaneous fistula etc. Confocal microscopy demonstrated biofilm accumulation on the ex vivo mesh implants, and fluorescent in situ hybridization was used to identify resident bacterial species. In vitro experiments showed that mesh material is hospitable to polymicrobial biofilm formation using recovered clinical bacterial strains. These observations demonstrate that bacteASM Conferences
SPEAKER ABSTRACTS rial biofilms on implanted surgical materials in the abdominal wall are important contributors to the morbidity of abdominal surgery.
S7:3
Human Oral Microbial Biofilms are Multi-species Communities P. Kolenbrander; National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD
Human oral bacteria form multi-species communities rather than mono-species communities. This activity is likely facilitated by their ability to coaggregate which is the cell-to-cell recognition between genetically distinct bacteria. Close proximity between cells in biofilms gives an opportunity for metabolic exchange. We have been investigating the ability of multispecies communities to grow on saliva as the sole source of nutrient. Specificity among species for mutualistic growth was evident. Initial colonizing species of tooth enamel interact with early, middle and late colonizers, as evidenced by their growth in two kinds of biofilm model systems: flow cell model and static model. The species studied included Streptococcus oralis, Streptococcus gordonii, and Actinomyces oris (all initial colonizers); Veillonella sp. PK1910 (early colonizer); Fusobacterium nucleatum and Porphyromonas gingivalis (middle colonizers), and Aggregatibacter actinomycetemcomitans (late colonizer). S. oralis often inhibited other species, but S. gordonii could overcome this inhibitory activity. A. actinomycetemcomitans could grow mutualistically with F. nucleatum and Veillonella sp. PK1910 in the absence of initial colonizers, supporting the idea of sequential colonization of enamel. P. gingivalis, which has been noted previously in samples taken 6 hours after enamel cleaning, grew mutualistically with the other tested species except S. oralis. Such specificity also supports sequential colonization. Collectively, many two-species and three-species communities exhibited mutualism, indicating a strong metabolic dependence among oral species for growth on saliva.
S7:4
In situ measurement of biofilm activity by FISH: implications for diagnosis in medical biofilms A. Moter1, A. Petrich1, C. Mallmann1, D. Schmiedel1, K. Rugor1, M. Musci2, R. Hetzer2, U. B. Göbel1; 1Charite, Berlin, GERMANY, 2 Deutsches Herzzentrum Berlin, Berlin, GERMANY Medical biofilms are difficult to treat because of their resistance to antibiotics. Common routine diagnostics comprise cultivation and testing for antibiotic susceptibility by determining the minimal inhibitory concentrations (MIC). These tests imply disintegration of the biofilm and growth of the bacteria in vitro. Therefore the routine procedures might miss bacteria in stationary phase, presumable persister cells and give no information about spatial distribution of the viable cells or active biofilm layers. We used Fluorescence in situ hybridization (FISH) to visualize vegetations on heart valves of endocarditis patients. We detected structured biofilms that were highly organized showing stratification with zones of elevated rRNA levels alternating with layers of only DAPI positive cells that were presumably dead. We also found FISH positive bacteria 5th ASM Conference on Biofilms
in culture negative samples and samples from patients under antibiotic therapy. The high signal intensity of FISH correlates to a high ribosomal content of the bacteria indicating metabolic activity at the time of surgery. To detect the activity of single bacterial cells more precisely we developed FISH probes for the 16S-23S internal transcribed spacer that is only present in actively transcribing cells. Using this spacer FISH we also detected positive cells in heart valves of patients under therapy. This technique might allow visualizing the effect of antimicrobial therapy on in vivo grown biofilms in situ. In summary, biofilm features like spatial organization and limited growth in vitro play an important role in human endocarditis. These findings confirm the higher resistance of bacteria toward antibiotic treatment in biofilms in the clinical setting. They stress the point that our current diagnostic techniques regarding cultivation and antibiotic resistance testing in vitro are not satisfactory. In situ techniques might further our knowledge about the efficiency of antibiotic treatment on biofilms in vivo.
S7:5
Antibiofilm activity of nanosized magnesium fluoride J. Lellouche, E. Kahana, S. Elias, A. Gedanken, E. Banin; Bar Ilan University, Ramat Gan, ISRAEL
The ability of bacteria to develop antibiotics resistances and colonize abiotic surfaces by forming biofilms is a major cause of medical implant-associated infections and results in prolonged hospitalization periods and patient mortality. This raises the urgent need to find novel approaches to inhibit bacterial colonization of surfaces. One approach comes from recent progress in nanotechnology, which offers an opportunity for the discovery of novel compounds with antimicrobial activity as well as the use of “nano-functionalization” surface techniques. In this study, we present a first demonstration of the antibiofilm activity of metal fluoride nanomaterials. Using an unreported microwave-based synthesis of MgF2 nanoparticles (MgF2 Nps) in ionic liquid, we demonstrate their ability to inhibit biofilm development of two common nosocomial biofilm-forming pathogens. E. coli biofilm formation decreased in a dose-dependent manner and resulted in approximately 75% reduction in biofilm biomass and S. aureus biofilm formation was completely inhibited. Scanning and transmission electron microscopic techniques indicated that the MgF2 Nps attach and penetrate into the cells. Flow cytometry analysis revealed that the Nps caused a disruption in the membrane potential. The MgF2 Nps also induced membrane lipid peroxidation and once internalized can interact with chromosomal DNA. Based on these findings we further explored the possibility of using the MgF2 Nps to coat surfaces and inhibit biofilm formation. A microwave synthesis and coating procedure was utilized to coat glass coupons. The MgF2 coated surfaces effectively restricted biofilm formation of the tested bacteria. This study emphasizes the potential of using metal fluoride nanoparticles as a new approach for the design of sterile surface coatings that may be useful for various medical applications.
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SPEAKER ABSTRACTS
S8:1
Altering Biofilm Cohesion as a Control Strategy P. S. Stewart1, E. R. Brindle1, D. A. Miller2, S. Aggarwal3, R. M. Hozalski3; 1Center for Biofilm Engineering, Bozeman, MT, 2Montana State University, Bozeman, MT, 3University of Minnesota, Minneapolis, MN
Bacteria in biofilms evade killing by antimicrobial agents and even when antimicrobials do kill biofilm cells they may fail to remove the biofilm. It is therefore attractive to develop control strategies based on disrupting biofilm cohesion. This strategy requires the development of techniques to measure biofilm material and mechanical properties and to assess the affect of treatments on these properties. Three approaches for characterizing the viscoelastic and failure properties of Staphylococcus epidermidis biofilms were developed and applied to demonstrate alterations in material properties resulting from chemical or enzymatic treatments. All three methods involve application of a force to the biofilm before and after treatment. The first system used video microscopy to quantify the time-dependent creep of a biofilm cell cluster in response to a defined increase in the fluid flow rate. The second system used atomic force microscope indentation to measure force versus distance curves in hydrated biofilm specimens. The third approach employed a calibrated microcantilever to pull biofilm apart and thereby quantify the failure strength. These methods demonstrated that: 1) biofilms behave as viscoelastic materials, 2) chemical and enzymatic treatments result in quantifiable alterations in biofilm material properties, 3) biofilm cohesive failure can be visualized and measured, and 4) significant biofilm removal can be achieved by combining sustained forces (e.g., fluid shear) with treatments that weaken biofilm cohesion. Treatment of S. epidermidis biofilms with iron chloride or chlorhexidine caused biofilms to become stiffer and behave more like a solid, elastic material. Treatment of these biofilms with urea or dispersin B caused the biofilms to become weaker and behave more like a viscous fluid.
S8:2
Biofilm matrix-degrading enzymes: useful research tools and potential anti-biofilm therapeutics J. Kaplan; New Jersey Dental School, Newark, NJ
Bacterial cells in a biofilm are encased in a self-synthesized extracellular matrix that holds the cells together in a mass and firmly attaches the bacterial mass to the underlying surface. The extracellular matrix of most biofilms contains polymers such as DNA and polysaccharides. Enzymes that degrade matrix polymers and destroy the physical integrity of the biofilm matrix have been shown to inhibit biofilm formation, detach pre-formed biofilms, and sensitize pre-formed biofilms to killing by antimicrobial agents, bacteriophages and macrophages in vitro. This talk will focus on two well-studied matrix-degrading enzymes, deoxyribonuclease I and the glycoside hydrolase dispersin B. These enzymes have proven to be useful tools for identifying the structural components of the biofilm matrix, for determining the physical and chemical properties of the matrix, and for elucidating the role of matrix polymers in 28
biofilm-mediated biocide resistance in a variety of bacterial species. Clinically, the use of matrix-degrading enzymes may be an attractive anti-biofilm strategy for the treatment and prophylaxis of biofilm-related infections. These enzymes could reduce or eliminate the need for conventional antibiotics, and may be less prone to the evolution of resistance because they do not kill bacteria or inhibit their growth.
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The Future Tool Box for Biofilm Control from Nanosciences to Transcriptomics H. M. Lappin-Scott1, S. K. Burton2; 1Bangor University, Bangor, UNITED KINGDOM, 2University of Exeter, Exeter, UNITED KINGDOM
The Future Tool Box for Biofilm Control from Nanosciences to Transcriptomics
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C-di-GMP Signaling and Biofilm Formation in vibrio cholerae F. Yildiz; University of California, Santa Cruz, Santa Cruz, CA
C-di-GMP production and degradation is controlled by proteins containing the GGDEF and EAL/HDGYP domains, respectively. Vibrio cholerae, the causative agent of the disease cholera, has 62 genes predicted to encode GGDEF/EAL/ HD-GYP domain proteins. I will discuss the molecular mechanisms through which c-diGMP signaling systems affect biofilm formation in V. cholerae.
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Signal Transduction of c-di-GMP in Pseudomonas aeruginosa S. Häußler1, J. Schmidt2, D. Bertinetti3, T. Becker1, M. Morr1, F. Schwede4, J. Wissing1, Z. Magnowska1, L. Jänsch1; 1Helmholtz Centre for Infection Research, Braunschweig, GERMANY, 2Helmholtz Centre for Infection Research, Branuschweig, GERMANY, 3 Institute for Biology, Biochemistry Division, University of Kassel, Kassel,, GERMANY, 4Research and Development, BIOLOG Life Science Institute,, Bremen, GERMANY
Living in matrix enclosed biofilms is a common lifestyle of many bacteria. One key component controlling the transition between a motile, single-cell way of life and community based biofilm formation is the second messenger bis-(3’-5’) cyclic di-GMP (c-di-GMP). Low intracellular levels of c-di-GMP promote a planktonic lifestyle, where bacteria are motile and virulence factors are expressed, and high c-di-GMP levels stimulate multicellular behavior, adherence and biofilm development. The synthesis of c-di-GMP is mediated by diguanylate cyclases containing a conserved GGDEF domain whereas the degradation is controlled by phosphodiesterases containing a conserved EAL domain. GGDEF and EAL domains are often fused to transmembrane and/or signal input domains. Thereby, c-di-GMP links environmental stimuli to an adaptive answer of the bacterial cell. However, little is known about the molecular details of the c-di-GMP signaling network. Up to date, ASM Conferences
SPEAKER ABSTRACTS only few types of c-di-GMP effector molecules are known. Among them are PilZ domain proteins and proteins containing a RXXD motif, which was first identified as an allosteric site of product feedback inhibition (I-site) in many diguanylate cyclases. In this work we used a c-di-GMP coupled sepharose for the isolation of c-di-GMP binding proteins of Pseudomonas aeruginosa PA01 which were subsequently identified by mass spectrometry. This approach yielded in seven putative c-di-GMP binding candidates, among others one protein with a PilZ domain and one containing a degenerated GGDEF-motif and the I-site motif RXXD. We report here on one further identified c-di-GMP binding protein that interferes with flagella driven motility in a c-di-GMP depending manner.
S9:3
How does c-di-GMP control biofilms in Pseudomonas aeruginosa? C. S. Harwood; University of Washington, Seattle, WA
High intracellular cyclic di-GMP [bis (3’-5’)-cyclic di-GMP] (cdi-GMP) promotes biofilm formation and reduces motility in many bacterial species. Conversely, low concentrations of intracellular c-di-GMP are correlated with increased motility. Such is the case for the opportunistic pathogen and model biofilm organism Pseudomonas aerugionsa. Like other gram-negative bacteria, P. aerguinosa has many genes encoding proteins with predicted GGDEF domains responsible for the synthesis of c-di-GMP. It also has genes with predicted EAL and HD-GYP domains for c-di-GMP hydrolysis. GGDEF-domains have diguanylate cyclase (DGC) activities that catalyze the synthesis of c-di-GMP from GTP, whereas EAL- and HD-GYP domains have phosphodiesterase (PDE) activities that catalyze the degradation of c-di-GMP to pGpG, which spontaneously converts to GTP. The general parameters of c-di-GMP activity have been established in P. aeruginosa, but environmental inputs that stimulate c-di-GMP synthesis, intracellular trafficking of c-di-GMP and mechanisms of c-di-GMP action are just beginning to be explored. As with other bacteria, only a small subset of P. aeruginosa EAL/GGDEF gene mutants has discernable mutant phenotypes. However such mutants have provided valuable clues about how cells produce and traffic cdi-GMP intracellularly. Also, several different novel c-di-GMP receptor proteins have been identified in P. aeruginosa and this has expanded our understanding of the range of functions that c-di-GMP can influence. In this presentation, I will describe our work on FleQ, a transcription factor that derepresses expression of exopolysaccharide (EPS) synthesis genes, as well as other genes important for biofilm formation, in response to c-di-GMP. In will also discuss our work the GGDEF protein WspR. WspR and its associated Wsp signal transduction system are important modulators of biofilm formation in P. aeruginosa. We are interested in fully understanding the in vivo and in vitro properties of WspR and how they influence Pel EPS synthesis.
5th ASM Conference on Biofilms
S9:4
Nitric oxide-induced dispersal in Pseudomonas aeruginosa biofilms involves cyclic di-GMP N. Barraud1, D. Schleheck1, J. Klebensberger1, J. S. Webb1, D. J. Hassett2, S. A. Rice1, S. Kjelleberg1; 1School of Biotechnology and Biomolecular Sciences and Centre for Marine Bio-Innovation, University of New South Wales, Sydney, AUSTRALIA, 2Department of Molecular Genetics, Biochemistry and Microbiology,, University of Cincinnati College of Medicine, OH
Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa, at low, non-toxic concentrations. NO was further shown to increase the motility of cells and the susceptibility of cells to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. Here, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE-specific inhibitor studies, (ii) enzymatic assays to measure c-di-GMP-PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-diGMP signaling in triggering the dispersal event induced by NO in P. aeruginosa biofilms, as dispersal involves increased PDE activity and an overall decrease in intracellular c-di-GMP levels. Furthermore, gene expression studies indicated global responses to low, non-toxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis-like regulator BdlA, which harbors two PAS domains and can indirectly modulate c-di-GMP levels is involved in the biofilm dispersal response induced by NO. This study provides evidence for a direct link between NO and c-di-GMP in the regulation of biofilm dispersal in P. aeruginosa. This regulatory pathway may be conserved across microbial species. Indeed, NO which is ubiquitous in nature, was found to induce dispersal also in biofilms of several Gram-positive and Gram-negative species, yeast as well as mixed species biofilms.
S9:5
Regulation of c-di-GMP signaling by O2 in E. coli and B. pertussis J. Tuckerman1, X. Wan2, M. Alam2, G. Gonzalez1, M. Gilles-Gonzalez1; 1University of Texas Southwestern Medical Center, Dallas, TX, 2 University of Hawaii, Honolulu, HI
The bacterial second messenger bis-(3’-5’)-cyclic diguanosine monophosphate (c-di-GMP) controls many aspects of bacterial adaptation to a planktonic versus a biofilm lifestyle. Our interests focus on two aspects of c-di-GMP signaling: 1) understanding how environmental signals, especially O2, regulate levels of c-di-GMP, and 2) identifying and characterizing novel molecular targets of c-di-GMP. We identified a pair of 29
SPEAKER ABSTRACTS O2-sensing phosphodiesterases nearly a decade ago, and to date O2 remains the only environmental signal shown to regulate c-di-GMP levels (Biochemistry (2000) 39:2685; Biochemistry (2001) 40:3420). These sensors contain heme held either in a PAS or a modified globin domain. We recently discovered BpeGReg, a globin-coupled diguanylate cyclase from Bordetella pertussis, and we demonstrated that BpeGReg synthesizes c-di-GMP when ligated to O2. We developed a phosphodiesterase-coupled assay of BpeGReg because this cyclase is product-inhibited by c-di-GMP. Our measurements allowed us to make precise determinations of the enzymatic activation of BpeGReg by O2. We show that expression of BpeGReg in Salmonella typhimurium enhances biofilm formation, while knockout of the BpeGReg gene of B. pertussis results in decreased biofilm formation. These results represent the first identification of a signal ligand for any diguanylate cyclase and provide definitive experimental evidence that a globin-coupled sensor regulates c-di-GMP synthesis and biofilm formation. E. coli contains a diguanylate cyclase highly homologous to BpeGReg, but this protein is closely associated with an O2-regulated c-di-GMP phosphodiesterase. Our enzymatic analyses of this E. coli c-di-GMP homeostatic mechanism led us to discover a novel and unexpected high-affinity target of c-di-GMP in E. coli. Targets of c-di-GMP have been long sought-after, though to date very few have been identified.
S10:1
Phenazines control gene expression and community behavior in L. E. Dietrich , A. Price-Whelan , T. K. Teal , D. K. Newman ; 1 Massachusetts Institute of Technolog, Cambridge, MA, 2Massachusetts Inst. of Tech., Cambridge, MA 1
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A variety of bacteria are known to release colorful, redox-active pigments. Generally these have been regarded as antibiotics that serve the sole purpose of inhibiting competing organisms. Using RNA arrays we found that one such compound, the endogenous antibiotic pyocyanin from Pseudomonas aeruginosa PA14, acts as a signaling molecule that activates the transcription factor SoxR. In Escherichia coli, SoxR regulates the superoxide stress response. Surprisingly, bioinformatic analysis coupled with gene expression studies in P. aeruginosa PA14 and Streptomyces coelicolor A3(2) revealed that the majority of SoxR regulons in bacteria lack the genes required for stress responses despite the fact that many of these organisms still produce redox-active small molecules. This finding indicated that redox-active pigments play a role independent of oxidative stress. Using P. aeruginosa and S. coelicolor mutants that are defective in biosynthesis of these pigments, we found that they have profound effects on the structural organization of colony biofilms: while wild-type cells form thick and smooth colonies when spotted onto agar plates, the absence of pigments causes the cells to aggregate into visually striking, wrinkled (rugose) morphologies. We are currently testing the idea that rugosity, which results in an increased surface to volume ratio, is an adaptation to electron acceptor limitation. Our results show that redoxactive pigments are not merely virulence factors or antibiotics, but also play important, conserved roles in the developmental process of bacterial communities.
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S10:3
Composition, Structure and Function of Hot Spring Cyanobacterial Mat Communities D. M. Ward; Montana State University, Bozeman, MT
The cyanobacterial mats of alkaline siliceous hot springs in Yellowstone National Park have been studied for many decades as natural, totally microbial, model systems for understanding the principles of microbial community ecology. Cultivation-independent molecular methods have provided an objective view of community composition, structure and function. Genomic and metagenomic analyses have enabled identification of, and an initial understanding of the physiological character of, all of the major phylogenetic groups inhabiting the community, some of which have no cultivated representatives. The patterning along environmental gradients of the genetic variation found within each major phylogenetic group, combined with evolutionary simulation modeling, have enabled definition of the ecologically distinct species within each group. Metatranscriptomic and metaproteomic analyses promise to reveal how each predominant species contributes uniquely to community function in space and time, thus enabling a true systems biology analysis of the composition, structure and function of the most abundant members of the community.
S10:4
Biogenic alteration of limestone by fungi isolated from epilithic biofilms associated with Mayan monuments at Chichen Itza S. Gómez-Cornelio1, O. Ortega-Morales1, S. De la Rosa-García1, P. Quintana2, J. Narváez-Zapata3, A. Oliva-Hernández3, H. Bullen4, H. Urcia4, W. Santiago2; 1Universidad Autónoma de Campeche, Campeche, MEXICO, 2CINVESTAV Mérida, Mérida, MEXICO, 3 Centro de Biotecnología Genómica, Tamaulipas, MEXICO, 4Northern Kentucky University, Kentucky, KY
Complex interactions occur between microbial communities and dimensional stone which may eventually lead to deterioration of the underlying material. Chichen Itza is a large archaeological Mayan site in Mexico. Its temple of Kukulkan is the largest and most emblematic structure, recently recognized as one of the New Seven Wonders of the World. A recent visual inspection has shown that extensive areas of microbial biofilm growth occur on walls, ceilings, stelae and carved Mayan statues on exterior surfaces in most buildings at this site. Preliminary microscopic examination and culturing showed that, as in other Mayan sites, these biofilms were dominated by phototrophic cyanobacterial and microalgal communities, followed by heterotrophic bacteria and fungi. Given that the profound activities of bacteria leading to limestone monuments deterioration have received considerable attention, we carried out a characterization of the biogeochemical consequences of metabolic activity on limestone by selected fungi isolated from biofilms at Chichen Itza. Our survey yielded 63 fungal isolates derived mainly from black epilithic black biofilms material inoculated using two different methods and two culture media. Three fungal isolates identified by ribosomal intergenic spacer analysis (RISA), Penicillium oxalicum TM1H52, Annulohypoxylon stygium ASM Conferences
SPEAKER ABSTRACTS TM1H19, Rosellinia sp TM1H05 and shown to be very active in calcium carbonate-dissolving activity plate assay with the subsequent formation of mygenic fabrics, were further incubated in liquid media containing sterile experimental limestone coupons. Electron scanning microscopy (SEM) and X-ray diffraction (XRD) of crystalline precipitates showed they were composed of a mixture of calcite (CaCO3) and calcium oxalates and abundant fungal biofilm growth on limestone. Furthermore, in addition to calcium oxalates, fourier transform infrared (ATRFTIR) spectroscopy showed spectral features that are consistent with the presence of citric acid on the limestone surface incubated in the presence of Penicillium oxalicum TM1H52, as evidenced by evolution of strong bands developing at 1590 cm-1 and 772 cm-1. Annulohypoxylon stygium TM1H19 showed similar bands to the Penicillium oxalicum TM1H52, however its bands are much weaker in intensity, which suggests that citric acid may develop at a lower concentration, with low surface coverage. These results provide experimental evidence for the formation of secondary mycogenic minerals by naturally occurring fungi derived from microbial biofilms associated with Chichen Itza buildings, suggesting a potential role in limestone transformation. As calcium oxalates may be metabolized by certain associated bacteria, fungi capable of synthesizing oxalic acids and other organic acids may influence in trophic terms the other microbial populations occurring in this calcareous environments.
S10:5
The effect of carbon limitation on biofilm activity, structure and planktonic cell yield. E. Bester1, O. Kroukamp2, G. M. Wolfaardt2; 1University of Toronto, Toronto, ON, CANADA, 2Ryerson University, Toronto, ON, CANADA
The production and release of significant numbers of planktonic cells is a widely accepted attribute of biofilms exposed to continuous flow and excess carbon conditions. While the reported response of biofilms exposed to carbon limited conditions varies widely, the associated effect on biofilm-derived planktonic cell yield has not been investigated. The goal was therefore to investigate how biofilm activity, structure and planktonic cell yield are affected by carbon limitation. Pseudomonas sp. CT07 biofilms were cultured under continuous flow in conventional flowcells and in silicone tubes of a CO2 evolution measurement system for 5 days on a defined growth medium containing 1mM citrate as the sole carbon source. This was followed by an 8-day incubation period without added carbon and a 4-day shift back to medium with carbon. The flow rates were too high to support planktonic cell division; it is therefore assumed that effluent cells were produced by the biofilm. Biofilm parameters were determined daily with confocal scanning laser microscopy and COMSTAT image analysis, as were whole-biofilm CO2 production rates and viable planktonic cell production rates. The biofilms reached a steady state with respect to CO2 production rate, biovolume, average thickness and planktonic cell yield within the initial 5 days. The switch to no-carbon medium led to a 90% reduction in biofilm respiration rate within 1 hr; biofilm activity further decreased to 3% of the previous steady state production rate for the ensuing 8 days. The shift to zero carbon 5th ASM Conference on Biofilms
reduced cell yield from the tube-grown biofilms by 40% within 1 hr (to 2.14 ± 2.04 x 106 CFU.cm-2.hr-1) with subsequent reductions to a level of 4.79 ± 3.82 x 104 CFU.cm-2.hr-1. The effect on biofilm structure was only evident after 6-7 days of carbon limitation, with significant decreases in biovolume and thickness and increases in roughness and surface to volume ratio, which would enhance nutrient capture from the bulk-liquid. Re-introduction of carbon led to a rapid increase in CO2 production rate and planktonic cell yield within 5 hrs, as well as biofilm volume and thickness after 24 hrs. This demonstrates that planktonic cell yield from biofilms is enhanced when the biofilm is active, and maintained at diminished levels when environmental conditions are less favourable. Biofilm formation could therefore be described as a simultaneous survival and proliferation strategy, where the dominant strategy will likely depend on the prevailing environmental conditions.
S11:2
Use of laser capture microdissection microscopy and quantitative RT-PCR to characterize localized gene expression in biofilms. M. Franklin, K. S. Williamson, K. McInnerney, A. C. Pérez-Osorio; Montana State University, Bozeman, MT
The local environmental conditions of biofilms are dependent on the impinging aqueous solution and on the metabolic activities of cells within the biofilm. As nutrients from the environment are consumed and waste products generated, chemical gradients are established in biofilms. These gradients of oxygen, nutrients, waste products, and signaling molecules may intersect or overlap, creating unique microenvironments at local sites within biofilms. Bacteria sense these local environmental conditions and respond through gene induction/repression. As a result, bacteria at defined sites in biofilms are not only physiologically distinct from planktonic cells, but may also differ from other cells within the same biofilm. To characterize cells at different regions within biofilms, we combined laser capture microdissection (LCM) and quantitative RT-PCR to study gene expression within Pseudomanas aeruginosa biofilms. Initial experiments were performed with an inducible gene for the green fluorescent protein (gfp), and correlated local gfp mRNA abundances with cell fluorescence. Next, we analyzed wild type P. aeruginosa biofilms for local abundance of acpP mRNA and 16S rRNA. The housekeeping gene acpP was solely expressed in the top 30 µm of the biofilm, while abundance of 16S rRNA was relatively uniform throughout the biofilm layers. In order to provide a normalization strategy for relative gene expression, we assayed genome copy number of LCM samples by qPCR, This approach allowed quantification of transcripts on a per cell basis, and an estimation of cell growth status by comparing the ratio of 16S rRNA to genome copy number. Cells at the top of these biofilms had 16S rRNA/genome ratios similar to cells in a transition between exponential and stationary phase. Cells in the middle and bottom layers of the biofilms had ratios that were not significantly different from stationary phase cells. Since much of the biofilm seemed to be in a stationary phase growth state, we analyzed cellular amounts of the stationary phase sigma factor, rpoS, and a quorum sensing regulator, rhlR. Surprisingly, both of these genes had the greatest abundance at the top of the bioflms, with less than one transcript per cell 31
SPEAKER ABSTRACTS in the middle or bottom of the biofilms. Finally, we combined LCM with microarray analyses on cells isolated from the top and bottom of the P. aeruginosa biofilms. The microarray results confirmed the qRT-PCR findings regarding localized expression of acpP, rhlR, rpoS at the top of the biofilms. The results also confirmed the relatively uniform abundance of 16S rRNA throughout the biofilm regions, and provided information on expression processes that may be unique to certain regions within the biofilms.
S11:3
Physiology of Shewanella oneidensis Biofilm Cells A. M. Spormann; Stanford University, Stanford, CA Stability and, in particular, dissolution of microbial biofilms is governed by environmental control of the physiology of biofilm microbes. In order to obtain molecular and energetic insight into biofilm stability, we developed an in situ assay to query whether transcription or translation is required for cell detachment from a biofilm and whether biofilm stability is an energy-dependent processes. In inhibition experiments of 12 hr old biofilms of the obligate respiring γ-proteobacterium Shewanella oneidensis MR-1 using the transcriptional and translational inhibitors rifampicin, tetracycline, and erythromycin we found that neither transcription nor translation is required for a detachment response. Importantly, a reduction in cellular ATP concentration, induced either by oxygen deprivation or addition of the inhibitors of oxidative phosphorylation CCCP, DNP or CN- resulted in massive detachment, suggesting that metabolic energy is required to maintain biofilm integrity. This sensitivity to uncouplers was also observed in deletion mutants defective in mshA and mxdB, representing the two dominant gene systems in S. oneidensis determining biofilm formation, which suggests that neither Type IV pili nor exopolysaccharides lock cells irreversibly in a biofilm at that stage. Concomitant to an ATP decrease in wild type biofilm cells, the concentration of the cellular second messenger c-di-GMP, which was previously shown to positively modulate biofilm stability, also deceased, suggesting a link between energy metabolism and c-di-GMP signaling. While 60 hr old biofilms maintained the same susceptibility to transcriptional and translational inhibitors, the extent of uncoupler-induced cell loss was strongly reduced suggesting that the majority of cells in older biofilms maintain biofilm integrity by other means than cells of younger biofilms. Overall our data show that cellular physiology profoundly affects biofilm phenotype.
S11:4
In Vivo RNA Labeling and Separation - a Novel Tool for Biofilm Research N. A. Stankiewicz, S. Berensmeier, U. Obst, T. Schwartz; Forschungszentrum Karlsruhe, Karlsruhe, GERMANY
Bacterial stress response to natural and technical stress factors enhances the fitness of bacterial populations such as natural biofilms in hostile environments. These responses range from stress induced gene expression to the secretion of beneficial exoenzymes. Moreover, the intra cellular stabilization of certain groups of stress related mRNAs also seem to play a role in bacterial fitness under adverse conditions. This in vivo RNA labeling and separation technique is based on the incorporation of digoxigenin-11-uridine-5`-triphosphate (DIG-11-UTP) in 32
the RNA of active bacteria. Without any chemical or physical treatment of the populations this nucleotide analog was taken up by bacteria and was incorporated specifically in the de novo synthesized RNA in their natural habitat. Depending on the growth conditions, the population’s composition and the gene expression activity the assembly rate of DIG-11-UTP ranged from 1.2% to 12.5% of the total intra cellular RNA. The labeling was shown for Gram-positive and Gram-negative reference bacteria as well as for natural biofilms from drinking water, surface water, and lake sediment. The separation of the DIG-RNA from total RNA extracts was performed with a biotinylated anti-digoxigenin antibody and streptavidin-functionalized magnetic particles. The average yield of separation was about 95% of labeled RNA from total RNA extracts. The unspecific bindings of non-labeled nucleic acids were smaller than 0.2%, evaluated by spiking experiments with an unmarked DNA template. Applicability of the developed method was demonstrated by rRNA-directed PCR-DGGE population analysis of natural biofilms and expression profiling of two stress induced genes (vanA and rpoS) in reference bacteria before and after the DIG-RNA separation. Population analyses demonstrated biofilm fingerprint patterns of active bacteria, which were neither influenced by labeling nor during magnetic bead separation. Even, induction rates of the mentioned stress genes were not altered during labeling and separation procedures. The presented in vivo labeling and separation method is a possible way analyzing the effects of stress response in natural bacterial populations. It could cover four mechanisms of stress response of bacterial populations: gene expression, interspecies interactions, shifts in the population’s composition and the stabilization of certain mRNAs. Therefore, the presented approach could be used as completion or alternative for metatranscriptome analyses putting a tighter focus on stress response of bacteria upon exogenious stimuli in their natural habitat. Analyzing up-regulated and stabilized gene products of active bacteria makes an examination of the entire transcriptome of the population unnecessary.
S12:2
Subpopulation interactions during Pseudomonas aeruginosa biofilm development L. Yang1, M. Nilsson2, M. Gjermansen2, M. Givskov2, T. TolkerNielsen2; 1Technical University of Denmark, Lyngby, DENMARK, 2 University of Copenhagen, Copenhagen, DENMARK
The structured nature of biofilms is thought to enable buildup and maintenance of physiologically distinct subpopulations in different micro-niches. Using flow-chamber-grown Pseudomonas aeruginosa biofilms as model system, we show in the present study that the development of heterogeneous biofilms may occur through mechanisms that involve complex subpopulation interactions. One example of this phenomenon is expression of the iron-siderophore pyoverdin in one subpopulation being necessary for development of another subpopulation which does not itself express the pyoverdin syntheses genes. Another example is quorum-sensing-controlled DNA-release in one subpopulation being necessary for development of another subpopulation which does not itself express the quorum-sensing genes. Mechanisms involved in the development of physiologically distinct subpopulations in biofilms may contribute ASM Conferences
SPEAKER ABSTRACTS to their innate tolerance towards biocides, protozoa, immune systems, and antibiotics, and therefore may be relevant as biofilm control targets.
S12:3
Natural transformation of Vibrio cholerae on chitin surfaces M. Blokesch; Swiss Federal Institute of Technology, Lausanne, SWITZERLAND
Vibrio cholerae is well adapted for life in the human intestine. However, it is also a normal member of aquatic habitats where it resides attached to the chitin-containing exoskeleton of crustaceans and their molts. Upon growth on chitin surfaces V. cholerae gains natural competence, which enables it to take up free DNA from the environment in order to stably integrate it into its genome (natural transformation). Thus, this environmental niche might represent an evolutionary hotspot for V. cholerae and other Vibrios. For V. cholerae we are only at the beginning of understanding the complex regulation circuit of natural competence. This distinct developmental program is coordinated by at least three major factors: chitin, quorum sensing and catabolite repression. The later is dependent on the secondary messenger cyclic adenosine monophosphate (cAMP) and CRP (cAMP receptor protein), which are concordantly necessary for chitin colonization and the induction of the competence program. The presentation will conclude with an overview of what is known so far about natural competence in Vibrio cholerae and propose a regulatory model taking into account some recent attempts of monitoring colonization of chitin surfaces using fluorescence microscopy.
S12:4
High-throughput biometric characterization of two distinct pili-driven surface motility modes in Pseudomonas aeruginosa M. L. Gibiansky1, J. C. Conrad1, V. D. Gordon1, D. A. Motto2, M. A. Mathewson1, W. G. Stopka1, D. C. Zelasko1, J. D. Shrout2, G. C. Wong1; 1University of Illinois, Urbana, IL, 2University of Notre Dame, Notre Dame, IN
Type IV pili are motility appendages primarily located at the bacterial poles and known to play a crucial role in ‘twitching’ motility, a surface motility mode which does not require flagella. In twitching, bacteria move by extending, attaching, and then retracting individual pili, which can be visualized as bacterial “grappling hooks.” Although pili-based motility is central to biofilm formation, a full characterization of pili-driven motility has been limited by measurement technology. Current techniques for investigating bacterial motility modes do not permit high-throughput, quantitative biometric studies of large numbers of motile bacteria. Moreover, the use of motility mutants does not allow us to see how bacteria switch from one motility appendage to another under different environmental conditions, nor elucidate the synergistic or antagonistic interactions between type IV pili and flagella. We develop and apply a high throughput biometric method to pili based motility: State of the art image recognition and particle tracking algorithms are used to identify and track large numbers of surface-associated 5th ASM Conference on Biofilms
bacteria as they use pili to move along a surface, up to ~800 cells per frame, over time periods up to 6 hrs. Examining wildtype (WT) and flagella-deficient (pilA) knockout strains of P. aeruginosa, we observe two distinct pili-driven motility modes: a “crawling” mode characterized by the bacteria lying flat on the surface, and a “walking” mode characterized by bacteria orienting themselves normal to the surface, and moving with one end interacting with the surface. From the measured data, we extract quantities such as bacterial velocity, orientation, and projected length, which can be used to distinguish computationally via custom-designed software the two motility modes and characterize their biometric signatures. In the flagella-deficient pilA mutant strain, we find that when “crawling,” bacteria preferentially move along their long axis and retain their orientation over time, in ‘large persistence length’ trajectories. In contrast, “walking” bacteria are able to move with rapid changes in direction, and the resulting ‘small persistence length’ trajectories are more convoluted than the relatively straight trajectories of “crawling” bacteria. Surprisingly, the average displacements are slightly greater for the “walking” motion, which allows the bacteria to sample more microenvironments before committing to a general direction. The maximum instantaneous velocities that bacteria can achieve in both modes are similar, and the bacteria are able to rapidly and repeatedly switch between them. We observe both “walking” and “crawling” in both the WT as well as the flagella-deficient pilA strain, with similar speed and orientation profiles to the mutants. This strongly suggests that flagella do not strongly interfere with this type of Type-IV pili based motion in the WT.
S12:5
Diversifying the bacterial portfolio: Identification of genetic pathways linked to the rugose small colony variant (RSCV) phenotype of Pseudomonas aeruginosa J. J. Harrison, Y. Irie, H. Almblad, J. Silverman, M. R. Parsek; University of Washington, Seattle, WA
Rugose small colony variants (RSCVs) of Pseudomonas aeruginosa are frequently isolated from infections as well as from laboratory biofilm reactors. RSCVs are known to overproduce extracellular polysaccharides, to aggregate in liquid culture, and to have a superior capacity to form multidrug tolerant biofilms in vitro. An understanding of the genetic pathways linked to this phenomenon is imperative, as many of these adaptations are likely to promote persistence of P. aeruginosa in chronically infected tissues, such as burn wounds and the Cystic Fibrosis lung. The best characterized pathway linked to RSCV formation is the wrinkly spreader phenotype (wsp) pathway. Mutations in the wsp pathway cause the RSCV phenotype by directly modulating intracellular levels of the second messenger 3’,5’-cyclic-dimeric-guanosine monophosphate (c-di-GMP), which ultimately occurs through the activity of an output diguanylate cylcase (wspR). However, the prevalence of wsp mutations among clinical and laboratory isolates as well as the occurrence of other genetic pathways linked to the P. aeruginosa RSCV phenotype remain poorly characterized. By assessing colony morphology diversity among P. aeruginosa PAO1 populations grown in biofilm reactors, we show that mutations in the wsp pathway account for 16). Fifty percent of isolates had a ratio 2 organisms) were more robust and showed more resistance to treatment than single species biofilms. Single species BF were most responsive to the antibiotics in general (particularly cipro and pip). However, 5-FU showed surprising anti-BF activity against all biofilms. Also for Pa, anti-BF activity was seen with all anti-tumor drugs. CONCLUSION: Anti-tumor drugs, highlighting 5-FU, showed suprizing activity vs. biofilms, suggesting a strategy of combined anti-tumor/ anti-antibiotic inhibition merits screening in this Poloxamer model. Biotyping grouped the biofilms into proposed categories (I-IV) which may be important distinctions for clinical treatments, as well as classification purposes.
A100
Asymptomatic bacterial biofilm communities on rhythm management devices differ considerably from virulent ones M. Pichlmaier1, C. Kühn1, A. Haverich1, W. Abraham2; 1Hanover Medical School, Hanover, GERMANY, 2Helmholtz Center for Infection Research, Braunschweig, GERMANY
Background: Biofilm infections are difficult to treat due to their antibiotic resistances. To prevent their formation especially on long-time implants a deeper understanding of the causes and 72
risk factors for their formation is urgently needed. Objectives: 1. Assessment of rhythm management devices from asymptomatic patients for bacterial biofilm communities. 2. Comparing biofilm communities found on symptomatic devices with those determined in asymptomatic ones. Methods: Amplicons of 16S rRNA genes from DNA extracted from the implants were analysed by SSCP; main bands were identified by sequencing and phylogenetic analyses. The results were correlated with known risk factors. Results: From 108 devices changed for battery depletion in asymptomatic patients, been in place for 64 months and remained asymptomatic 47% had bacterial DNA. The identified bacterial taxa were untypical for clinical device infections. Known risk factors for device infections did not correlate with the bacterial DNA in this population. Common cohabitation was identified among the strains found. The results were compared with rhythm management devices explanted due to acute infections and very different biofilm communities were found between these two types. The most common species in biofilm communities of symptomatic devices was Staphylococcus epidermis followed by S. aureus. Seldom two Staphylococci were found together. Stenotrophomonas maltophila or Propionibacterium acnes were preferably detected in asymptomatic biofilms but Staphylococcus spp., Micrococcus sp. or Pseudomonas lini were found in symptomatic ones. Conclusions: The results suggest that it is not essential to suppress any biofilm formation but only bacterial species characteristic for symptomatic biofilms.
B101
Streptococcus gordonii platelet-interactive proteins are determinants of biofilm formation on saliva coated surfaces H. J. Petersen1, L. Hartles1, C. Keane2, S. W. Kerrigan2, M. M. Vickerman3, H. F. Jenkinson1; 1Bristol University, Bristol, UNITED KINGDOM, 2Royal College of Surgeons in Ireland, Dublin, IRELAND, 3SUNY, Buffalo, NY
Viridans streptococci are early colonizers of saliva coated oral surfaces and provide the foundations for development of complex biofilm communities. The streptococci, and other micro-organisms within these communities, can potentially dissipate into the bloodstream, interact with platelets, and cause endocarditis. Two families of cell surface proteins found across the oral streptococci are known to be involved in platelet interactions. These are the serine-rich repeat glycoproteins, and the Antigen I/II (AgI/II) adhesins. In S. gordonii, the Hsa glycoprotein and the AgI/II proteins SspA and SspB have platelet interactive functions and are therefore potential virulence factors in infective endocarditis. Recently, a novel high molecular mass surface protein on Streptococcus gordonii involved in platelet interactions has been characterized, and designated Platelet adherence protein A (PadA). It is hypothesized that these polypeptides are principally associated with adherence and colonization of the oral cavity. Therefore we have determined their respective roles in biofilm formation on saliva-coated surfaces. Mutants in padA, hsa, and sspA/B were generated by allelic exchange and their abilities to support biofilm formation were investigated in static and under flow conditions. Interactions of streptococci with platelets and platelet receptors GPIb and GPIIb/IIIa were measured by in vitro binding ASM Conferences
POSTER ABSTRACTS assays. The ΔpadA mutant was ablated in production PadA protein, and was deficient in platelet adherence, and in binding platelet receptor GPIIb/IIIa. The Δhsa mutant showed reduction in platelet adherence and in binding to GPIb. The ΔpadA mutant was deficient in biofilm formation under static and flow conditions, while the Δhsa mutant was abrogated in biofilm formation under flow conditions only. Biofilms of the sspA/B mutant were similar to the wild-type in architecture. The results suggest that platelet-interactive polypeptides on the S. gordonii cell surface may have important roles in biofilm formation and in colonization of the oral cavity.
C102
Biofilms of Malassezia pachydermatis H. Suttie, H. Ceri, D. W. Morck; University of Calgary, Calgary, AB, CANADA
Malasszia pachydermatis is a zoonotic, opportunistic yeast commonly present in canine otitis externa infections, the most common condition presented to small animal veterinary clinics in North America. Infections with M.pachydermatis, and the subsequent common secondary bacterial infections associated with this disease, often results in severe chronic and recurring inflammation of the ear canal of dogs. We hypothesized based on presentation of the infection, anatomical structures of the outer ear canal, and chronic recurring clinical signs that this may be a biofilm-related infection. We therefore proposed to grow this yeast as a biofilm on the MBEC DeviceTM to determine if biofilms of M.pachydermatis form in vitro and to assess the MBEC and MIC of several antifungal agents commonly employed in treatment of dogs with infection due to this pathogenic yeast. M.pachydermatis was grown on the MBEC device using Sabouraud dextrose broth supplemented with 2.5% soybean oil. Nystatin (N), miconazole (M), clotrimazole (C), and thiabendazole (T) were assessed for ability to kill planktonic and biofilm yeast as monitored by regrowth in recovery media, plate count methods, SEM, CSLM, and Fun1/Calcofluor live:dead staining. Elaborate M.pachydermatis biofilms formed readily by 48h on the MBEC device as evidenced by microscopic assessment. It appeared conventional antifungal agents were largely ineffective at eradicating biofilm [MBECs (μg/ml): N=256, M=256, C=1024, T=512; MICs (μg/ml) N=32, M=64, C=1024, T=512]. Residual biofilm was particularly evident at the air:fluid:surface interface. Inclusion of a bacterial-origin surfactant (100μM) markedly improved the effectiveness of N (MBEC=4, MIC=8), M (MBEC=4, MIC=4), and T (MBEC=4, MIC=4), but not C (MBEC=1024, MIC=512) on 48h biofilms; however, viability staining indicated yeast cells attached to the MBEC pegs following such treatment remained viable and despite this antifungal:surfactant combination treatment they could potentially regrow biofilm under appropriate conditions. The biofilm mode of growth demonstrated by M.pachydermatis, and the resulting tolerance to conventionally employed antifungal agents, may be critical for the persistence of this canine infection in the presence of antifungal treatment. Further screening of antimicrobials with efficacy against fungal biofilms will be required to derive effective treatment.
5th ASM Conference on Biofilms
A103
The Investigational Lipoglycopeptide Oritavancin Exhibits Activity Against Mature In Vitro Biofilms Derived From Clinical Isolates of Staphylococcus aureus A. Belley, I. Sarmiento, T. Parr Jr, G. Moeck; The Medicines Company, St-Laurent, QC, CANADA
Background: Medical biofilms pose a serious clinical challenge because they exhibit remarkable tolerance to antimicrobial agents. Oritavancin is an investigational lipoglycopeptide that effects killing of gram-positive pathogens in part by perturbation of bacterial membrane integrity. The objective of this study was to elucidate the activity of oritavancin against in vitro biofilms derived from clinical isolates of S. aureus. Methods: Eighty-seven clinical isolates of S. aureus (37 methicillin-susceptible isolates; 50 methicillin-resistant) were studied; all isolates were obtained from patients in a phase-2 clinical study of complicated skin and skin structure infections and were susceptible to vancomycin. Seventy-two-hour biofilms were formed in MBEC Physiology & Genetics plates (Innovotech, Edmonton, Canada) in tryptic soy broth containing 1% glucose: MBEC plate lids were transferred daily to fresh medium. Following biofilm establishment, biofilms were either challenged overnight with oritavancin at 4, 8, 16 or 32 µg/ml or vancomycin at 128 µg/ml. Biofilm cell densities on MBEC peg lids were determined by sonication and serial dilution plating. Planktonic MICs against bacteria shed from the preformed biofilms were also determined. Minimal biofilm eradication concentrations (MBEC; the concentration of antibacterial agent needed to sterilize the biofilm) for oritavancin and vancomycin against each strain were determined after 72-hour incubation to recover surviving bacteria that tolerated exposure to the antibacterial agents. Results presented are from 4 independent experiments. Results: All 87 clinical isolates of S. aureus formed in vitro biofilms in MBEC plates after 72-hours incubation as evidenced by recovery of adherent bacteria on the MBEC plate pegs (mean cell density of 6.7 ± 0.9 log10 CFU/peg) and all having an MBEC90 >128 µg/ml for the prototypic glycopeptide cell wall inhibitor vancomycin. Under the stringent experimental conditions used in the assay, 88.5% (77 of 87 isolates) of the strains exhibited an oritavancin MBEC/ MIC ratio ≤2, highlighting that oritavancin is nearly as potent at sterilizing the in vitro biofilms as inhibiting planktonic bacteria: the 10 remaining isolates exhibited an oritavancin MBEC/ MIC ratio of at least 4. The mode of the MBECs obtained for oritavancin was 8 µg/ml whereas its MBEC90 was 32 µg/ml. Conclusion: These results demonstrate that oritavancin is active against in vitro biofilms derived from a significant collection of clinical isolates of S. aureus and thus warranting further investigation of oritavancin as a therapeutic agent for biofilmtype infections.
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POSTER ABSTRACTS
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Antimicrobial activity of silver carbene complexes against a broad spectrum of clinical bacterial and fungal isolates in the planktonic and biofilm mode of growth S. K. Parmenter1, D. M. Hudak1, J. P. Housholder1, A. I. Jones1, E. K. Cope1, B. D. Wright2, M. J. Panzner2, W. J. Youngs2, J. G. Leid1; 1 Northern Arizona University, Flagstaff, AZ, 2University of Akron, Akron, OH
Biofilms are inherently resistant to host immunity and antibiotics due to differential gene and gene product expression compared to planktonic bacteria. As a result, biofilm infections are difficult to treat and eradicate from patients. This aspect of biofilm infections can lead to chronic disease, augmented medical expenses, and higher rates of morbidity and mortality. The development of novel, efficacious, biofilm-specific antimicrobial compounds would lead to dramatic improvement of patient treatments and outcomes. Four N-heterocyclic silver-carbene complexes - SCC1, SCC8, SCC22 and SCC23, synthesized by our collaborators at the University of Akron, were tested for antimicrobial activity against clinical strains of bacteria and fungi. In order to establish the efficacy of the antimicrobials, the minimum inhibitory concentration (MIC) of the SCCs against planktonic bacterial and fungal samples was determined. All four of the SCCs displayed exceptional antimicrobial activity against a broad spectrum of gram-negative and gram-positive bacteria and medically relevant fungi. Therefore, we tested the efficacy of these compounds against the biofilm form of growth from a selection of these clinical strains. The strains tested as biofilms were, a mucoid strain of Pseudomonas aeruginosa, methicillin resistant Staphylococcus aureus (MRSA), Streptococcus pseudopneumoniae, Klebsiella pneumoniae, Acinetobacter baumannii, Burkholderia cepacia, Enterobacter intermedius and Haemophilus influenzae. Killing assays, as determined by viable bacterial counts after 24 hr exposure, were performed in order to determine the ability of the SCCs to effectively penetrate and kill the microbial communities. Analysis revealed that all four SCCs were effective against mucoid Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae biofilms. SCC1, SCC8, and SCC22 had little antimicrobial activity against MRSA, Streptococcus pseudopneumoniae, and Enterobacter intermedius biofilms. However, SCC23 in high concentrations was effective against all of the biofilms. Additional studies using SCCs encapsulated in nanoparticles to provide a sustained release of SCC over a period of days are currently being performed. These promising results show that silver carbene compounds have the potential to be used to treat biofilm-mediated infections.
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K. M. Stevens , D. D. Kerr , J. Amon , J. Piedimonte , J. Philippe , S. Koval2, D. G. Storey1; 1University of Calgary, Calgary, AB, CANADA, 2University of Western Ontario, London, ON, CANADA 1
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Burkholderia cenocepacia establishes opportunistic and difficult74
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Inflammatory response to biofilm vs. planktonic conditioned media of P. aeruginosa and S. aureus on the apical surface of mouse epithelial cell cultures J. M. Kofonow, B. Chen, A. G. Chiu, J. N. Palmer, N. A. Cohen; Hospital of the University of Pennsylvania, Philadelphia, PA
Tobramycin resistance is multi-factorial in Burkholderia cenocepacia. 1
to-treat infections in the lungs of cystic fibrosis patients. Very little is known about the factors that contribute to its intrinsic antibiotic resistance, but mechanisms include those associated with the stress response and those associated with the biofilm state, leading us to hypothesize that factors involved in both stress response and biofilm formation contribute to multifactorial antibiotic resistance of B. cenocepacia. To address this hypothesis a Tn5-mutant library was screened for tobramycin resistance and biofilm formation. Mutants demonstrating both properties were selected for analysis by genomic sequencing. Bioinformatic analysis revealed mutations in a broad array of genes including those encoding proteins involved in: flagella biosynthesis, lipid A biosynthesis, DNA synthesis and repair, oxidative stress, putative membrane proteins, and proteins of unknown function. Mutations involved in oxidative stress and flagella biosynthesis were further characterized, following verification of mutations by complementation. These mutants were analyzed for biofilm formation and tobramycin susceptibility using the MBECTM device and confocal laser scanning microscopy (CLSM) and for virulence using a Drosophila melanogaster fly nicking model of infection. CLSM imaging of biofilms verified that all the mutant biofilms were reduced in cell number and devoid of microcolonies when compared to the parental strain. All mutants were also verified to be more susceptible to tobramycin in both MIC and MBIC when compared to the parent. Fly nicking revealed that only the flhDC flagella mutant was avirulent, and that the oxidative stress mutants were equivalent in virulence to the parent. The oxidative stress mutants were also subjected to a hydrogen peroxide disc diffusion test, and only the mutant in cydA, encoding a Cytochrome bd ubiquinol oxidase subunit, had reduced resistance to this stressor. All mutants in flagellar biosynthesis genes were also analyzed for motility and presence of flagella by transmission electron microscopy. All these mutants were immotile and lacked flagella. Currently, all phenotypes are being verified using insertional, non-polar mutations in the genes of interest. To date this analysis has revealed that genes encoding the FlhDC regulator and the Cytochrome bd ubiquinol oxidase play a significant role in biofilm formation and antibiotic resistance. Further, FlhDC has been verified to regulate the expression of flagella and play a role in virulence, while Cytochrome bd ubiquinol oxidase plays a role in resistance against hydrogen peroxide. Overall, our results demonstrate that antibiotic resistance in B. cenocepacia involves a multitude of factors including those responsible for flagellar motility and stress response.
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Recent studies have demonstrated that mucosal biofilms are associated with a unique inflammatory profile compared to nonbiofilm inflamed mucosa of patients with Chronic Rhinosinusitis (CRS). Here we looked at a biofilm’s secretary effect on the immune response of epithelial cell cultures. Sterile conditioned media from Pseudomonas aeruginosa and Staphylococcus ASM Conferences
POSTER ABSTRACTS aureus 7-day-old biofilms and planktonic cultures were used to stimulate primary mouse nasal septal Air Liquid Interface (ALI) cultures (n=2 ALIs per condition). Each media was normalized to 500 ng protein and added to the apical surface of the ALI’s for 18 hours at 37°C with 5% CO2. The basal media was collected and run in a luminex assay for cytokine levels. P. aeruginosa biofilm conditioned media stimulated the ALI cultures to secrete more IL-6 compared to its planktonic media (p=0.043). This cytokine is produced in response to tissue damage leading to inflammation and may be an important cytokine contributing to the persistence of inflammation in biofilm-mediated CRS. S. aureus biofilm conditioned media stimulated the expression of GM-CSF (p=0.039) and MIP-1α (p=0.002) and decreased the expression of IP-10 (p=0.005) compared to its planktonic media. This suggests that S. aureus biofilms are associated with a pro-granulocytic immune response compared to its planktonic stimuli and also to gramnegative bacteria such as P. aeruginosa, a novelty that has been described before but never with specificity to the biofilm state of S. aureus. Conclusion: While previous studies have reported on biofilm-mediated inflammation of sinus tissue this model allowed us to determine the innate inflammatory role of the epithelium in response to secreted components of bacteria in a biofilm vs. planktonic state. The secreted components of P. aeruginosa and S. aureus are associated with an inflammatory milieu unique to the biofilm state and furthermore demonstrate differences in response to gram negative vs. gram positive microorganisms.
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Detection of biofilms in infective endocarditis by Fluorescence in situ Hybridization D. Schmiedel1, A. Petrich1, C. Mallmann1, K. Rugor1, M. Musci2, R. Hetzer2, U. B. Göbel1, A. Moter1; 1Charité University Hospital Berlin, Berlin, GERMANY, 2German Heart Institute Berlin, Berlin, GERMANY
Infective endocarditis is a rare but life-threatening disease associated with high mortality. Its characteristic histopathology is the presence of vegetations on the cardiac valves composed of both bacterial and host components. Early diagnosis of the causative microorganism is crucial for the timely choice of the antibiotic treatment and thus the patient’s outcome. Microbiological diagnosis is based mainly on culture techniques. However, these methods often fail due to previous antibiotic therapy or fastidious nature of the causative microorganisms. Our aim was to detect, visualize and identify the infectious agents in infective endocarditis and to study their biofilm formation using Fluorescence in situ Hybridization (FISH). Heart valve biopsies from 54 suspected endocarditis patients were obtained during valve surgery and submitted to FISH. Specimens were screened for the most common pathogens using a eubacterial probe along with genus- or species-specific probes for identification of streptococci, staphylococci, enterococci. In addition, probes were developed for detection of Bartonella quintana and Tropheryma whipplei that are infrequent causes of infective endocarditis but usually missed by conventional diagnostic techniques. Results were compared with those of culture-based diagnostics and clinical data. Discrepant results were subjected to comparative sequence analysis of PCR amplified 16S rRNA genes. 5th ASM Conference on Biofilms
FISH detected microorganisms in 26 of 54 heart valves, including rare pathogens such as Bartonella quintana, Tropheryma whipplei and Candida spp. It successfully diagnosed infective endocarditis in 5 culture-negative cases and showed the bacteria within their histological context. Biofilm architecture depended on the bacterial species. Strong FISH signals indicated recent activity of the cells and showed layers of different activity within the biofilm. In conclusion, FISH might be a valuable tool for diagnosis of endocarditis in particular in culture negative cases. FISH of in vivo grown biofilms allows the analysis of the biofilm mode of growth in endocarditis.
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Resolution of Staphylococcus aureus biofilm infection using vaccination and antibiotic treatment R. A. Brady1, G. O’May1, J. G. Leid2, J. W. Costerton3, M. E. Shirtliff1; 1Univ. of Maryland, Baltimore, Baltimore, MD, 2Northern Arizona University, Flagstaff, AZ, 3Allegheny Hospital, Pittsburgh, PA
Methicillin-resistant and sensitive Staphylococcus aureus (MRSA and MSSA) infections are initiated by systemic inoculation of staphylococci from colonized hosts where S. aureus exists as a sessile biofilm population. The bacteria may then initiate a chronic, degenerative biofilm infection by reattaching to host proteins, or remain planktonic, causing sepsis and death. Previous attempts of S. aureus vaccine development have had limited success since gene products up-regulated only in the septic and/or planktonic mode of growth were utilized. We designed biofilm-specific, quadrivalent vaccine by taking into account in vivo expression and the heterogeneous nature of protein expression within the biofilm. Antibiotic treatment was also administered to eradicate the remaining non-attached, sensitive planktonic cells. Vaccination coupled with vancomycin treatment effectively cleared an MRSA biofilm infection in a rabbit model, while antibiotics or vaccine alone failed. This approach to vaccine development can lead to the generation of protective antigens against the epidemic of MRSA and MSSA infections that result in over 40,000 deaths per year in the United States. In addition, since 56% of all infections are biofilm related, this approach may be used in vaccine development against other biofilm producing bacteria.
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Phenotypic diversity in Vibrio cholerae J. A. J. Haagensen1, J. C. Thøgersen1, G. K. Schoolnik2, A. M. Spormann1; 1Stanford University. Bio-X program, Stanford, CA, 2 Stanford University. School of Medicine, Stanford, CA
Vibrio cholerae is a pathogenic bacterium that causes severe diarrhoeal disease in man. If infections are left untreated, death may occur within a short time due to massive dehydration. During infection V. cholerae colonizes the intestinal tract where it excretes cholera toxin (CT) and produces toxin coregulated pili (TCP), two virulence factors that are central for pathogenicity. V. cholerae has the capacity to adapt to changing environments like the human host as well as the aquatic 75
POSTER ABSTRACTS reservoirs. Bistability, is an example of an adaptive strategy used by V. cholerae to diversify into different phenotypic cell subpopulations while being in the same environment. During this process tcpA, the gene encoding the principal repeating subunit of the TCP pilus filament is highly up-regulated in some cells and controlled in a bistable way. In this study we have been investigating how different environmental factors influence induction of virulence and bistibility in V. cholerae. A V. cholerae strain(1) in which the promoter of the toxin coregulated pilus gene, tcpA, is fused to a gfp gene encoding for a Gfp derivative with reduced half life and then inserted into the chromosome was used. By monitoring the intensity of green fluorescence emitted from the single cells expressing tcpA, using fluorescence activated cell sorting and confocal microscopy, virulent cells could be distinguished from non virulent cells, as there is a direct link between tcpA expression and virulence. Bistability in tcpA expression occurs in V. cholerae when exposed to bicarbonate. We have found that bistability is affected and induction of tcpA is significantly increased and maintained when glucose is introduced to a bicarbonate environment. Furthermore we find that a mutant strain(1) defective in CRP also show high expression of tcpA, without bicarbonate induction. These findings indicate that catabolic repression might play a role in bistibility and level of tcpA induction in V. cholerae. Bistable regulation of virulence in a structured environment was investigated using a laminar flow cell system. Under standard growth conditions biofilms of V. cholerae show dispersal of cells located in the core of microcolony structures. However if cells experience inducing conditions such as the presence of bicarbonate during the initial phase of biofilm development we find that cells in the middle region of microcolonies become induced, and stay induced also after removal of bicarbonate. Furthermore the cells loose their ability to disperse from the biofilm. In this very same region CRP and RpoS mutant cells show induction of tcpA expression and no dissolution. These findings point at the importance of structure in the persistence of virulence-induced populations. (1) A. T. Nielsen and G. Schoolnik. Submitted.
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Detection of the biofilm formation in Candida blood stream isolates F. A. Ruzicka1, V. Hola1, R. Kadlec2; 1Masaryk University, Faculty of Medicine, Brno; VUCHS, Vikýřovice, CZECH REPUBLIC, 2 VUCHS Rapotin, Brno, CZECH REPUBLIC
The yeasts of the genus Candida belong among important ethiological agents of the nosocomial bloodstream infections. Indwelling medical devices are considered one of the main predispositions to yeast infections. Therefore, the biofilm formation is important virulence factor in these microorganisms. The characteristic features of the biofilm infections, especially long-persisting focuses and high resistance to antifungal agents, complicate therapy. Therefore, the knowledge of presence of this virulence factor in bloodstream isolates is necessary for choice of optimal therapy. Unfortunately, in different researchers there are substantial differences among results of the biofilm formation testing in yeasts, especially in C. albicans. Some authors describe high biofilm-positivity in Candida sp. isolates but a number of other authors find the biofilm positivity only in a part of blood stream isolates. The aim of the study was to 76
assess how frequently this virulence factor occurred in yeast blood stream isolates. In this study, the 93 bloodstream isolates (C. albicans, 48, C. parapsilosis, 23, C. glabrata, 12, C. tropicalis, 8, C. krusei, 2, from patients hospitalised on various departments of St. Anne´s Faculty Hospital in Brno were tested. We assessed the biofilm formation in these strains by two commonly used methods, the polystyrene microtiter plate method (MTP) and the cultivation on PVC plastic disc. The biofilm layer structure on the plastic disc was examined by means of the scanning electron microscopy (SEM). The biofilm formation on the surface of the plastic discs was detected in all tested strains. On the other hand, we did not detect the biofilm formation in the number of strains by microtiter plate method. Only 38 strains, C. albicans (11), C. parapsilosis (12), C. tropicalis (8), C. glabrata (5), C. krusei (2) were biofilm-positive by the microtiter plate assay. This is probably due to both different culture surfaces and different manipulation with the sample. Our SEM observations showed that some strains formed multilayered and complex biofilm, whereas others (considered to be biofilm-negative by MTP) formed a thin rudimental biofilm layer on the PVC discs. Our data show that the biofilm formation is widespread in yeast invasive isolates but these strains differ in the biofilm quantity and structure. This work was supported by the grant LA09032.
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Cellular response to in vivo biofilm developing on silicone implants M. van Gennip, L. D. Christensen, K. Qvortrup, M. Alhede, P. Ø. Jensen, N. Høiby, M. Givskov, T. Bjarnsholt; University of Copenhagen, Copenhagen, DENMARK
Pseudomonas aeruginosa is associated with infections of medical implants in humans, where they are believed to form biofilms. The biofilm mode of growth contributes to the tolerance against treatment with antibiotics as well as to the host response. Polymorhonuclear leukocytes (PMNs), which are part of the innate immune defense and some of the first cell types to be recruited to the site of infection, most often fail to eradicate the biofilm infection. In general, the only way to eradicate the biofilm is to remove the medical implant from the host. By modifying the in vivo foreign-body infection model established by Christensen et al. in 2007 using 4 mm silicone tubes (id 4 mm, od 6 mm) instead of flat, square implants, we are able to study both the clearance of bacteria in vivo as well as the pathogen-host interaction. Although the ratio of live to dead PMNs have been determined in the bronchoalveolar lavage (BAL) fluid of infected animals, interactions of P. aeruginosa biofilms with PMNs have exclusively been studied in vitro in continuous-culture flow cells. To get a more nuanced view on the in vivo interactions, we followed a development of a biofilm in a foreign-body infection model by means of scanning electron microscopy (SEM). Wild-type P. aeruginosa (PAO1 Iglewski) bacterial pellet from a centrifuged overnight culture was resuspended in 0.9% NaCl to an OD600nm of 0.1. The tubes were colonized for 20-hours with shaking at 110 rpm. The tubes were inserted in the peritoneal cavity of BALB/c mice at day 0. At day one and two mice were euthanized and the tubes removed. The tubes were fixed in 2% glutaraldehyde, post-fixed in 1% OsO4, critical point dried using CO2 and sputter coated with gold according to standard procedures. ASM Conferences
POSTER ABSTRACTS Specimens were investigated with a FEI XL Feg30 SEM. Day 0: Before insertion the bacteria were evenly spread throughout the inside of the tube in a mono layer. Day 1: During the first day there was a significant increase in the number of bacteria and a biofilm had formed. An abundant number of PMNs had clearly been attracted and seemed to enter the tubes from the tube openings. Day 2: At day two an even thicker layer of biofilm had been formed. PMNs were now scattered over the entire biofilm area. However, most of them seemed to be under attack by bacteria as they appeared to be broken and embedded in the biofilm matrix. In this pilot study we show a SEM based inspection of the development of a P. aeruginosa biofilm on tubes inserted in the peritoneal cavity of mice and we also exhibit the interaction of the PMNs with the biofilm bacteria. The increase in the thickness of the biofilm is in correspondence with an increase in CFU per tube as observed in this mouse model. The model supports previous in vitro studies in flow cells with PMNs being killed by bacteria.
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Cyclic-di-GMP, GGDEF/EAL encoding genes in Escherichia coli and response to antimicrobial stress M. A. Stan, H. Ceri, R. J. Turner; University of Calgary, Calgary, AB, CANADA
Introduction: The multi-drug tolerance and decreased antimicrobial susceptibility typical of bacterial biofilms may be mediated through regulation of cyclic nucleotide c-di-GMP, which has been associated with the initiation of biofilm growth and the production of matrix exopolysaccharides. C-di-GMP acts as an integral messenger in global signaling networks, such as the σS general stress response pathway, to orchestrate physiological changes in response to environmental cues. Intracellular levels of c-di-GMP are regulated by the highly redundant GGDEF and EAL domains, which synthesize and degrade c-di-GMP, respectively. We hypothesized that deletion of specific GGDEF/ EAL genes may influence both biofilm formation and cell survival of biofilms exposed to antimicrobials. We tested 23 single mutant strains of E. coli missing either a GGDEF or EAL encoding gene to examine which genes may regulate biofilm formation and antimicrobial susceptibility. Methods: The ability of wild type E. coli K-12 and each of the mutant strains to form biofilms and to resist treatment with antibiotics and metals was quantified using the Calgary Biofilm Device, based on viable cell counting and confocal laser scanning microscopy. Results: We determined that the majority of single GGDEF/ EAL gene deletions resulted in minor, statistically insignificant, changes in biofilm cell densities. However, biofilms of the ΔyaiC and ΔyoaD mutants had lower cell densities compared to wild type as well as slow growing phenotypes in which these strains exhibited decreased growth at 24 hours but eventually surpassed wild type biofilm formation at 48 hours. Upon exposure of ΔyaiC and ΔyoaD biofilms to CuSO4 and tobramycin, we observed that there were substantial differences in the ability of these strains to withstand environmental stress compared to wild type. Loss of this GGDEF encoding gene provided a survival advantage to mature biofilms, as ΔyaiC biofilms were less susceptible to antimicrobial challenge. Conversely, the loss of the yoaD gene, encoding an EAL domain, resulted in increased sensitivity to both CuSO4 and tobramycin after 24 5th ASM Conference on Biofilms
hour exposure. Deletion of yaiC and yoaD in logarithmically growing E. coli also influenced susceptibility to aminoglycoside exposure, with a more than 50 fold increase in planktonic cell survival to 16 µg/mL tobramycin, compared to wild type. However, these differences disappear at lower concentrations of antibiotic. Conclusions: Our results demonstrated that the loss of specific c-di-GMP binding proteins may alter both biofilm growth and susceptibility to antimicrobial agents. These results also suggest that yaiC and yoaD, which are under positive σS control, may have a role in the antimicrobial tolerance of biofilm and logarithmically growing E. coli cells, in addition to their previously suggested function in the production of cellulose.
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Effects of disordered hemispherical micropatterns on Staphylococcus epidermidis biofilm formation A. Ihnen, J. Lee, W. Y. Lee; Stevens Institute of Technology, Hoboken, NJ
Hypothesis: Surfaces which have physical patterns in the shape and scale of bacteria cells have been shown to influence the microorganism’s adhesion and biofilm formation characteristics. With recognition of the spherical shape and the ~0.7 to 1 μm diameter of S. epidermidis, the aim of this investigation was to study the potential effects of hemispherical pattern features of 0.5, 1.0 and 2.0 μm on S. epidermidis adhesion and biofilm formation. Experimental Methods: Layer-by-layer self-assembly (LBLSA) was used to create disordered, hemispherical pattern features by: (1) self-assembling a monolayer of silica particles of the desired size on a silicon substrate and (2) using the silica particle self-assembly as a mold to pattern polymer surfaces. Poly(dimethylsiloxane) (PDMS) was used as a model polymeric material. S. epidermidis strain NJ9709 was cultured in tryptic soy broth with the inoculum concentration of 108 CFU/ml for a 5 hour period, with observations taken at 10 and 30 minutes; 1, 3 and 5 hours. After fixing and staining, the samples were imaged using scanning electron microscopy (SEM), confocal fluorescence and light microscopy. The effects of pattern size on the retention and biofilm formation of S. epidermidis was examined as a function of culture time. SEM was utilized to observe the attachment behavior of the bacteria on the different surfaces. Fluorescence and light microscopy was used to determine average surface coverage by biofilm. Results: The pattern features covered ~70% of each sample surface in a disordered manner with serpentine unpatterned regions of several microns in size. The 1.0 μm pattern reduced biofilm surface coverage by ~30% after 5 hours of culture in comparison to that on an unpatterned surface while the effect of the 0.5 and 2.0 μm patterns was negligible. On the 1.0 μm surface, the bacteria initially adhered on the unpatterned areas and subsequently developed into biofilms by spreading across the unpatterned areas while avoiding those covered by the pattern. Conversely, the 0.5 µm patterned surface had no significant effect on bacteria adhesion and biofilm formation. On the 2.0 µm patterned surface, the bacteria initially adhered within the surface features. However, during 5 hours of culture, biofilm colonies eventually grew out of the pattern and covered the entire sample surface. Conclusions: The results suggest that the size of surface pattern is an important factor in altering bacteria adhesion and biofilm formation characteristics. We 77
POSTER ABSTRACTS In Situ Hematologic Biofilm Community in 3 patients with Amyotrophic Lateral Sclerosis
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J. Ellis, M. Prochazka, S. E. Fry; Fry Laboratories, LLC, Scottsdale, AZ
The use of bacteriophages to minimize urinary catheter-associated biofilms S. M. Lehman, R. M. Donlan; Centers for Disease Control and Prevention, Atlanta, GA
Background: Catheter-associated urinary tract infections (CAUTI) are a common healthcare-associated infection, increasing patient morbidity, hospital stays, and costs of care. Catheter-associated microorganisms may also be a reservoir of antimicrobial resistance. Most CAUTI develop when bacteria from the patient or the environment colonize the urinary catheter and form extensive biofilms. Proteus mirabilis and Pseudomonas aeruginosa commonly co-colonize urinary catheters. P. aeruginosa has been implicated in CAUTI that progresses to bacteremia. P. mirabilis causes mineral-encrusted biofilms which increase the risk of pyelonephritis and bloodstream infection. The addition of bacteriophages (phages) to the patient’s bladder or to a catheter coating may reduce or prevent biofilm development. The purpose of this study is to evaluate phages for their ability to minimize biofilm formation by organisms that cause CAUTI. Methods: Clinical isolates of P. mirabilis and P. aeruginosa were evaluated for their ability to form biofilms. Seventy phages isolated from sewage were tested for their ability to lyse the best biofilm-forming isolates of these bacteria and to prevent biofilm formation in a microtiter plate system containing synthetic urine. Biofilm formation in untreated and phage-treated systems was assessed using the crystal violet staining method (O’Toole & Kolter, Mol Microbiol 1998). Phage lytic assays were performed by spotting phage suspensions onto bacterial lawns. Phage host ranges were tested on clinical isolates of related bacteria that are commonly isolated from urinary tract infections and catheter biofilms. Results: Many of the tested phages produced plaques with the expanding halo characteristic of a free polysaccharide depolymerase. Twentytwo of 33 P. mirabilis phages infected one or the other of two P. vulgaris isolates (7 tested), but no cross-genus host range was observed for any phage. In the crystal violet biofilm assay, 2 to 32 of the 35 tested phage strains were able to reduce the amount of biofilm formed by 4 P. aeruginosa strains (P80% of distributed genes are nonunique, ie. present in multiple genomes) than has been observed with all other bacterial species examined. This finding suggests that a high proportion of these distributed genes may be providing important survival traits. Neighbor grouping analyses based on distributed gene possession studies demonstrate two distinct clades separated by a very significant difference. These studies represent the first comparative genomic analyses performed for this important pathogen and support the hypothesis that individual strain differences in genomic makeup may underlie the wide range of clinical conditions with which it has been associated.
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Meningococci undergo transcriptional changes in the presence of a Neisseria lactamica biofilm K. Strand, A. Ritter, K. Compton, C. Newton, J. Drees, E. Aho; Concordia College, Moorhead, MN
Neisseria meningitidis and N. lactamica are Gram-negative diplococci that commonly colonize the upper respiratory tract of humans. Population studies examining neisserial carriage have shown N. lactamica is typically acquired at an earlier age than N. meningitidis, and sequential colonization by these two species has been documented in an individual host. While N. lactamica strains rarely cause disease, colonization by N. meningitidis can progress to septicemia and meningitis. Numerous investigators have examined bacterial virulence factors and host responses involved in neisserial colonization and invasive disease, however little is known about the roles biofilm formation and interspecies bacterial interactions play in these processes. In this study, we demonstrate in vitro biofilm formation by N. lactamica and investigate the ability of N. lactamica biofilm cultures to induce transcriptional changes in N. meningitidis. We examined interspecies communication among Neisseria using a co-culture system in which planktonic N. meningitidis and biofilm-grown N. lactamica are separated by a 0.4 µm porous polycarbonate membrane that prohibits the passage of bacterial cells but permits the exchange of molecules across the membrane. We established 34-hour static biofilms of N. lactamica strain NL4 in polystyrene tissue culture wells and added membrane inserts containing log-phase planktonic N. meningitidis strain FAM18 to the wells. The co-culture was incubated for two hours and total RNA was isolated from the biofilm-exposed meningococci. Differential gene expression in co-cultured meningococci compared to meningococci grown in the same system in the absence of a N. lactamica biofilm was measured using custom Affymetrix microarrays containing probsets specific to the N. meningitidis genome. N. meningitidis that were exposed to the N. lactamica biofilm displayed numerous transcriptional changes when compared to control meningococci. From a total of 36216 sequences represented on the microarray, 512 (1.4%) biofilm-exposed N. meningitidis targets displayed differential expression of +/- 1.5-fold (mean log2 ratios, p-value ≤ 0.05). Of these transcripts, 68% were upregulated and 32% were downregulated. The differentially regulated sequences ASM Conferences
POSTER ABSTRACTS included transcripts involved in cell growth and metabolism (42%), sequences of unknown function (27%), and sequences encoding well established neisserial virulence factors (8%). The virulence-associated transcripts included sequences involved in capsule production, piliation, and iron acquisition. These data suggest meningococci may undergo important transcriptional changes when they enter the human nasopharynx and encounter resident bacterial populations. The establishment of this model system will facilitate further study of intercellular communication among Neisseria species.
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Staphylococcus Vaccine Targets for Prevention of Biofilm Infections: Antigen Discovery and Efficacy Evaluation in Rat Challenge Models T. McNeely, T. E. Ebert, G. Pancari, S. Smith, S. Secore, D. Clark, J. Cook, L. A. R Surgery Team, L. D. Cope; Merck, Inc, West Point, PA
Background. Staphylococcus aureus and S. epidermidis are major causes of hospital acquired infections; a vaccine to prevent staphylococcal disease would relieve much suffering due to these infections. Indwelling central venous catheters are a common and important source of nosocomial staphylococcal infections. Methods. For antigen discovery, gene expression was compared between planktonic bacteria grown in vivo (in implanted dialysis tubing chambers) and adherent bacteria grown in vivo (on implanted gauze pads). RNA was collected from Staphylococcus obtained from the two growth conditions and micro-array analysis performed for each; the two expression sets were compared. Based on up-regulation in the adherent (biofilm) state, antigens were selected to test for immune protection against formation of an early biofilm. Sprague-Dawley rats were immunized 3X i.m. followed by surgery on day 35 to implant a catheter into the jugular vein. To test for vaccine efficacy, the rats were challenged on day 45, i.v. via the tail vein, with a sub-lethal dose of Staphylococcus. Indwelling catheters were evaluated for bacterial colonization at 24H post challenge. Results. Gene chip analysis revealed that multiple genes were upregulated in the 24H adherent (biofilm) versus planktonic cells. In the challenged cannulated rats, catheters were colonized by 1H post challenge. By 24H post challenge, bacteria formed a biofilm on the catheter, as evidenced by increased icaA, icaB and icaD mRNA upregulation, and PIA Ps production. Immunization with S. epidermidis antigen MRKA resulted in a 70-100% reduction in colonized catheters in vaccinated animals, compared to 0% reduction in controls. Reduction of catheter colonization was correlated with antibody titer. S. aureus antigen IsdB was among the genes upregulated under biofilm growth conditions. Immunization with IsdB led to prevention of catheter colonization in 73% of animals compared to only 22% for control animals. Conclusions. The discovery method and challenge model were utilized for evaluation of antigen targets for medically important Staphylococcus spp. This clinically relevant animal model will enable testing of efficacy of novel target antigens, for prevention of biofilm formation.
5th ASM Conference on Biofilms
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Synergistic antibacterial efficacy of tobramycin and a quorum sensing inhibitor against Pseudomonas aeruginosa in an in vivo intraperitoneal foreign-body infection model L. D. Christensen1, M. van Gennip1, M. Alhede1, N. Kumar2, S. Kjelleberg3, N. Høiby1, T. Bjarnsholt1, M. Givskov1; 1University of Copenhagen, Faculty Of Health Sciences, Copenhagen N, DENMARK, 2School of Chemistry, University of New South Wales, Sydney, AUSTRALIA, 3School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, AUSTRALIA
Pseudomonas aeruginosa growing as biofilms is frequently linked to infections on foreign-body implants, which can cause serious complications. P. aeruginosa uses its quorum sensing (QS) systems to regulate its virulence and the biofilm mode of growth contributes to P. aeruginosa´s tolerance towards the immune system and numerous antibiotics. QS deficient P. aeruginosa biofilms has however been shown to be sensitive toward tobramycin, in contrast to the QS proficient. Wild-type biofilms treated with either the QS inhibitor (QSI) or tobramycin alone had little effect on the eradication, thus a combination of treatments with a QSI and tobramycin was shown to have a synergistic effect on the killing of in vitro biofilm. The natural extension to this experiment was then to test if the synergistic effect also was true in an in vivo biofilm model. In the present study we used a modified version of the in vivo foreign-body infection model that we introduced in 2007. Here we insert silicone tube implants with a size of 4 mm (inner diameter 4 mm/ outer diameter 6 mm) instead of square implants. We precolonized the implants with wild-type P. aeruginosa and inserted them in the peritoneal cavity of BALB/c mice. After insertion the mice were divided into four groups where the treatments consisted of i.p. injection of: a) QSI furanone C-30 (1 mg/kg body weight (BW), dissolved in 2.6% ethanol) every 8 hour (6 injections), initiated one hour post-infection b) 2.6% ethanol every 8 hour, initiated one hour post-infection c) 30 mg/kg BW tobramycin once, initiated 24 hours post-infection d) 0.9% NaCl once, initiated 24 hours post-infection, administered as follows. Gr. 1 (combination treatment) received treatment a) and c) (n=11) Gr. 2 (furanone C-30 group) received treatment a) and d) (n=8) Gr. 3 (tobramycin group) received treatment b) and c) (n=10) Gr. 4 (placebo) received treatment b) and d) (n=9). Forty-eight hours after insertion the foreign-body implants were removed from the mice and the number of colony-forming units per implant was determined. By means of this model we found that treatment of wild-type P. aeruginosa with a combination of furanone C-30 and tobramycin (Gr. 1) resulted in a significant faster clearing of the implants as compared to both the placebo group and the single treatments with furanone C-30 (Gr. 2) or tobramycin (Gr. 3) (p=0.0002, p=0.0003 and p=0.001, respectively). A significant difference in clearing was also observed between the placebo group and the single treatment groups (Gr. 2: p=0.006 and Gr. 3: p= 0.0003). Additionally, we found a significant difference in clearing between the 83
POSTER ABSTRACTS two single treatment groups (Gr. 2 vs. Gr. 3, p=0.01). The present results show that a synergistic antimicrobial effect is achieved when using a combination treatment with QSI furanone C-30 and tobramycin against a P. aeruginosa foreign-body infection in mice.
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Role of Fimbriae in K penumoniae Biofilm Formation C. Schroll1, K. B. Barken2, K. A. Krogfelt1, C. Struve1; 1Statens Serum Institut, Copenhagen, DENMARK, 2Technical University of Denmark, Lyngby, DENMARK
Klebsiella pneumoniae is an important gram-negative opportunistic pathogen causing primarily urinary tract infections, respiratory infections, and bacteraemia.The ability of bacteria to form biofilms on medical devices, e.g. catheters, has a major role in development of many nosocomial infections. Most clinical K. pneumoniae isolates express two types of fimbrial adhesins, type 1 fimbriae and type 3 fimbiae. In this study, we have characterized the role of type 1 and type 3 fimbriae in K. pneumoniae biofilm formation. ell-defined isogenic fimbriae mutants of a clinical K. pneumoniae isolate were constructed, and their ability to form biofilm in a flow cell system was investigated by confocal scanning laser microscopy. The wildtype strain was found to form characteristic biofilms and it was revealed that development of K. pneumoniae biofilms primarily occurs by clonal growth and not by recruitment of planktonic cells. Expression of type 1 fimbriae did not influence K. pneumoniae biofilm formation, and by a phase-switch assay it was demonstrated that expression of type 1 fimbriae is in fact down-regulated in cells growing as biofilms. In contrast, expression of type 3 fimbriae was established to strongly promote biofilm formation. The high incidence of catheter associated infections caused by K. pneumoniae may be related to the ability of most isolates to express type 3 fimbriae
C129
BIOFILM FORMATION AND ANTIBIOTIC TOLERENCE OF NOVEL CYSTIC FIBROSIS PATHOGENS T. H. Jakobsen1, L. Poulsen1, C. R. Hansen2, K. Qvortrup1, H. K. Johansen2, N. Høiby2, M. Givskov1, T. Bjarnsholt1; 1Faculty of Health Sciences, University of Copenhagen, Copenhagen, DENMARK, 2Department of clinical microbiology, Rigshospitalet, Copenhagen, DENMARK
The general conception have for many years been that bacterial strains live as single cells, but slowly the picture is changing as an increasing number of bacterial species are revealed to be able to attach to each other and form aggregates. Much work has been done to describe the biofilm formation of several bacterial species. P. aeruginosa, which is usually the dominating pathogenic strain in the lungs of chronically infected Cystic Fibrosis (CF) patients, has been shown to persist in the lungs due to its biofilm forming capabilities. In vitro experiments suggest that bacteria are less susceptible to antibacterial agents when growing in biofilm, why the knowledge of the biofilm forming potential of other pathogenic bacteria is important. In the recent years many new groups of gram negative CF pathogens 84
have been identified concurrent with the development of more specific identification methods. Achromobacter xylosoxidans, Stenotrophomonas maltophilia, Ralstonia pickettii, Burkholderia multivorans and species from the relative newly identified genus Pandoraea are categorized as new CF pathogens. The present study was performed to investigate the biofilm forming capacity and elucidate the protective role of developed biofilm to antibiotic treatment of those novel CF pathogens. Clinical isolates from CF patients of A. xylosoxidans, R. pickettii, P. apista, S. maltophilia, and B. multivorans were investigated for biofilm formation under different conditions; (a) adherence to polymeric surfaces, (b) growth under stationary condition in a suspension and (c) flow cell system. Using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) techniques it was clear that the investigated bacteria aggregate in biofilms. The in vitro flow cell system was used to investigate the protective role of biofilm towards different antibiotics. Furthermore, different phenotypes as swimming, swarming, twitching motility and the presence of QS regulated virulence factors (protease, elastase, chitinase and rhamnolipid) of these 5 pathogens were investigated under different growth conditions. P. aeruginosa PAO1 was used as control in all the experiments. S. malthophilia and B. multivorans were capable of attaching to polymeric surfaces whereas all strains were able to form aggregates i.e. biofilm structures in flow cells and under stationary condition. It was also evident that the aggregated cells exhibited an increased tolerance to tobramycin, colistin and ceftazidime in concentration above the normal minimal inhibitory concentrations (MIC) levels. Biofilm formation makes treatment of a bacterial infection more difficult and the capabilities of the investigated strains should be taken into consideration when treatment procedure is initiated.
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Non-random distribution of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds M. Fazli1, T. Bjarnsholt1, K. Kirketerp-Møller2, B. Jørgensen2, A. S. Andersen3, K. A. Krogfelt3, M. Givskov1, T. Tolker-Nielsen1; 1 Department of International Health, Immunology and Microbiology. Faculty of Health Sciences, University of Copenhagen, Copenhagen, DENMARK, 2Copenhagen Wound Healing Center, Bispebjerg Hospital, Copenhagen, DENMARK, 3Statens Serum Institut, Copenhagen, DENMARK
Chronic wounds, such as diabetic foot ulcers, pressure ulcers and venous leg ulcers are an increasing problem world wide, and are associated with severe patient suffering, reduced life quality, and high costs to the health care systems. Microbial infection is believed to be one of the barriers to normal wound healing. Although the microflora of chronic wounds is multispecies and heterogeneous, Staphylococcus aureus and Pseudomonas aeruginosa are among the most frequently isolated bacteria from these wounds, and are widely known as causing persistent biofilm based infections in their hosts. We previously demonstrated that there is a lack of correlation between the bacteria detected by standard culturing and those detected directly by peptide nucleic acid-based fluorescence in situ hybridization (PNA-FISH) in chronic wound samples. While S. aureus was detected more frequently by swab sample cultivation than by ASM Conferences
POSTER ABSTRACTS PNA-FISH, the opposite was true for P. aeruginosa. This lack of correlation between detection by swab sample cultivation and PNA-FISH may be due to an ability of different bacterial species to colonize different regions of chronic wounds. We examined wound biopsies obtained from patients diagnosed as having chronic venous leg ulcers, and detected and located bacterial aggregates in these wounds by the use of PNA-FISH and confocal laser scanning microscopy (CLSM). We acquired CLSM images (15 for each wound) at 3 regions in 5 sections cut from five wounds containing P. aeruginosa and five wounds containing S. aureus. To get a measure of the distance of the detected bacteria to the wound surface we located the center of mass of the bacterial aggregates identified on each image by using the moment calculator tool of ImageJ software, and measured its distance to the wound surface. The distance of P. aeruginosa aggregates to the wound surface was significantly higher than that of S. aureus aggregates (P
