Biofilm Review

JOURNAL OF BACTERIOLOGY, Nov. 2007, p. 7948–7960 Vol. 189, No. 220021-9193/07/$08.00�0 doi:10.1128/JB.00787-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.

MEETING REVIEW

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, Que-bec, Canada, 25 to 29 March 2007) maintained the format sovalued at past conferences (keynote talks, invited speakers,talks selected from abstracts, evening specialty sessions, and ahands-on workshop) while increasing scientific diversity by del-egating the selection of invited speakers and of talks fromabstracts to session chairs appointed by the organizing com-mittee. In particular, the committee targeted the underrepre-sentation of research on medically relevant biofilms other thanthose of Pseudomonas aeruginosa and sought to increase thevisibility of clinical aspects of biofilm-based disease research;the fruits of these efforts will become apparent in the descrip-tions of the sessions that follow. Also, an effort was made toincrease participation by non-U.S. investigators. At the 3rdBiofilms Conference, also held in Canada, 34% of attendeescame from outside the United States. At the Quebec Citymeeting, 53% were non-U.S. delegates who hailed from theUnited Kingdom/Europe (22%), Canada (11%), Scandinavia(8%), Asia (4%), Australia/New Zealand (2%), and India,Central and South America, Israel, and Africa (6%). The num-ber of attendees (ca. 600) and the number of submitted ab-stracts (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 havenot changed significantly since the earlier meetings, new per-spectives are emerging. First is the recognition that Pseudomo-nas aeruginosa, while a superb model organism for numerousreasons, is only one of many bacteria important to theoreticalas well as applied aspects of biofilm research; other modelorganisms (e.g., Vibrio spp., Bacillus spp., oral bacteria, andstaphylococci) are now being investigated as natural and fittingalternatives to P. aeruginosa. Second, while flow cell work withmonocultures grown in laboratory media will continue to pro-vide critical baseline data and continue to be especially usefulin examining theoretical questions, it is becoming clear thatbiofilm behavior and physiology need to be studied in a man-ner that reflects the natural environment, whether within thehuman lung or on a soil particle. Experimental systems have

therefore become more sophisticated and employ environmen-tally relevant substrata and media; accordingly, in vivo sam-pling and experimentation are becoming more common. Last,moving to the fore is the recognition that the vast majority ofbacteria, including many of those involved in human disease,must associate with other genera of bacteria as part of theirdaily existence: multispecies communities are becoming a tar-geted research area.

This review summarizes the individual sessions through theirplatform talks, many of which highlighted work presented ingreater detail as poster presentations. We hope these descrip-tions (and the accompanying references, which give a flavor ofthe topics) will convey the overall high scientific quality notonly of the invited talks but also of the talks selected fromsubmitted abstracts. The consensus of the attendees was thatthis meeting elevated the already high reputation of this con-ference series. We hope this review will convey some of thatfeeling to those who have yet to attend the conference.

KEYNOTE TALK 1

E. Peter Greenberg (University of Washington) opened themeeting by speaking on the sociomicrobiology of biofilms: theapplication to biofilm microbiology of E. O. Wilson’s insectsociety-derived sociobiological concepts. In biofilms, bacterialive in close proximity, the populations are heterogeneous inactivity, and individuals exhibit distinct behaviors, all of whichcan be studied from an ethological perspective. Biofilms offera unique opportunity to examine the influence of environmen-tal 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 wellas in the natural environment, and motility and biofilm initia-tion in Pseudomonas aeruginosa are dependent on iron (4). Thehuman immune effector lactoferrin sequesters iron; in thepresence of lactoferrin (i.e., at low levels of available iron), P.aeruginosa maintains twitching motility, refuses to becomesessile, and is thus dramatically attenuated in biofilm forma-tion (58). Production of lactoferrin is seen as a way for the hostimmune system to slow the rate of biofilm formation so thatthe microbial intruders can be dealt with before reaching aclinically relevant population density. Greenberg continuedthis theme by showing that the iron chelator EDTA can dis-perse a P. aeruginosa biofilm (3), and that citrate as a carbonsource can result in a flat biofilm because of citrate’s ironchelation capability. Lastly he introduced the use of the iron

* Corresponding author. Mailing address: Oral Biofilm Communi-cation Unit, Oral Infections and Immunity Branch, National Instituteof 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 protected].

� Published ahead of print on 31 August 2007.

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surrogate gallium as a biofilm inhibitor, a subject explored ingreater detail by University of Washington colleagues (28).

DEVELOPMENT AND PHYSIOLOGY

The opening session was chaired by George O’Toole (Dart-mouth College) and Paula Watnick (Tufts—New EnglandMedical Center). The chairs put together a set of talks thatillustrated not only the diversity but also the commonality inthe mechanisms of biofilm development displayed by sevendifferent biofilm-forming species. Roberto Kolter (HarvardUniversity) presented evidence that differentiation within Ba-cillus subtilis biofilm populations gives rise to six specialist celltypes. Each specialist has a defined function, such as matrixbuilding, propagation, or swimming. Time lapse imaging offluorescent reporter constructs implied that the cells exhibitonly one phenotype at a time, although some cells could switchbetween phenotypes. Kolter suggested that, depending on thelocal conditions, a certain probability exists that any one cellwould switch. Also discussed in the session were various cell-signaling pathways and environmental triggers that result inthe modification of biofilm extracellular polymeric substances(EPS) in response to nutrient conditions. These include therelease of DNA to the matrix in response to iron starvation inP. aeruginosa, presented by Liang Yang (Danish TechnicalUniversity, Lyngby, Denmark) (74), and the production ofLapA adhesion protein in response to phosphate limitation viaa cyclic di-GMP (c-di-GMP) signaling pathway in Pseudomo-nas fluorescens, presented by Russell Monds (Dartmouth Col-lege) (42). Co-chair Watnick showed that Vibrio choleraeknockout mutants in the sugar phosphotransferase system canform a biofilm when grown on glucose but not when grown onmannose. Glucose excess results in the synthesis of EPS ratherthan of glycogen, whereas under glucose limitation, glycogen isformed and EPS are degraded, thereby releasing the biofilmfrom the substratum. Carol Kumamoto (Tufts University) useda simple but elegant agar system to show that the switch to aninvasive, filamentous phenotype via mitogen-activated-proteinkinase sensing in Candida albicans is a response to surfacecontact, not to oxygen concentration (35). Daniel Verhamme(University of Dundee) presented evidence that the transcrip-tion factor DegU regulates more biofilm properties in B. sub-tilis than previously thought, ranging from attachment andsocial behavior to colony architecture. Karin Sauer (Bingham-ton University) identified two phosphorylated proteins, Bfl1and Bfl2, which appear to regulate the early and late biofilmmaturation process; this observation strengthens the hypothe-sis that biofilm formation by P. aeruginosa occurs by a stageddevelopmental process. Using green fluorescent protein (GFP)reporter fusions and confocal microscopy, Olivier Brun (Uni-versity of Minnesota) identified biofilm-specific promoters inMycobacterium marinum; a putative attachment promoter isexpressed at the base of the biofilm. Yves Brun (Indiana Uni-versity) showed how attachment forces could be accuratelymeasured through micromanipulation: the bending of a mi-cropipette in response to a controlled pulling via an attachedbacterial cell (66). Using this technique it was shown thatCaulobacter crescentus first attached by the flagella and thenrotated until pilus tethers stopped the rotation long enough forthe organism to create a holdfast.

BIOFILM-HUMAN INTERACTIONS I

In the first of two sessions designed to emphasize the rela-tionships of bacteria to the human host, session chairs wereFriedrich Gotz (Tubingen University) and Ute Romling(Karolinska Institute). Gotz described the role of polysaccha-ride intercellular adhesin (PIA) in biofilm formation by Staph-ylococcus epidermidis and Staphylococcus aureus (17). Mutantsthat do not make PIA are defective in adherence and biofilmformation, and these mutants are much less virulent than theparent strains in animal models of infection. Homologous poly-saccharides are found in several other bacteria; thus, this mo-lecular architecture is conserved across bacterial phylogeny.Biofilms of S. aureus and S. epidermidis have transcriptome (50,51) and proteome profiles that differ from those of planktoniccells with respect to murein and PIA synthesis as well as thephysiology of ammonia and acid production. Phage releasediffers also (49). Holgar Rohde (University of Hamburg) spokeon non-PIA-mediated biofilm formation in S. epidermidis. Hediscussed the role of embp (extracellular matrix binding pro-tein) in PIA-negative biofilm formation. This newly discoveredprotein was identified and shown to be crucial for biofilmformation in clinically important PIA-negative S. epidermidisisolates through transposon mutagenesis and subsequentscreening for biofilm-negative mutants in a PIA-negative back-ground strain. Fragments of recombinant embp induce cellaggregation and biofilm formation in embp-negative S. epider-midis strains, and the fragments also influence binding to hostcells as well as to host components such as fibronectin, findingsthat underscore the role of the protein in pathogenesis. Theprotein is broadly distributed in S. epidermidis strains and thusmay be central to the colonization of host tissues.

Paul Stoodley (Center for Genomic Sciences, Pittsburgh,PA) presented work on in vivo imaging of bacterial biofilms ininfections. A combination of fluorescence in situ hybridization(FISH), antibodies, and general stains was used to show bio-films associated with infected sutures and an infected arthro-plastic remodeling (62), as well as in mucosal epithelia fromupper respiratory tract infections (adenoids and middle earmucosae) in clinical specimens (Fig. 1) and the chinchillamodel of otitis media (18). Several of these cases were culturenegative even though symptoms of bacterial infection werepresented. Despite the success in demonstrating biofilms,Stoodley cautioned that biofilm imaging as a routine clinicaldiagnosis practice is a long way from acceptance. Ed Swords(Wake Forest University) expanded on biofilm formation inthe chinchilla model of otitis media by describing the relation-ship between phosphorylcholine (PCho) modification of lipo-oligosaccharides in nontypeable Haemophilus influenzae (72)and the infection. A mutant that does not add PCho formedbiofilms of lower density than did the parent strain and alsocaused increased inflammation levels in the animal. Thus, itseems that PCho modification results in tolerance of the in-fection by the animal (reduced initial inflammation) as well asin stable growth and maturation of the biofilm.

Several talks focused on P. aeruginosa and cystic fibrosis,both from the laboratory perspective and through examinationin vivo. The development of a tissue culture model system forthe study of interactions between P. aeruginosa and cystic fi-brosis-affected human airway epithelial cells was described by

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Greg Anderson (Dartmouth College). Addition of the bacteriato confluent eukaryotic-cell cultures caused detachment of theeukaryotic cells within a few hours. Detachment could begreatly reduced by the presence of tobramycin in the medium;although the number of bacteria was reduced, the system re-tained some bacteria despite the antibiotic addition. The tox-icity of nitrite and its derivatives (e.g., nitric oxide) to anaero-bically grown P. aeruginosa biofilms was discussed by DanHassett (University of Cincinnati). He showed that mucA mu-tants (the mucoid strains that predominate in cystic fibrosislung infections) are extremely sensitive to NO at a pH of 6.5,the approximate pH of lung airway mucus according to hismeasurements in lungs recently removed from transplant pa-tients; however, he found limited NO toxicity at a pH of 7 orabove. He further related the toxicity to inefficient nitrite re-ductase and nitric oxide reductase activities in mucA mutants,and he suggested that nitrite or NO could be useful as atherapeutic agent for cystic fibrosis patients (76) if the bacteriain situ are growing anaerobically. Janus Haagensen (DanishTechnical University) used FISH to show the presence of P.

aeruginosa cell aggregates within the sputa of cystic fibrosispatients who had coinfections with mucoid and nonmucoidstrains; however, these aggregates were absent from the sputaof patients infected solely with nonmucoid strains. Further,aggregates were shown to contain other bacteria in addition toP. aeruginosa; antibiotic treatment of the patients resulted inreductions in mucoidy and aggregation. Niels Høiby (Univer-sity of Copenhagen) presented new ideas on P. aeruginosainfection that emphasized the focal nature of the infection andthe importance of understanding the role of oxygen tension inthe lung in bacterial growth and subsequent tissue destruction.On the basis of autopsy and sputum specimens, he describedhow the infection compartmentalizes in the lung. In the respi-ratory zone, where oxygen tension is high relative to that in thebronchi, no thick mucous layer is present but mucoid P. aerugi-nosa colonies are found surrounded by polymorphonuclearleukocytes (PMN); inflammation and tissue destruction occurin this zone, and Høiby believes this is where the infection hasits true genesis. These mucoid colonies, together with de-stroyed lung tissue, are then transported by coughing to thebronchi (conductive zone), where they become embedded inthe naturally occurring thick mucous layer. The mucous layercreates steep oxygen gradients; single P. aeruginosa cells, aswell as mucoid colonies, can be found in the mucus.

Co-chair Romling spoke on second-messenger c-di-GMPsignaling and phenotype switching (25, 57). Low levels of c-di-GMP encourage motility (twitching and swimming), whereashigh levels encourage a sessile phenotype through productionof exopolymers and extracellular appendages. GGDEF/EALdomain proteins have phosphodiesterase as well as guanylatecyclase activity and thus regulate c-di-GMP levels; these pro-teins are widespread in bacteria, which emphasizes the univer-sality of this regulatory mechanism. In Salmonella enterica se-rovar Typhimurium, which has 20 GGDEF/EAL proteins, the“rdar” (red, dry, and rough) phenotype is controlled by c-di-GMP levels. This phenotype is characterized by a rough colonymorphology, red colonies, and clumpy, adherent cells. Expres-sion of the transcriptional regulator cgsD, which is controlledby environmental signals (such as temperature) as well as bythe c-di-GMP level, results in production of the rdar pheno-type through synthesis of curli as well as by indirect activationof cellulose exopolymer synthesis; csgD knockouts have asmooth colony morphology and are nonadherent and non-clumping. AdrA is one of four GGDEF/EAL proteins thatinfluence the transcription of cgsD. These c-di-GMP “net-works” have been shown to be crucial to the development of abiofilm phenotype in several bacteria. Paul Rainey’s talk, dis-cussed later in this paper, explored c-di-GMP networks in anevolutionary context.

BIOFILMS IN NATURAL ENVIRONMENTS

Michael Kuhl (University of Copenhagen) and Steven Lin-dow (University of California—Berkeley) were the chairs of asession designed to examine the structure and function ofbiofilms in terrestrial and marine ecosystems, including bacte-ria as commensals or pathogens of plant or animal hosts. Kuhlspoke on spatiotemporally resolved techniques for imagingchemical species, particularly oxygen, within microbial matsand corals. Much of this work employs films that contain a

FIG. 1. Pediatric adenoid-associated biofilm that meets the fourcriteria suggested by Parsek and Singh (45a) for the determination ofa biofilm: (i) the pathogenic bacteria are surface associated or adher-ent to a substratum; (ii) direct examination reveals bacteria in clustersand encased in a matrix of bacterial or host constituents; (iii) theinfection is localized; (iv) the infection is resistant to antibiotic therapy.The first, second, and third criteria were determined by microscopicexamination. The fourth criterion was fulfilled by empirical evidence:unsuccessful resolution with antibiotic treatment. The fresh specimenwas stained with the nucleic acid stain Syto 9 (red), which stainedbacterial cocci (black arrows) as well as host nuclei (white arrows).Less defined nuclei are deeper in the tissue. The biofilm EPS wasstained with lectins (green), and the surface of the adenoid (blue)was imaged using reflected confocal microscopy. Scale symbol(black), 10 �m in each dimension. Image courtesy of Laura Nistico,Paul Stoodley, and Luanne Hall-Stoodley (Center for GenomicSciences, Pittsburgh, PA).

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photochemically active sensor species (21), which reports theconcentration of the soluble target species (Fig. 2). Lifetimeimages are produced that describe how a chemical parameterchanges in response to changes in the environment. Examplesinclude changes in photosynthetic O2 production as a functionof the light level (33) or changes in oxygen concentration as afunction of the flow rate in a bacterial biofilm (34).

Two talks on Bahamian marine stromatolithic mats high-lighted interesting properties of this system. Chris Dupraz(University of Lausanne) presented data on extracellular poly-saccharide inhibition of biogenic calcium carbonate formation(16). When mineralization does occur, changes in the local pHresult in the formation of differently shaped carbonate crystals,thus providing a snapshot of the pH at that site in the biofilm.Alan Decho (University of South Carolina) discussed layeringand microbial activity in the same biofilms. Microautoradio-graphic data together with FISH studies show that in thissystem, in sharp contrast to the spatial organization in other

mat systems, active sulfate reduction occurs at the very surfaceof the biofilm (6). Decho suggested that this may be set upthrough homoserine lactone signaling.

Plant-associated biofilms were introduced by Clay Fuqua(Indiana University), who related inorganic phosphate (Pi)deprivation to enhanced biofilm formation in Agrobacteriumtumefaciens. Pi is frequently limiting in the environment; thephosphate response regulator protein PhoB is activated duringphosphate starvation, and in vitro, phosphate-starved A.tumifaciens biofilms have an elevated biomass relative to thatof normal biofilms. When phoB is induced, the cells bind tosubstrata using their poles, and they also show wheat germagglutinin binding at their poles. A transposon mutant selectedfor the lack of polar binding is not recognized by wheat germagglutinin, and the mutated gene bears similarity to a gene inthe holdfast biosynthesis pathway of Caulobacter crescentus. InA. tumefaciens, low Pi levels may be a cue for the production ofa specialized polymer and thus for biofilm initiation. Co-chairSteve Lindow continued the theme of plant pathogen biofilmsin his talk on Xylella fastidiosa (43). This bacterium infectsplant xylem vessels, which then become blocked; the denselypacked biofilms in blocked vessels have restricted access tonutrients and thus have very low activity, as assessed by pro-pidium iodide staining. Solitary bacteria in unblocked vesselscan move from vessel to vessel. Bacterial biomass and the levelof the diffusible signal factor DSF (the product of rpfF) are lowearly in infection, and movement from vessel to vessel (biofilmmetastasis) is encouraged. Late in infection, biomass and signallevels increase and cue polymer production, with resultantvessel blockage. Lindow proposed to treat infection by intro-ducing signal-hyperproducing strains to push early infectionstoward the late-stage phenotype, thereby sacrificing a few xy-lem vessels but concomitantly inhibiting the spread of the in-fection and saving the plant. Susanne von Bodman (Universityof Connecticut) also spoke on a plant xylem pathogen: Pantoeastewartii (31). Cell density-dependent synthesis of EPS is re-quired for virulent biofilm formation; mutants with mutationsin the esaI and esaR quorum-sensing loci form less-dense bio-films and do not disperse effectively in planta. The bacteriumwas shown to specifically colonize annular rings and spiral wallthickenings within the xylem.

Black band disease in corals was discussed by Laurie Rich-ardson (Florida International University). Comparison of thebacterial community from diseased coral with that fromhealthy coral (54) has shown that the migration of certainbacteria, in particular that of Beggiatoa spp., produces sharpgradients of sulfide and oxygen that kill the coral. Further,toxin-producing cyanobacteria have been identified from thecommunity and may be responsible for regional differences inthe disease. Dhana Rao (University of the South Pacific, Fiji)spoke on the interaction between Pseudoalteromonas tunicataand Roseobacter gallaeciensis, two surface colonizers of themarine alga Ulva australis (47). P. tunicata can outcompeteother natural community members when grown as a biofilm innatural seawater, but R. gallaeciensis outcompetes P. tunicataunder the same circumstances. Likewise, P. tunicata cannotinvade an extant biofilm composed of other species, but R.gallaeciensis can invade and eventually comes to dominate sucha biofilm. Interestingly, P. tunicata is unable to persist in ster-ile-filtered seawater.

FIG. 2. Combined microscopic imaging of oxygen concentrationand bacterial colonization within a biofilm reprinted from reference 34with permission. (A) Projection and vertical slices through a confocalimage stack of a biofilm structure consisting of GFP-marked Pseudo-monas putida (green) and heterotrophic bacteria labeled with Syto 60(red) imaged through an O2 coverslip-sensor mounted on top of a flowchamber. (B) Corresponding O2 distribution at the base of the biofilm.The central cell cluster is outlined by the dashed curve. Scale bars,40 �m.


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Two talks on biofilms in terrestrial systems highlighted theimportance of water availability and of local chemistry. Waterpotential is critical to soil biofilm development, and LarryHalverson (Iowa State University) showed that production ofthe extracellular polysaccharide alginate is upregulated inPseudomonas putida biofilms under matric water stress but notunder osmotic stress. Biofilms with reduced alginate levels areless dense and flatter. Alginate seems to protect against des-iccation by maintaining a hydrated environment for the cells(11, 12, 70). Ryan Hunter (University of Guelph) showedthat in an in vitro P. aeruginosa biofilm, precipitated iron isheterogeneously distributed. This may be correlated withthe local pH, and the bacterium alters its lipopolysaccharidechemistry in response to pH (22, 23). Less precipitate wasfound associated with metabolically active cells than withcarbonyl cyanide m-chlorophenylhydrazone (CCCP)-inhib-ited cells; protons transported to the cell surface by respi-ration appear to compete with metal cations for reactive cellsurface sites.

CROSS-KINGDOM INTERACTIONS IN BIOFILMS

Leo Eberl (University of Zurich) chaired a session on solu-ble signals and interactions of bacteria with eukaryotes.Carsten Matz (Helmholtz Center for Infection Research,Braunschweig, Germany) spoke on protozoan grazing effectson biofilms (38, 39). Grazing by amoebae induces changes inbacterial gene regulation, e.g., rhamnolipid production is in-creased. Bacterial defense mechanisms against grazing includemicrocolony formation (protective shielding) and the produc-tion of soluble inhibitors of amoebal growth.

Soluble signaling molecules were the theme of several talks,and in a second example of cross-kingdom inhibition, BastiaanKrom (University Medical Center, Groningen, The Nether-lands) showed that homoserine lactone from P. aeruginosainhibits hyphal formation in Candida albicans. Further, break-down products of homoserine lactone inhibit germ tube for-mation. C12 and C14 homoserine lactones had the greatesteffect. In an example of beneficial cross-kingdom signaling,Miguel Camara (University of Nottingham) discussed how ho-moserine lactone signaling molecules produced by biofilmsinfluence the attachment of Ulva linza (marine alga) zoospores(63). Using lactones synthesized by Vibrio anguillarum asmodel compounds, he showed that spore attachment increaseswith increasing lactone concentration. Using natural bacterialisolates, three patterns of increasing zoospore attachment wereseen as bacterial numbers increased: a linear relationship, anexponential relationship, and a bell-shaped curve. These resultssuggest a role in nature for this signaling mechanism.

Two talks addressed cross-kingdom interactions in patho-genesis. Costi Sifri (University of Virginia Health System) dis-cussed the nematode Caenorhabditis elegans as a model organ-ism for bacterial infection (56). Virulence is measured as thereduction in the mean lifetime of the worm; S. aureus colonizesthe alimentary tract, and known virulence factors such as PIAhave been demonstrated to apply to the worm. Coagulase-negative staphylococci have also been shown to be virulent inthis model, and PIA-negative mutants do not kill as effectivelyas the parent strain. Thomas Rasmussen (Chr. Hansen A/S,Hørnsholm, Denmark) talked about quorum sensing in Pseudo-

monas aeruginosa biofilms and how it affects interactions be-tween the biofilm and PMN. Wild-type P. aeruginosa biofilmsupernatants lyse PMN, but supernatants of quorum-sensingmutant biofilms do not. Microarray analysis of wild-type bio-films exposed to PMN showed upregulation of PQS (Pseudo-monas quinolone signal), phenazine, phospholipase, elastase,and rhamnolipid synthesis genes, whereas quorum-sensing mu-tant biofilms upregulated catalase. These results suggest thatwild-type biofilms mount an aggressive attack on PMN andthat mutant biofilms respond to the PMN oxidative burst byshielding themselves with catalase.

EVOLUTION AND DIVERSITY

Paul Rainey (Massey University, New Zealand) chaired thesession on evolution and diversity and gave the opening talk,presenting biofilm development in Pseudomonas fluorescens asa process of mutation and subsequent selection. Numerousmutations that generate biofilm-forming genotypes were iden-tified; the majority occurred within negative regulators of var-ious diguanylate cyclases. The net effect was to reduce negativeregulator function, with concomitant overproduction of c-di-GMP. In turn, enzymes involved in the production of a cellu-lose-based polymer were activated, causing cells not only toremain attached after cell division but also to generate a mat ofpolymer-linked cells at the air-liquid interface in broth micro-cosms. Continued selection of mat-forming genotypes (known“wrinkly spreaders” [Fig. 3]) resulted in the evolution of ge-notypes that no longer overproduced the polymer but couldstill grow within the mat. These selfish (cheating) genotypesdid not contribute to mat strength or integrity and thus causedthe mat to collapse. Molecular analyses of cheaters revealeda range of different mutations whose net effect is to abolishthe activity of the previously activated diguanylate cyclases.The process can continue; mat-forming (cooperating) geno-types can once again evolve from the cheating types (5).Thus, diversity and development in biofilms may occur sim-ply by the environment selecting for “standard” mutations,not through a hard-wired developmental cycle particular tothe bacterium.

John Roth (University of California—Davis) addressed thequestion of whether stress (such as could occur during biofilmgrowth) increases mutation frequency. Problems in definingexactly how a mutation arises and in accurate counting ofmutations have left this question unresolved for decades, andstudies of mutations in biofilms underscore the uncertainty.Roth presented theoretical arguments against the existence ofa “hypermutable” state under any circumstances, as well as analternative interpretation of the data from the best-acceptedexample of hypermutation (10) whereby the observationscould be due to gene duplication (32). Roth believes thatselection is more important than the mutation rate.

Pradeep Singh (University of Washington) described exper-iments that suggest an increase in the occurrence of genetic/phenotypic variants in biofilm systems (8). Singh showed thatthese variants are generated by error-prone repair (recA andrecBC mutants) of double-stranded breaks in DNA arisingfrom oxidative stress; addition of an antioxidant to the biofilmreduces the occurrence of variants. Once the variants appear,they are selected for and come to dominate the biofilm. Con-


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tinuing the theme of variants, Alfred Spormann (Stanford Uni-versity) argued that the stability of Vibrio cholerae biofilms isrelated to the appearance of small-colony variants (SCV). Hedescribed how these biofilms fall apart after the appearance ofthe SCV and how the SCV can be sorted into four classesbased on motility. Characterization of SCV physiology is nowunder way using 2,000-parameter Biolog plates, and Spormannargued for an increased emphasis on physiology in biofilmexperiments.

Jaione Valle (Public University of Navarre, Pamplona,Spain) concluded the session by describing how insertion se-quence IS256 mediates phenotypic switching in Staphylococcusaureus (69). Knockouts of transcription factor �B have in-creased IS256 activity and produced biofilm-negative variantswith dramatically higher IS256 copy numbers in the chromo-some at a high frequency. Osmotic stress can complement thelack of �B and restore the wild-type phase variation frequency,suggesting that �B is not directly involved. �B appears to act asa regulator of IS256 activity and thereby as a controller ofbiofilm-negative phenotype occurrence.

KEYNOTE TALK 2

Ivan Matic (University of Paris) continued the discussion ofgenetic variation in biofilms from the perspective of mutationand repair. Observed mutation rates are typically low becausemost mutations are deleterious or neutral and because the cellhas extensive investments in repair mechanisms. However,populations of natural isolates contain strong mutators thatdisplay mutation rates 100-fold higher than those of laboratorystrains; these mutators are deficient in repair (15). They can beselected for in gnotobiotic mice by sequential multiple-antibi-otic treatments; the surviving cells are mutators. Also presentin natural populations are weak mutators whose mutation ratesare 4 to 5 times those of laboratory strains. The difference inthe mutation rate is a by-product of selection for improvedfitness, and mutation rates can be very different in the hostenvironment than in the laboratory.

THE SLIME MATRIX

Tony Romeo (Emory University) chaired the session focus-ing on the regulation, composition, and function of EPS. Thecharacterization of EPS chemistry and function has remainedelusive even though EPS is a hallmark that distinguishes bio-film populations from planktonic cultures. Romeo discussedthe regulation of poly-�1,6-N-acetyl-D-glucosamine (PGA),which is a common component of EPS in gram-positive as wellas gram-negative bacteria. Romeo discussed how the carbonstorage regulator (Csr) RNA-binding protein, which repressesPGA and biofilm formation, can be sequestered by the non-coding short RNAs csrB and csrC (2). In turn, csrB and csrCcan be degraded by csrD. Romeo concluded that this regula-tory network was adapted for fine-tuning homeostatic adjust-ment rather than being an “on-off” system. Matt Parsek (Uni-versity of Washington) discussed the contribution of the EPSgenes pel and psl in P. aeruginosa colony morphology variantsisolated from in vitro biofilm systems. Similar colony morphol-ogy variants were isolated from cystic fibrosis patients; thus,the in vitro system may reflect certain in vivo conditions. Byknocking out c-di-GMP in a sticky mutant, the wild-type mor-phology was recovered; c-diGMP signaling was important indirecting adhesion and cohesion (30). Jean-Marc Ghigo (Pas-teur Institute, Paris, France) presented evidence for secretionof valine into EPS. This secretion acts not only as a “carbondump” but also to provide an antimicrobial effect against in-vading planktonic bacteria, which have been shown to be moresensitive than biofilm cells to valine. Per Nielsen (AalborgUniversity, Aalborg, Denmark) introduced evidence for thepresence of amyloid protein (originating from fimbriae) as aubiquitous component of EPS produced by many differentspecies and found in freshwater, brackish-water, and seawaterenvironments. Nielsen used antibodies and FISH (Fig. 4) todemonstrate that this previously overlooked protein is pro-duced by as many as 50% of the species in a given environ-ment. Sarah Schooling (University of Guelph) concluded thesession by presenting evidence that membrane vesicles are animportant component of P. aeruginosa biofilm EPS (53). DNA

FIG. 3. Colony variants—including two different wrinkly spreader genotypes—of P. fluorescens that arise during selection in spatially structuredmicrocosms. (Inset) Niches occupied by different variants. Wrinkly spreader genotypes form a biofilm at the air-liquid interface (culture at right).Image courtesy of Paul Rainey (Massey University, New Zealand).


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from vesicles was estimated to make up at least 0.33% of theextracellular DNA. Vesicle production may play a significantrole in EPS function for biofilms. The data presented in thissession, the lively discussion at the specialty evening session onEPS chaired by Hans-Curt Flemming and Dan Wozniak, andthe final keynote address by Bill Costerton on structured EPSillustrate how EPS is no longer seen as an amorphous carbo-hydrate material that serves simply to provide a physical matrixfor cells but rather as a chemically complex mechanically andstructurally adaptive material that has multiple biological func-tions.

CELL SIGNALING IN BIOFILMS

A function for cell signaling in a biofilm context was firstdescribed for P. aeruginosa in 1998 (14). The concept that asessile bacterial population could coordinate its phenotype andorganization through quorum-sensing mechanisms quicklycaptured the imagination, and it was thought that signalingmight be a magic bullet for the control of biofilms. Since then,the field has gained in complexity, because the diversity ofsignal molecules and mechanisms has grown. Matt Parsek, whowas involved in the earliest biofilm signaling work, chaired thesession, which included examples of signaling in three differentexperimental systems. Susanne Haussler (Helmholtz Centerfor Infection Research, Braunschweig, Germany) discussedPQS in P. aeruginosa. She presented data extending the role ofPQS from iron chelation (9) to the release of DNA to the EPSmatrix in response to stress. Haussler suggested that PQS is aregulator of coordinated dispersal (movement of bacterial cellsfrom the biofilm to the bulk liquid), because of its promotionof detergent tolerance. Deborah Hogan (Dartmouth College)

presented an example of competitive survival of two organismsthat involves response to, as well as interference with, eachother’s signaling systems. P. aeruginosa and C. albicans arecommonly isolated in cases of opportunistic infections (71),and in coculture, P. aeruginosa can colonize and kill C. albicanshyphae through the production of virulence factors, such as thefungicide pyocyanin (29), which are regulated by the Las/Rhlsignaling system. However, the production by P. aeruginosa ofits virulence factor-stimulating signaling molecule OdDHL[N-(3-oxododecanoyl)-L-homoserine lactone] causes C. albi-cans to switch its morphotype to the colonization-resistant buthost cell-invasive yeast form. In addition to this defensive re-sponse, C. albicans mounts an offensive against P. aeruginosathrough the production of farnesol. Farnesol, a signal for C.albicans to block the formation of hyphae, also depresses PQSsignaling and pyocyanin production in P. aeruginosa. In thiscross-kingdom interaction, signals coordinate the phenotype ofthe producing organism while at the same time altering thephenotype of the competitor. Cell signaling in the social bac-terium Myxococcus xanthus was discussed by Heidi Kaplan(University of Texas Medical School). She presented evidencefor a quorum-sensing system sensitive to membrane integrity.M. xanthus responded to an accumulation of lysed cell wallcomponents, such as peptidoglycan, in the culture by initiatingsporulation. Intriguingly, this recognition and response todamage in the population is not unlike the “danger model” ofthe mammalian immune system response (40), in which rec-ognition of cell damage is the immune activator. It remains tobe seen how widespread sensing and response to self-damageis among prokaryotes. Finally, Jan Kreft (University of Bonn)presented modeling evidence that cell-signaling mechanisms ingram-negative bacteria may be adapted to regulate behavior

FIG. 4. Detection of amyloids in a brackish-water biofilm, with simultaneous labeling of amyloid adhesins by antibodies and identification ofbacteria by FISH. (A) Transmitted light image showing small rods attached to an empty sheath, presumably from a cyanobacterium (judging fromthe morphology and the ability to produce red fluorescent phycoerythrin). (B) Epifluorescence image of the same field of view as that in panel A.Green fluorescence marks antibodies raised against conformational epitopes of amyloid adhesins. (C) Epifluorescence image of the same field ofview as that in panel A. Shown are results of FISH using the red fluorescent oligonucleotide probe CFB719, targeting the class Bacteriodetes, andblue fluorescent EUBmix, targeting most members of the domain Bacteria. (D) Overlay of panels A, B, and C. Colocalization of the threefluorescent probes shows as white. Scale bar, 20 �m. Image courtesy of P. Larsen, J. L. Nielsen, D. Otzen, and P. H. Nielsen (Aalborg University,Aalborg, Denmark).


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over the small distances between adjacent cells and withinsmall clusters (19). Based on the observation that the diversityof signals in gram-negative species is small (primarily acetyl-homoserine lactones) but that a large number of species re-spond to these molecules, Kreft hypothesized that signaling inbiofilms is local. Signals are directed toward nearest-neighbor,closely related cells (i.e., toward clones), rather than towardspatially distant and therefore, presumably, less closely relatedcells. Kreft’s modeling suggests that current assumptions re-garding the distance over which signaling operates may need tobe reevaluated.

BIOFILM-HUMAN INTERACTIONS II

The second human microbiology session was chaired by Mi-chael Otto (NIH Rocky Mountain Labs) and David Stickler(University of Cardiff). The first three talks focused on biofilmsin the urinary tract. Stickler discussed Proteus mirabilis biofilmformation in urinary catheters and the resultant struvite crys-tallization (61). Multispecies biofilms attenuated crystallizationby modifying the pH; patient urine chemistry and biofilm spe-cies composition were important factors in the rate of catheterblockage. Barbara Trautner (Baylor College of Medicine) thenpresented evidence that biofilm infection of urinary catheterscould be reduced by a probiotic approach: seeding the catheterwith avirulent urinary tract isolates of Escherichia coli (64, 65).Seeding with these E. coli isolates reduced the incidence ofsymptomatic infection nearly 20-fold: from 2.7 per 100 catheterdays to 0.15 per 100 catheter days. Control of device-relatedbiofilm infections is perhaps best addressed not by attemptingto render the surface sterile, which has proved extremely chal-lenging and is possibly futile, but rather by prophylactic inoc-ulations. Trautner is currently designing E. coli strains throughgenetic modification that will be even more efficient at “com-petitive exclusion.” Mark Schembri (University of Queensland,Brisbane, Australia) discussed the role of antigen 43 as animportant adhesin and virulence factor for uropathogenic E.coli in a mouse model (68). Adhesins such as antigen 43 may beuseful targets for modification to create probiotic E. colistrains.

The next two presentations were on a rapidly growing areaof interest in medical biofilms: chronic wound infections. KlausKirketerp-Møller (Bispebjerg University Hospital, Copenha-gen, Denmark) presented evidence from clinical specimenssuggesting that the persistence of P. aeruginosa in inflamedchronic wound infections was due to its protected locationwithin microcolonies, where it remained impervious to, andcould kill, leukocytes. Garth James (Montana State University)used confocal microscopy and denaturing gradient gel electro-phoresis to show the broad diversity of infecting bacteria, evenin similar wound types. Importantly, 16S rRNA analysisshowed that, in addition to the routinely cultured aerobic andfacultatively anaerobic species, a number of uncultured obli-gate anaerobes were present. James hypothesized that strictanaerobes may inhabit anaerobic microniches created throughoxygen consumption by aerobic biofilms.

Session co-chair Michael Otto then gave the first of two talkson the molecular pathogenesis of staphylococcal infections. Heshowed that spontaneous mutation in the biofilm-repressingagr locus causes S. epidermidis to form different biofilm phe-

notypes. The difference in architecture between the heteroge-neous wild type and the thicker but flatter agr mutant wascaused by the phenol-soluble modulin (PSM) (75). GFP re-porters of PSM synthesis, together with exogenous PSM addi-tion, suggest that PSM is responsible for controlled detach-ment, possibly through surfactant properties. Anna Muench(University of Maryland Dental School) used microarrays toidentify genes unique to clinical isolates of S. aureus. Differ-ential expression showed that approximately 50% of the geneswere unique to clinical strains, that many of these genes wereinvolved in stress response and the production of virulencefactors, and that they were upregulated in biofilms. In contrast,only 10% of genes unique to laboratory strains were upregu-lated in biofilms of laboratory strains. These data indicate thatcontinued laboratory passage of clinical strains may cause aloss of virulence, because no selective pressure exists to main-tain virulence genes, and that many of these genes are impor-tant in the context of biofilms.

In addition to the infectious disease-related talks, two talkson oral biofilms were given. Rob Palmer (NIDCR, NIH) usedimmunofluorescence confocal microscopy and enterobacterialrepetitive intergenic consensus PCR to investigate phenotypicand genotypic diversity within a Veillonella sp. in natural dentalplaque biofilms from human volunteers. In as little as 4 h,changes in coaggregation, antibody reactivity, and enterobac-terial repetitive intergenic consensus fingerprinting patternsoccurred (44). Palmer concluded that the only way to fullyunderstand the complexity of the time-dependent microdiver-sity present in dental biofilms was through a polyphasic ap-proach. Mary Ellen Davey (Forsyth Institute, Harvard Univer-sity) discussed the role of capsule expression and biofilmformation in Porphyromonas gingivalis. Interestingly, Daveyfound that encapsulated P. gingivalis strains did not form bio-films, while strains lacking a capsule did (13). Further, capsuleexpression was found to be dependent on high nutrient levelsand rapid growth, explaining why encapsulated forms are morecommonly observed on culture plates. Bernd Kreikemeyer(University Clinic, Rostock, Germany) discussed the develop-ment of a probiotic approach to reduce the incidence of naso-pharyngeal infections by group A streptococci (37). In a simplecoculture, it was found that Streptococcus salivarius inhibitedthe development of Streptococcus pyogenes biofilms; however,in more-complex consortia, the effect was not as straightfor-ward, because other nonpathogenic species, including the pro-biotic E. coli Nissle strain, attenuated the interference effect.

BIOFILMS IN INDUSTRIAL ANDENGINEERED SYSTEMS

Much early pioneering work in biofilm research was con-ducted in the context of fouling prevention in industrial andmarine systems, and also for optimization of bioconversionprocesses in wastewater treatment and bioremediation. SatoshiOkabe (Hokkaido University, Japan) and Linda Blackall (Uni-versity of Queensland, Brisbane, Australia) were the chairs forthis session on biofilms in engineering applications. KorneelRabaey (University of Queensland) described the use of mi-crobial electron transfer systems to create microbial fuel cells.He showed how power output could be optimized by varyingspecies composition and growth conditions, while indicating


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that initial applications may be limited to low-current situa-tions such as sensors and feedback switches (1). ChristianPicioreanu (Delft University) and Andrew Kato-Marcus (Ari-zona State University) each presented mathematical modelsthat could be used to optimize these fuel cells. Picioreanu’smodel showed how biofilm structure may be an importantfactor for charge density on the anode, while Kato-Marcusmodeled the anode as an electron acceptor, with the biofilmitself as an integral part of the anode. Moshe Herzberg (YaleUniversity) illustrated how molecular, microscopic, and ex-pression techniques could be applied to identify biofilm activityin reverse osmosis systems.

Three talks focused on multispecies biofilms in wastewatertreatment. Co-chair Linda Blackall showed how the develop-ment of structured biofilm communities in flocs could be usedfor the simultaneous removal of ammonium, nitrate/nitrite,and phosphorus from wastewater. Electron microscopy sug-gested that the flocs may have structured EPS, which appearedsimilar to the honeycomb-like structures introduced later inthe meeting by Bill Costerton. Co-chair Satoshi Okabe used apolyphasic approach that employed FISH, microautoradiogra-phy, and microelectrodes to characterize the distribution andactivity of anaerobic ammonium-oxidizing (anammox) bacteriain a mixed wastewater biofilm (67). These bacteria were dis-tributed throughout the biofilm, but rates of ammonium andnitrite oxidation differed at different locations in the biofilm.Silvia Weber (Technical University of Munich) used similartechniques to investigate granular flocs. A FISH protocol wasoptimized for use in thick, dense biofilms; this approach mayhave applications in other biofilm systems such as microbialmats. Maria Giao (University of Minho, Portugal) showed theimportance of nutrients and hydrodynamics to Legionella pneu-mophila biofilm formation. Low carbon levels increased thenumber of viable but nonculturable bacteria in the biofilm,suggesting that environmental factors should be taken intoaccount when one is culturing from drinking water systems.Carsten Schwermer (Max Planck Institute for Marine Micro-biology, Bremen, Germany) presented industrially funded re-search that employed FISH and microelectrodes to determinethe influence of nitrate on a biofilm community from an oilfieldseawater injection system that contains sulfate-reducing bacte-ria (SRB). Nitrate achieved the desired effect of reducing SRBactivity and sulfide production and may see widespread usethroughout the industry as a more environmentally compatiblealternative to conventional biocides used for SRB control. Fi-nally, Chuanwu Xi (University of Michigan) presented work onoptical coherence tomography, a high-resolution imaging tech-nique that does not require staining, for real-time monitoringof living biofilms. The instrument offers two advantages overconventional microscopes, especially in process situations: itcan be positioned much further from the biofilm, and it canpenetrate opaque materials (73).

PREVENTION AND TREATMENT OF BIOFILMS

Michael Givskov (Danish Technical University) and PhilStewart (Montana State University) were session chairs. Stew-art’s talk on the visualization of antimicrobial action in biofilmsdescribed the use of calcein AM as a viability indicator. Whentreated with the quaternary amine Barquat, biofilm microcolo-

nies quickly lost calcein fluorescence at the edges but showedtime-dependent retention of fluorescence at the center (Fig.5). Treatment with chlorite caused loss of fluorescence only ina thin band at the edge of the colony. Treatment with nisin ledto a rapid and uniform fluorescence loss. This approach allowsspatiotemporally resolved measurement of toxicity and sug-gests that differences in susceptibility to particular agents existwithin the biofilm.

Thomas Neu (Helmholtz Center for Environmental Re-search, Magdeburg, Germany) described fluid dynamic gaug-ing, a method of monitoring biofilm stability by which the shearstrength of fouling layers can be measured (41). The baselayers of the biofilm were found to be highly stable, whereasthe upper layers were easily removed. Neu also showed mag-netic resonance imaging data that allow real-time, nondestruc-

FIG. 5. Visualization of antimicrobial action against a Staphylococ-cus epidermidis biofilm. Bacteria in a flow cell-grown biofilm wereloaded with the green fluorescent dye calcein AM and then exposed to50 mg liter�1 alkyl dimethyl benzyl ammonium chloride (ADBAC)under continuous flow. Time lapse confocal scanning laser microscopyexamination of fluorescence loss shows permeabilization of cells by thebiocide. The number in each panel is the time, in minutes, after theintroduction of ADBAC into the flow cell. In the first and last panels,biomass was imaged in transmission mode (gray scale). Image courtesyof W. Davison and P. Stewart (Center for Biofilm Engineering, Mon-tana State University).


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tive visualization of biofilm biomass, as well as of flow velocityand shear stress around the biofilm.

Persister cells, first reported by Spoering and Lewis (59), aredefined as a numerically small (�1%), antimicrobial-resistant,slow-growing subpopulation of cells that is maintained in bothplanktonic and biofilm cultures; these cells were the subject oftalks by Peter Gilbert (University of Manchester) and KimLewis (Northeastern University). Using high-throughput cellsorting, Gilbert showed that an Escherichia coli populationlabeled with GFP contained a very small fraction of poorlymetabolizing (weakly fluorescent) cells that also demonstratedmultidrug resistance. Large volumes of culture were processedto collect sufficient mRNA for microarray analysis. High levelsof prophage transcripts and a group of candidate “persister”genes including the putative regulator ykgK were identified.Antisense suppression of pin, ykgK, and ykgM eliminated per-sister cells, while overexpression increased persisters. Thesegenes may be regulators of the persistence state and thus poten-tial drug targets for recalcitrant biofilm infections. Lewis’ mainobservations on weakly metabolizing cells being persisters (55)are in agreement with those of Gilbert. Lewis also used transcrip-tional profiling to identify a number of candidate genes, but heidentified genes different from those discovered by Gilbert; Lewis’persister genes included the dehydrogenase-encoding gene glpDand the lipid biosynthesis gene plsB (60). Lewis thought that thedifferences between candidate genes from the two studies may bea result of strain differences. He also reported that Candida albi-cans, in contrast to bacteria, forms persister cells only in a biofilm;persisters are not part of the population during planktonic culture(36).

Jennifer Kofonow (Northern Arizona University) spoke onthe use of biofilm-specific antibodies to detect biofilm infec-tions in a rabbit model. Antigens are being identified by aproteomics approach as present in infected animals but absentin healthy animals; because the targets are eventually to beused in the diagnosis of human biofilm infections, humanblood must be screened as well, and one candidate protein has

emerged. The development of antimicrobials from marine in-vertebrates was reported on by Domenico Schillaci (Universityof Palermo). Cytosol of the sea urchin Paracentrotus lividus hasactivity against S. epidermidis and S. aureus. A 5-kDa peptidefraction inhibits bacterial adhesion and interferes with bacte-rial respiration in biofilms.

Co-chair Michael Givskov spoke on quorum-sensing block-ers as drugs. Advantages of this approach are (i) that devel-opment of resistance to compounds similar to those generatednaturally by the bacterium is highly unlikely and (ii) that block-ers can be used to modulate antibiotic resistance and to gen-erate synergistic effects with other antimicrobials (48). Givskovsuggested that rhamnolipid is a necrotic agent for PMN (7, 24)(Fig. 6) and that reduced rhamnolipid synthesis may partlyexplain the more rapid clearance of quorum-sensing-defectivePseudomonas aeruginosa than of the wild type in a mousemodel of lung infection (20). A promising garlic-based quo-rum-sensing blocker (46) has been synthesized and tested; it isless active than garlic extract, but it is possible that a mixture ofgarlic-based compounds can reach or exceed the efficacy ofnatural garlic extract.

The topic of predatory/parasitic bacteria as antibiofilmagents was covered by Daniel Kadouri (University of Medicineand Dentistry of New Jersey). Bdellovibrio bacteriovorus cangreatly diminish the biofilm biomass of E. coli as well as that ofP. fluorescens (26). The less well known exoparasitic bacteriumMicavibrio aeruginosavorus is likewise able to reduce the bio-film biomass of laboratory and clinical strains of P. aeruginosa,Klebsiella pneumoniae, and Burkholderia cepacia (27). Closingthe session, David Davies (Binghamton University) spoke on abiofilm-dispersing activity found in the organic fraction of P.aeruginosa spent culture medium. The activity reduces P.aeruginosa biofilm thickness by 50%; it also acts against mixed-species biofilms, and it has a synergistic effect with antibioticswhereby a lower antibiotic concentration is required to achievethe same level of killing as that seen in the absence of thedispersing activity.

FIG. 6. Quorum-sensing-controlled killing of PMN. Freshly isolated, Syto 62-stained PMN (red) interact in vitro with a GFP-tagged biofilm (green).(Left) Wild-type bacteria kill the PMN through quorum-sensing-controlled production of rhamnolipid. The PMN are lysed and disappear before theycan eliminate the biofilm. (Right) In the presence of the P. aeruginosa LasR/RhlR double-quorum-sensing mutant, the PMN readily traverse the biofilmand phagocytose the virulence-factor-deficient bacteria. Images courtesy of Thomas Bjarnsholt (Danish Technical University).


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KEYNOTE TALK 3

The prebanquet address was given by Bill Costerton, theperson who, beginning at the University of Calgary and later asthe director of Montana State University’s Center for BiofilmEngineering, brought biofilms to the forefront of microbiolog-ical research and who is now developing a biofilm researchcenter at the University of Southern California. Costerton in-troduced his ideas on structured biofilm EPS, which he termed“caserna,” named after the temporary stone forts built by sol-diers of ancient Rome. These are networks and honeycomb-shaped scaffolds with pore sizes of several micrometers (Fig. 7)which develop in several-day-old cultures of S. epidermidis iso-lates (52), although they have been seen in other bacterialcultures as well. Based on scanning electron microscopic,freeze-substitution transmission electron microscopic, andconfocal and light microscopic evidence, these structures ap-pear to form from the contents (especially ribosomes) of lysingcells that merge with one another. Costerton termed the pro-cess “bacterial coalescence” and noted that the repeated struc-ture, which can form macroscopic structures with dimensionsof millimeters, likely resulted from a genetically regulated pro-cess. As discussed in the “slime matrix” session, little is knownabout the structural and chemical composition of biofilm EPS;the possibility of higher-order structural organization, such asthat described for proteins, is provocative. Casernum produc-tion in natural environments and a role for caserna in biofilmfunction remain to be demonstrated. However, the possibilityof structured EPS hints at yet further levels of complexity inbiofilm systems.

CODA

In summary, the meeting elevated its reputation for show-casing high-quality, exciting science from a rapidly moving fieldand for demonstrating the broad applicability of biofilm biol-ogy to microbiological research. The organizing committee

took steps to attract an audience and a program that weredemographically as well as scientifically diverse; this bench-mark for the meeting seems to have been heartily embraced,and the committee hopes that this approach will continue.

ACKNOWLEDGMENTS

The committee (Niels Hoiby, University of Copenhagen; Søren Mo-lin, Technical University of Denmark; Robert J. Palmer, Jr., NIDCR,NIH; Matthew Parsek, University of Washington; Paul Stoodley,Allegheny-Singer Research Institute) thanks ASM for its continuedsupport and development of this conference series. In particular, wethank ASM Conferences staff members Lisa Nalker and Latonya Ni-chols for outstanding work in bringing the 4th Conference on Biofilmsto fruition.

Robert J. Palmer, Jr., is supported by the Intramural ResearchProgram of the National Institute of Dental and Craniofacial Researchat the National Institutes of Health. Paul Stoodley is supported byAllegheny-Singer Research Institute and Philips Oral Healthcare.

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