BIOFFILME

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August 11, 2000 11:2 Annual Reviews AR110-03
Annu. Rev. Microbiol. 2000. 54:49–79

Copyright c 2000 by Annual Reviews. All rights reserved
BIOFILM FORMATION AS MICROBIAL

DEVELOPMENT
George O’Toole,1 Heidi B. Kaplan,2 and Roberto Kolter3
1Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire
Access provided by Universidade Federal da Santa Maria on 05/23/23. For personal use only.
03755; e-mail: georgeo@Dartmouth.edu

Annu. Rev. Microbiol. 2000.54:49-79. Downloaded from www.annualreviews.org
2Department of Microbiology and Molecular Genetics, University of Texas Medical
School, Houston, Texas 77030; e-mail: hkaplan@utmmg.med.uth.tmc.edu

3Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston,
Massachusetts 02115; e-mail: kolter@mbcrr.harvard.edu
Key Words communities, life cycle, fungi, bacteria, environment, surface

■ Abstract Biofilms can be defined as communities of microorganisms attached
to a surface. It is clear that microorganisms undergo profound changes during their
transition from planktonic (free-swimming) organisms to cells that are part of a com-
plex, surface-attached community. These changes are reflected in the new phenotypic
characteristics developed by biofilm bacteria and occur in response to a variety of en-
vironmental signals. Recent genetic and molecular approaches used to study bacterial
and fungal biofilms have identified genes and regulatory circuits important for initial
cell-surface interactions, biofilm maturation, and the return of biofilm microorganisms
to a planktonic mode of growth. Studies to date suggest that the planktonic-biofilm
transition is a complex and highly regulated process. The results reviewed in this arti-
cle indicate that the formation of biofilms serves as a new model system for the study
of microbial development.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
A New Approach to Studying Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Does Biofilm Formation Constitute a Form of Microbial Development? . . . . . . . . 52
GRAM-NEGATIVE BACTERIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Regulation of Initial Attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Biofilm Formation Proceeds via Multiple Convergent Genetic Pathways . . . . . . . . 53
Early Attachment Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Maturation of the Biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Detachment and Return to the Planktonic Growth Mode . . . . . . . . . . . . . . . . . . . 62
Similarity of M. xanthus Fruiting Body Development
and P. aeruginosa Biofilm Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
GRAM-POSITIVE BACTERIA AND MULTISPECIES BIOFILMS . . . . . . . . . . . . 63
Clinical Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
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50 O’TOOLE ET AL
Early Attachment Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Leaving the Biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
FUNGAL BIOFILMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Initial Attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
SUMMARY AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
INTRODUCTION
Microorganisms are often viewed as simple creatures when compared with

“higher” organisms. The study of microbial development, however, has shown

that microorganisms are capable of complex differentiation and behaviors. Ex-
amples include the Caulobacter crescentus cell cycle-controlled swarmer-to-stalk
cell transition and Bacillus subtilis spore formation, in which individual vegetative
cells integrate multiple external and internal signals to successfully synthesize a
new morphological structure that is adapted to survival in a variety of harsh envi-
ronments. Myxococcus xanthus takes this complexity to another level by chore-
ographing the behavior of an entire cell population, which forms a multicellular
fruiting body that contains resistant spores. The results reviewed below indicate
that the formation of surface-attached microbial communities, known as biofilms,
serves as another excellent model system for the study of microbial development.
Biofilms can be defined simply and broadly as communities of microorgan-
isms that are attached to a surface. A concerted effort to study microbial biofilms
began only 2 decades ago with the rediscovery that, in natural aquatic systems,
bacteria are found predominately attached to surfaces (69). The first recorded
observation that we uncovered concerning biofilms, however, comes from a 1933
paper by Henrici, in which he states “. . . it is quite evident that for the most part
water bacteria are not free floating organisms, but grow upon submerged sur-
faces” (90). Furthermore, the fouling of ships’ hulls by microorganisms in marine
environments had already been recognized as a serious problem for a number
of years before Henrici’s publication (6). Although biofilm formation has been
a recognized and scientifically documented aspect of microbial physiology for
∼100 years, we are just beginning to understand this process at the molecular
level. In the past few years, simple screens for the isolation of biofilm-defective
mutants have been devised, thus making genetic analyses of biofilm development
possible (85, 88, 121, 142). These studies have provided important insights into
the genetic basis of biofilm development.
Biofilms can comprise a single microbial species or multiple microbial species
and can form on a range of biotic and abiotic surfaces. Although mixed-species
biofilms predominate in most environments, single-species biofilms exist in a
variety of infections and on the surface of medical implants (1, 11, 53). These
single-species biofilms are the focus of most current research. Pseudomonas aerug-
inosa has emerged as the most studied single-species, biofilm-forming gram-
negative bacterium, although, as detailed in this review, among the gram-negative
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BIOFILM FORMATION 51
bacteria, Pseudomonas fluorescens, Escherichia coli, and Vibrio cholerae have

also been studied in detail. The gram-positive biofilm-forming bacteria that have
been studied include Staphylococcus epidermidis, Staphylococcus aureus, and the
enterococci.
Studies indicate that biofilms are a stable point in a biological cycle that includes
initiation, maturation, maintenance, and dissolution (Figure 1, see color insert).
Bacteria seem to initiate biofilm development in response to specific environmental
cues, such as nutrient availability. Although these conditions vary widely, the gram-
negative organisms covered in this review, with the exception of Myxococcus
xanthus and E. coli O517:H7, undergo a transition from free-living, planktonic

cells to sessile, surface-attached cells in response to a nutrient-rich medium. These
biofilms continue to develop as long as fresh nutrients are provided, but when they
are nutrient deprived, they detach from the surface and return to a planktonic mode
of growth. Presumably, this starvation response allows the cells to search for a fresh
source of nutrients and is driven by well-studied adaptations that bacteria undergo
when nutrients become scarce (111). Therefore, we propose that the starvation
response pathway can be subsumed as a part of the overall biofilm developmental
cycle.
It is remarkable that most microorganisms seem able to make the transition to
life on a surface, irrespective of their physiological capabilities. Early studies sug-
gested that the overall hydrophobicity and/or surface charge of a bacterium could
serve as a good predictor of the surfaces that an organism might colonize (35).
Although these factors are clearly important in initial cell-surface and cell-cell
interactions, they are by no means the whole story. Bacterial surfaces are hetero-
geneous, and, what is most important, they can change dramatically in response to
changes in their environment. Therefore, a bacterium cannot be accurately mod-
eled as a sphere with a uniform surface. An in-depth understanding of the bacterial
components required for biofilm development and the mechanisms that regulate
their production and activity is needed for a fuller understanding of this ubiquitous
microbial phenomenon.
A New Approach to Studying Biofilms

To identify and characterize the bacterial elements and genetic determinants that
are necessary for biofilm development, a simple genetic screen has been used by a
number of groups (72, 85, 88, 121, 142). This screen uses the well of a microtiter
dish as the chamber in which a biofilm is established. Biofilms are visualized with
a variety of dyes (such as crystal violet and safarinin). The simplicity of the assay
has meant that high-throughput screens of many thousands of randomly generated
mutants have been carried out with relative ease. Mutants that are unable to form
a biofilm in gram-negative organisms under these conditions were termed surface
attachment defective or sad by our group. Sad is used as a generic designation for
non–biofilm-forming mutants that are isolated in the microtiter dish screens. It is
interesting that subsequent microscopic examination revealed that some of the sad
mutants undergo initial attachment normally, but are blocked in downstream steps
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52 O’TOOLE ET AL
in the developmental process. Therefore, as described in detail below, a simple

genetic screened has allowed the elucidation of a number of the steps that are
required for biofilm development. In addition, rapid progress has been made in
identifying certain bacterial structural components and sensory systems that are
necessary for the initiation of biofilm formation. The bacteria must be able to
attach to and move on surfaces, to sense their cell density, and ultimately to form
a three-dimensional structure of cells encased in exopolysaccharides.
Once microorganisms have established a foothold on a surface, they begin
to undergo a series of changes that adapt them to life on a surface. It is these
changes that will be one of the exciting new areas of investigation in the future
and that reflect the developmental nature of biofilm formation. Common adapta-
tions that have already been observed include the expression of large quantities
of exopolysaccarides that may protect the biofilm and lead to biocide resistance
(17, 21, 50, 65, 125). In addition, mature biofilms can have complex architectural
features. Initial studies of biofilms by electron microscopy led to the dehydra-
tion of samples and a deceivingly simplistic view of biofilms as cells piled atop
one another (35). Recent advances in confocal scanning laser microscopy have
allowed the visual inspection of fully hydrated biofilms, with a concomitant rad-
ical shift in how biofilm architecture is viewed and studied (115). For example,
Figure 1 (see color insert) shows a representation of the structure of a mature
P. aeruginosa biofilm. The biofilm developed comprises mushroom-shaped mi-
crocolonies of bacteria that are surrounded by an extracellular polysaccharide
matrix and separated by fluid-filled channels.
The striking similarity of the modeled three-dimensional structure of the mature
P. aeruginosa biofilm and that of a fruiting body formed by the soil bacterium
M. xanthus led us to compare these two systems more carefully. We found a
remarkable number of conserved elements and processes, from their type IV pili-
dependent movement on surfaces to the requirement for quorum sensing. We were
able to evaluate biofilm formation in the context of microbial development and to
consider that the M. xanthus fruiting body is akin to a single-species biofilm. In
addition, the reservoir of knowledge on M. xanthus fruiting-body formation should
prove useful for the further understanding of biofilm development.
Does Biofilm Formation Constitute a Form

of Microbial Development?
To determine whether biofilm formation constitutes a form of microbial devel-
opment, we must understand the term “microbial development.” In a recently
published book, Shimkets & Brun define development in microbes as involving
“changes in form and function that play a prominent role in the life cycle of the
organism” (161). Examples of changes in form are the transition from a free-
swimming swarmer cell to a replicating surface-attached stalk cell in C. crescen-
tus and sporulation in B. subtilis and M. xanthus (161). In biofilm development by
P. aeruginosa, such gross morphological changes in individual cells have not yet
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been observed. Perhaps the changes in form are subtle or are reflected in changes
in the relationships between individuals and groups of cells. That is, the bacteria
undergo a transition from a planktonic (“loner”) existence to a community-based
existence in which they must interact with many neighbors of various species in
close proximity. Functional changes are also evident in biofilm-grown cells. As
explored in detail in this review, the physiology, cell surfaces, resistance to envi-
ronmental insults, and other properties of biofilm cells are markedly different from
their planktonic counterparts. As for biofilm formation being a prominent part of
the lifestyle of microbes, this has been clear for nearly a century (90). Finally,
Shimkets & Brun point out that environmental signals are an essential driving
force for microbial development. Many studies have suggested that environmen-
tal cues play a role in biofilm development (as reviewed in 35). The goal of this
review is to present the evidence accumulated over the past few years that supports
the concept that biofilm formation is indeed a new model system for the study of
microbial development.
GRAM-NEGATIVE BACTERIA
Regulation of Initial Attachment

Biofilm formation is thought to begin when bacteria sense environmental condi-
tions that trigger the transition to life on a surface (62, 140–142, 145, 148, 168, 170,
178, 181, 184). These environmental signals vary among organisms. For example,
P. aeruginosa and P. fluorescens will form biofilms under almost any conditions
that allow growth (141). On the other hand, some strains of Escherichia coli K-12
and Vibrio cholerae will not form biofilms in minimal medium unless supple-
mented with amino acids (148, 180). In contrast, E. coli O517:H7 is reported to
make a biofilm only in low-nutrient media (51). In addition to the nutritional con-
tent of the medium, other environmental cues that can influence biofilm formation
include temperature, osmolarity, pH, iron, and oxygen (62, 140–142, 145, 148,
168, 170, 178, 181, 184).
Biofilm Formation Proceeds via Multiple Convergent

Genetic Pathways
The complexity of the signals triggering biofilm development is evident from the
fact that organisms have multiple genetic pathways that control this behavior. In
P. fluorescens, multiple pathways control biofilm formation and function under
different growth conditions. The first sad mutants in P. fluorescens were isolated
from screens that were performed by using cells grown on minimal glucose medium
plus casamino acids. It is surprising that the biofilm-defective phenotype of a
subset of these mutants could be suppressed by growth on citrate or glutamate
or in the presence of high iron concentrations. These data suggest that there are
at least two pathways involved in biofilm development in this organism. Similar
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54 O’TOOLE ET AL
results have been observed for P. aeruginosa (GA O’Toole, unpublished data). The
different medium conditions that promote biofilm formation and the subsets of
genes required under each environmental condition may simulate various niches
that are normally colonized by this organism (142).
V. cholerae may have at least three different means for adhering to surfaces,
depending on whether this organism is within its human host or in an aquatic en-
vironment. Tcp (toxin-coregulated pilus) is a type-IV pilus that has been shown
to be important for colonizing the gut of animals and is an important virulence
factor (91). A second type-IV pilus (Msh for mannose-sensitive hemagglutinin)
was also identified, but was shown not to be required for gut colonization and
pathogenesis (172). Watnick et al showed that Msh, but not Tcp, was required
for biofilm formation on non-nutritive abiotic surfaces such as plastic and glass
(180). It is interesting that V. cholerae is also known to colonize the chitin sur-
faces of shellfish, which may be a natural reservoir for this microorganism. Chitin
(a polymer of N-acetyl-D-glucosamine) can also serve as a source of carbon and
nitrogen for V. cholerae and is therefore considered a nutritive surface (33). Stains
carrying mutations in the mshA gene, although unable to colonize plastic, are in-
distinguishable from the wild type for attachment to chitin (180). This suggests
that as yet unidentified factors are necessary for chitin colonization. Therefore,
V. cholerae appears to adopt different strategies for biofilm formation, depending
on its surroundings. Furthermore, the ability to form biofilms on a variety of sur-
faces outside the host may contribute to its ability to survive in these hostile and
sometimes nutrient-limited environments. After all, V. cholerae primarily lives
outside a human host. Presumably, the better adapted this bacterium is to survival
in its aquatic environment, the more likely it is to cause subsequent outbreaks of
disease.
E. coli, the historical workhorse of bacterial genetics, has proven to be an excel-
lent model organism for the study of biofilm development. E. coli K-12 can form
biofilms on abiotic surfaces and can do so in a range of environmental conditions
(51, 72, 148, 176; PN Danese, LA Pratt, S Dove, R Kolter, manuscript in prepara-
tion). Genetic analyses have revealed that some functions are necessary for biofilm
formation under all environmental conditions tested, whereas other functions are
required for biofilm formation during growth in either minimal or rich medium,
but not both (PN Danese, LA Pratt, S Dove, R Kolter, manuscript in preparation).
However, there are some differences in biofilm formation phenotypes among dif-
ferent strains of E. coli K-12 (148, 176; PN Danese, LA Pratt, S Dove, R Kolter,
manuscript in preparation), suggesting that decades of laboratory domestication
may have led to the loss of some functions required for biofilm development.
Early Attachment Events

In a 1997 paper, Palmer & White (145) outlined the steps for the early stages of
biofilm formation that included cell-surface and cell-cell interactions, followed by
the development of the mature biofilm. At the time, the molecular determinants
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required for these steps had not yet been identified, but many aspects of their model
still hold true. Many early studies on the initial attachment of bacteria suggested
that simple chemical models could account for the behavior of bacteria during their
initial stages of attachment (61, 80, 124, 131). Although these chemical interactions
must contribute to cell-surface interactions, these early events are much more
complex. For example, it is possible that a variety of bacterial surface structures
involved in attachment (such as pili) are different from the overall surface character
of the microorganism. Furthermore, each surface structure may be specific to an
attachment surface of particular properties, and the expression of these structures
may change depending on the environment to which the bacterium is exposed.

P. aeruginosa By using phase-contrast microscopy to analyze a collection of mu-

tants that are defective for surface attachment, flagella and type-IV pili were shown
to play important roles in the early events in biofilm development by P. aeruginosa.
These observations are consistent with previous studies that strongly hinted at the
roles of these cell surface structures in the adhesion of bacteria to a wide variety
of surfaces (52, 54, 59, 79, 144, 150, 153, 154, 164, 165, 167, 187). In addition to
flagella and pili, Makin & Beveridge have reported that changes in lipopolysac-
carides (LPSs) result in altered attachment behavior (122). In particular, P. aerug-
inosa strains make two species of LPS (A and B band), and loss of B-band LPS
by mutation reduces attachment to hydrophilic surfaces and increases attachment
to hydrophobic surfaces. A-band mutants had only mild effects on attachment
(122).
Observing P. aeruginosa before attachment reveals that the organism swims
along the surface almost as if it is scanning for an appropriate location for initial
contact (Figure 2, see color insert). It appears that, once bacteria initiate surface
contact, they come to rest on the surface. However, time-lapse microscopy reveals
that, once P. aeruginosa forms a monolayer on an abiotic surface, the bacteria
continue to move (for time-lapse movies, refer to http://gasp.med.harvard.edu/bio-
films/annrev/movies.html). However, this movement occurs only on the surface
and uses twitching motility instead of swimming. P. aeruginosa and other bacteria
can move along a surface by using a type-IV pili–mediated mode of movement
that is ∼100-fold slower than the more familiar flagellar-dependent swimming.
Twitching motility is absolutely dependent on type-IV pili, and it has been pro-
posed that, by extending and retracting their pili, bacteria can push or pull them-
selves across a surface (22). Furthermore, there are indications that cells move by
twitching motility only when they are in contact with other cells, suggesting that
this is a community behavior (159). A similar set of observations has also been
made for M. xanthus, in which social motility also requires functional type-IV
pili (189). During the first few hours after formation of a biofilm, as shown by
time-lapse phase-contrast microscopy, the clusters [or microcolonies (Figure 2, see
color insert)] of cells form as the consequence of individual cells twitching across
the surface towards each other (141). In the early stages, these microcolonies
can also disperse and/or move as a unit across a surface, further emphasizing the
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56 O’TOOLE ET AL
dynamic and somewhat transient early interactions of bacteria with a surface and
each other.
In Figure 3 (see color insert), the structures of wild-type and mutant type-IV pili
are shown after 24 h of growth in a continuous-flow chamber (images courtesy of
M Parsek, Northwestern University). The difference in structure between the wild
type and mutant after 24 h is striking. Whereas the wild-type cells form character-
istic mounds, the mutant forms only small aggregates and/or a dense monolayer
of cells. These images strongly suggest that type-IV pili mutants are defective in
downstream developmental events that are necessary to form a mature biofilm. As
a result, these and similar mutants will be useful in the attempt to identify and
characterize the events that are necessary for the formation of the mature biofilm.
One of the sad mutants isolated on minimal medium supplemented with glucose
and casamino acids had a lesion in the crc (catabolite repression control) locus.
This gene was originally identified based on its global role in carbon metabolism
(186). P. aeruginosa will typically use organic acids such as succinate preferentially
over sugars such as glucose. A crc mutant, in contrast, will use sugars and organic
acids simultaneously. It is important that the presence of the various carbon sources
is still required for expression of catabolic functions, so the crc mutant is not simply
derepressed for all carbon use genes. Crc is also involved in twitching motility
(25, 140) and, in particular, in regulating the genes required for the synthesis of
type-IV pili (140). Therefore, Crc may be part of a signal transduction pathway
that relays input signals (such as carbon availability) and thereby regulates the
transition from planktonic to biofilm growth. To date, however, it is not known
how Crc regulates gene expression or responds to environmental cues (119).
It has been proposed that contact with a surface may induce changes in gene
expression, and there is evidence to support this idea in P. aeruginosa. Studies by
Davies and coworkers showed that one of the genes required for the synthesis of
the exopolysaccharide (EPS) alginate (algC ) is up-regulated three to fivefold in re-
cently attached cells vs their planktonic counterparts (43, 44). This result is not sur-
prising because alginate, whose regulation has been studied in depth (65, 78, 125),
has long been implicated as the extracellular matrix in biofilms of P. aeruginosa.
These experiments were among the first to show surface contact–induced gene
expression in P. aeruginosa. Recent studies in the laboratory of Wozniak have
taken this observation a step further. Wozniak and colleagues noticed that isolates
of P. aeruginosa from the cystic fibrosis (CF) lung that made large quantities of
alginate (mucoid strains) were also nonmotile, and these authors suspected a link
between the two phenotypes. In a series of genetic experiments, they showed that
expression of a sigma factor (AlgT/AlgU or σ 22) required for alginate synthesis
resulted in down-regulation of a key flagellar biosynthetic gene (68). These data
suggest that, on contacting the surface, flagellar synthesis is down-regulated and
alginate synthesis is up-regulated. This form of surface-mediated alteration in gene
expression is not unprecedented. Vibrio parahaemolyticus induces the synthesis
of laterally localized flagella for swarming motility upon contact with the surface
at the expense of expressing polarly localized flagella. Increasing the torque on
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the rotation of the polar flagellum, either by bringing the cell into contact with
a solid surface or by increasing the medium’s viscosity, results in initiation of
a signal for lateral flagella synthesis (127–129). Based on this surface-induced
gene expression, one could predict that one of the signals sensed by the cells and
required to induce alginate synthesis could be an alteration in the cell envelope
caused by physical interaction of bacteria with a solid surface. It is also not diffi-
cult to imagine that expression of other genes in P. aeruginosa is also altered as a
result of surface contact. Work by Brozel and colleagues supports this contention.
Using a nonbiased approach, these workers monitored changes in global protein
expression patterns in attached cells and found ≥11 proteins whose levels were
altered during various stages of attachment (27).

P. fluorescens A number of factors have been shown to affect the ability of

P. fluorescens to form a biofilm. For example, treatment with low levels of pro-
tease resulted in loss of attachment (142), which has also been observed in other
organisms (146). This suggested a role for extracellular proteins in biofilm devel-
opment. Consistent with this idea is the ability of exogenous protein to interfere
with attachment (60), possibly competing with bacterium-associated polypeptides.
Furthermore, attachment is dependent on new protein synthesis, suggesting that
biofilm development is a regulated process in this organism (142).
In a search for surface factors that are important for biofilm development,
Williams & Fletcher isolated transposon mutants with defects in the O-antigen of
LPS (183). These mutants showed an increased ability to attach to hydrophobic
surfaces and quartz sand and a decreased ability to attach to water wettable surfaces.
The authors proposed that this altered attachment was caused by the exposure of
the lipid moiety of the membrane leaflet upon loss of the O-antigen (183). In other
work, a selection for P. fluorescens strains with improved attachment properties
resulted in the isolation of two classes of spontaneous mutants (151). One class
was LPS mutants (these mutants were also altered for their outer membrane pro-
tein profile), and the other class included strains that overexpressed an EPS. We
have also isolated mutants with an increased ability to bind to poly(vinylchloride)
plastic, and they all map to LPS biosynthetic genes (GA O’Toole & R Kolter,
unpublished data). Furthermore, this increased attachment could be eliminated by
protease treatment, suggesting that it is not directly the loss of LPS that affects
biofilm development, but the role of LPS in modulating protein-mediated adhesion
that is important (GA O’Toole & R Kolter, unpublished data).
A large-scale screening for strains that are defective in biofilm formation on
poly(vinylchloride) resulted in the isolation of >30 sad mutants (142). Among the
mutants isolated were a number that were defective in flagella-mediated motility,
and sequence analysis of their mutations revealed that these mutations were lo-
cated in structural genes that are required for flagellar biosynthesis (142). DeFlaun
et al also isolated mutants with a decreased ability to adhere to quartz sand and
showed that these strains were defective in flagellum-mediated motility and lacked
a flagellum (47). The ClpP protease is also required for biofilm formation (142).
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58 O’TOOLE ET AL
These data suggest that there is a protease-sensitive regulator involved in biofilm

development, although the target of this protease is not known. This observa-
tion adds biofilm formation to the list of bacterial systems that are regulated by
proteases (38, 46, 98, 116, 134).
A large number of the isolated sad mutants appear to have defects in genes with
no matches to the P. aeruginosa genome in the Genbank database, which strongly
suggests that the molecular basis of biofilm formation in this organism may be
markedly different in even closely related pseudomonads. It is interesting that
some of the sequences of the genes disrupted in the sad mutants match the recently
released partial sequence of P. putida (GA O’Toole & R Kolter, unpublished data).
This idea of differences in biofilm development among various pseudomonads
is further reinforced by the fact that P. fluorescens is apparently not capable of

twitching motility, and no mutants that are defective in type-IV pili biogenesis
were isolated in the screen for sad mutants in this strain (142). This is in contrast
to P. aeruginosa, in which fully half of the sad mutants isolated had mutations in
genes required for type-IV pili biogenesis (GA O’Toole, unpublished data).
V. cholerae V. cholerae requires flagella-mediated motility, the mannose-sensi-

tive type-IV pilus encoded by the mshA locus, and synthesis of the major EPS, to
form a wild-type biofilm (180). Flagella and type-IV pili are not absolutely required
for initial attachment to the surface, but they greatly facilitate this process in
V. cholerae. Studies with phase-contrast microscopy suggested that, once bacteria
are on the surface, flagella-mediated motility might be necessary for cells to spread
out across this surface. It is interesting that, in the few areas where nonflagellated
mutants do manage to make an initial cell-surface contact, the biofilm they make
is essentially indistinguishable from the wild-type strain. Therefore, the flagella
promote initial attachment, but may not be required for downstream developmental
events (181). The mshA mutants are delayed by ∼1–2 days in formation of the
biofilm, but, once formed, the biofilm architecture of this strain is also identical to
the wild type, consistent with the idea that the Msh pili facilitate initial cell-surface
interactions, but not later events. The Msh pili are type-IV pili, which are known
to mediate twitching motility in other organisms. However, to date, no twitching
motility mediated by these or any other pili has been observed in this organism
(PI Watnick & R Kolter, unpublished data). It is possible that, in this organism,
the type-IV pili act solely as adhesins or, alternatively, that the proper conditions
to detect twitching motility have not yet been found.
E. coli Genevaux and colleagues screened a library of E. coli mutants for those
defective in biofilm formation, and they split the resulting mutants into two classes
based on the level of severity of their defect [<40% adherence and 40%–75%
adherence relative to the wild type (72)]. Phenotypic analysis revealed that 34
of 72 mutants that they isolated were defective in flagellum-mediated motility
(72). Pratt & Kolter (148) carried out a screen similar to the one above and de-
termined that type-I pili mutants are defective for biofilm formation. They also
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observed that E. coli requires flagellum-mediated motility to initiate early attach-

ment events (148). Using phase-contrast microscopy, they determined that type-I
pili were defective for initial cell-surface contact. There is evidence that type-I pili
retract and, thus, that they might also mediate a form of surface-dependent motil-
ity (136). Consistent with this possibility is our observation that E. coli K-12 is
capable of a slow twitching-like behavior (SE Finkel, unpublished data). Hultgren
and coworkers have presented evidence that type-I pili retraction might be impor-
tant for close associations with eukaryotic cells (136), but it remains unknown
whether type-I pili retraction is required for biofilm development. Flagellar mu-
tants, on the other hand, can make initial cell-surface contacts (albeit less well
than can the wild type), but once on the surface, they cannot spread out to cover
the surface. Pratt & Kolter extended the motility studies to show that, although
motility was required (e.g. the flagella must be present and not paralyzed), chemo-
taxis was not necessary for biofilm development in the microtiter dish system
(148).
An outer membrane protein, Ag43, facilitates both cell-surface and cell-cell
contacts when cells are grown on minimal medium, but apparently plays no role
when cells are grown on rich medium (PN Danese, LA Pratt, S Dove, R Kolter,
manuscript in preparation). Expression of Ag43 is under the control of both the
OxyR system, which regulates responses to oxidative stress, and Dam methylase,
which causes Ag43 expression to be phase variable (89). As mentioned above,
the expression of genes that are required for the production of Ag43, as well as
for biogenesis of type-I pili in E. coli ( fim), is phase variable. There may be a
selective advantage for E. coli to have a fraction of a bacterial population, but not
all members, “primed” for the initiation of biofilm development (148; PN Danese,
LA Pratt, S Dove, R Kolter, manuscript in preparation). As discussed below, phase
variation may also play a role in regulating biofilm formation in some gram-positive
organisms.
In subsequent studies, Genevaux and colleagues found that a subset of biofilm-
defective mutants was shown to be defective in the synthesis of LPS, with mutations
in the rfaG, rfaP, and galU genes (71). These were pleiotropic mutants with de-
fects in the cell membrane, motility, and type-I pili biogenesis. These workers
also found that a dsbA mutant was defective in biofilm formation, which was
probably caused by the poor growth of this strain as well as defects in motility,
biogenesis of type-I pili, and LPS structure in this strain (70). Two lines of evi-
dence from Lejeune and coworkers suggest that signals from the outer membrane
or periplasm play some role in regulating biofilm development. They showed a
role for curli (a proteinaceous cell surface structure) in the early stages of biofilm
formation by E. coli. They also isolated an allele of ompR (a regulator of mem-
brane protein synthesis) that resulted in increased expression of csgA, the gene
encoding the curlin subunit, and improved biofilm formation (176). Furthermore,
mutations in the cpxAR signal transduction pathway, which senses signals in the
bacterial envelope, decreased csgA expression and reduced the amount of biofilm
formed (55).
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60 O’TOOLE ET AL
Maturation of the Biofilm

After attachment to a surface, bacteria undergo further adaptation to life in a
biofilm. Two properties are often associated with surface-attached bacteria—
increased synthesis of EPS and the development of antibiotic resistance. These
features appear to create a protective environment and cause biofilms to be a
tenacious clinical problem. Biofilm bacteria may also develop other properties,
including increased resistance to UV light, increased rates of genetic exchange,
altered biodegradative capabilities, and increased secondary metabolite production
(7, 12, 24, 76, 81, 99, 123, 133, 156, 185, 194, 195). Until relatively recently, how-
ever, little was known about the molecular mechanisms underlying these changes.
P. aeruginosa In P. aeruginosa, the study of EPS has focused on alginate, a

polymer of uronic acid and guluronate, because of its presumed importance in the
CF lung (65, 77, 78). Isolates of P. aeruginosa from the CF lung (P. aeruginosa is
the predominant pathogen in the lungs of individuals with this disease) make large
amounts of this polysaccharide. Most natural isolates of P. aeruginosa, however,
are nonmucoid and make much less of this polymer. Therefore, this conversion
to a mucoid state in the CF lung has long been thought to be an essential part of
the pathogenesis of P. aeruginosa in this environment. The copious quantity of
alginate produced is thought to be an important factor in the resistance of P. aerug-
inosa to tobramycin, the primary therapy used to treat CF patients (94). Nickel and
colleagues showed that physically disrupting the structure of the biofilm was suf-
ficient to restore tobramycin susceptibility to the levels seen in planktonic bacteria
(137). They concluded that alginate prevented diffusion of the tobramycin into
the biofilm. However, the studies listed below suggest that it is the physiological
adaptation of bacteria in the biofilm that renders them resistant to antibiotics. The
phenotypic changes that might affect antibiotic resistance include slower growth,
expression of new multi-drug-resistant pumps, expression and/or increased local
concentrations of antibiotic-modifying or -degrading enzymes, or alterations in
antibiotic targets (8–10, 94, 137, 175, 188).
The regulation of alginate is a complex process involving a number of regulators
and responding to a variety of environmental cues (reviewed in 21, 65, 77, 125).
Two studies have shown the interesting result that the contact of bacteria with an
abiotic surface can lead to up-regulation of the alginate biosynthesis genes (43, 44).
This increased expression may be mediated through the regulator AlgT (68). One
crucial experiment that remains to be done is to assess the structure and antibiotic
resistance of a strain of P. aeruginosa that is blocked for alginate synthesis.
One of the most exciting recent results in biofilm biology has been the demon-
stration that the quorum-sensing molecules known as acylhomoserine lactones
(acyl-HSLs) are required for biofilm maturation (45). The lasI gene codes for an
enzyme that directs the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone,
one of two well-described acyl-HSLs that are produced by P. aeruginosa. Davies
and colleagues showed that a lasI mutant, although still capable of early cell-
surface interactions, does not develop the hallmark architecture of a P. aeruginosa
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biofilm. In fact, the phenotype of a lasI mutant after 24 h of growth in a flow cell
looks very much like a type-IV pili mutant (Figure 3, see color insert). These
authors measured this characteristic architecture by looking at the proximity of
cells to each other (lasI biofilms were much more crowded) and thickness of the
biofilm (lasI mutants were much thinner). These data suggest that formation of
biofilm architecture is not a stochastic process, but is controlled as part of a com-
plex regulatory system. Furthermore, the fact that lasI mutants were more crowded
suggests that quorum sensing may not act only to count the bacteria present, but
also to keep them from becoming too crowded. That is, acyl-HSLs may play
a role in a prokaryotic version of contact inhibition. Two other analyses of the

biofilm of a lasI mutant, when taken together, are also quite revealing. First, a
strain that is unable to produce acyl-HSL became extremely sensitive to a biocide

(sodium dodecyl sulfate) in comparison with the response of the wild-type strain,
suggesting that some aspect of the acyl-HSL–mediated development is important
for the troublesome biocide resistance developed by biofilm-grown bacteria. Fur-
thermore, this loss in biocide resistance by the lasI mutant occurred despite the
lack of any change in total amount of EPS in the wild type and lasI mutant. These
data strongly support the idea that, although EPSs may be necessary for antibiotic
resistance, they are clearly not sufficient for this characteristic biocide resistance
phenotype of biofilm-grown cells.
V. cholerae A strain that is defective in EPS synthesis in V. cholerae is defective

in the early stages of biofilm development (180, 191). EPS mutants are reduced in
initial attachment and, furthermore, do not develop the architecture that has been
observed for the wild-type strain (181), in contrast to an EPS mutant of P. aerug-
inosa (algD), which has no apparent defect in initial attachment (GA O’Toole,
unpublished data). These observations suggest that, although some structures or
extracellular factors may be in common between these two organisms, their pre-
cise roles in biofilm formation may differ. At this point, functions required for the
development of a mature V. cholerae biofilm are not clear.
E. coli As the best studied bacterium, E. coli should serve as an excellent model
for biofilm formation, but this organism has historically received little attention
from biofilm researchers, although this has changed with some exciting recent
studies. The three-dimensional architecture of an E. coli K-12 biofilm has been
analyzed and shown to have many of the characteristics of the classical P. aerug-
inosa biofilm, including microcolonies, water channels, marked heterogeneity of
structure, and significant thickness. Furthermore, colanic acid, the major EPS of
E. coli, is necessary for the formation of this characteristic architecture, but does not
play a role in the initial colonization of the abiotic surface (PN Danese, LA Pratt,
R Kolter, manuscript in preparation; Figure 4, see color insert). Prigent-Combaret
and colleagues have used a different approach to show that biofilm-grown E. coli
cells are distinct from their planktonic counterparts. In a screen for loci whose
expression is altered upon attachment, they observed that ∼40% of genes are al-
tered by at least twofold. Up-regulated genes include the OmpC porin and the
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62 O’TOOLE ET AL
wca locus (required for colanic acid synthesis), whereas the fliC gene (required
for flagellar biogenesis) is down-regulated (149; PN Danese, LA Pratt, R Kolter,
manuscript in preparation). Recalling that the down-regulation of flagellar synthe-
sis upon attachment has also been observed in P. aeruginosa (68), together, these
studies lay the essential groundwork for future advances in the study of E. coli
biofilm development.
Detachment and Return to the Planktonic Growth Mode

The portion of the biofilm developmental pathway that concerns detachment rep-
resents an important area of future research. One possible signal for detaching
may be starvation, although this has not been investigated in detail (GA O’Toole,
unpublished data). Boyd & Chakrabarty reported that the enzyme alginate lyase
may play a role in the detachment phase in P. aeruginosa (20). They showed that
overexpression of alginate lyase could speed detachment and cell sloughing from
biofilms (20). A recent study by Allison and colleagues showed that a P. fluo-
rescens biofilm decreased after extended incubation, which they attribute, at least
in part, to the loss of EPS (2). Furthermore, they presented evidence showing that
acyl-HSLs and/or another factor present in stationary-phase culture supernatants
mediated this effect (2). Little else is known about the functions or regulatory
pathways involved in release of bacteria from the biofilm.
Similarity of M. xanthus Fruiting Body Development

and P. aeruginosa Biofilm Formation
While analyzing early biofilm development with time-lapse phase-contrast mi-
croscopy, we noticed that microcolonies of P. aeruginosa formed and dispersed on
the surface (141). As shown in Figure 5, this microcolony formation was reminis-
cent of the early stages of fruiting-body formation in M. xanthus (82), and a closer
analysis of these two processes reveals additional similarities (Figure 6, see color
insert). For example, both organisms initiate their developmental pathways in re-
sponse to nutritional signals and in the presence of a solid surface (35, 102, 162).
The initial stage in biofilm development is the formation of a monolayer of cells
(141), and similarly, M. xanthus forms a cell mat growing on a surface (as re-
viewed in 56). After monolayer/mat formation, both organisms form clusters of
cells that are referred to as microcolonies for P. aeruginosa (141) and aggregates
for M. xanthus (Figure 5; reviewed in 82). Both of these behaviors require type-IV
pili (141, 189) and are very similar in appearance.
In P. aeruginosa, type-IV pili are required for a mode of surface translocation
known as twitching motility (22), whereas this behavior in M. xanthus is histori-
cally referred to as gliding social motility (reviewed in 82). A recent publication
from Mattick’s laboratory convincingly argues that twitching and gliding social
motility are in fact the same behavior (159). As for twitching motility in P. aerug-
inosa (3, 4, 41, 42), social gliding motility in M. xanthus requires cell-cell contact
(93), type-IV pili (177, 189, 190), and a set of chemotaxis-related genes (encoded
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Figure 5 The morphological similarity in the structure of a P. aeruginosa biofilm and a

Myxococcus fruiting body is evident in these top-down photographs. Both organisms form dis-
tinct aggregates of cells that are well separated from their neighbors. Left: 8-h-old biofilm of
P. aeruginosa grown on PVC plastic at 400× magnification. Right: Fruiting bodies of Myxococ-
cus xanthus after 6 h on starvation agar plates at 5× magnification. Microcolonies and fruiting
bodies are indicated by arrows.
by the frz locus) (126, 130). Thus, directed cell movement is required for both
biofilm and fruiting-body formation. After microcolony formation, P. aeruginosa
develops the complex architecture that is characteristic of this organism, in a
process shown to require extracellular signaling (45). Similarly, the M. xanthus
cells form a mushroomlike structure, reminiscent of those seen in P. aeruginosa
biofilms, that will eventually give rise to endospores. The formation of fruit-
ing bodies also requires extracellular signaling molecules known as A-signal [a
mixture of amino acids (113, 114)] and C-signal [a protein (100, 101)]. It is im-
portant that, despite the similarities in these processes, they occur in response to
very different environmental signals—biofilm formation in P. aeruginosa occurs
in a nutrient-rich environment, whereas spore formation is triggered by starvation
(35, 102, 141, 147, 163).
GRAM-POSITIVE BACTERIA
AND MULTISPECIES BIOFILMS
Clinical Relevance
A number of gram-positive infections, including those caused by Staphylococcus
epidermidis, S. aureus, and the enterococci, have proven to be particularly difficult
to treat with current antibiotic therapies, partly owing to their high-level natural
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64 O’TOOLE ET AL
resistance to antimicrobial compounds. Furthermore, these organisms become re-

sistant to the highest deliverable levels of antibiotics when growing in a biofilm
(37, 97, 152, 175). It is estimated that ≤60% of nosocomial infections are derived
from biofilm-related infections, many of which are caused by coagulase-negative
staphylococci (11, 64, 75, 95).
Early Attachment Events

S. epidermidis Mutants of S. epidermidis in biofilm formation on abiotic sur-
faces have been isolated with a microtiter dish-based assay (85, 86, 121, 135). The
ability to form a biofilm in microtiter dishes has been strongly correlated with
the ability of particular strains to cause disease in a clinical setting (49). Ge-
netic studies have led to the conclusion that biofilm development by this organism
occurs initially via cell-surface interactions (85, 86). These interactions may be
mediated through a number of factors, including uncharacterized surface proteins
(96), extracellular proteins (158), capsular polysaccharide/adhesin (PS/A) (132),
and the cell surface–localized autolysin encoded by the atlE gene (87). PS/A is a
high-molecular-mass (>250,000-kDa) polysaccharide composed of β-1,6-linked
glucosamine and substituted with succinate and acetate (132). AtlE is synthesized
as a proteolytically cleaved precursor of an ∼120-kDa protein and was reported
to have vitronectin-binding activity, suggesting that this protein is an adhesin for
binding to both biotic and abiotic surfaces (87). However, the detailed charac-
terization of most of the other factors and their role in biofilm formation has not
yet been reported. Furthermore, little is known about whether the formation of
biofilms by S. epidermidis is regulated and, if so, what signals induce biofilm
formation.
Subsequent to cell-surface interactions, these organisms enter the so-called
“accumulative phase” of biofilm formation. This involves cell-cell interactions and
the formation of cell aggregates on the surface. Numerous studies have implicated a
polysaccharide intercellular adhesin (ICA) in this process (92, 120, 121, 132, 135).
The ica genes code for the synthesis of this adhesin (120, 121, 132, 135), and recent
studies suggest that the ica locus may also code for the PS/A described above (132).
It is interesting that the expression of the ica locus has been shown to be phase
variable (192, 193). The molecular basis for this variation appears to be the insertion
and excision of an IS256 insertion element, although it is not clear whether these
events are in any way regulated in response to changes in cell physiology (193).
S. aureus Recent studies by Cramton and colleagues have shown that S. aureus,
like S. epidermidis, has the ica locus, which encodes the functions required for
intracellular adhesin. These data suggest that the early stages in biofilm formation
may be similar between these two organisms (36).
Enterococci Enterococi are important pathogens in device-related infections

and, like other gram-positive organisms, the formation of enterococcal biofilms
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on medical implants is an increasing clinical issue (15, 160, 169, 174). As for
other organisms, growth in a biofilm confers Enterococcus spp. with an increased
resistance to antibiotics (63). Essentially nothing is known about the underlying
molecular genetics controlling biofilm formation and maintenance in enterococci.
Dental Microbes All of the descriptions above focus on the development of a

biofilm in a pure culture system. Studying biofilms in the context of pure cul-
tures has proved fruitful because it allows the use of classical genetic approaches.
However, in the natural setting, bacteria are seldom found in biofilms composed
of a single species. Rather, the rule is to find many species coexisting within
biofilms. Studies of biofilms in the oral cavity, pioneered by Kolenbrander and
colleagues, have revealed a fascinating universe of specific interspecies interac-

tions (106, 107, 110).
Biofilms that form on teeth are more commonly called plaque and can comprise
hundreds of bacterial species. Dental biofilms are subjected to a number of harsh
environmental conditions such as nutrient availability (feast or famine), aerobic-
to-anaerobic transitions, and pH changes, as well as exposure to detergents every
time we brush our teeth, all of which may contribute to the regulation of biofilm
development (31). When a clean surface is present in the mouth (e.g. after periodic
cleaning), a well-defined series of events takes place. First, a coating of glycopro-
teins, mucins, and other proteins (known as the “acquired pellicle”) begins to cover
the surface almost immediately (73, 74). This conditioned surface is now prepared
for the adhesin of microorganisms known as the “primary colonizers,” the most
common of which are the streptococci, which are soon followed by the actino-
mycetes (19, 138, 166, 171). The streptococci bind to various components in the
pellicle, including proline-rich proteins and enzymes such as α-amylase (73, 157).
Bacterial surface structures that are common to biofilm formation in other systems,
such as pili and outer membrane proteins, play a role in the interactions with the
pellicle (5, 32, 66, 67, 106).
In addition to bacteria-pellicle interactions, these organisms are capable of
cell-cell interactions or coaggregation, either with themselves or with other gen-
era (103, 105, 106, 109). The coaggregation between streptococci can be blocked
by various sugars, including lactose and galactose, suggesting that lectinlike re-
ceptors are involved (104, 108, 109). A number of lipoproteins, including FimA
[a pili-associated adhesin (143)], ScaA [part of an ABC transporter that partic-
ipates specifically in interactions of streptococci with actinomycetes (5, 106)],
ScbA (34), PsaA (155), and SsaB [which has similarity to fimbrial adhesins
(66, 67)], are required for coaggregation, and it is interesting that these pro-
teins share extensive amino acid sequence similarity. Type-I pili, in addition
to their role in adherence to the pellicle, are also required for cell-cell interac-
tions in some organisms (182). For the most part, once coaggregation interactions
have occurred, it is not possible to competitively displace organisms already in
an aggregate by adding more of those organisms, suggesting tight interactions
and/or multiple interactions (106). However, some binding interactions can be
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66 O’TOOLE ET AL
interrupted by the addition of specific sugars, suggesting at least some simple

cell-cell interactions.
Once the initial colonizers have attached to the surface, cell-cell communica-
tion comes into play (18, 19, 118). In streptococci for example, the growth of the
bacteria is relatively slow up to a surface density of ∼2 × 106 cells/mm2. However,
above this threshold density, the growth rate of the bacteria apparently increases,
as measured by 3H-thymine uptake. This increased growth rate can be stimulated
in less dense communities by the addition of supernatants of stationary-phase
cultures, suggesting the requirement for an extracellular factor. Preliminary bio-
chemical analysis has shown that this compound, called START, has a molecular
weight of <3000 Mr (118). It is possible that this low-molecular-weight com-
pound acts as a quorum-sensing signaling molecule to accelerate the growth of

surface-attached communities.
Maturation
Gram-Positive Pathogens As was the case for gram-negative organisms, gram-
positive microbes also produce extracellular polysaccharides (often referred to as
“slime”) when they are growing on a surface. Little is known about how slime
affects the normal development of the biofilm and what role it plays in determining
the architecture of these biofilms. Deighton & Borland showed that S. epidermidis
increased slime production in iron-limited medium and late in the growth phase
when nutrients were presumably being exhausted. They suggested that these may
be an important signal in vivo, when iron and nutrient levels may be limiting
(48). Furthermore, Muller et al showed that production of the capsular PS/A was
closely associated with slime production, suggesting a coregulation of early steps
in attachment with downstream events (135).
S. aureus also makes an EPS or slime and does so in a phase-variable manner.
The increased EPS is correlated with the ability to form a biofilm in an in vitro
system, but the signal(s) regulating this process has not been identified (16). There
is evidence that σ B may play a role in cell-cell interactions in the biofilm, because
strains carrying a mutation in the gene that encodes this transcription factor tend
to aggregate much more than the wild type (112).
Dental Microbes Subsequent to initial cell-surface interactions, gram-negative

organisms undergo a series of changes that are precipitated by changes in gene
expression. Similar observations have been made in dental microbes. In experi-
ments performed in S. mutans, the expression of gtfBC (which encodes a glucosyl-
transferase required for the synthesis of glucan polymers) was 10- to 70-fold higher
in the biofilm than in planktonic culture, whereas ftf (also involved in polymer
synthesis) expression fell ∼100-fold (28). Furthermore, glycoprotein metabolism,
which is important in the mouth, is improved by a consortium of bacteria that
cometabolize these substrates (23, 173). In addition to changes in the physiology
of individual organisms, the nature of the community also begins to change. For
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example, gram-negative bacteria of the genus Fusobacterium act as a bridge be-

tween primary colonizers and later colonizers (107, 108). That is, primary colo-
nizing streptococci cannot aggregate with late colonizers directly, but can do so
via their ability to coaggregate with Fusobacterium spp. (106). Among the late
colonizing organisms are S. salivarius (166), propionibacteria, prevotellae, veil-
lonellae (110, 138), and Selenomonas flueggei (110). In general, these organisms
tend to coaggregate less than do the early colonizing organisms.
Leaving the Biofilm

Gram-Positive Pathogens Essentially nothing is known about whether staphy-

lococci or enterococci can detach from a biofilm and, if so, how this process is
mediated. As described above, expression of the ica locus may be phase variable,
and Ziebuhr and colleagues propose that the “switch-off” of ica by the IS256 in-
sertion element may be the mechanism by which individual S. epidermidis (and
possibly S. aureus) cells can leave the biofilm to colonize new surfaces (193).
Dental Microorganisms Little is known about how microorganisms detach from

a biofilm during development, although there are hints that it is an active process
in oral microbes. For example, S. mutans has a surface protease that cleaves its
own surface proteins (117), which may serve as a mechanism to release from the
biofilm.
FUNGAL BIOFILMS
Fungal infections, especially in immunocompromised individuals and those on

heavy courses of antibacterial compounds (i.e. subject to super infection), are a
growing concern. It has been recognized that, like bacteria, fungi such as Candida
albicans are also capable of biofilm formation on medical implants (26, 29, 30, 57).
Recent work has shed some insight into the molecular mechanisms that control
the development of fungal biofilms.
Initial Attachment
Hawser & Douglas reported the use of disks made of catheter material as a simple
assay for biofilm development in vitro (83). Using this system, they showed that
C. albicans could form biofilms on a range of abiotic surfaces and that biofilm
formation occurred best on latex, poly(vinylchloride), or silicone elastomer, but
less well on polyurethane or pure latex (83). Baillie & Douglas went on to use this
model system in an elegant series of experiments to show that the switch between
yeast form and hyphal growth plays an important role in biofilm development (14).
Using scanning electron microscopy as a tool, they observed that the biofilm layer
closest to the surface comprised primarily yeast form cells. Consistent with this
observation, strains unable to make hyphae formed only a thin biofilm of yeast
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68 O’TOOLE ET AL
cells on the catheter material. The upper portion of the biofilm (and presumably
the downstream event in this developmental process) is comprised of a layer of
hyphae. Strains that are unable to make the yeast form could still attach to the
surface and generate a structure that is similar to the upper layer of the wild-type
biofilm, except that this community was more easily removed from the surface.
This observation suggests that the yeast form cells are not absolutely required for
attachment, but can act as an anchor to keep the fungi firmly attached to the surface
(14). Baillie & Douglas also showed that the surface to which the yeast attached
could influence the structure of the biofilm and, furthermore, that the structure of
fungal biofilms is reminiscent of their bacterial counterparts (14).

Maturation As mentioned above, the architecture of the biofilms formed by C.

albicans looks similar to that of bacterial biofilms. Of greater interest, however, is
the question of whether these fungi develop new physiological properties that are
different from their planktonic counterparts. Two recent papers by Douglas and
colleagues have used classical assays to address this question. Using their stan-
dard assay for biofilm formation in conjunction with electron microscopy, they
showed that C. albicans biofilms became resistant to five antifungal compounds
that are used routinely in clinical settings (84). This drug resistance phenotype
is often associated with bacterial biofilms. Furthermore, this increase in antibi-
otic resistance is not due simply to the decrease in growth rate observed in fungi
growing on the surface (13). Clearly, these workers have gone a long way to-
wards establishing that a mode of life and developmental system long associated
with bacteria may be a common strategy used by eukaryotic microorganisms as
well.
SUMMARY AND FUTURE DIRECTIONS
A combination of genetic and molecular techniques, in conjunction with direct mi-

croscopic visualization, has been used to initiate investigations into the molecular
mechanisms that control biofilm development. Based on these and earlier studies,
biofilm formation can be viewed as a well-regulated developmental process that
results in the formation of a complex community of organisms. To form these
communities, microorganisms must integrate external and internal signals, take
stock of their neighbors by determining their density and type, and coordinate a
timed series of multicellular behaviors that are likely to be associated with mor-
phological changes. These behaviors and the gene expression that drives them will
continue to be an active area of investigation. Although some general concepts can
be applied to the formation of all biofilms, many species-specific behaviors exist
that reflect the unique needs of each microorganism. As we gain a deeper under-
standing of pure culture systems, it is important to keep in mind that most biofilms
are multispecies consortia. Multispecies biofilms almost assuredly demand the
ability to communicate between species and suggest the possibility of particular
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organisms performing specialized roles in the community. Understanding the

molecular interactions between two or more species within biofilms will also add
to our general understanding of the diverse strategies for survival in the microbial
world.
Visit the Annual Reviews home page at www.AnnualReviews.org
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14:57
Annual Reviews
AR110-CO
Figure 1 Model of biofilm development. Individual planktonic cells can form cell-to-surface and cel-to-cell contacts resulting in the formation of
microcolonies. The hallmark architecture of the biofilms form in an acylhomoserine lactone-dependent process. Cells in the biofilm can return to a
planktonic lifestyle to complete the cycle of biofilm development.
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September 11, 2000 14:57 Annual Reviews AR110-CO
Figure 2 Phase contrast microscopy of the early events in biofilm formation by P. aerug-
inosa. Within 30 minutes or less, a monolayer of bacteria forms on the abiotic surface.
By 3–4 hours, this monolayer almost completely covers the surface and is punctuated by
microcolonies. These microcolonies become more numerous and more easily visualized in
the 8-hour old biofilm.
Figure 3 Confocal microscopy of twenty four-hour biofilms formed in continuous flow

chambers by P. aeruginosa. (A) wild-type and (B) type IV pili-defective mutant. This image
shows a top-down view of the biofilm formed by the wild-type strain. The characteristic
mound-shaped architecture of the P. aeruginosa biofilm can be seen. In contrast, the type
IV pili mutant forms a biofilm lacking this normal architecture. Images courtesy of Matt
Parsek, Northwestern University.
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September 11, 2000 14:57 Annual Reviews AR110-CO
Figure 4 Deconvoluted sagittal view of biofilms formed by (A) wild-type (illus-

trating the normal architecture of the biofilm formed by these organisms) and (B)
colanic acid defective strains of E. coli K-12 (which completely lack this architec-
ture). For movies that better illustrate these differences in biofilm architecture go to:
http//gasp.med.harvard.edu/biofilms/ecoli/colanic.html
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September 11, 2000
14:57
Annual Reviews
AR110-CO
Figure 6 Parallels between P. aeruginosa biofilm formation and fruiting body formation in M. xanthus. See text for an in-depth discussion of the
similarities between these developmental pathways. This figure represents a modification of a previous figure (35a).
Annual Review of Microbiology
Volume 54, 2000
CONTENTS
THE LIFE AND TIMES OF A CLINICAL MICROBIOLOGIST, Albert
Balows 1
ROLE OF CYTOTOXIC T LYMPHOCYTES IN EPSTEIN-BARR
VIRUS-ASSOCIATED DISEASES, Rajiv Khanna, Scott R. Burrows 19
BIOFILM FORMATION AS MICROBIAL DEVELOPMENT, George
O'Toole, Heidi B. Kaplan, Roberto Kolter 49
MICROBIOLOGICAL SAFETY OF DRINKING WATER, U. Szewzyk,
R. Szewzyk, W. Manz, K.-H. Schleifer 81
THE ADAPTATIVE MECHANISMS OF TRYPANOSOMA BRUCEI
FOR STEROL HOMEOSTASIS IN ITS DIFFERENT LIFE-CYCLE
ENVIRONMENTS, I. Coppens, P. J. Courtoy 129
THE DEVELOPMENT OF GENETIC TOOLS FOR DISSECTING THE

BIOLOGY OF MALARIA PARASITES, Tania F. de Koning-Ward,

Chris J. Janse, Andrew P. Waters 157
NUCLEIC ACID TRANSPORT IN PLANT-MICROBE
INTERACTIONS: The Molecules That Walk Through the Walls, Tzvi
Tzfira, Yoon Rhee, Min-Huei Chen, Talya Kunik, Vitaly Citovsky
187
PHYTOPLASMA: Phytopathogenic Mollicutes, Ing-Ming Lee, Robert E.
Davis, Dawn E. Gundersen-Rindal 221
ROOT NODULATION AND INFECTION FACTORS PRODUCED BY
RHIZOBIAL BACTERIA, Herman P. Spaink 257
ALGINATE LYASE: Review of Major Sources and Enzyme
Characteristics, Structure-Function Analysis, Biological Roles, and
Applications, Thiang Yian Wong, Lori A. Preston, Neal L. Schiller 289
INTERIM REPORT ON GENOMICS OF ESCHERICHIA COLI, M.
Riley, M. H. Serres 341
ORAL MICROBIAL COMMUNITIES: Biofilms, Interactions, and
Genetic Systems, Paul E. Kolenbrander 413
ROLES OF THE GLUTATHIONE- AND THIOREDOXIN-
DEPENDENT REDUCTION SYSTEMS IN THE ESCHERICHIA COLI
AND SACCHAROMYCES CEREVISIAE RESPONSES TO OXIDATIVE
STRESS, Orna Carmel-Harel, Gisela Storz 439
RECENT DEVELOPMENTS IN MOLECULAR GENETICS OF
CANDIDA ALBICANS, Marianne D. De Backer, Paul T. Magee, Jesus
Pla 463
FUNCTIONAL MODULATION OF ESCHERICHIA COLI RNA
POLYMERASE, Akira Ishihama 499
BACTERIAL VIRULENCE GENE REGULATION: An Evolutionary
Perspective, Peggy A. Cotter, Victor J. DiRita 519
LEGIONELLA PNEUMOPHILA PATHOGENESIS: A Fateful Journey
from Amoebae to Macrophages, M. S. Swanson, B. K. Hammer
567
THE DISEASE SPECTRUM OF HELICOBACTER PYLORI : The
Immunopathogenesis of Gastroduodenal Ulcer and Gastric Cancer, Peter
B. Ernst, Benjamin D. Gold 615
PATHOGENICITY ISLANDS AND THE EVOLUTION OF
MICROBES, Jörg Hacker, James B. Kaper 641
DNA SEGREGATION IN BACTERIA, Gideon Scott Gordon, Andrew
Wright 681
POLYPHOSPHATE AND PHOSPHATE PUMP, I. Kulaev, T.
Kulakovskaya 709
ASSEMBLY AND FUNCTION OF TYPE III SECRETORY SYSTEMS,
Guy R. Cornelis, Frédérique Van Gijsegem 735
PROTEINS SHARED BY THE TRANSCRIPTION AND
TRANSLATION MACHINES, Catherine L. Squires, Dmitry Zaporojets
775
HOLINS: The Protein Clocks of Bacteriophage Infections, Ing-Nang
Wang, David L. Smith, Ry Young 799
OXYGEN RESPIRATION BY DESULFOVIBRIO SPECIES, Heribert
Cypionka 827
REGULATION OF CARBON CATABOLISM IN BACILLUS
SPECIES, J. Stülke, W. Hillen 849
IRON METABOLISM IN PATHOGENIC BACTERIA, Colin Ratledge,
Lynn G Dover 881

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August 11, 2000 11:2 Annual Reviews AR110-03

Annu. Rev. Microbiol. 2000. 54:49–79

BIOFILM FORMATION AS MICROBIAL

03755; e-mail: georgeo@Dartmouth.edu

2Department of Microbiology and Molecular Genetics, University of Texas Medical

School, Houston, Texas 77030; e-mail: hkaplan@utmmg.med.uth.tmc.edu

Massachusetts 02115; e-mail: kolter@mbcrr.harvard.edu

Key Words communities, life cycle, fungi, bacteria, environment, surface

Early Attachment Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Microorganisms are often viewed as simple creatures when compared with

“higher” organisms. The study of microbial development, however, has shown

bacteria, Pseudomonas fluorescens, Escherichia coli, and Vibrio cholerae have

xanthus and E. coli O517:H7, undergo a transition from free-living, planktonic

A New Approach to Studying Biofilms

in the developmental process. Therefore, as described in detail below, a simple

Does Biofilm Formation Constitute a Form

Regulation of Initial Attachment

Biofilm Formation Proceeds via Multiple Convergent

Early Attachment Events

may change depending on the environment to which the bacterium is exposed.

P. aeruginosa By using phase-contrast microscopy to analyze a collection of mu-

altered during various stages of attachment (27).

P. fluorescens A number of factors have been shown to affect the ability of

These data suggest that there is a protease-sensitive regulator involved in biofilm

is further reinforced by the fact that P. fluorescens is apparently not capable of

V. cholerae V. cholerae requires flagella-mediated motility, the mannose-sensi-

observed that E. coli requires flagellum-mediated motility to initiate early attach-

Maturation of the Biofilm

P. aeruginosa In P. aeruginosa, the study of EPS has focused on alginate, a

a role in a prokaryotic version of contact inhibition. Two other analyses of the

strain that is unable to produce acyl-HSL became extremely sensitive to a biocide

V. cholerae A strain that is defective in EPS synthesis in V. cholerae is defective

Detachment and Return to the Planktonic Growth Mode

Similarity of M. xanthus Fruiting Body Development

Figure 5 The morphological similarity in the structure of a P. aeruginosa biofilm and a

resistance to antimicrobial compounds. Furthermore, these organisms become re-

Early Attachment Events

Enterococci Enterococi are important pathogens in device-related infections

Dental Microbes All of the descriptions above focus on the development of a

colleagues, have revealed a fascinating universe of specific interspecies interac-

interrupted by the addition of specific sugars, suggesting at least some simple

pound acts as a quorum-sensing signaling molecule to accelerate the growth of

Dental Microbes Subsequent to initial cell-surface interactions, gram-negative

example, gram-negative bacteria of the genus Fusobacterium act as a bridge be-

Leaving the Biofilm

Gram-Positive Pathogens Essentially nothing is known about whether staphy-

Dental Microorganisms Little is known about how microorganisms detach from

Fungal infections, especially in immunocompromised individuals and those on

fungal biofilms is reminiscent of their bacterial counterparts (14).

Maturation As mentioned above, the architecture of the biofilms formed by C.

SUMMARY AND FUTURE DIRECTIONS

A combination of genetic and molecular techniques, in conjunction with direct mi-

organisms performing specialized roles in the community. Understanding the

Visit the Annual Reviews home page at www.AnnualReviews.org

tion 12(3):208–13 10. Anwar H, Strap JL, Costerton WJ. 1992.

antibiotic-induced and polymorphonu- A method for the study of de novo protein

ulation. J. Bacteriol. 178:1172–77 30. Busscher HJ, van Hoogmoed CG,

Staphylococcus aureus and is required and physiological differentiation in Strep-

starvation proteins in Escherichia coli. J. dermidis by iron limitation. Infect. Immun.

39. Deleted in proof 49. Deighton MA, Balkau B. 1990. Adherence

of Pseudomonas aeruginosa to unworn scopic examination of natural sessile bac-

12:1067–71 Can. J. Microbiol. 23(12):1733–36

under conditions of nutrient depletion in formation. Infect. Immun. 64(1):277–