FEMS Microbiology Letters 236 (2004) 163–173

www.fems-microbiology.org

MiniReview

What drives bacteria to produce a biofilm?

Kimberly K. Jefferson *

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The Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA
Received 26 March 2004; received in revised form 1 June 2004; accepted 3 June 2004

First published online 15 June 2004

Abstract

Nearly 40 years ago, Dr. R.J. Gibbons made the first reports of the clinical relevance of what we now know as bacterial biofilms
when he published his observations of the role of polysaccharide glycocalyx formation on teeth by Streptococcus mutans [Sci. Am.
238 (1978) 86]. As the clinical relevance of bacterial biofilm formation became increasingly apparent, interest in the phenomenon
exploded. Studies are rapidly shedding light on the biomolecular pathways leading to this sessile mode of growth but many fun-
damental questions remain. The intent of this review is to consider the reasons why bacteria switch from a free-floating to a biofilm
mode of growth. The currently available wealth of data pertaining to the molecular genetics of biofilm formation in commonly
studied, clinically relevant, single-species biofilms will be discussed in an effort to decipher the motivation behind the transition from
planktonic to sessile growth in the human body. Four potential incentives behind the formation of biofilms by bacteria during
infection are considered: (1) protection from harmful conditions in the host (defense), (2) sequestration to a nutrient-rich area
(colonization), (3) utilization of cooperative benefits (community), (4) biofilms normally grow as biofilms and planktonic cultures
are an in vitro artifact (biofilms as the default mode of growth).
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Biofilm; Genetics; Microbial communities

1. Introduction ticularly important and clinically relevant example of

Genetic adaptation is the cornerstone of fitness and
survival and can ensue from mutations and recombi-
nation within genes, acquisition of new genetic material,
or from the regulated expression of existing genetic
material. Flexibility in bacterial gene expression permits
survival in environments with rapidly changing condi-
tions, and bacteria, being particularly adaptable, have
flourished in nearly every environmental niche on our
planet. Bacterial species that are able to colonize hu-
mans are especially creative in their regulatory pro-
cesses. Many pathogenic and commensal bacteria are
capable of transitioning between life in the environment
and in the human host, and all must be able to adapt to
sudden shifts in nutrient availability as well as to pri-
mary and secondary host immune defenses. One par-
*
Tel.: +1-617-525-2680; fax: +1-617-731-1541.
E-mail address: kjefferson@rics.bwh.harvard.edu.
bacterial adaptation through systematized gene expres-
sion is the ability to grow as part of a sessile, exopoly-
mer-enshrouded community referred to as a biofilm.
Scientific interest in the process of bacterial biofilm
formation has erupted in recent years and studies of the
molecular genetics of biofilm formation have begun to
shed light on the driving forces behind the transition to
the biofilm mode of existence.
It is now recognized that biofilm formation is an
important aspect of many, if not most bacterial diseases,
including native valve endocarditis, osteomyelitis, dental
caries, middle ear infections, medical device-related in-
fections, ocular implant infections, and chronic lung
infections in cystic fibrosis patients [2]. Established
biofilms can tolerate antimicrobial agents at concentra-
tions of 10–1000-times that needed to kill genetically
equivalent planktonic bacteria, and are also extraordi-
narily resistant to phagocytosis, making biofilms
extremely difficult to eradicate from living hosts [3].

0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.06.005
164 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173

Consequently, biofilm-related infections that appear to
respond to a therapeutic course of antibiotics may re-
lapse weeks or even months later, making surgical re-
moval and replacement of the infected tissue or medical
device a frequent and unfortunate necessity.
The serious and pervasive clinical impact of bacterial
biofilms has inspired many researchers to investigate the
regulatory mechanisms behind their formation and dis-
solution, with the ultimate goal of pinpointing specific
targets for chemotherapeutic agents. A number of target
quired for biofilm formation. The formation of a biofilm
in turn alters the microenvironment of its own inhabit-
ants which then leads to additional alterations in gene
expression and further maturation of the biofilm, and so
on. Complicating the study of gene expression in bio-
films even further, biofilm inhabitants are heteroge-
neous. Disease-related biofilms can be multi-species or
even multi-kingdom, such as the biofilms involved in
tooth decay, or single-species such as those involved in
endocarditis, but even bacteria within single-species

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gene-directed as well as global proteomics- and ge-
nomics-based studies have lead to the identification of a
plethora of genes associated with biofilm development.
Sorting out the roles of all of these genes is however, an
exceedingly complex task. For one thing, the regulatory
processes of biofilm elaboration are cyclical and dy-
namic. In other words, external conditions trigger
alterations in the expression of a subset of genes re-
biofilms are heterogeneous with respect to gene expres-
sion. This is due to diffusion limitations imparted by the
biofilm, which result in local variations in pH, nutrient
and oxygen availability, and concentrations of bacterial
metabolites. On top of all of these complications, bio-
film studies are confounded by the intrinsic limitations
of the in vitro biofilm models and the techniques avail-
able to study the roles of these genes.

Table 1
Genes required for biofilm formation
Gene Protein/function Species References
Adhesion
abpA Amylase binding S. gordonii [7]
sspA/B Human salivary protein and collagen binding S. gordonii [7]
gbpA Polysaccharide formation S. mutans [7]
tarC Regulator of glucosyltransferase S and glucan binding protein S. mutans [50]
icaADBC Intercellular adhesin synthesis S. aureus, S. epidermidis [8]
hla Hemolytic toxin S. aureus [51]
clfA Clumping factor A, fibrinogen binding protein S. aureus [52]
dltA D -alanine esterification of teichoic acids S. aureus [53]
atlE Autolysin/adhesin S. epidermidis [8]
aap Accumulation associated protein S. epidermidis [54]
bopABCD Biofilm on plastic surfaces operon Enterococcus faecalis [55]
esp Enterococcal surface protein E. faecalis [56]
agn43 Antigen protein involved aggregation E. coli [57]
Quorum sensing
comX Competence S. gordonii [58]
comABCDE Competence S. mutans [12]
luxS? Quorum sensing S. mutans [33]
lasI Synthesis of 3OC12-HSL quorum-sensing signal P. aeruginosa [35]
Cell wall
PBP2B Peptidoglycan synthesis S. gordonii [7]
PBP5 Peptidoglycan synthesis S. gordonii [7]
glmM Peptidoglycan synthesis S. gordonii [7]
bacA Peptidoglycan synthesis S. gordonii [7]
brpA Possible regulator of autolysis S. mutans [34]
Metabolism
ccpA Carbon catabolite control protein S. mutans [34]
crc Global carbon metabolism regulator P. aeruginosa [59]

Stress response
dgk Stress response regulator, lantibiotic regulator S. mutans [12]
rB ? Alternate sigma factor-stress response S. aureus, S. epidermidis [13,14]
purR Regulator of purine synthesis, metabolism S. epidermidis [60]
rpoS? Regulator involved in slow growth E. coli [15,18]
mutT DNA mismatch repair S. gordonii [7]
Plasmids
tra Conjugative pilus of F plasmid E. coli [41]
 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173 165

Despite the complications associated with the study truths become unveiled. Tables 1 and 2 present a com-
of biofilms, comparative and comprehensive analysis of pilation of genes from various clinically relevant bacte-
all of the existing data can enable one to elucidate ria that have been implicated in the biofilm mode of
congruencies and subsequently, globally significant growth. Table 1 lists genes that appear to be required for

Table 2
Gene expression UP or DOWN in biofilms with respect to planktonic cells
Gene Protein/function Regulation Species References
Adhesion
algC Alginate synthesis UP P. aeruginosa [6]

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wcaB Colanic acid synthesis UP E. coli [5]
csgA Curli UP E. coli [61]
clfA Clumping factor/fibrinogen binding DOWN S. aureus [62]
scaA Co-aggregation DOWN S. gordonii [63]
abpA Amylase binding DOWN S. gordonii [63]
rggD Glucosyltransferase inducer UP S. gordonii [63]
int/CoA Intrageneric co-aggregation-relevant adhesin DOWN S. gordonii [63]
flpA Fibronectin-binding DOWN S. gordonii [63]
has Streptococcal hemagglutinin DOWN S. gordonii [63]
Quorum sensing
pA4296 Probable two-component response regulator DOWN P. aeruginosa [19]
comD,E Competence factors UP S. mutans [64]
Cell wall
mreC Cell morphology UP P. aeruginosa [19]
dltA D -Alanine-D -Alanyl carrier DOWN S. gordonii [63]
ddl D -Alanine: D -Alanine ligase DOWN S. gordonii [63]
Stress response
rpoH Stress/stationary phase s factor UP P. aeruginosa [19]
rpoS Heat shock s factor DOWN P. aeruginosa [19]
proU Transport-osmotic adaptation UP E. coli [5]
DnaK: Protein folding(heat shock) UP S. mutans [65]
Grpe: folding(heat shock) UP S. mutans [65]
60 kDa chaperonin: heat shock DOWN S. mutans [65]
htgX Putative heat shock protein DOWN S. gordonii [63]

Carbohydrate metabolism
Fructose bisphosphate aldolase DOWN S. mutans [65]
Pyruvate kinase DOWN S. mutans [65]
6-Phospho-B-galactosidase DOWN S. mutans [65]
pbg Phospho-b-glucosidase UP S. gordonii [63]
P02925 D -ribose-binding periplasmic protein UP E. coli [66]
P02927 D -galactose-binding protein UP E. coli [66]
Q6150 Malate dehydrogenase UP E. coli [66]
Division
minicell associated protein Div IVA: cell division UP S. mutans [65]
FTSZ: septum formation UP S. mutans [65]
ATP-dependent DNA helicase RECG: DNA DOWN S. mutans [65]
replication
H-NS: DNA binding UP E. coli [66]

Motility
PA2128 Probable fimbrial protein DOWN P. aeruginosa [19]
pilA Pilin protein DOWN P. aeruginosa [19]
flgD Flagellar basal-body rod modification protein DOWN P. aeruginosa [19]
PA1092 Flagellin type B DOWN P. aeruginosa [19]
fliD Flagellar capping protein DOWN P. aeruginosa [19]
flgE Flagellar hook protein DOWN P. aeruginosa [19]
fliC Flagellar synthesis DOWN E. coli [5]
Phage
Coat protein of bacteriophage Pf1 UP P. aeruginosa [19]
Helix destabilizing protein of bacteriophage Pf1 UP P. aeruginosa [19]
Probable coat protein of bacteriophage Pf1 UP P. aeruginosa [19]
166 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173
biofilm formation (the cause genes) and Table 2 lists the
genes that are differentially regulated in an established
biofilm (the effect genes). Answers from many questions
can be extricated from trends apparent in these tables.
For example, the conspicuous presence of surface ad-
hesions in Table 1 addresses the question ‘‘How do
biofilms adhere to solid surfaces?’’. Answering such
mechanistic questions is, inarguably, critical to our un-
derstanding of the biofilm mode of growth. However,
excellent reviews dedicated to understanding the
‘‘How?’’ of biofilm formation have already been pub-
lished [2,4], and I would like to venture instead, to
address the fundamental, albeit somewhat philosophical
question ‘‘Why do bacteria form biofilms in the human
host?’’.
2. Why do bacteria form biofilms?
According to Darwin’s theory of evolution, the only
true driving force behind the course of action of any
organism is reproductive fitness. Any action that in-
creases proliferation will endure within a species.
Therefore, when we talk about the driving force behind
biofilm formation we are asking the question ‘‘How does
the biofilm mode of growth promote survival and
propagation of the cell?’’ It almost seems counter-intu-
itive that the biofilm mode of growth could confer a
reproductive fitness advantage when one considers that
biofilm bacteria have a reduced rate of growth relative
to bacteria growing planktonically in broth culture.
Outside of the laboratory, however, bacteria rarely, if
ever, find themselves in an environment as nutrient rich
as culture media, and in these less-than-ideal conditions,
there are a number of fitness advantages imparted by the
biofilm mode of growth. This review proposes four ad-
vantages which are illustrated in Fig. 1. The more we
learn about the genetic regulation of biofilm formation,
the more we understand about the relative roles of these
benefits and about the forces that drive the switch to the
biofilm mode of growth.
2.1. Defense: biofilm formation as a stress response
Biofilms are resistant to physical forces such as the
shear forces produced by blood flow and the washing
action of saliva. Organisms within biofilms can with-
stand nutrient deprivation, pH changes, oxygen radicals,
disinfectants, and antibiotics better than planktonic or-
ganisms. Biofilms are also resistant to phagocytosis, and
the phagocytes that attempt an assault on the biofilm
may actually do more harm to surrounding tissues than
to the biofilm itself. The chronic nature of certain in-
fections is inarguably due to the development of a re-
silient biofilm. The invulnerability of biofilms is not
completely understood but is likely dependent upon a
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Fig. 1. Dr. Sean D. Taverna’s artistic interpretation of the four driving
forces behind bacterial biofilm formation that are discussed in this
review.
number of biofilm-specific characteristics including slow
growth and physiologic heterogeneity of the inhabitants.
Another important trait that fortifies biofilm resistance
is the sticky matrix which may contain DNA and other
polymers but in general, is predominantly composed of
exopolysaccharides.
The important role of exopolysaccharide (EPS) in
both the early and late stages of biofilm formation is
exemplified by the conspicuous presence of genes in-
volved in polysaccharide synthesis in Tables 1 and 2. In
Escherichia coli, csgA, which encodes a protein involved
in the synthesis of colanic acid, is involved in aggrega-
tion and algC, the gene required for alginate synthesis
plays a role in Pseudomonas aeruginosa biofilms [4–6].
EPS synthesis is important in the development of gram-
positive biofilms as well. Glucan binding protein GbpA
is a glucosyltransferase that has been implicated in su-
crose-dependent polysaccharide production and biofilm
formation in S. mutans [7]. In addition, the intercellular
adhesin locus (icaADBC) in Staphylococcus aureus and
Staphylococcus epidermidis encodes the gene products
responsible for the synthesis of a b-1-6-linked poly-N-
acetylglucosamine polymer called PNAG or PIA
(polysaccharide intercellular adhesin) [8]. Fig. 2 illus-
trates the important role for the exopolysaccharide
PNAG in the morphology of S. aureus biofilms. A weak
PNAG-producing strain retains the simple morphology
of a young biofilm whereas a strong PNAG-producing
strain forms tight mushroom-like microcolonies sepa-
rated by wide channels. In addition to its roles in ag-
gregation and biofilm structure, EPS plays a part in
defense, enabling biofilms to resist shear forces and
 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173 167

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Fig. 2. Confocal microscopic imaging demonstrates the influence of exopolysaccharide elaboration on the structure of S. aureus biofilms. S. aureus
clinical isolate strain MN8, and the isogenic, constitutive PNAG over-producing derivative strain MN8m were allowed to form biofilms on collagen-
coated glass coverslips for 48 h under flow conditions. The biofilms were stained using the BacLight Live/Dead kit which stains live bacteria green
and dead bacteria red. These confocal images demonstrate that the level of PNAG synthesis plays a critical role in biofilm structure. MN8 formed a
somewhat unstructured biofilm, whereas the PNAG-overproducing strain MN8m formed a highly structured biofilm with dense mushroom-shaped
microcolonies separated by large channels.

phagocytosis by inflammatory cells [2]. Some evidence
suggests that EPS may also be involved in tolerance of
biofilms to antimicrobial agents but this is still a matter
of debate [3]. However, if the sole force driving EPS
production and biofilm formation is resistance to dan-
gers encountered in the body, then what are the envi-
ronmental cues that warn bacteria that they need to
bolster their defenses? Certain bacterial species may
have evolved to switch on transcription of genes re-
quired for EPS synthesis in response to certain envi-
ronmental stimuli that are encountered upon host entry,
before the immune system mounts a specific attack [9].
For example, certain stimuli that mirror the physiology
of the human body, such as iron deprivation and
osmotic stress, induce the expression of genes encoding
proteins that synthesize EPS in staphylococci and
enterococci [10,11].
The involvement of stress response regulators in
biofilm formation would support the hypothesis that the
motivation behind biofilm formation is defense, but the
contribution of these regulators is somewhat unclear. As
indicated in Table 1, studies have implicated the stress
response regulators Dgk, rB , and RpoS, of S. mutans,
S. aureus, and E. coli (respectively) in biofilm formation
[12–15]. Other studies however, dispute the roles of rB
and RpoS in biofilm formation [16–18]. In P. aeruginosa
RpoS expression is down in biofilms but expression of
RpoH, a sigma factor that has been linked to the sta-
tionary phase and stress, is elevated [19]. The role of the
stress response regulators may depend upon the condi-
tions under which biofilm formation is triggered or upon
the genetic background of the bacterial cell, again sug-
gesting that what we categorize as a biofilm actually
represents a collection of different growth states. Bio-
films are inarguably resilient, but if defense is the major
driving force behind the bacterial mode of growth in the
human body, then why would the bacteria form a sessile
community in such an inhospitable place? In sum, it is
apparent that the stress is not the only trigger for this
mode of growth.
2.2. Colonization: biofilm formation as a mechanism to
remain in a favorable niche
Humans and other animals have developed intricate
immune systems for one critical reason: microorgan-
isms are continually trying to inhabit their bodies. The
body, or at least parts of it, is nutrient rich and rela-
tively stable with respect to water content, oxygen
availability, and temperature. Consequently, there is a
never-ending race between the development of the host
immune system and the progression of bacterial strat-
egies to evade it. In some cases, a compromise has been
made, and as such, the body is inhabited by a large
number of commensals, many of which exist as bio-
films. As the body is obviously an appealing place for
bacteria to live, it may be that the primary motivation
for switching to the biofilm mode of growth is to re-
main fixed.
Bacteria have a number of strategies to ensure that
they remain fixed in the human body. Bacterial surface
proteins that bind to host extracellular matrix proteins
such as fibronectin, fibrinogen, vitronectin, and elastin
are referred to as MSCRAMMs (microbial surface
component recognizing adhesive matrix molecules) and
often play a key role in initial adherence of bacteria to
solid surfaces within the host [20]. S. aureus is partic-
ularly notable for the abundance of MSCRAMMs that
168 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173
it can produce, including clumping factors A and B
(ClfA/B), fibronectin binding factors A and B (FnBA/
B), and a collagen binding protein (Cna). S. epidermidis
produces at least two autolysin–adhesins that bind to
fibronectin and the fibrinogen binding protein Fbe [8].
Streptococcus pyogenes contains genes for fibronectin
(prtF) and fibrinogen (emm) binding proteins [20]. The
oral streptococci have evolved to bind to the pellicle or
conditioning film on tooth surfaces which is composed
of salivary glycoproteins and lipids. The salivary ag-
glutinin glycoprotein binding proteins (SspA and SspB
in Streptococcus gordonii and SpaP in S. mutans), and
salivary amylase binding proteins of various oral
streptococci aid in binding to the pellicle on the surface
of teeth [7,21]. Flagella, pili, and fimbriae have also
been implicated in adherence of Vibrio cholerae, E. coli,
P. aeruginosa, and Salmonella enterica [22]. Interest-
ingly, Table 2 indicates that once the biofilm is estab-
lished, expression of a number of the adhesins and
motility factors is suppressed. This suggests that the
main role of adhesins, pili, and flagella is in initial
attachment, and that once the development of the
biofilm has passed this stage, the proteins are no longer
needed and their expression is inhibited. Overall, bac-
teria produce an impressive array of adhesins that
appear to have evolved as a means to inhabit the hu-
man body.
Also in support of the hypothesis that biofilm for-
mation is a mechanism for organisms to stay put in the
favorable environment of the human host, is the finding
that carbon catabolite induced gene regulation plays a
critical role in biofilm formation (Table 1). Exopoly-
saccharide expression and biofilm elaboration are
markedly enhanced in certain bacteria, including the
pseudomonads, V. cholerae, and E. coli, the staphylo-
cocci and the streptococci, when glucose or another
readily utilizable carbon source is abundant [4,23–25].
When nutrient sources are depleted, the bacteria detach
and become planktonic, suggesting that nutrient de-
privation is a trigger to move on, in search of a better
habitat. Glucose-induced exopolysaccharide production
may be multi-functional. It is possible that glucose
simply serves as a substrate in the EPS synthesis
pathway but studies with S. aureus in our laboratory
suggest that, at least for this organism, this is not the
case. Glucose appears to augment EPS elaboration at
the level of transcriptional regulation rather than at the
level of EPS synthesis [25]. A second possibility, which
supports the biofilm as a mode of defense is that bac-
teria may have evolved to interpret elevated glucose
levels as a cue that it is in the bloodstream, and that it
needs to form a biofilm to remove itself from circula-
tion and protect itself from the immune system. Alter-
natively, polysaccharide production may function as a
mechanism of glucose storage during times of plenty,
and/or as a mechanism to augment the accumulation
phase so that when the organism finds itself in an en-
vironment rich in nutrients it can occupy that niche.
With all of the complex mechanisms that pathogenic
and commensal bacteria have evolved to survive in the
human body, it is clear that the benefits that we afford
them outweigh the hurdles imparted by our immune
systems.
3. Community: biofilms and communal behavior
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3.1. Are biofilms multicellular organisms?
The idea that bacteria often exist within nature as
biofilms rather than individual, free-floating cells was
brought into wide acceptance by the ideas and intuition
of Dr. J. William Costerton [1]. Following the excite-
ment generated by his trailblazing revolution of long-
standing scientific dogma, other revolutionary
hypotheses about the nature of bacterial behavior be-
gan to emerge. One such hypothesis is that biofilms
should be regarded as multicellular organisms and that
biofilm bacteria exhibit cooperative, unselfish behavior
[26]. The behavior of bacteria within biofilms has even
sparked skepticism about the Darwinian theories of
evolution [27]. Hypothetical challenges to well-accepted
theories are entertaining but without scientific evidence
to back them up they remain purely philosophical, and
there has not yet been an effort to scientifically validate
the challenge to our current ideas about evolution.
Recently however, mathematical modeling of biofilm
systems and scientific experiments are being designed to
test whether cooperative, altruistic behavior in bacteria
is compatible with the mainstream theory of evolution
[28].
There are indeed similarities between biofilm bacteria
and multicellular organisms. For instance, bacteria (in-
cluding planktonic bacteria) can sense their surround-
ings, and this enables them to adjust their metabolic
processes to maximize the use of available substrates
and to protect themselves from detrimental conditions.
When bacteria are growing within a biofilm, these
changes in gene expression result in phenotypic hetero-
geneity within the biofilm which can be interpreted as
specialization or division of labor similar to cellular
differentiation seen in multicellular organisms. In addi-
tion, bacteria secrete substances referred to as autoin-
ducing signals, which influence gene expression and may
be a means by which cells communicate with one an-
other. There is even a growing body of evidence that
bacteria exhibit altruistic behavior and can undergo a
process similar to programmed cell death, again sug-
gestive of multicellularity [29]. However, there are fun-
damental distinctions between bacteria and multicellular
organisms. For example, while bacterial cells can react
and adapt to their environmental surroundings, they do
 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173
not permanently differentiate. For instance, one can
isolate colonic epithelial cells from a human and grow
them in tissue culture medium, and even though the cells
are suddenly faced with radical changes in their sur-
rounding milieu, they continue to grow as colonic epi-
thelial cells and can even form a polarized monolayer
similar to colonic epithelium. Scientists have devoted
much effort to developing methods to ‘‘undifferentiate’’
differentiated cells, but the process of cellular differen-
tiation, even in simple multicellular organisms, is not
easily reversed by any natural means. This is because
their genetic regulatory patterns have been permanently
altered. If however, you remove bacterial cells from a
biofilm and radically change their environmental con-
ditions then they will quickly adapt to their new envi-
ronmental surroundings and exhibit phenotypic
changes. Depending on the conditions in which they are
grown, they will even convert back to the planktonic
mode of growth. Bacterial cells do not differentiate, ra-
ther they respond to their environmental surroundings
by adapting their gene expression to suit their own needs
for survival. For this reason, it is more accurate to refer
to biofilms as interactive communities rather than
comparing them to multicellular organisms. Nonethe-
less, the community lifestyle is likely an important mo-
tivation for biofilm formation and provides its members
with a number of benefits. In addition to the advantage
of resistance to environmental changes, which is dis-
cussed in the defense section, the biofilm may benefit
from a number of properties of a communal existence
including division of the metabolic burden, gene trans-
fer, and selfless behavior.
3.1.1. Division of the metabolic burden
Diffusion limitations imparted by the biofilm struc-
ture result in local variations in nutrient availability, pH,
and oxygen tension. Therefore, the bacteria within bio-
films are inevitably heterogeneous with respect to gene
expression. Many biofilms are made up of a variety of
bacterial species and some even contain mixtures of
bacteria and fungi. The members of these mixed biofilms
have different requirements and perform different met-
abolic functions making commensalism a widespread
phenomenon in biofilms [26]. For example, whereas
early colonizers of the oral cavity are aerobic or facul-
tatively anaerobic, limited oxygen diffusion through the
biofilm provides an environmental niche allowing for
later colonization by obligate anaerobes [7]. A study in
which promoter activity was monitored as a function of
the expression of a fluorophore indicated that hetero-
geneity in the gene expression profiles of the individual
cells exists even within single-species biofilms [30]. It is
likely that this heterogeneity translates into specialized
functions of cells within a biofilm [30]. Fruiting body
formation by Myxobacteria is frequently cited as an
example of cell specialization in bacteria and it is an
169
attractive idea that this phenomenon occurs in other
bacteria as well [26]. Although it has not been defini-
tively proven, the heterogeneity within biofilms may
indeed result in a ‘‘division of labor’’ of sorts and cer-
tainly increases the metabolic efficiency of the popula-
tion as a whole.
A popular notion is that such division of labor is
coordinately regulated within biofilms through inter-
cellular communication. Autoinducing signals are small
molecules, generally homoserine lactones in gram-neg-
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atives and peptides in gram-positives, that are consti-
tutively released by bacteria and which, when present at
a critical concentration, will induce the expression of
certain genes. Autoinducing signals are frequently re-
ferred to as quorum-sensing signals because when a
bacterial population reaches a high enough density, the
local concentrations reach threshold levels and alter
gene expression. However, it has not been shown con-
clusively that bacteria actually respond to the accumu-
lation of a quorum, and it has been suggested that the
more biologically significant role of the autoinducing
signals is to relay information to the bacterial cell about
local diffusion rates [31]. In her provocative review,
Dr. Rosemary Redfield suggests that expression of se-
creted proteins are induced in the presence of elevated
levels of autoinducing signals not because the bacteria
have evolved to work cooperatively, but because the
benefits of secreted proteins are realized by an individual
bacterial cell when local conditions limit diffusion and
mixing [31]. One example used is the secretion of a
protease which is required to degrade exogenous pro-
teins so that the bacteria can assimilate amino acids.
Under conditions of reduced diffusion or mixing, se-
creted proteases and the proteins degraded by them
would remain in the vicinity of, and benefit the cell. It is
therefore logical that bacteria would restrict expression
of secreted proteins under conditions of high mixing and
diffusion. There are also examples of autoinducer effects
that do not readily fit this model. The diffusion sensing
model suggests that a bacterial cell responds to it’s own
secreted signals, but recognition of and response to
signals secreted by heterologous species has been well-
documented [32]. It is likely that both diffusion-sensing
and quorum-sensing are aspects of autoinducing signals
but a more precise answer incorporating roles for both
of these phenomena awaits further investigation.
While the primary function of autoinducing signals
remains unclear, their role in biofilm development is
even more ambiguous. As is indicated in Table 1, one
study found a role for the LuxS system in S. mutans but
two additional studies indicated that LuxS was not re-
quired for biofilm formation [12,33,34]. Equally con-
founding results were obtained when different
investigators studied the role of the lasRlasI quorum-
sensing system in P. aeruginosa [35]. Furthermore, there
is evidence that the accessory gene regulator (Agr) which
170 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173
is involved in quorum-sensing in the staphylococci ac-
tually inhibits biofilm formation [36]. A more recent
study suggests that the Agr effect is dependent upon the
flow strength over the biofilm and that under static
conditions, Agr reduces biofilm formation, under low to
moderate flow it does not affect biofilm formation, and
under very strong flow it increases biofilm formation
[30]. These results may support the diffusion-sensing
theory if one considers that under rapid flow, the auto-
inducing signals may rapidly diffuse out of the biofilm.
Overall, a biofilm represents both a quorum and
imparts restrictions on diffusion, so regardless of whe-
ther one accepts the diffusion-sensing or the quorum-
sensing hypothesis, it would seem, at least superficially,
that auto-inducing signals influence its development. In
parts of the human body however, especially in places
such as heart valves and teeth, biofilms are subjected to
strong shear forces which may keep autoinducing signal
levels low. A recent report indicates that in the human
lung, the acyl-homoserine lactone quorum-sensing sig-
nal of P. aeruginosa is inactivated by some unidentified
host cell-associated factor [37]. Does this indicate the
dispensability of quorum-sensing and mean that
P. aeruginosa can establish a biofilm in the lung despite
immune-mediated inhibition of quorum-sensing, or is
the finding that the immune system targets this signal
suggestive of its importance in biofilm formation? The
answer is unclear and the role of quorum-sensing in
biofilm formation remains elusive.
3.1.2. Gene transfer
Nucleic acid is the basis for evolution and conse-
quently its one true purpose is to replicate and perpet-
uate its own specific code. A reproductive fitness
advantage will perpetuate the genetic material of an
individual. The same rules apply to obligate infectious
agents such as viruses and plasmids, and as these agents
can not replicate their own genetic material, they would
cease to exist without a means to spread from one or-
ganism to another. Bacteriophage and plasmids have
therefore both evolved mechanisms to promote their
maintenance within a bacterium and their spread to
other bacteria. For example, bacteriophage slow down
their replicative machinery and integrate into the bac-
terial chromosome so that their genome is replicated as
the cell divides. Plasmid replication is an expensive
process for a bacterial cell and plasmids are quickly lost
unless there are required or beneficial. Plasmids have
therefore evolved a rather elaborate method for survival.
They carry toxin–antitoxin gene pairs which make their
maintenance necessary for bacterial survival [38]. The
plasmid-encoded toxin is a stable protein and the anti-
toxin is a labile protein. When a bacterial cell divides the
daughter cell inherits both toxin and antitoxin. If the cell
fails to replicate the plasmid as it divides or if the
daughter cell fails to inherit the plasmid, then the supply
of labile antitoxin rapidly declines whereas the toxin
lingers and destroys the plasmid-free daughter cell.
These cunning maintenance methods ensure vertical
transfer of phages and plasmids but in order to be suc-
cessful, a phage or infectious plasmid must also utilize
horizontal transfer. A biofilm is the ideal environment
for horizontal exchange of genetic material [39]. The
close proximity fosters rapid spread of phage as well as
conjugation and uptake of plasmid DNA by competent
bacteria. Plasmids and phage have consequently devel-
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oped methods to induce the transition to the biofilm
mode of growth in their host so that they can spread to
uninfected bacteria and sometimes even to cross the
species barrier [40]. Dr. Jean-Marc Ghigo found that the
pili encoded in a number of conjugative plasmids adhere
nonspecifically to solid surfaces and to other bacteria
leading to a dramatic increase in biofilm formation of E.
coli and other Gram-negative bacteria [41]. By inducing
the transition to the biofilm mode of growth, the plas-
mid is likely increasing its chances for horizontal
transmission. In addition, a number of phage genes,
including phage coat protein genes, are activated in
P. aeruginosa biofilms, supporting the idea that an ef-
fective strategy for horizontal phage transmission is to
re-enter the infectious cycle during the biofilm mode of
growth [19]. In effect, we may need to consider that
biofilm formation benefits not only bacterial fitness but
the proliferative potential of bacteriophage and plas-
mids as well.
Horizontal gene transfer within biofilms may also
directly benefit the bacteria through the exchange of
antibiotic resistance determinants. In S. gordonii, the
expression of competence factors has been implicated as
both a cause and an effect of biofilm formation sup-
porting a role for exchange of genetic material in bio-
films [39,42]. Whether the primary function of
competence factors is to assimilate external DNA as a
means to increase their genetic diversity or simply to use
it as a nutrient source is controversial, but the end result
is that the biofilm is an ideal environment for the ex-
change of genetic material. The motivation of bacteria
themselves as well as plasmids and bacteriophage to
exchange genetic material may all play an important
role in the process of biofilm development.
3.1.3. Selfless behavior
Overall, bacteria enjoy a number of benefits due to
their community mode of growth but are bacteria truly
cooperative and capable of exhibiting unselfish or even
altruistic behavior? Indeed, experimental evidence from
mathematical modeling supports the concept that bac-
teria can exhibit altruistic behavior [28]. While this may
initially appear to defy the rules of survival of the fittest,
the models indicate that unselfish behavior in biofilm
inhabitants can increase the overall growth yield.
Therefore, altruistic bacteria, despite a sacrifice in
 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173
growth rate are, under certain conditions, actually more
fit. Results from these studies also suggest that altruism
is more efficient when the bacteria exist in small, clonal
clusters, perhaps explaining the microcolony formation
characteristic of biofilms. Fitness need not entail ag-
gression, beneficial symbiotic relationships can increase
yield, and Darwin’s theories about evolution through
competition and fitness can be aptly applied to unselfish
behavior.
There is mounting evidence that a process similar to
apoptosis may actually occur in bacteria. Homologues
of pro-apoptotic genes such as caspases are widespread
among bacteria [43]. Additionally, several examples of
toxin–antitoxin cassettes, similar to those that ensure
plasmid maintenance, have been found within the
chromosomes of gram-negative and gram-positive bac-
teria, and it has been suggested that programmed cell
death may occur within the biofilm community [38]. One
rationalization for altruistic behavior in bacteria is that
it reduces the metabolic load and increases nutrient
availability to the survivors. This sort of unselfish be-
havior could be explained in a clonal population of
bacteria where death of some still results in perpetuation
of the genetic code. This type of phenomenon is ratio-
nalized by the kin selection model [43]. However, bac-
terial biofilms are rarely clonal, and surviving bacteria
would quickly out-compete sacrificial bacteria. A more
plausible hypothesis regarding the chromosomal toxin–
antitoxin cassettes, has recently been proposed by
Dr. Kim Lewis. He suggests that the cassettes induce, in
a fraction of the bacterial population, not death, but a
quiescent, persister state which enables survival in the
event of a sudden unfavorable environmental change
[3]. Overall, the idea that bacterial cells can undergo
programmed cell death, while appealing, does not
make immediate sense with respect to the laws of evo-
lution, but only further experimentation will resolve this
question.
4. The biofilm as the default mode of growth
In the laboratory, bacteria are generally grown
planktonically, but the utopian microcosms created in
culture vessels are designed to maximize bacterial
growth rates, not to replicate natural growth conditions
of the bacteria. In fact, some bacterial species appear to
constitutively utilize the biofilm mode outside of the lab.
The oral streptococci are very highly adapted to sessile
growth on the surface of teeth. Most of the oral bacterial
species lack an environmental niche and are found al-
most exclusively within the mouth [24]. For these bac-
teria, planktonic growth would cause them to be quickly
washed away by saliva, swallowed and destroyed within
the acidic juices of the stomach. These bacteria likely
spend the majority of their natural existence growing as
171
a biofilm. And yet they are often grown planktonically
in the laboratory.
It is possible that the presence of a suitable substrate
for attachment is all that is required to trigger biofilm
formation. There is mounting evidence that immediately
subsequent to the initial adherence of bacterial to a solid
surface, changes in gene regulation begin to occur [4,44].
This suggests that cells actually sense the solid surface to
which they are attached and that this sensing system
triggers a signaling cascade that may lead to some of the
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early gene expression patterns necessary for biofilm
formation. For example, in P. aeruginosa, expression of
algC, a gene required for alginate synthesis, is increased
within minutes of attachment, and when S. epidermidis
makes contact with a solid surface, the normally
spherical cell forms a leg-like appendage [45,46]. These
findings suggest that, similar to eukaryotic cells, bacte-
rial cells possess surface-sensing systems that induce
intracellular signals powerful enough to result in tran-
scriptional and morphologic changes.
The sensing mechanisms utilized by bacteria to detect
adherence are not well understood. Changes in the
perceived osmolarity caused by charges on solid surfaces
may be an important cue for bacteria to recognize sur-
faces [22]. The EnvZ–OmpR two-component system
which is involved in sensing environmental osmolarity,
has also been shown to regulate expression of curli and
colanic acid [47,48]. The fibrillar surface structure curli
plays a role in adherence and colanic acid is an exo-
polysaccharide involved in aggregation. The role of os-
molarity in biofilm regulation has also been noted in
staphylococci [13] and Pseudomonas fluorescens [49].
Overall, the biofilm mode of growth may be the default
mode of growth for at least some bacterial species sug-
gesting that we should be questioning what triggers the
planktonic mode of growth rather than what motivates
the biofilm mode of growth.
5. Conclusions
Scientific interest in biofilms has exploded in the past
decade. This fascination with biofilms is due in large
part to their presumed clinical relevance. But it is also,
undeniably, due to the appeal of projecting traits of
higher organisms on these life forms that were once
thought to be so simple and autonomous. The ability of
prokaryotes to adapt to their surroundings is indeed
remarkable, but whether they actually communicate,
coordinate, and specialize within biofilms for the benefit
of the community, as opposed to simply reacting to their
environments in order to selfishly promote their own
survival, has not yet been sufficiently established. Un-
fortunately, we often make the erroneous conclusion
that certain questions are purely philosophical and can
not be tested scientifically. In fact, such questions can
172 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173
often, if not always, be objectively examined, and sci-
entists have recently begun to develop mathematical
models that predict the relative impacts of altruistic vs.
selfish behavior on the survival and propagation of
bacteria [28].
In conclusion, it is evident that bacteria reap a
number of benefits from the biofilm mode of growth and
it is likely that different forces motivate bacteria to
transition to one of a variety of biofilm states depending
on the genetic makeup of the organism and its envi-
ronment. The alternative motives for biofilm formation
presented in this review are by no means exhaustive or
mutually exclusive and because it is such a complex
process, they may all have a role.
Acknowledgement
I thank Dr. Sean Taverna (Rockefeller University)
for his artistic contribution. I would also like to ac-
knowledge that there are a number of excellent publi-
cations on the molecular genetics of biofilm formation
which could not be referenced here due to space
limitations.
References
[1] Costerton, J.W., Geesey, G.G. and Cheng, K.J. (1978) How
bacteria stick. Sci. Am. 238, 86–95.
[2] Donlan, R.M. and Costerton, J.W. (2002) Biofilms: survival
mechanisms of clinically relevant microorganisms. Clin. Micro-
biol. Rev. 15, 167–193.
[3] Lewis, K. (2001) Riddle of biofilm resistance. Antimicrob. Agents
Chemother. 45, 999–1007.
[4] O’Toole, G., Kaplan, H.B. and Kolter, R. (2000) Biofilm
formation as microbial development. Annu. Rev. Microbiol. 54,
49–79.
[5] Prigent-Combaret, C. and Lejeune, P. (1999) Monitoring gene
expression in biofilms. Methods Enzymol. 310, 56–79.
[6] Davies, D.G., Chakrabarty, A.M. and Geesey, G.G. (1993)
Exopolysaccharide production in biofilms: substratum activation
of alginate gene expression by Pseudomonas aeruginosa. Appl.
Environ. Microbiol. 59, 1181–1186.
[7] Loo, C.Y. (2003) Oral Streptococcal genes that encode biofilm
formation. In: Medical Implications of Biofilms (Wilson, M. and
Devine, D., Eds.), vol. 1, pp. 212–227.
[8] Heilmann, C. (2003) Molecular basis of biofilm formation by
Staphylococcus epidermidis. In: Medical Implications of
Biofilms (Wilson, M. and Devine, D., Eds.), vol. 1, pp.
110–135, .
[9] Shirtliff, M.E., Mader, J.T. and Camper, A.K. (2002) Molecular
interactions in biofilms. Chem. Biol. 9, 859–871.
[10] Deighton, M. and Borland, R. (1993) Regulation of slime
production in Staphylococcus epidermidis by iron limitation.
Infect. Immun. 61, 4473–4479.
[11] Baldassarri, L., Cecchini, R., Bertuccini, L., Ammendolia, M.G.,
Iosi, F., Arciola, C.R., Montanaro, L., Di Rosa, R., Gherardi, G.,
Dicuonzo, G., Orefici, G. and Creti, R. (2001) Enterococcus spp.
produces slime and survives in rat peritoneal macrophages. Med.
Microbiol. Immunol. 190, 113–120.
[12] Yoshida, A. and Kuramitsu, H.K. (2002) Multiple Streptococcus
mutans genes are involved in biofilm formation. Appl. Environ.
Microbiol. 68, 6283–6291.
[13] Knobloch, J.K., Bartscht, K., Sabottke, A., Rohde, H., Feucht,
H.H. and Mack, D. (2001) Biofilm formation by Staphylococcus
epidermidis depends on functional RsbU, an activator of the sigB
operon: differential activation mechanisms due to ethanol and salt
stress. J. Bacteriol. 183, 2624–2633.
[14] Rachid, S., Ohlsen, K., Wallner, U., Hacker, J., Hecker, M. and
Ziebuhr, W. (2000) Alternative transcription factor sigma(B) is
involved in regulation of biofilm expression in a Staphylococcus
aureus mucosal isolate. J. Bacteriol. 182, 6824–6826.
Downloaded from https://academic.oup.com/femsle/article-abstract/236/2/163/535288 by guest on 09 September 2019
[15] Adams, J.L. and McLean, R.J. (1999) Impact of rpoS deletion on
Escherichia coli biofilms. Appl. Environ. Microbiol. 65, 4285–4287.
[16] Kies, S., Otto, M., Vuong, C. and G€ otz, F. (2001) Identification of
the sigB operon in Staphylococcus epidermidis: construction and
characterization of a sigB deletion mutant. Infect. Immun. 69,
7933–7936.
[17] Valle, J., Toledo-Arana, A., Berasain, C., Ghigo, J.M., Amorena,
B., Penades, J.R. and Lasa, I. (2003) SarA and not sigma(B) is
essential for biofilm development by Staphylococcus aureus. Mol.
Microbiol. 48, 1075–1087.
[18] Corona-Izquierdo, F.P. and Membrillo-Hernandez, J. (2002) A
mutation in rpoS enhances biofilm formation in Escherichia coli
during exponential phase of growth. FEMS Microbiol. Lett. 211,
105–110.
[19] Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R.,
Teitzel, G.M., Lory, S. and Greenberg, E.P. (2001) Gene
expression in Pseudomonas aeruginosa biofilms. Nature 413,
860–864.
[20] Patti, J.M., Allen, B.L., McGavin, M.J. and Hook, M. (1994)
MSCRAMM-mediated adherence of microorganisms to host
tissues. Annu. Rev. Microbiol. 48, 585–617.
[21] Burne, R.A. (1998) Oral streptococci... products of their environ-
ment. J. Dent. Res. 77, 445–452.
[22] Lejeune, P. (2003) Contamination of abiotic surfaces: what a
colonizing bacterium sees and how to blur it. Trends Microbiol.
11, 179–184.
[23] Ammendolia, M.G., Di Rosa, R., Montanaro, L., Arciola, C.R.
and Baldassarri, L. (1999) Slime production and expression of the
slime-associated antigen by staphylococcal clinical isolates. J.
Clin. Microbiol. 37, 3235–3238.
[24] Burne, R.A., Chen, Y.M., Li, Y., Bhagwat, S. and Wen, Z. (2003)
Gene expression in oral biofilms. In: Medical Implications of
Biofilms (Wilson, M. and Devine, D., Eds.), vol. 1, pp. 212–227, .
[25] Jefferson, K.K., Pier, D.B., Goldmann, D.A. and Pier, G.B. (2004)
The teicoplanin-associated locus regulator (TcaR) and the inter-
cellular adhesin locus regulator (IcaR) are transcriptional inhib-
itors of the ica locus in Staphylococcus aureus. J. Bacteriol. 186,
2449–2456.
[26] Shapiro, J.A. (1998) Thinking about bacterial populations as
multicellular organisms. Annu. Rev. Microbiol. 52,
81–104.
[27] Caldwell, D.E. (1999) Post-modern ecology – is the environment
the organism? Environ. Microbiol. 1, 279–281.
[28] Kreft, J.U. (2003) Biofilms promote altruism. In: ASM Confer-
ences 2003, American Society for Microbiolgy Eds.
[29] Rice, K.C. and Bayles, K.W. (2003) Death’s toolbox: examining
the molecular components of bacterial programmed cell death.
Mol. Microbiol. 50, 729–738.
[30] Yarwood, J.M., Bartels, D.J., Volper, E.M. and Greenberg, E.P.
(2004) Quorum sensing in Staphylococcus aureus biofilms. J.
Bacteriol. 186, 1838–1850.
[31] Redfield, R.J. (2002) Is quorum sensing a side effect of diffusion
sensing? Trends Microbiol. 10, 365–370.
[32] Federle, M.J. and Bassler, B.L. (2003) Interspecies communica-
tion in bacteria. J. Clin. Invest. 112, 1291–1299.
 K.K. Jefferson / FEMS Microbiology Letters 236 (2004) 163–173
[33] Merritt, J., Qi, F., Goodman, S.D., Anderson, M.H. and Shi, W.
(2003) Mutation of luxS affects biofilm formation in Streptococcus
mutans. Infect. Immun. 71, 1972–1979.
[34] Wen, Z.T. and Burne, R.A. (2002) Functional genomics approach
to identifying genes required for biofilm development by Strep-
tococcus mutans. Appl. Environ. Microbiol. 68, 1196–1203.
[35] Davies, D.G., Parsek, M.R., Pearson, J.P., Iglewski, B.H.,
Costerton, J.W. and Greenberg, E.P. (1998) The involvement of
cell-to-cell signals in the development of a bacterial biofilm.
Science 280, 295–298.
[36] Vuong, C., Gerke, C., Somerville, G.A., Fischer, E.R. and Otto,
M. (2003) Quorum-sensing control of biofilm factors in Staphy-
lococcus epidermidis. J. Infect. Dis. 188, 706–718.
[37] Chun, C.K., Ozer, E.A., Welsh, M.J., Zabner, J. and Greenberg,
E.P. (2004) Inactivation of a Pseudomonas aeruginosa quorum-
sensing signal by human airway epithelia. Proc. Natl. Acad. Sci.
USA 101, 3587–3590.
[38] Hayes, F. (2003) Toxins-antitoxins: plasmid maintenance, pro-
grammed cell death, and cell cycle arrest. Science 301, 1496–1499.
[39] Cvitkovitch, D.G., Li, Y.H. and Ellen, R.P. (2003) Quorum
sensing and biofilm formation in Streptococcal infections. J. Clin.
Invest. 112, 1626–1632.
[40] Roberts, A.P., Pratten, J., Wilson, M. and Mullany, P. (1999)
Transfer of a conjugative transposon, Tn5397 in a model oral
biofilm. FEMS Microbiol. Lett. 177, 63–66.
[41] Ghigo, J.M. (2001) Natural conjugative plasmids induce bacterial
biofilm development. Nature 412, 442–445.
[42] Loo, C.Y., Corliss, D.A. and Ganeshkumar, N. (2000) Strepto-
coccus gordonii biofilm formation: identification of genes that code
for biofilm phenotypes. J. Bacteriol. 182, 1374–1382.
[43] Bayles, K.W. (2003) Are the molecular strategies that control
apoptosis conserved in bacteria? Trends Microbiol. 11, 306–311.
[44] Kuchma, S.L. and O’Toole, G.A. (2000) Surface-induced and
biofilm-induced changes in gene expression. Curr. Opin. Biotech-
nol. 11, 429–433.
[45] Davies, D.G. and Geesey, G.G. (1995) Regulation of the alginate
biosynthesis gene algC in Pseudomonas aeruginosa during biofilm
development in continuous culture. Appl. Environ. Microbiol. 61,
860–867.
[46] Kodjikian, L., Burillon, C., Lina, G., Roques, C., Pellon, G.,
Freney, J. and Renaud, F.N. (2003) Biofilm formation on
intraocular lenses by a clinical strain encoding the ica locus: a
scanning electron microscopy study. Invest. Ophthalmol. Vis. Sci.
44, 4382–4387.
[47] Prigent-Combaret, C., Vidal, O., Dorel, C. and Lejeune, P. (1999)
Abiotic surface sensing and biofilm-dependent regulation of gene
expression in Escherichia coli. J. Bacteriol. 181, 5993–6002.
[48] Vidal, O., Longin, R., Prigent-Combaret, C., Dorel, C., Hoor-
eman, M. and Lejeune, P. (1998) Isolation of an Escherichia coli
K-12 mutant strain able to form biofilms on inert surfaces:
involvement of a new ompR allele that increases curli expression.
J. Bacteriol. 180, 2442–2449.
[49] O’Toole, G.A. and Kolter, R. (1998) Initiation of biofilm
formation in Pseudomonas fluorescens WCS365 proceeds via
multiple, convergent signalling pathways: a genetic analysis.
Mol. Microbiol. 28, 449–461.
[50] Idone, V., Brendtro, S., Gillespie, R., Kocaj, S., Peterson, E.,
Rendi, M., Warren, W., Michalek, S., Krastel, K., Cvitkovitch, D.
and Spatafora, G. (2003) Effect of an orphan response regulator
on Streptococcus mutans sucrose-dependent adherence and cario-
genesis. Infect. Immun. 71, 4351–4360.
[51] Caiazza, N.C. and O’Toole, G.A. (2003) Alpha-toxin is required
for biofilm formation by Staphylococcus aureus. J. Bacteriol. 185,
3214–3217.
173
[52] Vaudaux, P.E., Francois, P., Proctor, R.A., McDevitt, D., Foster,
T.J., Albrecht, R.M., Lew, D.P., Wabers, H. and Cooper, S.L.
(1995) Use of adhesion-defective mutants of Staphylococcus aureus
to define the role of specific plasma proteins in promoting
bacterial adhesion to canine arteriovenous shunts. Infect. Immun.
63, 585–590.
[53] Gross, M., Cramton, S.E., G€ otz, F. and Peschel, A. (2001) Key
role of teichoic acid net charge in Staphylococcus aureus coloni-
zation of artificial surfaces. Infect. Immun. 69, 3423–3426.
[54] Hussain, M., Herrmann, M., von Eiff, C., Perdreau-Remington,
F. and Peters, G. (1997) A 140-kilodalton extracellular protein is
essential for the accumulation of Staphylococcus epidermidis
Downloaded from https://academic.oup.com/femsle/article-abstract/236/2/163/535288 by guest on 09 September 2019
strains on surfaces. Infect. Immun. 65, 519–524.
[55] Hufnagel, M., Koch, S., Creti, R., Baldassarri, L. and Huebner, J.
(2004) A putative sugar-binding transcriptional regulator in a
novel gene locus in Enterococcus faecalis contributes to produc-
tion of biofilm and prolonged bacteremia in mice. J. Infect. Dis.
189, 420–430.
[56] Toledo-Arana, A., Valle, J., Solano, C., Arrizubieta, M.J.,
Cucarella, C., Lamata, M., Amorena, B., Leiva, J., Penades,
J.R. and Lasa, I. (2001) The enterococcal surface protein, Esp, is
involved in Enterococcus faecalis biofilm formation. Appl. Envi-
ron. Microbiol. 67, 4538–4545.
[57] Kjaergaard, K., Schembri, M.A., Ramos, C., Molin, S. and
Klemm, P. (2000) Antigen 43 facilitates formation of multispecies
biofilms. Environ. Microbiol. 2, 695–702.
[58] Lunsford, R.D. and London, J. (1996) Natural genetic transfor-
mation in Streptococcus gordonii: comX imparts spontaneous
competence on strain wicky. J. Bacteriol. 178, 5831–5835.
[59] O’Toole, G.A., Gibbs, K.A., Hager, P.W., Phibbs Jr., P.V. and
Kolter, R. (2000) The global carbon metabolism regulator Crc is a
component of a signal transduction pathway required for biofilm
development by Pseudomonas aeruginosa. J. Bacteriol. 182, 425–
431.
[60] Knobloch, J.K., Nedelmann, M., Kiel, K., Bartscht, K., Hors-
tkotte, M.A., Dobinsky, S., Rohde, H. and Mack, D. (2003)
Establishment of an arbitrary PCR for rapid identification of
Tn917 insertion sites in Staphylococcus epidermidis: characteriza-
tion of biofilm-negative and nonmucoid mutants. Appl. Environ.
Microbiol. 69, 5812–5818.
[61] Prigent-Combaret, C., Prensier, G., Le Thi, T.T., Vidal, O.,
Lejeune, P. and Dorel, C. (2000) Developmental pathway for
biofilm formation in curli-producing Escherichia coli strains: role
of flagella, curli and colanic acid. Environ. Microbiol. 2, 450–464.
[62] Wolz, C., Goerke, C., Landmann, R., Zimmerli, W. and Fluck-
iger, U. (2002) Transcription of clumping factor A in attached and
unattached Staphylococcus aureus in vitro and during device-
related infection. Infect. Immun. 70, 2758–2762.
[63] Gilmore, K.S., Srinivas, P., Akins, D.R., Hatter, K.L. and
Gilmore, M.S. (2003) Growth, development, and gene expression
in a persistent Streptococcus gordonii biofilm. Infect. Immun. 71,
4759–4766.
[64] Li, Y.H., Tang, N., Aspiras, M.B., Lau, P.C., Lee, J.H., Ellen,
R.P. and Cvitkovitch, D.G. (2002) A quorum-sensing signaling
system essential for genetic competence in Streptococcus mutans is
involved in biofilm formation. J. Bacteriol. 184, 2699–2708.
[65] Svensater, G., Welin, J., Wilkins, J.C., Beighton, D. and Ham-
ilton, I.R. (2001) Protein expression by planktonic and biofilm
cells of Streptococcus mutans. FEMS Microbiol. Lett. 205, 139–
146.
[66] Tremoulet, F., Duche, O., Namane, A., Martinie, B. and Labadie,
J.C. (2002) A proteomic study of Escherichia coli O157:H7 NCTC
12900 cultivated in biofilm or in planktonic growth mode. FEMS
Microbiol. Lett. 215, 7–14.