JOURNAL OF BACTERIOLOGY, Nov. 2007, p. 7948–7960 0021-9193/07/$08.00 0 doi:10.1128/JB.
00787-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 22
Bioﬁlms 2007: Broadened Horizons and New Emphases
Robert J. Palmer, Jr.,1* and Paul Stoodley2
Oral Bioﬁlm Communication Unit, Oral Infections and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892,1 and Center for Genomic Sciences, 320 E. North Avenue, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania 150902 The 4th ASM Conference on Bioﬁlms (Quebec City, Quebec, Canada, 25 to 29 March 2007) maintained the format so valued at past conferences (keynote talks, invited speakers, talks selected from abstracts, evening specialty sessions, and a hands-on workshop) while increasing scientiﬁc diversity by delegating the selection of invited speakers and of talks from abstracts to session chairs appointed by the organizing committee. In particular, the committee targeted the underrepresentation of research on medically relevant bioﬁlms other than those of Pseudomonas aeruginosa and sought to increase the visibility of clinical aspects of bioﬁlm-based disease research; the fruits of these efforts will become apparent in the descriptions of the sessions that follow. Also, an effort was made to increase participation by non-U.S. investigators. At the 3rd Bioﬁlms Conference, also held in Canada, 34% of attendees came from outside the United States. At the Quebec City meeting, 53% were non-U.S. delegates who hailed from the United Kingdom/Europe (22%), Canada (11%), Scandinavia (8%), Asia (4%), Australia/New Zealand (2%), and India, Central and South America, Israel, and Africa (6%). The number of attendees (ca. 600) and the number of submitted abstracts (ca. 400) did not change from the previous meeting, which suggests that, after three meetings with steady growth, the target population is now well represented at the meeting. While the major questions and areas of investigation have not changed signiﬁcantly since the earlier meetings, new perspectives are emerging. First is the recognition that Pseudomonas aeruginosa, while a superb model organism for numerous reasons, is only one of many bacteria important to theoretical as well as applied aspects of bioﬁlm research; other model organisms (e.g., Vibrio spp., Bacillus spp., oral bacteria, and staphylococci) are now being investigated as natural and ﬁtting alternatives to P. aeruginosa. Second, while ﬂow cell work with monocultures grown in laboratory media will continue to provide critical baseline data and continue to be especially useful in examining theoretical questions, it is becoming clear that bioﬁlm behavior and physiology need to be studied in a manner that reﬂects the natural environment, whether within the human lung or on a soil particle. Experimental systems have therefore become more sophisticated and employ environmentally relevant substrata and media; accordingly, in vivo sampling and experimentation are becoming more common. Last, moving to the fore is the recognition that the vast majority of bacteria, including many of those involved in human disease, must associate with other genera of bacteria as part of their daily existence: multispecies communities are becoming a targeted research area. This review summarizes the individual sessions through their platform talks, many of which highlighted work presented in greater detail as poster presentations. We hope these descriptions (and the accompanying references, which give a ﬂavor of the topics) will convey the overall high scientiﬁc quality not only of the invited talks but also of the talks selected from submitted abstracts. The consensus of the attendees was that this meeting elevated the already high reputation of this conference series. We hope this review will convey some of that feeling to those who have yet to attend the conference. KEYNOTE TALK 1 E. Peter Greenberg (University of Washington) opened the meeting by speaking on the sociomicrobiology of bioﬁlms: the application to bioﬁlm microbiology of E. O. Wilson’s insect society-derived sociobiological concepts. In bioﬁlms, bacteria live in close proximity, the populations are heterogeneous in activity, and individuals exhibit distinct behaviors, all of which can be studied from an ethological perspective. Bioﬁlms offer a unique opportunity to examine the inﬂuence of environmental manipulation on gene expression and heritability (45). Greenberg focused on the role of iron in bioﬁlm formation. Iron is frequently a limiting nutrient in the human body as well as in the natural environment, and motility and bioﬁlm initiation in Pseudomonas aeruginosa are dependent on iron (4). The human immune effector lactoferrin sequesters iron; in the presence of lactoferrin (i.e., at low levels of available iron), P. aeruginosa maintains twitching motility, refuses to become sessile, and is thus dramatically attenuated in bioﬁlm formation (58). Production of lactoferrin is seen as a way for the host immune system to slow the rate of bioﬁlm formation so that the microbial intruders can be dealt with before reaching a clinically relevant population density. Greenberg continued this theme by showing that the iron chelator EDTA can disperse a P. aeruginosa bioﬁlm (3), and that citrate as a carbon source can result in a ﬂat bioﬁlm because of citrate’s iron chelation capability. Lastly he introduced the use of the iron
Downloaded from jb.asm.org by on December 23, 2009
* Corresponding author. Mailing address: Oral Bioﬁlm Communication Unit, Oral Infections and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bldg. 30, Room 310, 30 Convent Drive, Bethesda MD 20892. Phone: (301) 594-0025. Fax: (301) 402-0396. E-mail: email@example.com. Published ahead of print on 31 August 2007.
Bﬂ1 and Bﬂ2. Stoodley cautioned that bioﬁlm imaging as a routine clinical diagnosis practice is a long way from acceptance. this observation strengthens the hypothesis that bioﬁlm formation by P. Each specialist has a deﬁned function.org by on December 23. depending on the local conditions. both from the laboratory perspective and through examination in vivo. Ed Swords (Wake Forest University) expanded on bioﬁlm formation in the chinchilla model of otitis media by describing the relationship between phosphorylcholine (PCho) modiﬁcation of lipooligosaccharides in nontypeable Haemophilus inﬂuenzae (72) and the infection. DEVELOPMENT AND PHYSIOLOGY The opening session was chaired by George O’Toole (Dartmouth College) and Paula Watnick (Tufts—New England Medical Center). or swimming. ﬁlamentous phenotype via mitogen-activated-protein kinase sensing in Candida albicans is a response to surface contact. This newly discovered protein was identiﬁed and shown to be crucial for bioﬁlm formation in clinically important PIA-negative S. Paul Stoodley (Center for Genomic Sciences. Also discussed in the session were various cellsignaling pathways and environmental triggers that result in the modiﬁcation of bioﬁlm extracellular polymeric substances (EPS) in response to nutrient conditions. as well as in mucosal epithelia from upper respiratory tract infections (adenoids and middle ear mucosae) in clinical specimens (Fig. Several of these cases were culture negative even though symptoms of bacterial infection were presented. Gotz described the role of polysaccha¨ ride intercellular adhesin (PIA) in bioﬁlm formation by Staphylococcus epidermidis and Staphylococcus aureus (17). 1) and the chinchilla model of otitis media (18).
BIOFILM-HUMAN INTERACTIONS I In the ﬁrst of two sessions designed to emphasize the relationships of bacteria to the human host. and the production of LapA adhesion protein in response to phosphate limitation via a cyclic di-GMP (c-di-GMP) signaling pathway in Pseudomonas ﬂuorescens. and these mutants are much less virulent than the parent strains in animal models of infection. Time lapse imaging of ﬂuorescent reporter constructs implied that the cells exhibit only one phenotype at a time. He discussed the role of embp (extracellular matrix binding protein) in PIA-negative bioﬁlm formation. Roberto Kolter (Harvard University) presented evidence that differentiation within Bacillus subtilis bioﬁlm populations gives rise to six specialist cell types. The protein is broadly distributed in S. epidermidis strains. Homologous polysaccharides are found in several other bacteria. subtilis than previously thought. Glucose excess results in the synthesis of EPS rather than of glycogen. thus.asm. thereby releasing the bioﬁlm from the substratum. Olivier Brun (University of Minnesota) identiﬁed bioﬁlm-speciﬁc promoters in Mycobacterium marinum. presented by Russell Monds (Dartmouth College) (42). a putative attachment promoter is expressed at the base of the bioﬁlm. which appear to regulate the early and late bioﬁlm maturation process. antibodies. aeruginosa occurs by a staged developmental process. Several talks focused on P. presented by Liang Yang (Danish Technical University. session chairs were Friedrich Gotz (Tubingen University) and Ute Romling ¨ ¨ ¨ (Karolinska Institute). These include the release of DNA to the matrix in response to iron starvation in P. The chairs put together a set of talks that illustrated not only the diversity but also the commonality in the mechanisms of bioﬁlm development displayed by seven different bioﬁlm-forming species. glycogen is formed and EPS are degraded. Yves Brun (Indiana University) showed how attachment forces could be accurately measured through micromanipulation: the bending of a micropipette in response to a controlled pulling via an attached bacterial cell (66). epidermidis have transcriptome (50. epidermidis isolates through transposon mutagenesis and subsequent screening for bioﬁlm-negative mutants in a PIA-negative background strain. 189. Co-chair Watnick showed that Vibrio cholerae knockout mutants in the sugar phosphotransferase system can form a bioﬁlm when grown on glucose but not when grown on mannose. Daniel Verhamme (University of Dundee) presented evidence that the transcription factor DegU regulates more bioﬁlm properties in B. Despite the success in demonstrating bioﬁlms. and general stains was used to show bioﬁlms associated with infected sutures and an infected arthroplastic remodeling (62). A mutant that does not add PCho formed bioﬁlms of lower density than did the parent strain and also caused increased inﬂammation levels in the animal. PA) presented work on in vivo imaging of bacterial bioﬁlms in infections. A combination of ﬂuorescence in situ hybridization (FISH). Using this technique it was shown that Caulobacter crescentus ﬁrst attached by the ﬂagella and then rotated until pilus tethers stopped the rotation long enough for the organism to create a holdfast. aureus and S. this molecular architecture is conserved across bacterial phylogeny. ranging from attachment and social behavior to colony architecture. Phage release differs also (49). The development of a tissue culture model system for the study of interactions between P. epidermidis. Pittsburgh. aeruginosa and cystic ﬁbrosis-affected human airway epithelial cells was described by
Downloaded from jb. Kolter suggested that. 2007
surrogate gallium as a bioﬁlm inhibitor. Fragments of recombinant embp induce cell aggregation and bioﬁlm formation in embp-negative S. although some cells could switch between phenotypes. and the fragments also inﬂuence binding to host cells as well as to host components such as ﬁbronectin. Karin Sauer (Binghamton University) identiﬁed two phosphorylated proteins. ﬁndings that underscore the role of the protein in pathogenesis. a certain probability exists that any one cell would switch. Mutants that do not make PIA are defective in adherence and bioﬁlm formation. not to oxygen concentration (35).VOL. it seems that PCho modiﬁcation results in tolerance of the infection by the animal (reduced initial inﬂammation) as well as in stable growth and maturation of the bioﬁlm. a subject explored in greater detail by University of Washington colleagues (28). 2009
. Holgar Rohde (University of Hamburg) spoke on non-PIA-mediated bioﬁlm formation in S. Using green ﬂuorescent protein (GFP) reporter fusions and confocal microscopy. epidermidis strains and thus may be central to the colonization of host tissues. Denmark) (74). propagation. Bioﬁlms of S. Lyngby. Thus. whereas under glucose limitation. such as matrix building. 51) and proteome proﬁles that differ from those of planktonic cells with respect to murein and PIA synthesis as well as the physiology of ammonia and acid production. aeruginosa and cystic ﬁbrosis. Carol Kumamoto (Tufts University) used a simple but elegant agar system to show that the switch to an invasive. aeruginosa.
these aggregates were absent from the sputa of patients infected solely with nonmucoid strains. BIOFILMS IN NATURAL ENVIRONMENTS Michael Kuhl (University of Copenhagen) and Steven Lin¨ dow (University of California—Berkeley) were the chairs of a session designed to examine the structure and function of bioﬁlms in terrestrial and marine ecosystems. Low levels of c-diGMP encourage motility (twitching and swimming). Addition of the bacteria to conﬂuent eukaryotic-cell cultures caused detachment of the eukaryotic cells within a few hours. antibiotic treatment of the patients resulted in reductions in mucoidy and aggregation. (iv) the infection is resistant to antibiotic therapy. Scale symbol (black). Pediatric adenoid-associated bioﬁlm that meets the four criteria suggested by Parsek and Singh (45a) for the determination of a bioﬁlm: (i) the pathogenic bacteria are surface associated or adherent to a substratum.asm. results in production of the rdar phenotype through synthesis of curli as well as by indirect activation of cellulose exopolymer synthesis. and Høiby believes this is where the infection has its true genesis. 10 m in each dimension. (ii) direct examination reveals bacteria in clusters and encased in a matrix of bacterial or host constituents. BACTERIOL. within microbial mats and corals. (iii) the infection is localized. The bioﬁlm EPS was stained with lectins (green). red colonies. second. Much of this work employs ﬁlms that contain a
Downloaded from jb. AdrA is one of four GGDEF/EAL proteins that inﬂuence the transcription of cgsD. Image courtesy of Laura Nistico. aeruginosa. and rough) phenotype is controlled by c-diGMP levels. particularly oxygen.org by on December 23. the system retained some bacteria despite the antibiotic addition. Pittsburgh. whereas high levels encourage a sessile phenotype through production of exopolymers and extracellular appendages. 2009
. The mucous layer creates steep oxygen gradients. which emphasizes the universality of this regulatory mechanism. aeruginosa colonies are found surrounded by polymorphonuclear leukocytes (PMN). aeruginosa cells. however. He further related the toxicity to inefﬁcient nitrite reductase and nitric oxide reductase activities in mucA mutants. discussed later in this paper. nitric oxide) to anaerobically grown P. On the basis of autopsy and sputum specimens. Janus Haagensen (Danish Technical University) used FISH to show the presence of P. together with destroyed lung tissue. The fourth criterion was fulﬁlled by empirical evidence: unsuccessful resolution with antibiotic treatment. Kuhl ¨ spoke on spatiotemporally resolved techniques for imaging chemical species.. In Salmonella enterica serovar Typhimurium.
FIG. dry. where oxygen tension is high relative to that in the bronchi. and Luanne Hall-Stoodley (Center for Genomic Sciences.7950
J. which stained bacterial cocci (black arrows) as well as host nuclei (white arrows). the approximate pH of lung airway mucus according to his measurements in lungs recently removed from transplant patients. adherent cells. as well as mucoid colonies. aeruginosa infection that emphasized the focal nature of the infection and the importance of understanding the role of oxygen tension in the lung in bacterial growth and subsequent tissue destruction. inﬂammation and tissue destruction occur in this zone. 57).g. Co-chair Romling spoke on second-messenger c-di-GMP ¨ signaling and phenotype switching (25. are then transported by coughing to the bronchi (conductive zone). the “rdar” (red. In the respiratory zone. The ﬁrst. These mucoid colonies. and he suggested that nitrite or NO could be useful as a therapeutic agent for cystic ﬁbrosis patients (76) if the bacteria in situ are growing anaerobically.
aeruginosa cell aggregates within the sputa of cystic ﬁbrosis patients who had coinfections with mucoid and nonmucoid strains. single P. where they become embedded in the naturally occurring thick mucous layer. which is controlled by environmental signals (such as temperature) as well as by the c-di-GMP level. This phenotype is characterized by a rough colony morphology. Expression of the transcriptional regulator cgsD. he described how the infection compartmentalizes in the lung. however. Paul Rainey’s talk.
Greg Anderson (Dartmouth College). Further. which has 20 GGDEF/EAL proteins. Detachment could be greatly reduced by the presence of tobramycin in the medium. aeruginosa bioﬁlms was discussed by Dan Hassett (University of Cincinnati). explored c-di-GMP networks in an evolutionary context. The toxicity of nitrite and its derivatives (e. Less deﬁned nuclei are deeper in the tissue. He showed that mucA mutants (the mucoid strains that predominate in cystic ﬁbrosis lung infections) are extremely sensitive to NO at a pH of 6. can be found in the mucus. and clumpy. no thick mucous layer is present but mucoid P. and the surface of the adenoid (blue) was imaged using reﬂected confocal microscopy. The fresh specimen was stained with the nucleic acid stain Syto 9 (red). and third criteria were determined by microscopic examination. csgD knockouts have a smooth colony morphology and are nonadherent and nonclumping. These c-di-GMP “networks” have been shown to be crucial to the development of a bioﬁlm phenotype in several bacteria. these proteins are widespread in bacteria. aggregates were shown to contain other bacteria in addition to P. although the number of bacteria was reduced.5. PA). including bacteria as commensals or pathogens of plant or animal hosts. 1. Paul Stoodley. Niels Høiby (University of Copenhagen) presented new ideas on P. he found limited NO toxicity at a pH of 7 or above. GGDEF/EAL domain proteins have phosphodiesterase as well as guanylate cyclase activity and thus regulate c-di-GMP levels.
Examples include changes in photosynthetic O2 production as a function of the light level (33) or changes in oxygen concentration as a function of the ﬂow rate in a bacterial bioﬁlm (34). but R. thus providing a snapshot of the pH at that site in the bioﬁlm. the cells bind to substrata using their poles. Cell density-dependent synthesis of EPS is required for virulent bioﬁlm formation. toxin-producing cyanobacteria have been identiﬁed from the community and may be responsible for regional differences in the disease. P. Decho suggested that this may be set up through homoserine lactone signaling. 2009
. and movement from vessel to vessel (bioﬁlm metastasis) is encouraged. In A. two surface colonizers of the marine alga Ulva australis (47). Fiji) spoke on the interaction between Pseudoalteromonas tunicata and Roseobacter gallaeciensis. tunicata can outcompete other natural community members when grown as a bioﬁlm in natural seawater. the phosphate response regulator protein PhoB is activated during phosphate starvation. P.. When mineralization does occur. but R. (A) Projection and vertical slices through a confocal image stack of a bioﬁlm structure consisting of GFP-marked Pseudomonas putida (green) and heterotrophic bacteria labeled with Syto 60 (red) imaged through an O2 coverslip-sensor mounted on top of a ﬂow chamber. Comparison of the bacterial community from diseased coral with that from healthy coral (54) has shown that the migration of certain bacteria. mutants with mutations in the esaI and esaR quorum-sensing loci form less-dense bioﬁlms and do not disperse effectively in planta. Solitary bacteria in unblocked vessels can move from vessel to vessel. 2. active sulfate reduction occurs at the very surface of the bioﬁlm (6). which then become blocked. gallaeciensis can invade and eventually comes to dominate such a bioﬁlm. the densely packed bioﬁlms in blocked vessels have restricted access to nutrients and thus have very low activity.org by on December 23. Likewise. Bacterial biomass and the level of the diffusible signal factor DSF (the product of rpfF) are low early in infection. who related inorganic phosphate (Pi) deprivation to enhanced bioﬁlm formation in Agrobacterium tumefaciens. The bacterium was shown to speciﬁcally colonize annular rings and spiral wall thickenings within the xylem. low Pi levels may be a cue for the production of a specialized polymer and thus for bioﬁlm initiation. as assessed by propidium iodide staining. tumifaciens bioﬁlms have an elevated biomass relative to that of normal bioﬁlms. P. tunicata cannot invade an extant bioﬁlm composed of other species. 2007
FIG. with resultant vessel blockage. Combined microscopic imaging of oxygen concentration and bacterial colonization within a bioﬁlm reprinted from reference 34 with permission. biomass and signal levels increase and cue polymer production. Chris Dupraz (University of Lausanne) presented data on extracellular polysaccharide inhibition of biogenic calcium carbonate formation (16). Interestingly. Pi is frequently limiting in the environment. The central cell cluster is outlined by the dashed curve. 40 m. phosphate-starved A. produces sharp gradients of sulﬁde and oxygen that kill the coral.
photochemically active sensor species (21). Further. Lifetime images are produced that describe how a chemical parameter changes in response to changes in the environment.
Downloaded from jb. in sharp contrast to the spatial organization in other
mat systems. thereby sacriﬁcing a few xylem vessels but concomitantly inhibiting the spread of the infection and saving the plant. (B) Corresponding O2 distribution at the base of the bioﬁlm. and in vitro. 189. Lindow proposed to treat infection by introducing signal-hyperproducing strains to push early infections toward the late-stage phenotype. When phoB is induced. Scale bars. and the mutated gene bears similarity to a gene in the holdfast biosynthesis pathway of Caulobacter crescentus. Co-chair Steve Lindow continued the theme of plant pathogen bioﬁlms in his talk on Xylella fastidiosa (43). 2). tumefaciens. Late in infection. Alan Decho (University of South Carolina) discussed layering and microbial activity in the same bioﬁlms. Two talks on Bahamian marine stromatolithic mats highlighted interesting properties of this system. changes in the local pH result in the formation of differently shaped carbonate crystals.VOL. and they also show wheat germ agglutinin binding at their poles. which reports the concentration of the soluble target species (Fig. Dhana Rao (University of the South Paciﬁc. This bacterium infects plant xylem vessels. tunicata under the same circumstances.asm. tunicata is unable to persist in sterile-ﬁltered seawater. Susanne von Bodman (University of Connecticut) also spoke on a plant xylem pathogen: Pantoea stewartii (31). in particular that of Beggiatoa spp. Black band disease in corals was discussed by Laurie Richardson (Florida International University). Plant-associated bioﬁlms were introduced by Clay Fuqua (Indiana University). A transposon mutant selected for the lack of polar binding is not recognized by wheat germ agglutinin. Microautoradiographic data together with FISH studies show that in this system. gallaeciensis outcompetes P.
Thus. an exponential relationship. phospholipase. 39). 12. 70). he showed that spore attachment increases with increasing lactone concentration. Hørnsholm. Using natural bacterial isolates. Thomas Rasmussen (Chr. they are selected for and come to dominate the bioﬁlm. and studies of mutations in bioﬁlms underscore the uncertainty. phenazine. Problems in deﬁning exactly how a mutation arises and in accurate counting of mutations have left this question unresolved for decades. Less precipitate was found associated with metabolically active cells than with carbonyl cyanide m-chlorophenylhydrazone (CCCP)-inhibited cells. but supernatants of quorum-sensing mutant bioﬁlms do not. the majority occurred within negative regulators of various diguanylate cyclases. aeruginosa inhibits hyphal formation in Candida albicans. breakdown products of homoserine lactone inhibit germ tube formation. In an example of beneﬁcial cross-kingdom signaling. EVOLUTION AND DIVERSITY Paul Rainey (Massey University. whereas quorum-sensing mutant bioﬁlms upregulated catalase. 2009
. aureus colonizes the alimentary tract. Denmark) talked about quorum sensing in Pseudo-
monas aeruginosa bioﬁlms and how it affects interactions between the bioﬁlm and PMN.g. Microarray analysis of wild-type bioﬁlms exposed to PMN showed upregulation of PQS (Pseudomonas quinolone signal). precipitated iron is heterogeneously distributed. Continued selection of mat-forming genotypes (known “wrinkly spreaders” [Fig. Once the variants appear. three patterns of increasing zoospore attachment were seen as bacterial numbers increased: a linear relationship. mat-forming (cooperating) genotypes can once again evolve from the cheating types (5). Roth presented theoretical arguments against the existence of a “hypermutable” state under any circumstances. 3]) resulted in the evolution of genotypes that no longer overproduced the polymer but could still grow within the mat.
Two talks on bioﬁlms in terrestrial systems highlighted the importance of water availability and of local chemistry. and a bell-shaped curve. and PIA-negative mutants do not kill as effectively as the parent strain. as well as an alternative interpretation of the data from the best-accepted example of hypermutation (10) whereby the observations could be due to gene duplication (32). Hansen A/S. These results suggest that wild-type bioﬁlms mount an aggressive attack on PMN and that mutant bioﬁlms respond to the PMN oxidative burst by shielding themselves with catalase. Miguel Camara (University of Nottingham) discussed how ho´ moserine lactone signaling molecules produced by bioﬁlms inﬂuence the attachment of Ulva linza (marine alga) zoospores (63). and in a second example of cross-kingdom inhibition. Pradeep Singh (University of Washington) described experiments that suggest an increase in the occurrence of genetic/ phenotypic variants in bioﬁlm systems (8). causing cells not only to remain attached after cell division but also to generate a mat of polymer-linked cells at the air-liquid interface in broth microcosms. addition of an antioxidant to the bioﬁlm reduces the occurrence of variants. Groningen. New Zealand) chaired the session on evolution and diversity and gave the opening talk. The net effect was to reduce negative regulator function. Braunschweig. Germany) spoke on protozoan grazing effects on bioﬁlms (38. Using lactones synthesized by Vibrio anguillarum as model compounds.7952
J. C12 and C14 homoserine lactones had the greatest effect. Molecular analyses of cheaters revealed a range of different mutations whose net effect is to abolish the activity of the previously activated diguanylate cyclases. BACTERIOL. S.asm. Bastiaan Krom (University Medical Center. e. rhamnolipid production is increased. and known virulence factors such as PIA have been demonstrated to apply to the worm. Singh showed that these variants are generated by error-prone repair (recA and recBC mutants) of double-stranded breaks in DNA arising from oxidative stress. Numerous mutations that generate bioﬁlm-forming genotypes were identiﬁed. aeruginosa bioﬁlm supernatants lyse PMN. Bacterial defense mechanisms against grazing include microcolony formation (protective shielding) and the production of soluble inhibitors of amoebal growth. presenting bioﬁlm development in Pseudomonas ﬂuorescens as a process of mutation and subsequent selection. and Larry Halverson (Iowa State University) showed that production of the extracellular polysaccharide alginate is upregulated in Pseudomonas putida bioﬁlms under matric water stress but not under osmotic stress. CROSS-KINGDOM INTERACTIONS IN BIOFILMS Leo Eberl (University of Zurich) chaired a session on soluble signals and interactions of bacteria with eukaryotes. with concomitant overproduction of c-diGMP. Further. not through a hard-wired developmental cycle particular to the bacterium. In turn. and the bacterium alters its lipopolysaccharide chemistry in response to pH (22. and rhamnolipid synthesis genes. diversity and development in bioﬁlms may occur simply by the environment selecting for “standard” mutations. Water potential is critical to soil bioﬁlm development. enzymes involved in the production of a cellulose-based polymer were activated. These selﬁsh (cheating) genotypes did not contribute to mat strength or integrity and thus caused the mat to collapse. Alginate seems to protect against desiccation by maintaining a hydrated environment for the cells (11. elastase. John Roth (University of California—Davis) addressed the question of whether stress (such as could occur during bioﬁlm growth) increases mutation frequency. Carsten Matz (Helmholtz Center for Infection Research. This may be correlated with the local pH. Roth believes that selection is more important than the mutation rate. The process can continue. Virulence is measured as the reduction in the mean lifetime of the worm. These results suggest a role in nature for this signaling mechanism. Soluble signaling molecules were the theme of several talks. protons transported to the cell surface by respiration appear to compete with metal cations for reactive cell surface sites. Coagulasenegative staphylococci have also been shown to be virulent in this model. Wild-type P. The Netherlands) showed that homoserine lactone from P. Two talks addressed cross-kingdom interactions in pathogenesis. aeruginosa bioﬁlm. Bioﬁlms with reduced alginate levels are less dense and ﬂatter. Costi Sifri (University of Virginia Health System) discussed the nematode Caenorhabditis elegans as a model organism for bacterial infection (56).org by on December 23. Con-
Downloaded from jb. Ryan Hunter (University of Guelph) showed that in an in vitro P. Grazing by amoebae induces changes in bacterial gene regulation. 23)..
000-parameter Biolog plates. Denmark) introduced evidence for the presence of amyloid protein (originating from ﬁmbriae) as a ubiquitous component of EPS produced by many different species and found in freshwater.
Downloaded from jb. these mutators are deﬁcient in repair (15). He described how these bioﬁlms fall apart after the appearance of the SCV and how the SCV can be sorted into four classes based on motility. Romeo concluded that this regulatory network was adapted for ﬁne-tuning homeostatic adjustment rather than being an “on-off” system. The difference in the mutation rate is a by-product of selection for improved ﬁtness. Per Nielsen (Aalborg University. Spain) concluded the session by describing how insertion sequence IS256 mediates phenotypic switching in Staphylococcus aureus (69). Nielsen used antibodies and FISH (Fig. (Inset) Niches occupied by different variants. and function of EPS.
THE SLIME MATRIX Tony Romeo (Emory University) chaired the session focusing on the regulation. Romeo discussed the regulation of poly. aeruginosa colony morphology variants isolated from in vitro bioﬁlm systems. Pamplona. Colony variants—including two different wrinkly spreader genotypes—of P.asm. 2009
tinuing the theme of variants. Osmotic stress can complement the lack of B and restore the wild-type phase variation frequency. 189. 2007
FIG. Wrinkly spreader genotypes form a bioﬁlm at the air-liquid interface (culture at right). Also present in natural populations are weak mutators whose mutation rates are 4 to 5 times those of laboratory strains. In turn. csrB and csrC can be degraded by csrD. 3. Sarah Schooling (University of Guelph) concluded the session by presenting evidence that membrane vesicles are an important component of P. Knockouts of transcription factor B have increased IS256 activity and produced bioﬁlm-negative variants with dramatically higher IS256 copy numbers in the chromosome at a high frequency. Jean-Marc Ghigo (Pasteur Institute. However. Image courtesy of Paul Rainey (Massey University. Observed mutation rates are typically low because most mutations are deleterious or neutral and because the cell has extensive investments in repair mechanisms. aeruginosa bioﬁlm EPS (53). 4) to demonstrate that this previously overlooked protein is produced by as many as 50% of the species in a given environment. which represses PGA and bioﬁlm formation. which is a common component of EPS in gram-positive as well as gram-negative bacteria.VOL.1. They can be selected for in gnotobiotic mice by sequential multiple-antibiotic treatments. suggesting that B is not directly involved. B appears to act as a regulator of IS256 activity and thereby as a controller of bioﬁlm-negative phenotype occurrence. the wild-type morphology was recovered. and Spormann argued for an increased emphasis on physiology in bioﬁlm experiments. and seawater environments. ﬂuorescens that arise during selection in spatially structured microcosms.6-N-acetyl-D-glucosamine (PGA). which have been shown to be more sensitive than bioﬁlm cells to valine. Alfred Spormann (Stanford University) argued that the stability of Vibrio cholerae bioﬁlms is related to the appearance of small-colony variants (SCV). the surviving cells are mutators. This secretion acts not only as a “carbon dump” but also to provide an antimicrobial effect against invading planktonic bacteria. Aalborg. composition. France) presented evidence for secretion of valine into EPS. Paris. can be sequestered by the noncoding short RNAs csrB and csrC (2). The characterization of EPS chemistry and function has remained elusive even though EPS is a hallmark that distinguishes bioﬁlm populations from planktonic cultures.
. and mutation rates can be very different in the host environment than in the laboratory. c-diGMP signaling was important in directing adhesion and cohesion (30). Characterization of SCV physiology is now under way using 2. the in vitro system may reﬂect certain in vivo conditions. DNA
KEYNOTE TALK 2 Ivan Matic (University of Paris) continued the discussion of genetic variation in bioﬁlms from the perspective of mutation and repair. By knocking out c-di-GMP in a sticky mutant. thus. Romeo discussed how the carbon storage regulator (Csr) RNA-binding protein. Jaione Valle (Public University of Navarre. populations of natural isolates contain strong mutators that display mutation rates 100-fold higher than those of laboratory strains. Matt Parsek (University of Washington) discussed the contribution of the EPS genes pel and psl in P. New Zealand). Similar colony morphology variants were isolated from cystic ﬁbrosis patients.org by on December 23. brackish-water.
Matt Parsek. 4. which are regulated by the Las/Rhl signaling system. (D) Overlay of panels A. In this cross-kingdom interaction. M. targeting the class Bacteriodetes. P. Braunschweig. CELL SIGNALING IN BIOFILMS A function for cell signaling in a bioﬁlm context was ﬁrst described for P. Since then. aeruginosa in 1998 (14). Scale bar. targeting most members of the domain Bacteria. and it was thought that signaling might be a magic bullet for the control of bioﬁlms. albicans to block the formation of hyphae. L. It remains to be seen how widespread sensing and response to self-damage is among prokaryotes. Cell signaling in the social bacterium Myxococcus xanthus was discussed by Heidi Kaplan (University of Texas Medical School).7954
J. Green ﬂuorescence marks antibodies raised against conformational epitopes of amyloid adhesins. The concept that a sessile bacterial population could coordinate its phenotype and organization through quorum-sensing mechanisms quickly captured the imagination. Intriguingly. Deborah Hogan (Dartmouth College)
presented an example of competitive survival of two organisms that involves response to. (C) Epiﬂuorescence image of the same ﬁeld of view as that in panel A. BACTERIOL. because the diversity of signal molecules and mechanisms has grown. Nielsen (Aalborg University. She presented data extending the role of PQS from iron chelation (9) to the release of DNA to the EPS matrix in response to stress. such as peptidoglycan. 20 m. (B) Epiﬂuorescence image of the same ﬁeld of view as that in panel A.asm. C. and blue ﬂuorescent EUBmix.org by on December 23. xanthus responded to an accumulation of lysed cell wall components. albicans are commonly isolated in cases of opportunistic infections (71). aeruginosa through the production of farnesol. In addition to this defensive response. However. aeruginosa of its virulence factor-stimulating signaling molecule OdDHL [N-(3-oxododecanoyl)-L-homoserine lactone] causes C. and in coculture. Denmark). Shown are results of FISH using the red ﬂuorescent oligonucleotide probe CFB719. presumably from a cyanobacterium (judging from the morphology and the ability to produce red ﬂuorescent phycoerythrin). each other’s signaling systems. a signal for C.33% of the extracellular DNA. Jan Kreft (University of Bonn) presented modeling evidence that cell-signaling mechanisms in gram-negative bacteria may be adapted to regulate behavior
. with simultaneous labeling of amyloid adhesins by antibodies and identiﬁcation of bacteria by FISH. chaired the session. Vesicle production may play a signiﬁcant role in EPS function for bioﬁlms. aeruginosa can colonize and kill C. Otzen. Germany) discussed PQS in P. Image courtesy of P. and P. the ﬁeld has gained in complexity. Farnesol. H. in the culture by initiating sporulation. Colocalization of the three ﬂuorescent probes shows as white. who was involved in the earliest bioﬁlm signaling work. which included examples of signaling in three different experimental systems. Detection of amyloids in a brackish-water bioﬁlm. aeruginosa.
Downloaded from jb. the lively discussion at the specialty evening session on EPS chaired by Hans-Curt Flemming and Dan Wozniak. the production by P. Larsen. P. albicans to switch its morphotype to the colonization-resistant but host cell-invasive yeast form. The data presented in this session. D. aeruginosa. in which recognition of cell damage is the immune activator. Susanne Haussler (Helmholtz Center ¨ for Infection Research. and C. because of its promotion of detergent tolerance. as well as interference with. Nielsen. albicans hyphae through the production of virulence factors. signals coordinate the phenotype of the producing organism while at the same time altering the phenotype of the competitor. She presented evidence for a quorum-sensing system sensitive to membrane integrity. Aalborg. B. 2009
from vesicles was estimated to make up at least 0. (A) Transmitted light image showing small rods attached to an empty sheath. Haussler suggested that PQS is a ¨ regulator of coordinated dispersal (movement of bacterial cells from the bioﬁlm to the bulk liquid). such as the fungicide pyocyanin (29). this recognition and response to damage in the population is not unlike the “danger model” of the mammalian immune system response (40). albicans mounts an offensive against P. Finally. also depresses PQS signaling and pyocyanin production in P.
FIG. J. and the ﬁnal keynote address by Bill Costerton on structured EPS illustrate how EPS is no longer seen as an amorphous carbohydrate material that serves simply to provide a physical matrix for cells but rather as a chemically complex mechanically and structurally adaptive material that has multiple biological functions. aeruginosa and C.
BIOFILM-HUMAN INTERACTIONS II The second human microbiology session was chaired by Michael Otto (NIH Rocky Mountain Labs) and David Stickler (University of Cardiff). Brisbane. antibody reactivity. Copenhagen. Mary Ellen Davey (Forsyth Institute. Denmark) presented evidence from clinical specimens suggesting that the persistence of P. in addition to the routinely cultured aerobic and facultatively anaerobic species. epidermidis to form different bioﬁlm phe-
notypes. Rostock. Further.VOL. rather than toward spatially distant and therefore.15 per 100 catheter days. Satoshi Okabe (Hokkaido University. possibly through surfactant properties.e. coli in a mouse model (68). while indicating
Downloaded from jb. including the probiotic E. Klaus Kirketerp-Møller (Bispebjerg University Hospital. together with exogenous PSM addition. Barbara Trautner (Baylor College of Medicine) then presented evidence that bioﬁlm infection of urinary catheters could be reduced by a probiotic approach: seeding the catheter with avirulent urinary tract isolates of Escherichia coli (64. Kreft’s modeling suggests that current assumptions regarding the distance over which signaling operates may need to be reevaluated. Stickler discussed Proteus mirabilis bioﬁlm formation in urinary catheters and the resultant struvite crystallization (61). less closely related cells. Bernd Kreikemeyer (University Clinic. coli strains. in more-complex consortia. and that they were upregulated in bioﬁlms. Seeding with these E. a number of uncultured obligate anaerobes were present. gingivalis strains did not form bioﬁlms. coli Nissle strain. Session co-chair Michael Otto then gave the ﬁrst of two talks on the molecular pathogenesis of staphylococcal infections.7 per 100 catheter days to 0. capsule expression was found to be dependent on high nutrient levels and rapid growth. suggest that PSM is responsible for controlled detachment. and that many of these genes are important in the context of bioﬁlms. it was found that Streptococcus salivarius inhibited the development of Streptococcus pyogenes bioﬁlms. and enterobacterial repetitive intergenic consensus ﬁngerprinting patterns occurred (44). Signals are directed toward nearest-neighbor. Based on the observation that the diversity of signals in gram-negative species is small (primarily acetylhomoserine lactones) but that a large number of species respond to these molecules. Control of device-related bioﬁlm infections is perhaps best addressed not by attempting to render the surface sterile. closely related cells (i. In contrast. but rather by prophylactic inoculations. and could kill. while strains lacking a capsule did (13). The ﬁrst three talks focused on bioﬁlms in the urinary tract. Differential expression showed that approximately 50% of the genes were unique to clinical strains. 65). He showed how power output could be optimized by varying species composition and growth conditions. changes in coaggregation. Trautner is currently designing E. two talks on oral bioﬁlms were given. In addition to the infectious disease-related talks. however. Rob Palmer (NIDCR. because other nonpathogenic species. Palmer concluded that the only way to fully understand the complexity of the time-dependent microdiversity present in dental bioﬁlms was through a polyphasic approach. 2007
over the small distances between adjacent cells and within small clusters (19). toward clones). Garth James (Montana State University) used confocal microscopy and denaturing gradient gel electrophoresis to show the broad diversity of infecting bacteria. Australia) discussed the role of antigen 43 as an important adhesin and virulence factor for uropathogenic E. He showed that spontaneous mutation in the bioﬁlm-repressing agr locus causes S. aeruginosa in inﬂamed chronic wound infections was due to its protected location within microcolonies. NIH) used immunoﬂuorescence confocal microscopy and enterobacterial repetitive intergenic consensus PCR to investigate phenotypic and genotypic diversity within a Veillonella sp. BIOFILMS IN INDUSTRIAL AND ENGINEERED SYSTEMS Much early pioneering work in bioﬁlm research was conducted in the context of fouling prevention in industrial and marine systems. even in similar wound types. coli strains through genetic modiﬁcation that will be even more efﬁcient at “competitive exclusion. because no selective pressure exists to maintain virulence genes. where it remained impervious to. These data indicate that continued laboratory passage of clinical strains may cause a loss of virulence. GFP reporters of PSM synthesis. The difference in architecture between the heterogeneous wild type and the thicker but ﬂatter agr mutant was caused by the phenol-soluble modulin (PSM) (75). Anna Muench (University of Maryland Dental School) used microarrays to identify genes unique to clinical isolates of S. Brisbane. 189. Australia) were the chairs for this session on bioﬁlms in engineering applications. Davey found that encapsulated P. Adhesins such as antigen 43 may be useful targets for modiﬁcation to create probiotic E. The next two presentations were on a rapidly growing area of interest in medical bioﬁlms: chronic wound infections.. presumably. In as little as 4 h. 2009
. only 10% of genes unique to laboratory strains were upregulated in bioﬁlms of laboratory strains. Germany) discussed the development of a probiotic approach to reduce the incidence of nasopharyngeal infections by group A streptococci (37). In a simple coculture. James hypothesized that strict anaerobes may inhabit anaerobic microniches created through oxygen consumption by aerobic bioﬁlms. aureus. Interestingly. in natural dental plaque bioﬁlms from human volunteers. patient urine chemistry and bioﬁlm species composition were important factors in the rate of catheter blockage. explaining why encapsulated forms are more commonly observed on culture plates. Multispecies bioﬁlms attenuated crystallization by modifying the pH. which has proved extremely challenging and is possibly futile.” Mark Schembri (University of Queensland. attenuated the interference effect. Kreft hypothesized that signaling in bioﬁlms is local. Harvard University) discussed the role of capsule expression and bioﬁlm formation in Porphyromonas gingivalis. 16S rRNA analysis showed that.asm. coli isolates reduced the incidence of symptomatic infection nearly 20-fold: from 2. Korneel Rabaey (University of Queensland) described the use of microbial electron transfer systems to create microbial fuel cells.org by on December 23. that many of these genes were involved in stress response and the production of virulence factors. Japan) and Linda Blackall (University of Queensland. the effect was not as straightforward. leukocytes. Importantly. and also for optimization of bioconversion processes in wastewater treatment and bioremediation.
Downloaded from jb. which appeared similar to the honeycomb-like structures introduced later in the meeting by Bill Costerton. This approach allows spatiotemporally resolved measurement of toxicity and suggests that differences in susceptibility to particular agents exist within the bioﬁlm. Stewart (Center for Bioﬁlm Engineering. The number in each panel is the time. Picioreanu’s model showed how bioﬁlm structure may be an important factor for charge density on the anode. A FISH protocol was optimized for use in thick. Thomas Neu (Helmholtz Center for Environmental Research. in minutes. Davison and P. especially in process situations: it can be positioned much further from the bioﬁlm. Bacteria in a ﬂow cell-grown bioﬁlm were loaded with the green ﬂuorescent dye calcein AM and then exposed to 50 mg liter 1 alkyl dimethyl benzyl ammonium chloride (ADBAC) under continuous ﬂow. and expression techniques could be applied to identify bioﬁlm activity in reverse osmosis systems. and it can penetrate opaque materials (73). Carsten Schwermer (Max Planck Institute for Marine Microbiology. after the introduction of ADBAC into the ﬂow cell. 5). Christian Picioreanu (Delft University) and Andrew Kato-Marcus (Arizona State University) each presented mathematical models that could be used to optimize these fuel cells. this approach may have applications in other bioﬁlm systems such as microbial mats. BACTERIOL.7956
J. and microelectrodes to characterize the distribution and activity of anaerobic ammonium-oxidizing (anammox) bacteria in a mixed wastewater bioﬁlm (67). Germany) described ﬂuid dynamic gauging. Finally. PREVENTION AND TREATMENT OF BIOFILMS Michael Givskov (Danish Technical University) and Phil Stewart (Montana State University) were session chairs. dense bioﬁlms. These bacteria were distributed throughout the bioﬁlm. Low carbon levels increased the number of viable but nonculturable bacteria in the bioﬁlm. Moshe Herzberg (Yale University) illustrated how molecular. and phosphorus from wastewater. Image courtesy of W. 5. nondestruc-
. Bremen.asm. 2009
FIG. while Kato-Marcus modeled the anode as an electron acceptor. Co-chair Linda Blackall showed how the development of structured bioﬁlm communities in ﬂocs could be used for the simultaneous removal of ammonium. When treated with the quaternary amine Barquat. Treatment with chlorite caused loss of ﬂuorescence only in a thin band at the edge of the colony. Magdeburg. a method of monitoring bioﬁlm stability by which the shear strength of fouling layers can be measured (41). Germany) presented industrially funded research that employed FISH and microelectrodes to determine the inﬂuence of nitrate on a bioﬁlm community from an oilﬁeld seawater injection system that contains sulfate-reducing bacteria (SRB). Treatment with nisin led to a rapid and uniform ﬂuorescence loss. In the ﬁrst and last panels. Chuanwu Xi (University of Michigan) presented work on optical coherence tomography. Neu also showed magnetic resonance imaging data that allow real-time. suggesting that environmental factors should be taken into account when one is culturing from drinking water systems. Co-chair Satoshi Okabe used a polyphasic approach that employed FISH. Electron microscopy suggested that the ﬂocs may have structured EPS. Montana State University). nitrate/nitrite. Portugal) showed the importance of nutrients and hydrodynamics to Legionella pneumophila bioﬁlm formation. The instrument offers two advantages over conventional microscopes. The base layers of the bioﬁlm were found to be highly stable. a high-resolution imaging technique that does not require staining. Three talks focused on multispecies bioﬁlms in wastewater treatment. Visualization of antimicrobial action against a Staphylococcus epidermidis bioﬁlm. Silvia Weber (Technical University of Munich) used similar techniques to investigate granular ﬂocs. Stewart’s talk on the visualization of antimicrobial action in bioﬁlms described the use of calcein AM as a viability indicator.
that initial applications may be limited to low-current situations such as sensors and feedback switches (1). Maria Giao (University of Minho. with the bioﬁlm itself as an integral part of the anode. but rates of ammonium and nitrite oxidation differed at different locations in the bioﬁlm.
nies quickly lost calcein ﬂuorescence at the edges but showed time-dependent retention of ﬂuorescence at the center (Fig. biomass was imaged in transmission mode (gray scale). microautoradiography. Time lapse confocal scanning laser microscopy examination of ﬂuorescence loss shows permeabilization of cells by the biocide.org by on December 23. for real-time monitoring of living bioﬁlms. Nitrate achieved the desired effect of reducing SRB activity and sulﬁde production and may see widespread use throughout the industry as a more environmentally compatible alternative to conventional biocides used for SRB control. microscopic. whereas the upper layers were easily removed.
and Burkholderia cepacia (27). because the targets are eventually to be used in the diagnosis of human bioﬁlm infections. Using high-throughput cell sorting. 2007
FIG. High levels of prophage transcripts and a group of candidate “persister” genes including the putative regulator ykgK were identiﬁed. in contrast to bacteria. it is less active than garlic extract. Givskov suggested that rhamnolipid is a necrotic agent for PMN (7. Gilbert showed that an Escherichia coli population labeled with GFP contained a very small fraction of poorly metabolizing (weakly ﬂuorescent) cells that also demonstrated multidrug resistance. aeruginosa bioﬁlm thickness by 50%. but it is possible that a mixture of garlic-based compounds can reach or exceed the efﬁcacy of natural garlic extract. Antisense suppression of pin. ﬁrst reported by Spoering and Lewis (59). Quorum-sensing-controlled killing of PMN. Lewis also used transcriptional proﬁling to identify a number of candidate genes. (Right) In the presence of the P. These genes may be regulators of the persistence state and thus potential drug targets for recalcitrant bioﬁlm infections. Syto 62-stained PMN (red) interact in vitro with a GFP-tagged bioﬁlm (green).asm. these cells were the subject of talks by Peter Gilbert (University of Manchester) and Kim Lewis (Northeastern University). while overexpression increased persisters. Lewis thought that the differences between candidate genes from the two studies may be a result of strain differences.org by on December 23. 189. antimicrobial-resistant. human blood must be screened as well. ykgK. aureus. Bdellovibrio bacteriovorus can greatly diminish the bioﬁlm biomass of E. coli as well as that of P. The development of antimicrobials from marine invertebrates was reported on by Domenico Schillaci (University of Palermo). A promising garlic-based quorum-sensing blocker (46) has been synthesized and tested. aeruginosa LasR/RhlR double-quorum-sensing mutant. aeruginosa. Images courtesy of Thomas Bjarnsholt (Danish Technical University). forms persister cells only in a bioﬁlm. The PMN are lysed and disappear before they can eliminate the bioﬁlm. the PMN readily traverse the bioﬁlm and phagocytose the virulence-factor-deﬁcient bacteria. epidermidis and S. as well as of ﬂow velocity and shear stress around the bioﬁlm. Jennifer Kofonow (Northern Arizona University) spoke on the use of bioﬁlm-speciﬁc antibodies to detect bioﬁlm infections in a rabbit model. are deﬁned as a numerically small ( 1%). Lewis’ persister genes included the dehydrogenase-encoding gene glpD and the lipid biosynthesis gene plsB (60). The less well known exoparasitic bacterium Micavibrio aeruginosavorus is likewise able to reduce the bioﬁlm biomass of laboratory and clinical strains of P. Klebsiella pneumoniae. 6) and that reduced rhamnolipid synthesis may partly explain the more rapid clearance of quorum-sensing-defective Pseudomonas aeruginosa than of the wild type in a mouse model of lung infection (20). 6. He also reported that Candida albicans. A 5-kDa peptide fraction inhibits bacterial adhesion and interferes with bacterial respiration in bioﬁlms. ﬂuorescens (26). Cytosol of the sea urchin Paracentrotus lividus has activity against S. but he identiﬁed genes different from those discovered by Gilbert. and it has a synergistic effect with antibiotics whereby a lower antibiotic concentration is required to achieve the same level of killing as that seen in the absence of the dispersing activity. Antigens are being identiﬁed by a proteomics approach as present in infected animals but absent in healthy animals. Large volumes of culture were processed to collect sufﬁcient mRNA for microarray analysis. 24) (Fig. Lewis’ main observations on weakly metabolizing cells being persisters (55) are in agreement with those of Gilbert. Persister cells. 2009
tive visualization of bioﬁlm biomass.VOL. David Davies (Binghamton University) spoke on a bioﬁlm-dispersing activity found in the organic fraction of P. persisters are not part of the population during planktonic culture (36). Downloaded from jb. aeruginosa spent culture medium. and ykgM eliminated persister cells. (Left) Wild-type bacteria kill the PMN through quorum-sensing-controlled production of rhamnolipid. Co-chair Michael Givskov spoke on quorum-sensing blockers as drugs. Freshly isolated. and one candidate protein has
emerged. Advantages of this approach are (i) that development of resistance to compounds similar to those generated naturally by the bacterium is highly unlikely and (ii) that blockers can be used to modulate antibiotic resistance and to generate synergistic effects with other antimicrobials (48). The activity reduces P. slow-growing subpopulation of cells that is maintained in both planktonic and bioﬁlm cultures. it also acts against mixedspecies bioﬁlms.
. The topic of predatory/parasitic bacteria as antibioﬁlm agents was covered by Daniel Kadouri (University of Medicine and Dentistry of New Jersey). Closing the session.
USA 101:16630–16635. Forbes. Costerton termed the process “bacterial coalescence” and noted that the repeated structure. Z. S. P. and P.. Hartmann. Wackym.. 2005.
ACKNOWLEDGMENTS The committee (Niels Hoiby. F. R. N. beginning at the University of Calgary and later as the director of Montana State University’s Center for Bioﬁlm Engineering. J. P. J. M. Matic. 5:230–239. NIH. Duncan. Appl. T. P. is provocative. Hall-Stoodley. Jr. M. Bacteriol. Chang. Verstraete. 2003.-S. Spiers. L. J. R. CODA In summary. Jensen. Vasil. Bacteriol. Kerschner. these structures appear to form from the contents (especially ribosomes) of lysing cells that merge with one another. J. 2005. Halverson. 3. Ehrlich. J.. 4. S.. D. Palmer. Haagensen. As discussed in the “slime matrix” session. the person who. Singh. K. J. 2007. development. JAMA 296:202–211. Geol. Microbial fuel cells for wastewater treatment.
took steps to attract an audience and a program that were demographically as well as scientiﬁcally diverse. and P. Romeo.. Microbiol. B. Sci. A. P. which can form macroscopic structures with dimensions of millimeters. 20. 2006. B. Acad.asm.. Boles. 17. L. Allegheny-Singer Research Institute) thanks ASM for its continued support and development of this conference series. Gotz. Jr. NIDCR. Kuttler. R.
REFERENCES 1. K. Mol. Greenberg. O. and M. D. Clauwaert. little is known about the structural and chemical composition of bioﬁlm EPS. Proc. C. de Guzman.. and E. and M.. 11. 2. Chang. L. H. 43:1367–1378. and the committee hopes that this approach will continue. J. and L. 2007. and S. Mol. Dupraz. A. Greenberg. Hougen. N. Paul Stoodley is supported by Allegheny-Singer Research Institute and Philips Oral Healthcare. G. Stoodley. B. A. Trends Microbiol. W. E. M. Note the coalescence of cells to form the parallel planar walls of the basic honeycomb structure. R.. Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a bioﬁlm. J. M. A. and J. Davey. The involvement of cell-to-cell signals in the development of a bacterial bioﬁlm. Nimtz. 16. 14. D. Iron and Pseudomonas aeruginosa bioﬁlm formation. ¨ 18. van de Mortel.. 10:156–163. Matthew Parsek. J. C. 2006. Hoffmann. Gieseke. C. Dupraz. D.. Proc.. M. J. Scanning electron microscopy of a part of a macroscopic casernum formed in liquid culture by the MH strain of S. K. Geffers. Z. 12. Cairns. M. 6. J. Burmolle. exciting science from a rapidly moving ﬁeld and for demonstrating the broad applicability of bioﬁlm biology to microbiological research. III. Søren Molin. Parsek. E. G. 2009
KEYNOTE TALK 3 The prebanquet address was given by Bill Costerton. A. Sulfate reducing bacteria in microbial mats: changing paradigms. J. Muller.. and J. new discoveries. we thank ASM Conferences staff members Lisa Nalker and Latonya Nichols for outstanding work in bringing the 4th Conference on Bioﬁlms to fruition. Banin. Environ. Haussler. 2007. O. Microbiol. J. Babitzke. Visscher. hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. Molin. 10. and confocal and light microscopic evidence. Rasmussen. Casernum production in natural environments and a role for caserna in bioﬁlm function remain to be demonstrated. and W. C. G.” named after the temporary stone forts built by soldiers of ancient Rome. P. 2006. L. Calum. J. M. likely resulted from a genetically regulated process. freeze-substitution transmission electron microscopic. J. Bacteriol. Greenberg. Nielsen. The ¨ Pseudomonas aeruginosa quinolone signal (PQS) has an iron-chelating activity. Banin. Reid. FIG. Iglewski. Adaptive divergence in experimental populations of Pseudomonas ﬂuorescens.7958
J. and P. Reduced water availability inﬂuences the dynamics. Greenberg. Acad. USA 102:11076–11081. and P. epidermidis. M. Knight. Self-generated diversity produces “insurance effects” in bioﬁlm communities. Hense. T.. ¨ Kreft. C. Microbiology 151:373–383. which he termed “caserna. Pearson. L. Mutational origins of wrinkly spreader diversity. epidermidis isolates (52). such as that described for proteins. Przekop. 2006. P.. and T. P. Microbiol. Direct detection of bacterial bioﬁlms on the middle-ear mucosa of children with chronic otitis media. Rothballer. These are networks and honeycombshaped scaffolds with pore sizes of several micrometers (Fig. the possibility of higher-order structural organization. Sediment. P. N.. K. T. brought bioﬁlms to the forefront of microbiological research and who is now developing a bioﬁlm research center at the University of Southern California. Bantinaki. Hentzer. E. 5. 19. C. E. Post. Burrows. Technol. Davies. 72:2064–2069. G. Hentzer. W. Denamur. A. Aelterman. Stub.. Buer. W. Microbiol. 2006. K. 1991. Microbial lithiﬁcation in marine stromatolites and hypersaline mats.org by on December 23.. the meeting elevated its reputation for showcasing high-quality.. R. However. and E. Evolution of mutation rates in bacteria. this benchmark for the meeting seems to have been heartily embraced. 1998. 185: 131–145. Rev. Hoiby. CsrB sRNA family: sequestration of RNA-binding regulatory proteins. W. Robert J. Water Sci. Spear. Rabaey. Kassen. Genetics 128:695–701. 7) which develop in several-day-old cultures of S. T. 185:6199–6204. Robert J. M. 7. 2004. M. H. S. Jensen. and E. 8:1318–1329. Thoendel. and L. M. Does efﬁciency sensing unify diffusion and quorum sensing? Nat. Based on scanning electron microscopic. Bjarnsholt. Alginate production by Pseudomonas putida creates a hydrated microenvironment and contributes to bioﬁlm architecture and stress tolerance under water-limiting conditions. BACTERIOL. K. Technical University of Denmark. Nguyen. Genetics 176:441–453. Bredenbruch. P.. 60:820–827. 7. A. R. C.. and ultrastructural properties of Pseudomonas putida bioﬁlms. Environ. 54:9–15. Costerton introduced his ideas on structured bioﬁlm EPS. P. Adaptive reversion of a frameshift mutation in Escherichia coli. G. 2006. Microbiol. P. Givskov. Rainey. and I. Curr. 8. Li. Foster. Nistico. H. F. Baumgartner. although they have been seen in other bacterial cultures as well. Science 280:295–298. In particular. P. The organizing committee
. B. Costerton. 2006. Image courtesy of Christoph Schaudinn and Bill Costerton (University of Southern California). Hayes. Paul Stoodley. X.
Downloaded from jb. and P. Visscher. University of Copenhagen. H. Dice. J. 189:8290–8299. Madsen. 13:429–438. E. M. Buckley. Molin. B. Pseudomonas aeruginosa tolerance to tobramycin. Palmer. Staphylococcus and bioﬁlms. 9. E. F. B. Microbiol. the possibility of structured EPS hints at yet further levels of complexity in bioﬁlm systems. Opin. 188:5510–5523. P. Natl.-U. Halverson. L. Robinson. J. 2002. Hu. Decho. University of Washington. A. 13. is supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research at the National Institutes of Health. Sci. P. 2007. 2005. D. 15. Brady. Enhanced bioﬁlm formation and loss of capsule synthesis: deletion of a putative glycosyltransferase in Porphyromonas gingivalis. M. Moser. Natl.
50. Gross. Kainovic. Environ. Hempel. T.
33. W. Li. Resch. and G.. Antimicrob. A. M. Pathol. and J.. T. A. McDougald. 2005. E. H.. B. Kuhl. 2005. Lewis. BJU Int. H. 2007. The danger model: a renewed sense of self. G. A. K. Schooling. 50:3839–3846.. J. D. 2009
. 2006. 2006. M. E. 2005. K. Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants. Proc. Persisters: a distinct physiological state of E. Microbiology 153:1329–1338. 2007. 188:4117–4124. Microbiol... Ehrlich.
37. and T. Trautner. J.. 47. Schaudinn. 48. Richter. and K. G. T. Romling. A. A. S. 2003. Phosphatedependent modulation of c-di-GMP levels regulates Pseudomonas ﬂuorescens Pf0-1 bioﬁlm formation by controlling secretion of the adhesin LapA. Rasmussen. and A. Hierar¨ chical involvement of various GGDEF domain proteins in rdar morphotype development of Salmonella enterica serovar Typhimurium. Molecular and imaging techniques for bacterial bioﬁlms in joint arthroplasty infections. D. Characterization of colony morphology variants isolated from Pseudomonas aeruginosa bioﬁlms. Lewis. T. Bacteriol. Erdos. Neu. 189. Hunter. C. 2006.. Moreno. Valle. E. J. Hunter. Trends Microbiol. Darouiche. G. High-resolution visualization of Pseudomonas aeruginosa PAO1 bioﬁlms by freeze-substitution transmission electron microscopy. Simm. D. Resch. Ulett. Parsek. Rasmussen. O’Toole. Trends Microbiol.. and K. Kirisits. Bacteriol.. 60.
31. D. F. T. Y. Functional analysis of antigen 43 in uropathogenic Escherichia coli reveals a role in long-term persistence in the urinary tract. Stoodley. 51. D.
25. and P. Environ. 57:677–701. C. A. P. D. L.. Proc. D. 9 April 2007. Thornby. Role of ¨ EAL-containing proteins in multicellular behavior of Salmonella enterica serovar Typhimurium. A. 2007. Microbiol. 57.
21. 2005. 2007. O’Toole. Sci. Matz. Y. Nistico. Williams. 188:5945–5957. B. Levenson. R. Calum.
32. T. Rapid succession within the Veillonella population of a developing human oral bioﬁlm in situ. Eisele. Appl. Kolenbrander. Application of a pH-sensitive ﬂuoroprobe (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa bioﬁlms.. H. 2006. Baum.
34. 2006. and N. Jensen. Microbiol. and G. Andersson. R. Kerr. and F. N. Matz. 2005. M. R. 60:602–616. Resch. 67. USA 102: 5576–5581. Holst. Science 296:301–305. Clin. Hanski. Mehta.. Hall-Stoodley. Daykin. Microbiol. Dice. Welsh.. E. Microbial colonization and competition on the marine alga Ulva australis. Bacteriol. P.. 2005. Microbe 2:231–237.. Proc. B. C. F. L. 73:6289–6295. Beveridge. R. Investig.. Adusei. Parsek. L. Tsaltas.1128/IAI. Kumamoto. B. ¨ D. USA 103:17319–17324. Kofoid. Multiple pathways of selected gene ampliﬁcation during adaptive mutation. Water Res. R. B. A.21448. Chem. Sci. J. Hansen. Environ. J. Natl. P. BMC Microbiol. Pressler. Bacteriol. 68. Nerz. J. Infect. Bioﬁlm formation and phenotypic variation enhance predation-driven persistence of Vibrio cholerae. L. bioﬁlm development. Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth. 2006. C. Sherlock. Appl. Hoiby. Parsek. Ogasawara. and R. Kreikemeyer. C. Saric. 2001. Kathju.. P. 52. Agents Chemother. and E. 183:6746–6751.. J. 189:2886–2896. K.. Mills.org by on December 23. S. 2005. C.
24.. Haesner. Bioﬁlms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. and S.. and T. Hull. Lewis. I. 2006.. R. D. M. Venzon. L. N. Roth. and G. and R. in press. Structure and shear strength of microbial bioﬁlms as determined with confocal laser scanning microscopy and ﬂuid dynamic gauging using a novel rotating disc bioﬁlm reactor. N. 71:2501–2510. J. Newman. A.. Natl. J. Microbiol. Khodursky. Acad.. 2007.. Reams. Remily. A.. doi:10. 71:2663–2676. Rutman. R. Proc. and U. A. Olakanmi. Costerton. Beloin. Sifri. Phage release from bioﬁlm and planktonic Staphylococcus aureus cells.1002/bit. Bacteriol. 28:92–94. Relat. Environ. and F.asm.. J.
Downloaded from jb. Gotz. 39. L. L. 98:1244–1249. D. 2005. Zhang. 72:5547–5555.. Microbiol.. and K. Romling. J. J. P. Greenberg. Johnson. Sci. O’Keeffe. Ausubel. 71:4044–4051. K. C. and R.. Rasmussen. Appl. Tsang. 9 April 2007. I. 2005. Microbiol. Stickler.. and M. S. C.Parsek. J.
43.. Bjarnsholt. Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa. 41. Singh. Feneley. T. 188:5136–5144. 189:3613–3623. T. Klimant. Freund. J. L. Nielsen. 117:877–888. Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic ﬁbrosis. 63:656–679. 2006. Sci. T. Acad. Control Hosp. Hoiby. coli. E. P. J.
40. Clin. Podbielski. Webster. Microbiol. Valle. L. Sci. A. M.. O. B. Voss.
38. C. and I. Gerstel. Candida albicans bioﬁlms produce antifungal-tolerant persister cells. Vulnerability of pathogenic bioﬁlms to Micavibrio aeruginosavorus. Nordheim. 64. Mol. Robinson. Proc. 2007. Microbiol. Microbiol. M. Sci. C. 13:27–33. M. Scholl. 59.
42. Rational design and synthesis of new quorum-sensing inhibitors derived from acylated homoserine lactones and natural products from garlic. Webb. Lembke. A. and S. Sociomicrobiology: the connections between quorum sensing and bioﬁlms. Mohle. FEMS Microbiol. 63. and S. Gotz. O. M. Natl. Palmer. A. E. 2006. 56. Tang. Persson. and F. A clinical assessment of the performance of a sensor to detect crystalline bioﬁlm formation on indwelling bladder catheters. Appl. R. O. G. O.. P.
28. Microbial communities in the surface mucopolysaccharide layer and the black band microbial mat of black band-diseased Siderastrea siderea. Taylor. B. 2005. Post. L. Kindaichi.
45. 58. R. Waters. Bacterial bioﬁlms.
44. V. Environ. Environ. and P. Environ. 187:7619–7630.. 2007. C.. L. Acad.
36. D. X. Diaz. Characterization of bioﬁlm formation by clinically relevant serotypes of group A streptococci. R. P. Mapping of the ¨ oxygen distribution and its dynamics within the endolithic algal community of the massive coral Porites lobata (Dana). A. J. Lasa. S. Sotereanos. 2005. Starkey. M. 27. Matzinger. K. Hu. B. M. Microbiol. 3:253–262. R. N. Ghigo.
23. E. and K. H. Johansen. Z. Ehrlich. Thar. Off the hook—how bacteria survive protozoan grazing. P. Kjelleberg. Singh. and H. M. Combined imaging of oxygen and ¨ bacteria in bioﬁlms. A. Minogue. Hidalgo-Grass. Differential gene expression proﬁling of Staphylococcus aureus cultivated under bioﬁlm and planktonic conditions. Andersson. Natl. J. Y. Beveridge. J. 2006. 52:385–387. USA 103:5764–5768. Rao. Horn. and P. Infect. Camara. Escherichia coli 83972 inhibits catheter adherence by a broad spectrum of uropathogens. Trautner. Susceptibility of bioﬁlms to Bdellovibrio bacteriovorus attack. K. P. and S. 1999. Givskov. Givskov. Almeida. O’Toole. Biotechnol. S. Bioeng. Rosenstein. Disruption of quorum sensing in seawater abolishes attraction of zoospores of the green alga Ulva to bacterial bioﬁlms. R. Koutsoudis. 2006. R. Rickelt. A. Appl. and U.
26. Mathur. Spoering. Schaller. Immun. Urology 61:1059–1062. Simm. D. Leicht. O. and B. P. Brun. Kuhl. S. Development of high-rate anaerobic ammonium-oxidizing (anammox) bioﬁlm reactors. Lewis. and M.-M. and R. Clin. Penades. Orthop. Ciofu. M. Environ. D. Proc. 13:302–307. G. Greenberg. Environ. Jonas. M. Jr. B. Membrane vesicles: an overlooked component of the matrices of bioﬁlms. Jakob. 2006. I. I. Epidemiol. F. Jones. 1998. Thoendel. C. C. Appl. 2005. C. M. Kohls. J. D. R. D. E. Yung. 2005:31–40. Nature 417:552–555. E. doi:10. Britigan. Phipps. 53.. 71:4809–4821. Tsushima. B regulates IS256-mediated Staphylococcus aureus bioﬁlm phenotypic variation. B. 2002. Yildiz. P. Williams. D.. P. J. Konig. G. J. H. T. Augustin.. Skinderso.. and P. Kuhl. 69. Microbiol. and J. M. A component of innate immunity prevents bacterial bioﬁlm development. 65. Infect. J. R. 2006. C. Kjelleberg. Trends Microbiol. U. A. and N. Comparative proteome analysis of Staphylococcus aureus bioﬁlm and planktonic cells and correlation with transcriptome proﬁling. 6:53. J. G. J. 61. R. Lindow. Z. 2006. A contact-activated kinase signals Candida albicans invasive growth and bioﬁlm development. Hull. W. Bacteriol. Satoh. Bacterial bioﬁlms: an emerging link to disease pathogenesis. G. 45a. P. M. Kurg. B. and R. Fehrenbacher.. A. and T. C. Phycol. H. Singh. M. 13:119–127. T. Y. 62. Kadouri. K. Kaldalu.01952-06. Appl. and J. and host colonization in Pantoea stewartii subspecies stewartii. 46. N. M. 49. A. 41:1623–1634. B. K. P. J. 54. Prost. I.. D. L. Morr. D. Appl. Begun.. M. 72:2864– 2875. M. M. R. Cole. Kugelberg. J. and L. J. other structures seen as mainstream concepts. R. Spoering. R. L. and G.. Sewecke.VOL. M. Langemann. and T. M. Lett. I. Biomol. Darouiche. and M. B. USA 103:5983–5988. S. Larkum. 252:89–96. Wilson. 72:5963–5973. Monds. Givskov. M. 73:2504–2514. Rev. M. Sensors Actuators B 51:163–170. 2005. Natl. D. R. A. Acad. USA 102:16819–16824. 2002. 2007. 2007. Immun. Mol. Cloete. Tait. N. Gotz. K. M. S. Natl. Vergara-Irigaray. Coating urinary catheters with an avirulent strain of Escherichia coli as a means to establish asymptomatic colonization. A. The worm has turned— microbial virulence modeled in Caenorhabditis elegans. Milton. Annu. Quorum sensing inhibitors: a bargain of effects. R. G. Christoffersen. Holst. Kader.. Lusch. Purcell. C. Kumamoto. von Bodman. LaFleur. A. Moser. Microbiol. J. D. Environ. Vulic. 73: 605–614.. R. R. 35. Givskov. 2006. Okabe. Res. P. GlpD and PlsB participate in persister cell formation in Escherichia coli. 66. 2007. 55. Org. Richardson. O. J. T. Bacteriol. M. B.
22. J. Kaneko. C. Stoodley. Acad. Pasztor.. A modular luminescence lifetime imaging system for mapping oxygen distribution in biological samples. 2003. Microbiology 152:895–904. 2007
O. and S. Adhesion of single bacterial cells in the micronewton range. and M. Hoiby. M. H. Appl. and S. L. W. J. The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibioﬁlm activity. USA 101:1737–1742. Joint. and P. Shah.
29. W. E. A. Schembri. D. 2004. Newell. D. L. A. 7:229–240.. Proteomics 6:1867–1877. Acad.. H. O. 2005. G. Quorum-sensing regulation governs bacterial adhesion. Ralph. Kadouri. Merino. Kjelleberg. F. Kader. and M. Beveridge. Sekar. 2005.
2006.. Coakley. Yoon.asm.. Dis. Marks. D. 2004.7960
J. Tolker-Nielsen. B. Cell envelope components contributing to bioﬁlm growth and survival of Pseudomonas putida in lowwater-content habitats. Hennigan. C. S. and L. L. and S. K. J. and D. E. Microbiology 153:1318–1328. Biomed. J. Hogan. Y. C. Christensen. Mol. 116:436–446. Infect. 2009
. Bioﬁlm growth increases phosphorylcholine content and decreases potency of nontypeable Haemophilus inﬂuenzae endotoxins. epidermidis bioﬁlms and the role of phenol-soluble modulins in formation of bioﬁlms. J. A. Speert. Effects of iron on DNA release and bioﬁlm development by Pseudomonas aeruginosa. R. 52:735–750. Microbiol. 72.
70. J. Lau. E. Swords. Barken. Karabulut. J. M. A. S. 75. Opin. Otto. Clin. Optics 11:034001... 71. Boucher. H. J. D. F. Hwang. S. Luo.
Downloaded from jb. R. Yang. Fungal-bacterial interactions: a mixed bag of mingling microbes. 9:359–364. Buettner. Mortensen. Burns. 2007. E. 74. Rockel. 2006. S. Curr. 2006. D. BACTERIOL. 2005. A. A. Anaerobic killing of mucoid Pseudomonas aeruginosa by acidiﬁed nitrite derivatives under cystic ﬁbrosis airway conditions. Gaston. Schurr. 2006. Lymar. G. Schlachter.. Investig.. J. C. and W. 74:1828–1836.
and T. M. E. Infect. Immun. Genomewide analysis of gene expression in Staphylococcus epidermidis bioﬁlms: insights into the pathophysiology of S. W. W. Skindersoe. M. Sturdevant. and M. M. F.. Wargo. Xi. J. 76. van de Mortel. R. M. Microbiol. B. 73. L. S. Hassett. Halverson. B. Highresolution three-dimensional imaging of bioﬁlm development using optical coherence tomography. Yao. 191:289–298. West-Barnette. A. Givskov.org by on December 23. J. V. S. Boppart. and D. G.