JOURNAL OF BACTERIOLOGY, Nov. 2007, p. 7948–7960
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0021-9193/07/$08.00⫹0 doi:10.1128/JB.00787-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
MEETING REVIEW
Biofilms 2007: Broadened Horizons and New Emphases䌤
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 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 del-
egating the selection of invited speakers and of talks from
abstracts to session chairs appointed by the organizing com-
mittee. In particular, the committee targeted the underrepre-
sentation 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 descrip-
tions 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 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 have
not 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 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 pro-
vide 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 man-
ner that reflects the natural environment, whether within the
human lung or on a soil particle. Experimental systems have
* Corresponding author. Mailing address: Oral Biofilm Communi-
cation 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: rjpalmer@mail.nih.gov.
䌤
Published ahead of print on 31 August 2007.
7948
Vol. 189, No.
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 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 tar-
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geted 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 descrip-
tions (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 con-
ference 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 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 well
as in the natural environment, and motility and biofilm initia-
tion 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 forma-
tion (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 dis-
perse 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
VOL. 189, 2007
surrogate gallium as a biofilm inhibitor, a subject explored in
greater 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 England
Medical Center). 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. Roberto Kolter (Harvard
University) presented evidence that differentiation within Ba-
cillus subtilis biofilm populations gives rise to six specialist cell
types. Each specialist has a defined function, such as matrix
building, propagation, or swimming. Time lapse imaging of
fluorescent reporter constructs implied that the cells exhibit
only one phenotype at a time, although some cells could switch
between phenotypes. Kolter suggested that, depending on the
local conditions, a certain probability exists that any one cell
would switch. Also discussed in the session were various cell-
signaling pathways and environmental triggers that result in
the modification of biofilm extracellular polymeric substances
(EPS) in response to nutrient conditions. These include the
release of DNA to the matrix in response to iron starvation in
P. aeruginosa, presented by Liang Yang (Danish Technical
University, Lyngby, Denmark) (74), and the production of
LapA adhesion protein in response to phosphate limitation via
a 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 cholerae
knockout mutants in the sugar phosphotransferase system can
form a biofilm when grown on glucose but not when grown on
mannose. Glucose excess results in the synthesis of EPS rather
than of glycogen, whereas under glucose limitation, glycogen is
formed and EPS are degraded, thereby releasing the biofilm
from the substratum. Carol Kumamoto (Tufts University) used
a simple but elegant agar system to show that the switch to an
invasive, filamentous phenotype via mitogen-activated-protein
kinase sensing in Candida albicans is a response to surface
contact, 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 and
social behavior to colony architecture. Karin Sauer (Bingham-
ton University) identified two phosphorylated proteins, Bfl1
and Bfl2, which appear to regulate the early and late biofilm
maturation process; this observation strengthens the hypothe-
sis that biofilm formation by P. aeruginosa occurs by a staged
developmental process. Using green fluorescent protein (GFP)
reporter fusions and confocal microscopy, Olivier Brun (Uni-
versity of Minnesota) identified biofilm-specific promoters in
Mycobacterium marinum; a putative attachment promoter is
expressed at the base of the biofilm. Yves Brun (Indiana Uni-
versity) showed how attachment forces could be accurately
measured through micromanipulation: the bending of a mi-
cropipette in response to a controlled pulling via an attached
bacterial cell (66). 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.
MEETING REVIEW 7949
BIOFILM-HUMAN INTERACTIONS I
In the first of two sessions designed to emphasize the rela-
tionships of bacteria to the human host, session chairs were
Friedrich Götz (Tübingen University) and Ute Römling
(Karolinska Institute). Götz described the role of polysaccha-
ride intercellular adhesin (PIA) in biofilm formation by Staph-
ylococcus epidermidis and Staphylococcus aureus (17). Mutants
that do not make PIA are defective in adherence and biofilm
formation, and these mutants are much less virulent than the
parent 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 planktonic
cells with respect to murein and PIA synthesis as well as the
physiology of ammonia and acid production. Phage release
differs also (49). Holgar Rohde (University of Hamburg) spoke
on non-PIA-mediated biofilm formation in S. epidermidis. He
discussed the role of embp (extracellular matrix binding pro-
tein) in PIA-negative biofilm formation. This newly discovered
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protein was identified and shown to be crucial for biofilm
formation in clinically important PIA-negative S. epidermidis
isolates through transposon mutagenesis and subsequent
screening for biofilm-negative mutants in a PIA-negative back-
ground strain. Fragments of recombinant embp induce cell
aggregation and biofilm formation in embp-negative S. epider-
midis strains, and the fragments also influence binding to host
cells as well as to host components such as fibronectin, findings
that underscore the role of the protein in pathogenesis. The
protein is broadly distributed in S. epidermidis strains and thus
may 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 in
infections. 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 from
upper respiratory tract infections (adenoids and middle ear
mucosae) in clinical specimens (Fig. 1) and the chinchilla
model of otitis media (18). Several of these cases were culture
negative even though symptoms of bacterial infection were
presented. Despite the success in demonstrating biofilms,
Stoodley cautioned that biofilm imaging as a routine clinical
diagnosis practice is a long way from acceptance. Ed Swords
(Wake Forest University) expanded on biofilm formation in
the 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 formed
biofilms of lower density than did the parent strain and also
caused increased inflammation levels in the animal. Thus, it
seems that PCho modification results in tolerance of the in-
fection by the animal (reduced initial inflammation) as well as
in stable growth and maturation of the biofilm.
Several talks focused on P. aeruginosa and cystic fibrosis,
both from the laboratory perspective and through examination
in vivo. The development of a tissue culture model system for
the study of interactions between P. aeruginosa and cystic fi-
brosis-affected human airway epithelial cells was described by
7950 MEETING REVIEW
FIG. 1. 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 adher-
ent to a substratum; (ii) direct examination reveals bacteria in clusters
and encased in a matrix of bacterial or host constituents; (iii) the
infection is localized; (iv) the infection is resistant to antibiotic therapy.
The first, second, and third criteria were determined by microscopic
examination. The fourth criterion was fulfilled by empirical evidence:
unsuccessful resolution with antibiotic treatment. The fresh specimen
was stained with the nucleic acid stain Syto 9 (red), which stained
bacterial cocci (black arrows) as well as host nuclei (white arrows).
Less defined nuclei are deeper in the tissue. The biofilm EPS was
stained 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 Genomic
Sciences, Pittsburgh, PA).
Greg Anderson (Dartmouth College). Addition of the bacteria
to confluent eukaryotic-cell cultures caused detachment of the
eukaryotic cells within a few hours. Detachment could be
greatly 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 Dan
Hassett (University of Cincinnati). He showed that mucA mu-
tants (the mucoid strains that predominate in cystic fibrosis
lung infections) are extremely sensitive to NO at a pH of 6.5,
the approximate pH of lung airway mucus according to his
measurements in lungs recently removed from transplant pa-
tients; however, he found limited NO toxicity at a pH of 7 or
above. 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 a
therapeutic agent for cystic fibrosis patients (76) if the bacteria
in situ are growing anaerobically. Janus Haagensen (Danish
Technical University) used FISH to show the presence of P.
J. BACTERIOL.
aeruginosa cell aggregates within the sputa of cystic fibrosis
patients who had coinfections with mucoid and nonmucoid
strains; however, these aggregates were absent from the sputa
of patients infected solely with nonmucoid strains. Further,
aggregates were shown to contain other bacteria in addition to
P. aeruginosa; antibiotic treatment of the patients resulted in
reductions in mucoidy and aggregation. Niels Høiby (Univer-
sity of Copenhagen) presented new ideas on P. 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.
On the basis of autopsy and sputum specimens, he described
how the infection compartmentalizes in the lung. In the respi-
ratory zone, where oxygen tension is high relative to that in the
bronchi, no thick mucous layer is present but mucoid P. aerugi-
nosa colonies are found surrounded by polymorphonuclear
leukocytes (PMN); inflammation and tissue destruction occur
in this zone, and Høiby believes this is where the infection has
its true genesis. These mucoid colonies, together with de-
stroyed lung tissue, are then transported by coughing to the
bronchi (conductive zone), where they become embedded in
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the naturally occurring thick mucous layer. The mucous layer
creates steep oxygen gradients; single P. aeruginosa cells, as
well as mucoid colonies, can be found in the mucus.
Co-chair Römling spoke on second-messenger c-di-GMP
signaling and phenotype switching (25, 57). Low levels of c-di-
GMP encourage motility (twitching and swimming), whereas
high levels encourage a sessile phenotype through production
of exopolymers and extracellular appendages. GGDEF/EAL
domain proteins have phosphodiesterase as well as guanylate
cyclase 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 colony
morphology, red colonies, and clumpy, adherent cells. Expres-
sion of the transcriptional regulator cgsD, which is controlled
by environmental signals (such as temperature) as well as by
the c-di-GMP level, results in production of the rdar pheno-
type through synthesis of curli as well as by indirect activation
of cellulose exopolymer synthesis; csgD knockouts have a
smooth colony morphology and are nonadherent and non-
clumping. AdrA is one of four GGDEF/EAL proteins that
influence the transcription of cgsD. These c-di-GMP “net-
works” have been shown to be crucial to the development of a
biofilm phenotype in several bacteria. Paul Rainey’s talk, dis-
cussed later in this paper, explored c-di-GMP networks in an
evolutionary context.
BIOFILMS IN NATURAL ENVIRONMENTS
Michael Kühl (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, including bacte-
ria as commensals or pathogens of plant or animal hosts. Kühl
spoke on spatiotemporally resolved techniques for imaging
chemical species, particularly oxygen, within microbial mats
and corals. Much of this work employs films that contain a
VOL. 189, 2007
FIG. 2. Combined microscopic imaging of oxygen concentration
and bacterial colonization within a biofilm reprinted from reference 34
with permission. (A) Projection and vertical slices through a confocal
image 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 flow
chamber. (B) Corresponding O2 distribution at the base of the biofilm.
The central cell cluster is outlined by the dashed curve. Scale bars,
40 ␮m.
photochemically active sensor species (21), which reports the
concentration of the soluble target species (Fig. 2). Lifetime
images are produced that describe how a chemical parameter
changes in response to changes in the environment. 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).
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 pH
result 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 layering
and microbial activity in the same biofilms. Microautoradio-
graphic data together with FISH studies show that in this
system, in sharp contrast to the spatial organization in other
MEETING REVIEW 7951
mat systems, active sulfate reduction occurs at the very surface
of the biofilm (6). Decho suggested that this may be set up
through homoserine lactone signaling.
Plant-associated biofilms were introduced by Clay Fuqua
(Indiana University), who related inorganic phosphate (Pi)
deprivation to enhanced biofilm formation in Agrobacterium
tumefaciens. Pi is frequently limiting in the environment; the
phosphate response regulator protein PhoB is activated during
phosphate starvation, and in vitro, phosphate-starved A.
tumifaciens biofilms have an elevated biomass relative to that
of normal biofilms. When phoB is induced, the cells bind to
substrata using their poles, and they also show wheat germ
agglutinin binding at their poles. A transposon mutant selected
for the lack of polar binding is not recognized by wheat germ
agglutinin, and the mutated gene bears similarity to a gene in
the holdfast biosynthesis pathway of Caulobacter crescentus. In
A. tumefaciens, low Pi levels may be a cue for the production of
a specialized polymer and thus for biofilm initiation. Co-chair
Steve Lindow continued the theme of plant pathogen biofilms
in his talk on Xylella fastidiosa (43). This bacterium infects
plant xylem vessels, which then become blocked; the densely
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packed biofilms in blocked vessels have restricted access to
nutrients and thus have very low activity, as assessed by pro-
pidium iodide staining. Solitary bacteria in unblocked vessels
can move from vessel to vessel. Bacterial biomass and the level
of the diffusible signal factor DSF (the product of rpfF) are low
early in infection, and movement from vessel to vessel (biofilm
metastasis) is encouraged. Late in infection, biomass and signal
levels increase and cue polymer production, with resultant
vessel blockage. Lindow proposed to treat infection by intro-
ducing signal-hyperproducing strains to push early infections
toward 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 (University
of Connecticut) also spoke on a plant xylem pathogen: Pantoea
stewartii (31). Cell density-dependent synthesis of EPS is re-
quired for virulent biofilm formation; mutants with mutations
in the esaI and esaR quorum-sensing loci form less-dense bio-
films and do not disperse effectively in planta. The bacterium
was shown to specifically colonize annular rings and spiral wall
thickenings within the xylem.
Black band disease in corals was discussed by Laurie Rich-
ardson (Florida International University). Comparison of the
bacterial community from diseased coral with that from
healthy coral (54) has shown that the migration of certain
bacteria, in particular that of Beggiatoa spp., produces sharp
gradients of sulfide and oxygen that kill the coral. Further,
toxin-producing cyanobacteria have been identified from the
community and may be responsible for regional differences in
the disease. Dhana Rao (University of the South Pacific, Fiji)
spoke on the interaction between Pseudoalteromonas tunicata
and Roseobacter gallaeciensis, two surface colonizers of the
marine alga Ulva australis (47). P. tunicata can outcompete
other natural community members when grown as a biofilm in
natural seawater, but R. gallaeciensis outcompetes P. tunicata
under the same circumstances. Likewise, P. tunicata cannot
invade an extant biofilm composed of other species, but R.
gallaeciensis can invade and eventually comes to dominate such
a biofilm. Interestingly, P. tunicata is unable to persist in ster-
ile-filtered seawater.
7952 MEETING REVIEW
Two talks on biofilms in terrestrial systems highlighted the
importance of water availability and of local chemistry. Water
potential is critical to soil biofilm development, 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. Biofilms with reduced alginate levels are
less 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) showed
that in an in vitro P. aeruginosa biofilm, precipitated iron is
heterogeneously distributed. This may be correlated with
the local pH, and the bacterium alters its lipopolysaccharide
chemistry in response to pH (22, 23). Less precipitate was
found associated with metabolically active cells than with
carbonyl cyanide m-chlorophenylhydrazone (CCCP)-inhib-
ited cells; protons transported to the cell surface by respi-
ration appear to compete with metal cations for reactive cell
surface 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 effects
on biofilms (38, 39). Grazing by amoebae induces changes in
bacterial gene regulation, e.g., rhamnolipid production is in-
creased. Bacterial defense mechanisms against grazing include
microcolony 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, Bastiaan
Krom (University Medical Center, Groningen, The Nether-
lands) showed that homoserine lactone from P. aeruginosa
inhibits hyphal formation in Candida albicans. Further, break-
down products of homoserine lactone inhibit germ tube for-
mation. C12 and C14 homoserine lactones had the greatest
effect. In an example of beneficial cross-kingdom signaling,
Miguel Cámara (University of Nottingham) discussed how ho-
moserine lactone signaling molecules produced by biofilms
influence the attachment of Ulva linza (marine alga) zoospores
(63). Using lactones synthesized by Vibrio anguillarum as
model compounds, he showed that spore attachment increases
with increasing lactone concentration. Using natural bacterial
isolates, three patterns of increasing zoospore attachment were
seen as bacterial numbers increased: a linear relationship, an
exponential relationship, and a bell-shaped curve. These results
suggest 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 the
reduction in the mean lifetime of the worm; S. aureus colonizes
the alimentary tract, and known virulence factors such as PIA
have been demonstrated to apply to the worm. Coagulase-
negative staphylococci have also been shown to be virulent in
this model, and PIA-negative mutants do not kill as effectively
as the parent strain. Thomas Rasmussen (Chr. Hansen A/S,
Hørnsholm, Denmark) talked about quorum sensing in Pseudo-
J. BACTERIOL.
monas aeruginosa biofilms and how it affects interactions be-
tween the biofilm and PMN. Wild-type P. aeruginosa biofilm
supernatants lyse PMN, but supernatants of quorum-sensing
mutant 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 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.
EVOLUTION AND DIVERSITY
Paul Rainey (Massey University, New Zealand) chaired the
session on evolution and diversity and gave the opening talk,
presenting biofilm development in Pseudomonas fluorescens as
a process of mutation and subsequent selection. Numerous
mutations 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 negative
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regulator 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 to
remain attached after cell division but also to generate a mat of
polymer-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 could
still grow within the mat. These selfish (cheating) genotypes
did not contribute to mat strength or integrity and thus caused
the mat to collapse. Molecular analyses of cheaters revealed
a range of different mutations whose net effect is to abolish
the 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 to
the bacterium.
John Roth (University of California—Davis) addressed the
question of whether stress (such as could occur during biofilm
growth) increases mutation frequency. Problems in defining
exactly how a mutation arises and in accurate counting of
mutations have left this question unresolved for decades, and
studies of mutations in biofilms underscore the uncertainty.
Roth presented theoretical arguments against the existence of
a “hypermutable” state under any circumstances, 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). Roth believes that
selection 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 that
these variants are generated by error-prone repair (recA and
recBC mutants) of double-stranded breaks in DNA arising
from oxidative stress; addition of an antioxidant to the biofilm
reduces the occurrence of variants. Once the variants appear,
they are selected for and come to dominate the biofilm. Con-
VOL. 189, 2007
FIG. 3. Colony variants—including two different wrinkly spreader genotypes—of P. fluorescens that arise during selection in spatially structured
microcosms. (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).
tinuing the theme of variants, Alfred Spormann (Stanford Uni-
versity) argued that the stability of Vibrio cholerae biofilms is
related to the appearance of small-colony variants (SCV). 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. Characterization of SCV physiology is now
under way using 2,000-parameter Biolog plates, and Spormann
argued for an increased emphasis on physiology in biofilm
experiments.
Jaione Valle (Public University of Navarre, Pamplona,
Spain) concluded the session by describing how insertion se-
quence IS256 mediates phenotypic switching in Staphylococcus
aureus (69). Knockouts of transcription factor ␴B have in-
creased IS256 activity and produced biofilm-negative variants
with dramatically higher IS256 copy numbers in the chromo-
some at a high frequency. Osmotic stress can complement the
lack of ␴B and restore the wild-type phase variation frequency,
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.
KEYNOTE TALK 2
Ivan Matic (University of Paris) continued the discussion of
genetic variation in biofilms from the perspective of mutation
and repair. Observed mutation rates are typically low because
most mutations are deleterious or neutral and because the cell
has extensive investments in repair mechanisms. However,
populations of natural isolates contain strong mutators that
display mutation rates 100-fold higher than those of laboratory
strains; these mutators are deficient in repair (15). They can be
selected for in gnotobiotic mice by sequential multiple-antibi-
otic treatments; the surviving cells are mutators. Also present
in natural populations are weak mutators whose mutation rates
are 4 to 5 times those of laboratory strains. The difference in
the mutation rate is a by-product of selection for improved
fitness, and mutation rates can be very different in the host
environment than in the laboratory.
MEETING REVIEW 7953
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THE SLIME MATRIX
Tony Romeo (Emory University) chaired the session focus-
ing on the regulation, composition, and function of EPS. The
characterization of EPS chemistry and function has remained
elusive even though EPS is a hallmark that distinguishes bio-
film populations from planktonic cultures. Romeo discussed
the regulation of poly-␤1,6-N-acetyl-D-glucosamine (PGA),
which is a common component of EPS in gram-positive as well
as gram-negative bacteria. Romeo discussed how the carbon
storage regulator (Csr) RNA-binding protein, which represses
PGA and biofilm formation, can be sequestered by the non-
coding short RNAs csrB and csrC (2). In turn, csrB and csrC
can 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 EPS
genes pel and psl in P. aeruginosa colony morphology variants
isolated 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. By
knocking out c-di-GMP in a sticky mutant, the wild-type mor-
phology was recovered; c-diGMP signaling was important in
directing adhesion and cohesion (30). Jean-Marc Ghigo (Pas-
teur Institute, Paris, France) presented evidence for secretion
of valine into EPS. This secretion acts not only as a “carbon
dump” but also to provide an antimicrobial effect against in-
vading planktonic bacteria, which have been shown to be more
sensitive than biofilm cells to valine. Per Nielsen (Aalborg
University, Aalborg, 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, brackish-water, and seawater
environments. Nielsen used antibodies and FISH (Fig. 4) to
demonstrate 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 the
session by presenting evidence that membrane vesicles are an
important component of P. aeruginosa biofilm EPS (53). DNA
7954 MEETING REVIEW J. BACTERIOL.

FIG. 4. Detection of amyloids in a brackish-water biofilm, with simultaneous labeling of amyloid adhesins by antibodies and identification of
bacteria by FISH. (A) Transmitted light image showing small rods attached to an empty sheath, presumably from a cyanobacterium (judging from

Downloaded from jb.asm.org by on December 23, 2009

the 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 of
view as that in panel A. Shown are results of FISH using the red fluorescent oligonucleotide probe CFB719, targeting the class Bacteriodetes, and
blue fluorescent EUBmix, targeting most members of the domain Bacteria. (D) Overlay of panels A, B, and C. Colocalization of the three
fluorescent 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).

from vesicles was estimated to make up at least 0.33% of the
extracellular DNA. Vesicle production may play a significant
role in EPS function for biofilms. The data presented in this
session, the lively discussion at the specialty evening session on
EPS chaired by Hans-Curt Flemming and Dan Wozniak, and
the final keynote address by Bill Costerton on structured EPS
illustrate how EPS is no longer seen as an amorphous carbo-
hydrate 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 func-
tions.
CELL SIGNALING IN BIOFILMS
A function for cell signaling in a biofilm context was first
described for P. aeruginosa in 1998 (14). The concept that a
sessile bacterial population could coordinate its phenotype and
organization through quorum-sensing mechanisms quickly
captured the imagination, and it was thought that signaling
might be a magic bullet for the control of biofilms. Since then,
the field has gained in complexity, because the diversity of
signal molecules and mechanisms has grown. Matt Parsek, who
was involved in the earliest biofilm signaling work, chaired the
session, which included examples of signaling in three different
experimental systems. Susanne Häussler (Helmholtz Center
for Infection Research, Braunschweig, Germany) discussed
PQS in P. aeruginosa. 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. Häussler suggested that PQS is a
regulator of coordinated dispersal (movement of bacterial cells
from the biofilm to the bulk liquid), because of its promotion
of detergent tolerance. Deborah Hogan (Dartmouth College)
presented an example of competitive survival of two organisms
that involves response to, as well as interference with, each
other’s signaling systems. P. aeruginosa and C. albicans are
commonly isolated in cases of opportunistic infections (71),
and in coculture, P. aeruginosa can colonize and kill C. albicans
hyphae through the production of virulence factors, such as the
fungicide pyocyanin (29), which are regulated by the Las/Rhl
signaling system. However, the production by P. aeruginosa of
its virulence factor-stimulating signaling molecule OdDHL
[N-(3-oxododecanoyl)-L-homoserine lactone] causes C. albi-
cans to switch its morphotype to the colonization-resistant but
host cell-invasive yeast form. In addition to this defensive re-
sponse, C. albicans mounts an offensive against P. aeruginosa
through the production of farnesol. Farnesol, a signal for C.
albicans to block the formation of hyphae, also depresses PQS
signaling and pyocyanin production in P. aeruginosa. In this
cross-kingdom interaction, signals coordinate the phenotype of
the producing organism while at the same time altering the
phenotype of the competitor. Cell signaling in the social bac-
terium Myxococcus xanthus was discussed by Heidi Kaplan
(University of Texas Medical School). She presented evidence
for a quorum-sensing system sensitive to membrane integrity.
M. xanthus responded to an accumulation of lysed cell wall
components, such as peptidoglycan, in the culture by initiating
sporulation. Intriguingly, this recognition and response to
damage in the population is not unlike the “danger model” of
the mammalian immune system response (40), in which rec-
ognition of cell damage is the immune activator. It remains to
be seen how widespread sensing and response to self-damage
is among prokaryotes. Finally, Jan Kreft (University of Bonn)
presented modeling evidence that cell-signaling mechanisms in
gram-negative bacteria may be adapted to regulate behavior
VOL. 189, 2007
over the small distances between adjacent cells and within
small clusters (19). Based on the observation that the diversity
of 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 in
biofilms is local. Signals are directed toward nearest-neighbor,
closely related cells (i.e., toward clones), rather than toward
spatially distant and therefore, presumably, less closely related
cells. Kreft’s modeling suggests that current assumptions re-
garding the distance over which signaling operates may need to
be 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 biofilms
in the urinary tract. Stickler discussed Proteus mirabilis biofilm
formation in urinary catheters and the resultant struvite crys-
tallization (61). Multispecies biofilms attenuated crystallization
by modifying the pH; patient urine chemistry and biofilm spe-
cies composition were important factors in the rate of catheter
blockage. 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, 65).
Seeding with these E. coli isolates reduced the incidence of
symptomatic infection nearly 20-fold: from 2.7 per 100 catheter
days to 0.15 per 100 catheter days. Control of device-related
biofilm infections is perhaps best addressed not by attempting
to 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 through
genetic 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 an
important adhesin and virulence factor for uropathogenic E.
coli in a mouse model (68). Adhesins such as antigen 43 may be
useful targets for modification to create probiotic E. coli
strains.
The next two presentations were on a rapidly growing area
of interest in medical biofilms: chronic wound infections. Klaus
Kirketerp-Møller (Bispebjerg University Hospital, Copenha-
gen, Denmark) presented evidence from clinical specimens
suggesting that the persistence of P. aeruginosa in inflamed
chronic wound infections was due to its protected location
within microcolonies, where it remained impervious to, and
could kill, leukocytes. Garth James (Montana State University)
used confocal microscopy and denaturing gradient gel electro-
phoresis to show the broad diversity of infecting bacteria, even
in similar wound types. Importantly, 16S rRNA analysis
showed that, in addition to the routinely cultured aerobic and
facultatively anaerobic species, a number of uncultured obli-
gate anaerobes were present. James hypothesized that strict
anaerobes may inhabit anaerobic microniches created through
oxygen consumption by aerobic biofilms.
Session co-chair Michael Otto then gave the first of two talks
on the molecular pathogenesis of staphylococcal infections. He
showed that spontaneous mutation in the biofilm-repressing
agr locus causes S. epidermidis to form different biofilm phe-
MEETING REVIEW 7955
notypes. The difference in architecture between the heteroge-
neous wild type and the thicker but flatter agr mutant was
caused 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 to
identify genes unique to clinical isolates of S. aureus. Differ-
ential expression showed that approximately 50% of the genes
were unique to clinical strains, that many of these genes were
involved in stress response and the production of virulence
factors, 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 that
continued laboratory passage of clinical strains may cause a
loss 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 talks
on oral biofilms were given. Rob Palmer (NIDCR, NIH) used
immunofluorescence confocal microscopy and enterobacterial
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repetitive intergenic consensus PCR to investigate phenotypic
and genotypic diversity within a Veillonella sp. in natural dental
plaque biofilms from human volunteers. In as little as 4 h,
changes in coaggregation, antibody reactivity, and enterobac-
terial repetitive intergenic consensus fingerprinting patterns
occurred (44). Palmer concluded that the only way to fully
understand 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 biofilm
formation in Porphyromonas gingivalis. Interestingly, Davey
found that encapsulated P. gingivalis strains did not form bio-
films, while strains lacking a capsule did (13). Further, capsule
expression was found to be dependent on high nutrient levels
and rapid growth, explaining why encapsulated forms are more
commonly 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 simple
coculture, it was found that Streptococcus salivarius inhibited
the 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 AND
ENGINEERED SYSTEMS
Much early pioneering work in biofilm research was con-
ducted in the context of fouling prevention in industrial and
marine systems, and also for optimization of bioconversion
processes in wastewater treatment and bioremediation. Satoshi
Okabe (Hokkaido University, Japan) and Linda Blackall (Uni-
versity of Queensland, Brisbane, Australia) were the chairs for
this session on biofilms in engineering applications. Korneel
Rabaey (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 varying
species composition and growth conditions, while indicating
7956 MEETING REVIEW J. BACTERIOL.

that initial applications may be limited to low-current situa-

tions such as sensors and feedback switches (1). Christian
Picioreanu (Delft University) and Andrew Kato-Marcus (Ari-
zona State University) each presented mathematical models
that could be used to optimize these fuel cells. Picioreanu’s
model showed how biofilm structure may be an important
factor for charge density on the anode, while Kato-Marcus
modeled the anode as an electron acceptor, with the biofilm
itself as an integral part of the anode. Moshe Herzberg (Yale
University) illustrated how molecular, microscopic, and ex-
pression techniques could be applied to identify biofilm activity
in reverse osmosis systems.
Three talks focused on multispecies biofilms in wastewater
treatment. Co-chair Linda Blackall showed how the develop-
ment of structured biofilm communities in flocs could be used
for the simultaneous removal of ammonium, nitrate/nitrite,
and phosphorus from wastewater. Electron microscopy sug-
gested that the flocs may have structured EPS, which appeared
similar to the honeycomb-like structures introduced later in
the meeting by Bill Costerton. Co-chair Satoshi Okabe used a
polyphasic approach that employed FISH, microautoradiogra-

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phy, and microelectrodes to characterize the distribution and
activity of anaerobic ammonium-oxidizing (anammox) bacteria
in a mixed wastewater biofilm (67). These bacteria were dis-
tributed throughout the biofilm, but rates of ammonium and
nitrite oxidation differed at different locations in the biofilm.
Silvia Weber (Technical University of Munich) used similar
techniques to investigate granular flocs. A FISH protocol was
optimized for use in thick, dense biofilms; this approach may
have applications in other biofilm systems such as microbial
mats. Maria Giao (University of Minho, Portugal) showed the
importance of nutrients and hydrodynamics to Legionella pneu-
mophila biofilm formation. Low carbon levels increased the
number of viable but nonculturable bacteria in the biofilm,
suggesting that environmental factors should be taken into
account 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 determine
the influence of nitrate on a biofilm community from an oilfield
seawater injection system that contains sulfate-reducing bacte-
ria (SRB). 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. Fi-
nally, Chuanwu Xi (University of Michigan) presented work on
optical coherence tomography, a high-resolution imaging tech-
nique that does not require staining, for real-time monitoring
of living biofilms. The instrument offers two advantages over
conventional microscopes, especially in process situations: it
can be positioned much further from the biofilm, and it can
penetrate opaque materials (73).
PREVENTION AND TREATMENT OF BIOFILMS
Michael Givskov (Danish Technical University) and Phil
Stewart (Montana State University) were session chairs. Stew-
art’s talk on the visualization of antimicrobial action in biofilms
described the use of calcein AM as a viability indicator. When
treated with the quaternary amine Barquat, biofilm microcolo-
FIG. 5. Visualization of antimicrobial action against a Staphylococ-
cus epidermidis 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. Time lapse confocal scanning laser microscopy
examination of fluorescence loss shows permeabilization of cells by the
biocide. The number in each panel is the time, in minutes, after the
introduction of ADBAC into the flow cell. In the first and last panels,
biomass was imaged in transmission mode (gray scale). Image courtesy
of W. Davison and P. Stewart (Center for Biofilm Engineering, Mon-
tana State University).
nies quickly lost calcein fluorescence at the edges but showed
time-dependent retention of fluorescence at the center (Fig.
5). Treatment with chlorite caused loss of fluorescence only in
a thin band at the edge of the colony. Treatment with nisin led
to a rapid and uniform fluorescence loss. This approach allows
spatiotemporally resolved measurement of toxicity and sug-
gests that differences in susceptibility to particular agents exist
within 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 shear
strength of fouling layers can be measured (41). The base
layers of the biofilm were found to be highly stable, whereas
the upper layers were easily removed. Neu also showed mag-
netic resonance imaging data that allow real-time, nondestruc-
VOL. 189, 2007
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 they
can eliminate the biofilm. (Right) In the presence of the P. aeruginosa LasR/RhlR double-quorum-sensing mutant, the PMN readily traverse the biofilm
and phagocytose the virulence-factor-deficient bacteria. Images courtesy of Thomas Bjarnsholt (Danish Technical University).
tive visualization of biofilm biomass, as well as of flow velocity
and shear stress around the biofilm.
Persister cells, first reported by Spoering and Lewis (59), are
defined as a numerically small (⬍1%), antimicrobial-resistant,
slow-growing subpopulation of cells that is maintained in both
planktonic and biofilm cultures; these cells were the subject of
talks by Peter Gilbert (University of Manchester) and Kim
Lewis (Northeastern University). Using high-throughput cell
sorting, 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. Large volumes of culture were processed
to collect sufficient mRNA for microarray analysis. High levels
of 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. These
genes may be regulators of the persistence state and thus poten-
tial drug targets for recalcitrant biofilm infections. Lewis’ main
observations 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 he
identified genes different from those discovered by Gilbert; Lewis’
persister genes included the dehydrogenase-encoding gene glpD
and the lipid biosynthesis gene plsB (60). Lewis thought that the
differences between candidate genes from the two studies may be
a 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 on
the use of biofilm-specific antibodies to detect biofilm infec-
tions in a rabbit model. Antigens are being identified by a
proteomics approach as present in infected animals but absent
in healthy animals; because the targets are eventually to be
used in the diagnosis of human biofilm infections, human
blood must be screened as well, and one candidate protein has
MEETING REVIEW 7957
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emerged. The development of antimicrobials from marine in-
vertebrates was reported on by Domenico Schillaci (University
of Palermo). Cytosol of the sea urchin Paracentrotus lividus has
activity against S. epidermidis and S. aureus. A 5-kDa peptide
fraction 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 generated
naturally 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). Givskov
suggested that rhamnolipid is a necrotic agent for PMN (7, 24)
(Fig. 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). A promising garlic-based quo-
rum-sensing blocker (46) has been synthesized and tested; it is
less active than garlic extract, but it is possible that a mixture of
garlic-based compounds can reach or exceed the efficacy of
natural garlic extract.
The topic of predatory/parasitic bacteria as antibiofilm
agents was covered by Daniel Kadouri (University of Medicine
and Dentistry of New Jersey). Bdellovibrio bacteriovorus can
greatly diminish the biofilm biomass of E. coli as well as that of
P. fluorescens (26). The less well known exoparasitic bacterium
Micavibrio aeruginosavorus is likewise able to reduce the bio-
film biomass of laboratory and clinical strains of P. aeruginosa,
Klebsiella pneumoniae, and Burkholderia cepacia (27). Closing
the session, David Davies (Binghamton University) spoke on a
biofilm-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 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.
7958 MEETING REVIEW
FIG. 7. Scanning electron microscopy of a part of a macroscopic
casernum formed in liquid culture by the MH strain of S. epidermidis.
Note the coalescence of cells to form the parallel planar walls of the
basic honeycomb structure. Image courtesy of Christoph Schaudinn
and Bill Costerton (University of Southern California).
KEYNOTE TALK 3
The prebanquet address was given by Bill Costerton, the
person who, beginning at the University of Calgary and later as
the director of Montana State University’s Center for Biofilm
Engineering, brought biofilms to the forefront of microbiolog-
ical research and who is now developing a biofilm research
center 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 bacterial
cultures as well. Based on scanning electron microscopic,
freeze-substitution transmission electron microscopic, and
confocal and light microscopic evidence, these structures ap-
pear to form from the contents (especially ribosomes) of lysing
cells 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 dimensions
of millimeters, likely resulted from a genetically regulated pro-
cess. As discussed in the “slime matrix” session, little is known
about the structural and chemical composition of biofilm EPS;
the possibility of higher-order structural organization, such as
that described for proteins, is provocative. Casernum produc-
tion in natural environments and a role for caserna in biofilm
function remain to be demonstrated. However, the possibility
of structured EPS hints at yet further levels of complexity in
biofilm systems.
CODA
In summary, the meeting elevated its reputation for show-
casing high-quality, exciting science from a rapidly moving field
and for demonstrating the broad applicability of biofilm biol-
ogy to microbiological research. The organizing committee
J. BACTERIOL.
took steps to attract an audience and a program that were
demographically 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 continued
support and development of this conference series. In particular, we
thank ASM Conferences staff members Lisa Nalker and Latonya Ni-
chols for outstanding work in bringing the 4th Conference on Biofilms
to fruition.
Robert J. Palmer, Jr., is supported by the Intramural Research
Program of the National Institute of Dental and Craniofacial Research
at the National Institutes of Health. Paul Stoodley is supported by
Allegheny-Singer Research Institute and Philips Oral Healthcare.
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