Current Topic Workshop: Biofilms and Infectious Disease

(March 22,2010 - March 25,2010 )

Organizers


Nick Cogan
Department of Mathematics, Florida State University
John Gunn
Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University
Daniel Wozniak
Infectious Diseases, The Ohio State University

Accepted Speakers

Nick Ashbolt
National Exposure Research Laboratory (MD-564), U. S. Environmental Protection Agency
David Davies
Department of Biological Sciences, State University of New York at Binghamton
Jack Dockery
Department of Mathematical Sciences, Montana State University
Hermann Eberl
Dept. Mathematics and Statistics, University of Guelph
Mahmoud Ghannoum
Department of Dermatology, University Hospitals Case Medical Center
Kim Lewis
Department of Biology, Northeastern University
Robert Palmer
Oral Infection and Immunity Branch, Natl Inst Dental Craniofacial Res - Natl Insts Health
Matt Parsek
Department of Microbiology, University of Washington
Karin Sauer
Dept. Biological Sciences, Binghamton University (SUNY)
Martin Schuster
Department of Microbiology, Oregon State University
Hal Smith
Department of Mathematics & Statistics, Arizona State University
Fitnat Yildiz
Microbiology & Environmental Toxicology, UC Santa Cruz
Monday, March 22, 2010
Time Session
03:30 PM
04:30 PM
Nick Cogan - What can a model of persister formation tell you?
What can a model of persister formation tell you?
Tuesday, March 23, 2010
Time Session
09:00 AM
10:00 AM
Matt Parsek - The Pel polysaccharide of P. aeruginosa
P. aeruginosa produces at least 3 types of exopolysaccaride (EPS): alginate, Pel and Psl. The Pel polysaccharide is a glucose-rich polymer and contributes to the formation of surface-associated biofilm. It has been reported that increasing the level of cyclic diguanylate (c-di-GMP) also results in the increased transcription of pel expression. This is due to derepression of pel expression by the c-di-GMP binding regulator, FleQ. In this work, we focused on regulation of pel expression and its relationship to c-di-GMP in biofilm communities. To measure the pel transcription in a biofilm, we used a tube biofilm culturing system. P. aeruginosa PAO1 was injected into the tube and incubated for 30 min. Control cells were incubated statically. Biofilms were grown at room temperature at 50 ml/h flow rate for 48 hours. Total RNA was isolated from biofilm cells and quantitative RT PCR was performed.

In a tube biofilm, pelA transcription increased about 8 times higher than that of control after only one hour post attachment and after 12 hours it was 20 times higher. This induction appeared to be Psl-specific, psl transcription was not induced under these conditions. It was shown that the transcription of PA4625 was elevated under high c-di-GMP conditions. In this tube biofilm, the transcription of PA4625 was increased 10 times higher, too. With the fleQ mutant, pelA transcription was still high and it was not induced by surface attachment. From these results, we hypothesized that c-di-GMP is elevated by initial attachment in biofilms. To test this, c-di-GMP levels of attached and planktonic cells were measured by HPLC/MS/MS system. The c-di-GMP levels of attached cells were found to be about 3 times higher than that of planktonic cells. Our data suggested that surface attachment induced the accumulation of c-di-GMP in the cell through FleQ dissociation from the pel promoter region, activating pel transcription and biofilm formation.
10:30 AM
11:30 AM
Nick Ashbolt - Pipe biofilm pathogens: Are they just a nosocomal risk?
Respiratory infections from bacteria that develop within hospital drinking water systems are not uncommon, such as Legionella pneumophila and Mycobacterium avium complex pathogens. Colonization success by any pathogen results from an array of complex interactions between biotic and abiotic factors. Low level seeding of drinking water distribution systems may occur periodically, most likely due to pathogens being associated with particulate matter breaking through water treatment, some of which may be within amoebae cysts. However, pathogen numbers only reach an important threshold once replication occurs, most of which is thought to occur within pipe biofilms (mostly within premise plumbing). For example, occurrence of L. pneumophila in hospital plumbing systems and outbreaks of legionellosis within hospitals around the world support its ubiquitous occurrence, no matter how good municipal drinking water treatment may be. The outstanding unknown is how much sporadic legionellosis and other biofilm-related disease occurs from non-institutional drinking water exposures? Hence, mathematical models are being developed to aid in identifying research needs to better estimate community disease risks from aerosolized drinking waters. Key parameters seem to be the density of pathogens within amoeba/biofilms were they replicate, and the proportion that may be released from the biofilm in respiratory-sized (< 10 m) aerosols. Shower heads would appear to be important sites for pathogen development and release, but upstream plumbing could be equally important, depending on the residual disinfectant type and concentration in the drinking water.
01:30 PM
02:30 PM
Bill Costerton - Role of microbial biofilms in device-related and other chronic diseases
Direct observations have revealed that the bacteria that cause device-related and other chronic diseases grow in matrix-enclosed biofilms, adherent to the surfaces of biomaterials and tissues. In this biofilm mode-of-growth, the organisms are virtually impervious to antibiotics, and to the antibodies and phagocytes that constitute the defense systems of virtually all mammals. Within the biofilm community the cells communicate by means of chemical and (possibly) electrical signals, so that these sessile communities can coordinate their responses to host countermeasures, and persist for months or even for years. Biofilm communities can withstand the attacks of antibacterial agents (e.g. antibiotics) that would readily kill planktonic cells of the same strain, and many medical specialties surgically remove the biofilms and their living or inert substrata, as their rational basis of anti-biofilm therapy. In general, biofilms cause damage to the affected tissues by their persistence. When host defenses and antibiotic therapy fail to resolve chronic infections, the inflammation that they stimulate becomes the predominant factor in damage to affected tissues.

Because of recent advances in Biofilm Microbiology, the clinical pendulum is swinging away form frontal attacks with antibiotics, towards the use of immune modulation to minimize the effects of inflammation on the host tissues. These same data have stimulated interest in the use of signals to minimize biofilm formation, and even to stimulate the dissolution of existing biofilms by promoting detachment. The notion of using physical forces (e.g. DC fields, and ultrasonic waves) to disrupt the internal communications within biofilms is also gaining traction, and a more complete understanding of the structure and function of whole microbial communities will engender new and practical technologies for biofilm control.
03:00 PM
04:00 PM
Martin Schuster - Communication and cooperation in bacterial populations: Mechanistic and evolutionary perspectives
There has been an explosion in research directed at understanding the mechanisms of how bacteria communicate and cooperate to perform a variety of multicellular behaviors, including biofilm formation. Not until very recently have microbiologists also begun to investigate these behaviors from the perspective of social evolution. Our goal is to integrate mechanistic and evolutionary approaches to investigate communication, also termed quorum sensing (QS), and cooperation in the model bacterium and opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa communicates via diffusible acyl-homoserine lactone signals to coordinate the expression of hundreds of genes, many of which encode extracellular virulence factors. On a mechanistic level, we have utilized a variety of different approaches, including transcriptomics, ChIP-chip, and mutagenesis, to identify directly and indirectly regulated genes, and to characterize additional regulators of the QS system. With respect to sociobiology, we have utilized in vitro evolution and analysis of natural P. aeruginosa populations to gain insight into the propensity of cheating in bacterial populations, which is a threat common to social systems across all domains of life. We identified variants that ceased production of shared extracellular factors and took advantage of their production by the group. The existence of these cheaters demonstrates the sociality of microbes, and provides a compelling resolution to the long-standing paradox in P. aeruginosa pathogenesis that although QS is required for infection in animal models, QS-deficient variants are commonly associated with infections. In addition to cheating, our evolution-in-a-test-tube experiment also revealed a mechanism of cheater control. Before cheating became detrimental to the population, a novel type of cooperator with superior fitness had evolved from a cheating ancestor. Experiments are underway to define the underlying mechanism. As an extension of our own work, an attempt will be made to compare and contrast current mechanistic and sociobiological views on biofilm formation. A combination of both perspectives appears necessary to build a complete model of biofilm formation and guide appropriate treatment strategies.
04:00 PM
05:00 PM
Kim Lewis - Persister Cells and the Paradox of Relapsing Chronic Infection
Pathogen populations produce persisters, specialized survivor cells that are dormant and highly tolerant to all known antibiotics. Molecular mechanisms of persister formation will be discussed, as well as their role in disease, such as biofilm infections of catheters, cystic fibrosis, and oropharyngeal candidiasis. Approaches to eradicating persisters will be discussed as well.
Wednesday, March 24, 2010
Time Session
09:00 AM
10:00 AM
Jack Dockery - Quorum Sensing and Biofilm Modeling
Many bacteria use the size and density of their colonies to regulate the production of a large variety of substances. This phenomenon is called quorum sensing. We present a review of mathematical models of quorum sensing and their use in biofilm Modeling.
01:30 PM
02:30 PM
David Davies - Biofilm Dispersion as a Novel Method to Control Chronic Biofilm Infections
Biofilm Dispersion as a Novel Method to Control Chronic Biofilm Infections
03:00 PM
04:00 PM
Fitnat Yildiz - Biofilm formation in Vibrio cholerae
Vibrio cholerae causes the disease cholera and is a natural inhabitant of aquatic environments. V. cholerae's ability to form biofilms, matrix-enclosed, surface-associated communities, is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. I will discuss production of biofilm matrix components and regulation of biofilm formation in V. cholerae.
04:00 PM
05:00 PM
Karin Sauer - A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development
The important human pathogen Pseudomonas aeruginosa has been linked to numerous biofilm-related chronic infections 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 known about the underlying regulatory events that lead to the formation of biofilms, the primary cause of many chronic and persistent human infections. By mapping the phosphoproteome over the course of P. aeruginosa biofilm development, demonstrated that biofilm formation following the transition to the surface attached lifestyle is regulated by three previously undescribed two-component systems: BfiSR (PA4196-4197) harboring an RpoD-like domain, an OmpR-like BfmSR (PA4101-4102), and MifSR (PA5511-5512) belonging to the family of NtrC-like transcriptional regulators. we identified three novel two-component regulatory systems that were required for the development and maturation of P. aeruginosa biofilms. 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. Further, we demonstrate the interdependency of this two-component systems network with GacS (PA0928), which was found to play a dual role in biofilm formation. This work describes a novel signal transduction network regulating committed biofilm developmental steps following attachment, in which phosphorelays and two sigma factor-dependent response regulators appear to be key components of the regulatory machinery that coordinates gene expression during P. aeruginosa biofilm development in response to environmental cues.
Thursday, March 25, 2010
Time Session
09:00 AM
10:00 AM
Hal Smith - The Freter Model of Biofilm Formation
Driven by recent advances in noninvasive microscopy, staining techniques, and genetic probes, there has been enormous increase in our understanding of biofilms. Along with this increase in understanding, has been increasing interest in mathematical models of biofilms to get at important mechanisms. Most recent modeling in the field has been directed towards understanding the mechanisms underlying the remarkable spatial structure of biofilms which has become evident through the use of modern imaging techniques. Most of these models are so complex that they can be investigated only using sophisticated numerical simulations.

On the other hand, there are relatively few simple, conceptual biofilm models which are amenable to mathematical analysis yet which yield significant and useful results. Here, we speak of models which do not attempt to provide much detail on the spatial structure of biofilms but which provide information on conditions suitable for biofilm formation and maintenance and which model the formation of biofilms directly, starting from an inoculum of planktonic bacteria. Freter et al. formulated a mathematical model to understand the phenomena of colonization resistance in the mammalian gut (stability of resident microflora to colonization). Essentially, their model can be viewed as a crude biofilm model. In contrast to state of the art biofilm models, the Freter model completely ignores the three-dimensional spatial structure of the biofilm. Yet it can give useful results. The model and its implications will be surveyed.
Name Email Affiliation
Abedon, Stephen abedon.1@osu.edu Microbiology, The Ohio State University
Ahmer, Brian ahmer.1@osu.edu Department of Microbiology, The Ohio State University
Ashbolt , Nick Ashbolt.Nick@epa.gov National Exposure Research Laboratory (MD-564), U. S. Environmental Protection Agency
Bakaletz, Lauren bakaletz.1@osu.edu; Director, Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital
Calhoun, Jason jason.calhoun@osumc.edu Department of Orthopaedics, The Ohio State University Medical Center
Chou, Thomas tomchou@ucla.edu Dept. of Biomathematics/Mathematics, UCLA
Cogan, Nick cogan@math.fsu.edu Department of Mathematics, Florida State University
Costerton, Bill costerto@usc.edu Department of Orthopedic Surgery, Allegheny-Singer Research Institute
Crary, Monica crary.4@buckeyemail..osu.edu Molecular Genetics, The Ohio State University
Das, Subinoy das.68@osu.edu Department of Otolaryngology-Head and Neck Surgery, The Ohio State University
Davies , David dgdavies@binghamton.edu Department of Biological Sciences, State University of New York at Binghamton
De Leenheer, Patrick deleenhe@math.ufl.edu Department of Mathematics, University of Florida
Dockery, Jack umsfjdoc@math.montana.edu; Department of Mathematical Sciences, Montana State University
Du, Huijing hdu@nd.edu Department of Mathematics, University of Notre Dame
Eberl , Hermann heberl@uoguelph.ca Dept. Mathematics and Statistics, University of Guelph
Faust, Russell russell.faust@nationwidechildrens.org Nationwide Childrens Hospital
French, Donald french@math.uc.edu Department of Mathematical Sciences, University of Cincinnati
Ghannoum, Mahmoud abigail.williams@uhhospitals.org Department of Dermatology, University Hospitals Case Medical Center
Gonzalez, Geoffrey gonzalez-escobed.1@osu.edu Microbiology (CMIB), The Ohio State University
Griffen, Ann griffen.1@osu.edu Dept of Pediatric Dentistry, Ohio State University
Gunn, John gunn.43@osu.edu Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University
Harvey, Cameron charvey2nd.edu Physics Department, University of Notre Dame
Hesse, David Hesse@Battelle.org Applied Biology and Aerosol Technology, Battelle Memorial Institute
Janies, Dan Daniel.Janies@osumc.edu Dept. of Biomedical Informatics, The Ohio State University
Joo, Jaewook jjoo1@utk.edu Physics, University of Tennessee
Klapper, Isaac klapper@math.montana.edu Dept. of Mathematical Sciences, Montana State Univ.
Lee, Jiyoung jlee@cph.osu.edu Environmental Health Sciences, The Ohio State University
Lewis, Kim k.lewis@neu.edu Department of Biology, Northeastern University
Leys, Gene leys.1@osu.edu College of Dentistry, The Ohio State University
Lim, Sookkyung limsk@math.uc.edu Department of Mathematical Sciences, University of Cincinnati
Limoli, Dominique limoli.1@osu.edu CMIB, The Ohio State University
Lower, Steven lower.9@osu.edu School of Earth Sciences, The Ohio State University
Ma, Luyan luyan.ma@osumc.edu Center for Microbial Interface Biology, The Ohio State University
Marshall, Joanna joanna.m.marshall@gmail.com Infectious Diseases, The Ohio State University College of Medicine
Masic, Alma alma.masic@mah.se School of Technology, Malmo University
Mason, Kevin kevin.mason@nationwidechildrens.org Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital
Mishra, Meenu meenu.mishra@osumc.edu CMIB, The Ohio State University
Munson, Robert munson.10@osu.edu The Research Institute at Nationwide Children's Hospital, The Ohio State University
Palmer, Robert rjpalmer@dir.nidcr.nih.gov Oral Infection and Immunity Branch, Natl Inst Dental Craniofacial Res - Natl Insts Health
Parsek, Matt parsem@u.washington.edu Department of Microbiology, University of Washington
Paul, Prabasaj paulp@denison.edu Physics, Denison University
Quan, David dquan@umd.edu Bioengineering, UMCP/UMBI/CBR
Rodriguez-Palacios, Alexander rodriguez-palacios.1@buckeyemail.osu.edu Food Animal Health Research Program, The Ohio State University
Ryder, Cynthia cynthia.ryder@osumc.edu Center for Microbial Interface Biology, The Ohio State University
Sauer, Karin ksauer@binghamton.edu Dept. Biological Sciences, Binghamton University (SUNY)
Schuster, Martin martin.schuster@oregonstate.edu Department of Microbiology, Oregon State University
Shirtliff, Mark mshirtliff@umaryland.edu Department of Microbial Pathogenesis, Univ. of Maryland, Baltimore
Shrout, Joshua joshua.shrout@nd.edu Civil Engineering and Geological Sciences, University of Notre Dame
Smith, Hal Department of Mathematics & Statistics, Arizona State University
Swords, Ed wswords@wfubmc.edu Department of Microbiology and Immunology, Wake Forest University Health Sciences
Szomolay, Barbara b.szomolay@imperial.ac.uk Biomathematical Sciences Group, Imperial College
Wang, Hua wang.707@osu.edu Dept. of Food Science & Technology, The Ohio State University
Wewers, Mark mark.wewers@osumc.edu Internal Medicine: Davis Heart and Lung Research Institute, The Ohio State University
Won, Gayeon won.35@osu.edu Veterinary Preventive Medicine, The Ohio State University
Wozniak, Daniel Daniel.wozniak@osumc.edu Infectious Diseases, The Ohio State University
Xu, Zhiliang zxu2@nd.edu Mathematics, University of Notre Dame
Yildiz, Fitnat yildiz@etox.ucsc.edu Microbiology & Environmental Toxicology, UC Santa Cruz
Yin, Li-Yan yin.92@osu.edu Orhtopaedic Department, OSUMC
Younger, John jyounger@umich.edu Department of Emergency Medicine, University of Michigan
Zhang, Tianyu zhang@math.montana.edu Department of Mathematical Sciences, Montana State University
Pipe biofilm pathogens: Are they just a nosocomal risk?
Respiratory infections from bacteria that develop within hospital drinking water systems are not uncommon, such as Legionella pneumophila and Mycobacterium avium complex pathogens. Colonization success by any pathogen results from an array of complex interactions between biotic and abiotic factors. Low level seeding of drinking water distribution systems may occur periodically, most likely due to pathogens being associated with particulate matter breaking through water treatment, some of which may be within amoebae cysts. However, pathogen numbers only reach an important threshold once replication occurs, most of which is thought to occur within pipe biofilms (mostly within premise plumbing). For example, occurrence of L. pneumophila in hospital plumbing systems and outbreaks of legionellosis within hospitals around the world support its ubiquitous occurrence, no matter how good municipal drinking water treatment may be. The outstanding unknown is how much sporadic legionellosis and other biofilm-related disease occurs from non-institutional drinking water exposures? Hence, mathematical models are being developed to aid in identifying research needs to better estimate community disease risks from aerosolized drinking waters. Key parameters seem to be the density of pathogens within amoeba/biofilms were they replicate, and the proportion that may be released from the biofilm in respiratory-sized (< 10 m) aerosols. Shower heads would appear to be important sites for pathogen development and release, but upstream plumbing could be equally important, depending on the residual disinfectant type and concentration in the drinking water.
What can a model of persister formation tell you?
What can a model of persister formation tell you?
Role of microbial biofilms in device-related and other chronic diseases
Direct observations have revealed that the bacteria that cause device-related and other chronic diseases grow in matrix-enclosed biofilms, adherent to the surfaces of biomaterials and tissues. In this biofilm mode-of-growth, the organisms are virtually impervious to antibiotics, and to the antibodies and phagocytes that constitute the defense systems of virtually all mammals. Within the biofilm community the cells communicate by means of chemical and (possibly) electrical signals, so that these sessile communities can coordinate their responses to host countermeasures, and persist for months or even for years. Biofilm communities can withstand the attacks of antibacterial agents (e.g. antibiotics) that would readily kill planktonic cells of the same strain, and many medical specialties surgically remove the biofilms and their living or inert substrata, as their rational basis of anti-biofilm therapy. In general, biofilms cause damage to the affected tissues by their persistence. When host defenses and antibiotic therapy fail to resolve chronic infections, the inflammation that they stimulate becomes the predominant factor in damage to affected tissues.

Because of recent advances in Biofilm Microbiology, the clinical pendulum is swinging away form frontal attacks with antibiotics, towards the use of immune modulation to minimize the effects of inflammation on the host tissues. These same data have stimulated interest in the use of signals to minimize biofilm formation, and even to stimulate the dissolution of existing biofilms by promoting detachment. The notion of using physical forces (e.g. DC fields, and ultrasonic waves) to disrupt the internal communications within biofilms is also gaining traction, and a more complete understanding of the structure and function of whole microbial communities will engender new and practical technologies for biofilm control.
Biofilm Dispersion as a Novel Method to Control Chronic Biofilm Infections
Biofilm Dispersion as a Novel Method to Control Chronic Biofilm Infections
Quorum Sensing and Biofilm Modeling
Many bacteria use the size and density of their colonies to regulate the production of a large variety of substances. This phenomenon is called quorum sensing. We present a review of mathematical models of quorum sensing and their use in biofilm Modeling.
Persister Cells and the Paradox of Relapsing Chronic Infection
Pathogen populations produce persisters, specialized survivor cells that are dormant and highly tolerant to all known antibiotics. Molecular mechanisms of persister formation will be discussed, as well as their role in disease, such as biofilm infections of catheters, cystic fibrosis, and oropharyngeal candidiasis. Approaches to eradicating persisters will be discussed as well.
The Pel polysaccharide of P. aeruginosa
P. aeruginosa produces at least 3 types of exopolysaccaride (EPS): alginate, Pel and Psl. The Pel polysaccharide is a glucose-rich polymer and contributes to the formation of surface-associated biofilm. It has been reported that increasing the level of cyclic diguanylate (c-di-GMP) also results in the increased transcription of pel expression. This is due to derepression of pel expression by the c-di-GMP binding regulator, FleQ. In this work, we focused on regulation of pel expression and its relationship to c-di-GMP in biofilm communities. To measure the pel transcription in a biofilm, we used a tube biofilm culturing system. P. aeruginosa PAO1 was injected into the tube and incubated for 30 min. Control cells were incubated statically. Biofilms were grown at room temperature at 50 ml/h flow rate for 48 hours. Total RNA was isolated from biofilm cells and quantitative RT PCR was performed.

In a tube biofilm, pelA transcription increased about 8 times higher than that of control after only one hour post attachment and after 12 hours it was 20 times higher. This induction appeared to be Psl-specific, psl transcription was not induced under these conditions. It was shown that the transcription of PA4625 was elevated under high c-di-GMP conditions. In this tube biofilm, the transcription of PA4625 was increased 10 times higher, too. With the fleQ mutant, pelA transcription was still high and it was not induced by surface attachment. From these results, we hypothesized that c-di-GMP is elevated by initial attachment in biofilms. To test this, c-di-GMP levels of attached and planktonic cells were measured by HPLC/MS/MS system. The c-di-GMP levels of attached cells were found to be about 3 times higher than that of planktonic cells. Our data suggested that surface attachment induced the accumulation of c-di-GMP in the cell through FleQ dissociation from the pel promoter region, activating pel transcription and biofilm formation.
A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development
The important human pathogen Pseudomonas aeruginosa has been linked to numerous biofilm-related chronic infections 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 known about the underlying regulatory events that lead to the formation of biofilms, the primary cause of many chronic and persistent human infections. By mapping the phosphoproteome over the course of P. aeruginosa biofilm development, demonstrated that biofilm formation following the transition to the surface attached lifestyle is regulated by three previously undescribed two-component systems: BfiSR (PA4196-4197) harboring an RpoD-like domain, an OmpR-like BfmSR (PA4101-4102), and MifSR (PA5511-5512) belonging to the family of NtrC-like transcriptional regulators. we identified three novel two-component regulatory systems that were required for the development and maturation of P. aeruginosa biofilms. 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. Further, we demonstrate the interdependency of this two-component systems network with GacS (PA0928), which was found to play a dual role in biofilm formation. This work describes a novel signal transduction network regulating committed biofilm developmental steps following attachment, in which phosphorelays and two sigma factor-dependent response regulators appear to be key components of the regulatory machinery that coordinates gene expression during P. aeruginosa biofilm development in response to environmental cues.
Communication and cooperation in bacterial populations: Mechanistic and evolutionary perspectives
There has been an explosion in research directed at understanding the mechanisms of how bacteria communicate and cooperate to perform a variety of multicellular behaviors, including biofilm formation. Not until very recently have microbiologists also begun to investigate these behaviors from the perspective of social evolution. Our goal is to integrate mechanistic and evolutionary approaches to investigate communication, also termed quorum sensing (QS), and cooperation in the model bacterium and opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa communicates via diffusible acyl-homoserine lactone signals to coordinate the expression of hundreds of genes, many of which encode extracellular virulence factors. On a mechanistic level, we have utilized a variety of different approaches, including transcriptomics, ChIP-chip, and mutagenesis, to identify directly and indirectly regulated genes, and to characterize additional regulators of the QS system. With respect to sociobiology, we have utilized in vitro evolution and analysis of natural P. aeruginosa populations to gain insight into the propensity of cheating in bacterial populations, which is a threat common to social systems across all domains of life. We identified variants that ceased production of shared extracellular factors and took advantage of their production by the group. The existence of these cheaters demonstrates the sociality of microbes, and provides a compelling resolution to the long-standing paradox in P. aeruginosa pathogenesis that although QS is required for infection in animal models, QS-deficient variants are commonly associated with infections. In addition to cheating, our evolution-in-a-test-tube experiment also revealed a mechanism of cheater control. Before cheating became detrimental to the population, a novel type of cooperator with superior fitness had evolved from a cheating ancestor. Experiments are underway to define the underlying mechanism. As an extension of our own work, an attempt will be made to compare and contrast current mechanistic and sociobiological views on biofilm formation. A combination of both perspectives appears necessary to build a complete model of biofilm formation and guide appropriate treatment strategies.
The Freter Model of Biofilm Formation
Driven by recent advances in noninvasive microscopy, staining techniques, and genetic probes, there has been enormous increase in our understanding of biofilms. Along with this increase in understanding, has been increasing interest in mathematical models of biofilms to get at important mechanisms. Most recent modeling in the field has been directed towards understanding the mechanisms underlying the remarkable spatial structure of biofilms which has become evident through the use of modern imaging techniques. Most of these models are so complex that they can be investigated only using sophisticated numerical simulations.

On the other hand, there are relatively few simple, conceptual biofilm models which are amenable to mathematical analysis yet which yield significant and useful results. Here, we speak of models which do not attempt to provide much detail on the spatial structure of biofilms but which provide information on conditions suitable for biofilm formation and maintenance and which model the formation of biofilms directly, starting from an inoculum of planktonic bacteria. Freter et al. formulated a mathematical model to understand the phenomena of colonization resistance in the mammalian gut (stability of resident microflora to colonization). Essentially, their model can be viewed as a crude biofilm model. In contrast to state of the art biofilm models, the Freter model completely ignores the three-dimensional spatial structure of the biofilm. Yet it can give useful results. The model and its implications will be surveyed.
Biofilm formation in Vibrio cholerae
Vibrio cholerae causes the disease cholera and is a natural inhabitant of aquatic environments. V. cholerae's ability to form biofilms, matrix-enclosed, surface-associated communities, is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. I will discuss production of biofilm matrix components and regulation of biofilm formation in V. cholerae.
video image

Role of microbial biofilms in device-related and other chronic diseases
Bill Costerton Direct observations have revealed that the bacteria that cause device-related and other chronic diseases grow in matrix-enclosed biofilms, adherent to the surfaces of biomaterials and tissues. In this biofilm mode-of-growth, the organisms are virtual

video image

Communication and cooperation in bacterial populations: Mechanistic and evolutionary perspectives
Martin Schuster There has been an explosion in research directed at understanding the mechanisms of how bacteria communicate and cooperate to perform a variety of multicellular behaviors, including biofilm formation. Not until very recently have microbiologists also

video image

Persister Cells and the Paradox of Relapsing Chronic Infection
Kim Lewis Pathogen populations produce persisters, specialized survivor cells that are dormant and highly tolerant to all known antibiotics. Molecular mechanisms of persister formation will be discussed, as well as their role in disease, such as biofilm infect

video image

A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development
Karin Sauer The important human pathogen Pseudomonas aeruginosa has been linked to numerous biofilm-related chronic infections Biofilms are complex communities of microorganisms encased in a matrix and attached to surfaces. It is well recognized that biofilm cel

video image

The Freter Model of Biofilm Formation
Hal Smith Driven by recent advances in noninvasive microscopy, staining techniques, and genetic probes, there has been enormous increase in our understanding of biofilms. Along with this increase in understanding, has been increasing interest in mathematical m