Bacterial biolm:

structure, function, and

antimicrobial resistance
DELPHINE DUFOUR, VINCENT LEUNG & CLINE M. LVESQUE
Biolms are microbial communities attached to surfaces and encased in an extracellular matrix of microbial origin.
They represent the predominant form of microbial life. Biolms are everywhere and can develop on virtually every
natural and man-made surface. Biolms are also ubiquitous in both normal and pathogenic human processes.
Biolm formation has been demonstrated for numerous pathogens and is clearly one of the main strategies for
bacterial survival in a variety of sites within the human body. In almost all instances, the biolm lifestyle helps
bacteria survive and persist within the environment. This review discusses the fundamental biology of microbial
biolm and how biolms impact the pathogenesis of human infections. The different mechanisms involved in the
reduced antimicrobial susceptibility of microorganisms in pathogenic biolms are discussed in detail in this review.
Possible approaches that could be explored in the search for new anti-biolm strategies to eradicate medically
relevant biolms are also presented.
Received 20 May 2011; accepted 13 November 2011.
Introduction
A brief history of biolms
A little more than 300 years ago, the Dutch scientist
Anthony van Leeuwenhoek scraped material from his
own teeth and, using his simple microscopes, noticed
a vast accumulation of moving objects not visible to
the naked eye. He called these microscopic bodies
animalcules, because he thought they were tiny living
animals. In a report to the British Royal Society, he
wrote, The number of these animalcules in the scurf
of a mans teeth is so many that I believe they exceed
the number of men in a kingdom. This observation
made him the rst biolm experimenter.
More than two centuries later, the American bacte-
riologist Arthur Henrici observed a growth of algae on
the walls of an aquarium in his laboratory. He placed
some microscopic slides in the aquarium, expecting to
see growth of algae on the slides and that permanent
specimen preparations could be used to observe the
microorganisms in situ. He was surprised to nd, in
addition to the algae, a thin and uniform coating of
bacteria of various forms, some of unusual morphol-
ogy. Henrici reported in 1933 that It is quite evident
that for the most part the water bacteria are not free
oating organisms, but grow on submerged surfaces;
they are of the benthos rather than the plankton (1).
Only a few years later, Heukelekian and Heller, two
scientists investigating the technology of aerobic and
anaerobic decomposition of sewage solids, made the
following observation: Surfaces enable bacteria to
develop in substrates otherwise too dilute for growth.
Development takes place either as bacterial slime or
colonial growth attached to surfaces (2). Finally,
during the same decade, the pioneer microbial ecolo-
gist Claude ZoBell made important contributions to
biolm microbiology. ZoBell found that when seawa-
ter was collected in sterile glass bottles, there were
more bacteria present on the surface of the glass than
in the seawater, highlighting the growth of sessile
bacteria (3,4).
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What these pioneering scientists were describing was
what we now call biolm. A biolm is presently seen as
a community of microorganisms held together by
a self-produced extracellular matrix and associated
with a surface (5). Development of microscopy and
molecular genetic techniques promoted the under-
standing that natural bacterial populations exist mostly
in biolm communities.
Biolm: a new perception of
microbial existence
Historically, many studies have assumed that microbes
lead solitary, asocial lives. On the contrary, bacteria
predominantly live in dense biolm populations inter-
acting extensively with each other (6). Indeed, most
bacteria in nature do not exist independently but
persist in co-ordinated, spatially organized, and meta-
bolically integrated biolm communities. Biolms are
ubiquitous and can develop on virtually every natural
and man-made surface. In natural habitats, biolms
generally provide homeostasis in the face of uctuating
and harsh environmental conditions (7). They can
develop under extreme environmental conditions. For
example, a research group has recently reported the
presence of microbial biolms inhabiting thermal
waters (3550C) more than 60 meters below sea level
in Italy (8). Other biolms have been found living on
frozen glaciers. In 2009, a glacier sample collection in
the Antarctic coast revealed the presence of a seawater
biolm composed of a novel bacterial species, Antarc-
ticimonas ava (9). Other than these very hot and cold
environments, biolms can also be observed in extreme
acidic environments, characterized by a high metal
content and lack of nutrients. Moreover, bacterial bio-
lms are present in areas with various hydration levels,
ranging from extremely low (e.g. deserts) to extremely
high (e.g. rainforests) saturation levels (10,11).
In industry, biolms cause food and drinking water
contamination, metal surface corrosion, and clogging
(12,13). Due to the possibility of water contaminated
with pathogens, biolms present a serious hazard to
the drinking supply. In the petrochemical industry,
biolms decrease the efciency of equipment such as
oil pipelines and platforms. Biolms can also develop
on ship hulls, shing traps, and nets, representing a
major economic problem. Although biolms can be
extremely costly to industry, positive aspects of the
practical application of biolms exist. For example,
benecial biolms are used in the production of indus-
trial chemicals (e.g. ethanol, vinegar) and in the
removal of toxic compounds during the treatment of
wastewater and oily seawater.
Biolms are also greatly associated with vegetable
life. The role of symbiotic nitrogen xers and bacterial
biolms in legumeRhizobium interactions and metal-
tolerant microbes in the improvement of legumes is of
great interest to professionals working in the eld of
agronomy. In contrast, plant pathogens such as
Pseudomonas syringae display detrimental effects on
plant growth, development, and crop yield by infect-
ing a wide range of plant species (e.g. olive, tomato,
rice, soybean) and causing chlorosis, necrosis, or
canker (14).
Biolms are also ubiquitous in both normal and
pathogenic human processes. Microorganisms com-
prising the human-associated microbiota are essential
for host development and health. Bacteria play impor-
tant roles such as in the production of vitamins and
essential amino acids. They can also help prevent colo-
nization by exogenous pathogens. One such example
is the Escherichia coli strain Nissle 1917. This biolm
organism has been used for many decades as a probi-
otic against a variety of intestinal disorders (15).
Biolm formation has been demonstrated for numer-
ous pathogens and is clearly one of the main strategies
for bacterial survival in a variety of sites within the
human body (16). Therefore, biolms provide a new
perspective through which to view infectious diseases.
Biolms in infectious diseases
Biolms are a major cause of human infections (17).
The majority of hospital-acquired infections are due to
biolms because they can be life-threatening coloniz-
ers of biomedical devices. Indeed, a large proportion
of nosocomial infections are related to the coloniza-
tion of pathogens on the surface of implanted medical
devices such as respirators, catheters (central venous,
urinary), prosthetic heart valves, and orthopedic
devices (Table 1). In fact, 95% of urinary tract infec-
tions are associated with a urinary catheter, 80% of
pneumonias are associated with mechanical ventila-
tion, and 87% of bloodstream infections are associated
with intravascular devices. Staphylococci and entero-
cocci are responsible for the frequent cases of
hospitalization-acquired infections. These two bacte-
rial genera are commensal inhabitants of the human
Bacterial biolm
3
ora of the skin, upper respiratory tract, lower gastro-
intestinal tract, and urogenital tract. For this reason,
they are amongst the most likely organisms to colonize
implanted medical devices (18).
Coagulase-negative staphylococci (CoNS; S. epider-
midis, S. lugdunensis, S. haemolyticus) and Staphylococ-
cus aureus are the predominant bacterial species
connected to device-associated infections (19). CoNS
are an important cause of endogenous infection of
intravascular catheters, either by tracking along their
external surface or by entering via the catheter hub
during manipulation by healthcare workers. S. epider-
midis nosocomial infections tend to be chronic and
less acute, while S. aureus is involved in acute infec-
tions because of its ability to elicit a more acute
immune response from the host (20).
Enterococci have emerged in recent years as patho-
gens associated with serious nosocomial infections
despite their lowlevel of virulence. They cause infection
almost exclusively in hospitalized patients who have
signicantly compromised immune defenses. Two
major bacterial species account for the vast majority of
biolm infections; Enterococcus faecalis is the most
common and causes 8090%of enterococcal infections,
while Enterococcus faecium causes 1015% (21).
Enterococci are among the most frequent cause of
nosocomial infections, particularly in intensive care
units where enterococci can be transmitted from one
patient to another via the hands of clinical staff. The
primary sites of infection are the urinary tract and soft
tissues adjacent to the intestinal ora where enterococci
are resident. Patients with extensive abdominal surgery,
indwelling devices, or who are undergoing abdominal
procedures are at greatest risk. Antibiotic resistance,
especially vancomycin resistance, has become a major
problem in enterococcal infections (22).
Several pathogens associated with chronic infections
are linked to biolm infections, including periodonti-
tis, cystic brosis pneumonia, chronic urinary tract
infections, chronic otitis media, and chronic wound
infections (Table 2). Contrary to acute infections,
which are mostly the result of planktonic (free-
oating) growth, in chronic bacterial infections, the
planktonic phenotype generally exists only transiently,
and usually as a minor population. Since chronic infec-
tions are fundamentally different from acute infec-
tions, different strategies are necessary to treat the
biolm infections more efciently. The fact that micro-
bial biolms represent a protected mode of growth
that allows cells to survive hostile environments pre-
sents a challenge for treating chronic biolm infections
(23,24). Chronic infections are thus important clini-
cally because bacteria in biolms resist host immune
responses and antibiotic treatment and these charac-
teristics are often cited as the reason bacteria have the
ability to persist for a long time in the human body.
Biolm lifestyle
Biolm formation
The development of a microbial biolm can be
described as a dynamic process involving successive
steps (Fig. 1). The rst step is the attachment of the
bacterial cells to the selected abiotic or biotic surface.
Bacteria usually adhere to a conditioning lm typically
composed of organic molecules (e.g. nutrients, sali-
vary proteins, large macromolecules) that can promote
the adherence of bacteria to the surface. Initial attach-
ment is mediated through weak reversible van der
Waals interactions between the cell surface and the
substratum, which can lead to stronger adhesion-
receptor mediated attachment (25). Bacterial cell
surface structures such as agella, mbriae, LPS, and
exopolysaccharides participate in irreversible interac-
tions. These can be dipole, hydrogen, ionic, or
hydrophobic. The second step corresponds to the
development of micro-colonies promoted by the
growth and division of the rst attached cells (primary
colonizers). The micro-colonies progressively enlarge
Table 1: Biolms of indwelling medical devices
Medical device Principal microorganisms
Contact lens P. aeruginosa, Gram-positive cocci
Denture Candida spp.
Urinary catheter E. coli, Candida spp., E. faecalis,
P. mirabilis, K. pneumoniae
Central venous catheter CoNS*, S. aureus
Mechanical heart valve CoNS, S. aureus
Articial hip prosthesis CoNS, S. aureus, Enterococcus spp.
Voice prostheses C. albicans, CoNS
Endotracheal tubes Enteric Gram-negative species
* CoNS: coagulase-negative staphylococci.
Dufour et al.
4
Table 2: Diversity of human infections involving biolms
Anatomic location Infectious disease Microorganisms
Eye Ocular infections S. aureus
Ear Otitis media Non-typeable H. inuenzae
Mouth Dental caries Acidogenic Gram-positive cocci
Endodontic infections Gram-positive anaerobic species, Bacteroides,
Neisseria, Fusobacterium
Periodontitis Gram-negative anaerobic oral bacteria
Respiratory tract CF pneumonia P. aeruginosa, B. cepacia
Chronic sinusitis S. aureus and CoNS*
Heart Endocarditis Viridans streptococci
Muscle Musculoskeletal infections Gram-positive cocci
Bone Osteomyelitis Various bacterial species
Skin Necrotizing fasciitis Group A streptococci
Foot Diabetic foot infection Corynebacterium spp. and various obligate anaerobes
Prostate gland Bacterial prostatis E. coli and other Gram-negative species
Vagina Staphylococcal toxic shock syndrome S. aureus
Biliary system Gallstone Enteric bacteria
Urinary tract Urinary tract infection E. coli, Enterococci, Klebsiella
Large intestine Persistent diarrhea E. coli
* CoNS: coagulase-negative staphylococci.
Fig. 1. Schematic representation of the distinct steps in microbial biolm development. The different stages in biolm
formation include initial attachment to the surface, formation of a monolayer along the surface with formation of
micro-colonies, biolm maturation with formation of a three-dimensional structure, and cell dispersion.
Bacterial biolm
5
and coalesce to form the rst layer of cells covering the
surface. When multiple layers of cells pile up on the
surface, the third step of the formation is obtained,
indicated by the presence of a mature biolm charac-
terized by the presence of macro-colonies surrounded
by water channels that help distribute nutrients and
signaling molecules. Finally, to survive when nutrients
become limited, or simply to spread and colonize to
other niches, some biolm cells can detach individually
or in clumps. In general, biolm dispersion occurs in
response to environmental changes and is dependent
on growing conditions (26).
The molecular mechanisms that regulate the distinct
developmental steps in biolm formation vary greatly
between different bacterial species, and even depend
on the environmental conditions for the same given
species. Nevertheless, biolms display a common
attribute, the biolm matrix. Contrary to free-oating
planktonic cells, biolm cells are embedded in a self-
produced extracellular matrix, the extracellular poly-
meric substance (EPS), that holds them together (27).
Biolms are composed of about 8085% EPS (by
volume) and only 1520% cells (by volume) (28).
Although the EPS may vary in chemical and physical
properties, its major components are polysaccharides
(homo- and heteropolysaccharides), proteins, and
extracellular DNA. The EPS plays a major role in
maintaining the integrity of the biolm and can confer
other benecial properties. Since the EPS is also highly
hydrated, it can prevent desiccation in some natural
biolms. The EPS can also act as a diffusion barrier,
preventing toxic substances such as antibiotics and
disinfectants from reaching their target (25,27,29).
Cell heterogeneity within biolms
Most biolms found in nature are polymicrobials,
where diverse species expressing multiple phenotypes
are involved. The human oral dental plaque biolm
and the biolms of periodontal diseases are perhaps
the best described multi-species microbial biolms
(see next section). Microbial biolms are thus very
complex and heterogeneous environments character-
ized by a wide physical, chemical, and biotic diversity.
The most amazing fact is that even in a mono-species
biolm, phenotypic heterogeneity exists. Cells of the
same bacterial species can exhibit different phenotypes
in a biolm even though they are separated by as little
as 10 mm (30). Three processes have been proposed to
explain this intra-species heterogeneity within a mono-
species microbial biolm and they are illustrated in
Figure 2.
Mono-species biolm communities are often com-
posed of phenotypically distinct subpopulations. Cell
differentiation in biolms may depend on the local
environmental conditions surrounding the cells. Dif-
ferent concentration gradients of oxygen, nutrients,
ions, and chemicals create a wide variety of microhabi-
tats providing conditions suitable for bacterial coloni-
zation. Stewart & Franklin (31) provided a nice
picture of phenotypic cell differentiation related to the
oxygen and nutrient gradients found in a mono-
species biolm of a facultative anaerobic bacterial
species. In their model, three different physiological
states are anticipated. Cells located in the upper
biolm layers consume all available oxygen and grow
aerobically, while an anaerobic micro-niche developed
underneath the aerobic layer. Oxygen- and nutrient-
depleted regions are found at the bottom layers of the
biolm structure and under these circumstances, most
of the sessile cells are metabolically inactive or dead.
Consequently, the individual bacterial cell response
to the local microenvironment leads to phenotypic
heterogeneity.
Switching to generate alternate phenotypes can also
occur spontaneously. A clonal population may have
two or more subpopulations with distinct phenotypic
properties, arising due to noise in gene expression
patterns. Stochastic noise arises from random uctua-
tions in protein abundance among individual cells due
to inherent stochasticity in gene expression (uctua-
tions in transcription initiation or mRNA degrada-
tion). Stochastic gene expression equips cells to face
environmental perturbations, an insurance against
future catastrophe, whether or not it occurs (32).
Genetic mutations in biolms may also be a direct
cause of phenotypic heterogeneity. Indeed, genetic
variations occurring through point mutation, inser-
tion, or deletion produce genetic variants and contrib-
ute to the increased phenotypic variability of the
biolm. Adaptive mutation is the term for mutations
that originate in a slowly growing or dormant popu-
lation under environmental stress and counteract the
factors causing the stress (33). Although adaptive
mutations are spontaneous, they result in the pheno-
typic variations conferring a tness advantage and pro-
moting survival of the population as a whole under
stressful conditions.
Dufour et al.
6
Dental plaque biolm
Oral microbial communities are some of the most
complex human microbiota. A study done by Zaura
et al. (34), analyzing the microbiomes of several intra-
oral niches (tooth surface, cheek, hard palate, tongue,
and saliva) from three healthy subjects using pyrose-
quencing, highlighted the presence of over 3,600
unique nucleotidic sequences per individual. These
sequences corresponded to specic variable regions of
the small subunit ribosomal RNA, enabling inter- and
intra-species distinctions in the bacterial world. Among
them, over 500 different species-level phylotypes, and
88104 higher taxa (genus or more inclusive taxon)
were found. The taxa belonging to Firmicutes, Proteo-
bacteria, Actinobacteria, Bacteroidetes, and Fusobac-
teria were predominant. The highest diversity was
associated with tooth samples, while the lowest diver-
sity was observed in the cheek samples. Recent esti-
mates of the number of bacterial species using
massively parallel DNAsequencing have suggested that
the oral cavity may contain as many as 19,000 bacterial
phylotypes, while each individual will only have a pro-
portion of these microbes (35).
Several factors may contribute to the microbial
diversity of the oral biolm, such as abundance of
nutrients, moisture, hospitable temperature, and the
availability of different surfaces (tongue, epithelial cells,
enamel). The community of microorganisms that
forms on the tooth surface is called the dental plaque
biolm. Dental plaque is perhaps the best-studied
biolm and serves as a good model for the study of
other microbial biolms. Dental plaque forms within
minutes or a few hours after a professional cleaning,
and is composed of more than 700 different bacterial
species. Extensive clinical studies have indicated that
the oral microbial ora is responsible for two major
biolm-related diseases: dental caries and periodontal
diseases (36). The bacteria comprising the dental
plaque biolm are not randomly distributed. Indeed,
dental plaque shows a high degree of organization and
is formed by sequential and ordered colonization of
Fig. 2. Processes leading to intra-species heterogeneity within a mono-species microbial biolm. Phenotypically
distinct subpopulations may arise following individual bacterial cell response to the local microenvironment, the
stochastic switching due to noise in gene expression patterns, and genetic mutations occurring through point
mutations (see text for additional details).
Bacterial biolm
7
multiple species of bacteria (37). During the process of
dental plaque formation, some oral bacterial species,
known as early or primary colonizers, adhere to the
acquired pellicle coating the tooth surface, which is
composed of salivary proteins (sialyted mucins,
a-amylase, prolin-rich proteins, agglutinin, statherin)
and bacterial cell fragments. Late colonizers then bind
to the already-attached primary colonizers. Although
streptococci make up 4782% of the microbiota colo-
nizing the tooth surface, studies by Socransky et al.
(38,39) identied six specic microbial groups or bac-
terial complexes within dental plaque biolm
(Table 3). The supragingival plaque is dominated by
Gram-positive bacteria (Streptococcus sp., Actinomyces
sp.) while the subgingival plaque is made up primarily
by Gram-negative anaerobic bacteria (A. actinomy-
cetemcomitans, F. nucleatum, Camphylobacter spp.,
Capnocytophaga spp., E. corrodens, P. gingivalis, P. in-
termedia, T. forsythia, oral spirochetes).
Under normal circumstances, the dental plaque
remains relatively stable (microbial homeostasis), con-
tributing to dental health (40,41). There were two
main schools of thought on the role of plaque bacteria
in relation to health and disease: the non-specic
plaque hypothesis, and the specic plaque hypothesis.
In the non-specic plaque hypothesis, the disease is
considered to be an outcome of the overall activity of
the total oral biolm community. Indeed, the non-
specic plaque hypothesis proposed that a heteroge-
neous mixture of microorganisms could play a role in
disease and that disease onset and progression are
thought to result from the increase in plaque beyond a
threshold level at which the host defenses were no
longer able to neutralize the plaque bacteria and their
toxic products. Several studies have been undertaken
to determine the microbial composition of dental
plaque from diseased sites to identify the pathogenic
organisms responsible, which led to the proposal of
the specic plaque hypothesis. This hypothesis states
that only a few species of the dental plaque biolm are
actively involved in disease. Although distinct differ-
ences in the composition of plaque at sites of health
and disease are described, disease occurs in the appar-
ent absence of these putative pathogens, while these
organisms may be recovered from healthy sites.
Recently, an alternative hypothesis has been proposed
that integrates the key elements of both the specic
and non-specic plaque hypotheses. The ecological
plaque hypothesis proposes that potentially patho-
genic organisms can be present in health but at levels
that are not clinically relevant. In the ecological plaque
hypothesis, the disease is a result of a shift in the
balance of the resident microora due to a response to
a change in local environmental conditions (e.g. sugar-
rich diet, low saliva ow, immune suppression) leading
to major ecological pressure. These conditions disrupt
the natural homeostasis present in plaque during
health, leading to an environment of organisms that
can cause disease. For example, a sucrose-rich diet
produces prolonged conditions of acidic pH. This
environmental change in dental plaque favors the
growth of acid-tolerating bacteria, which are capable
of demineralizing enamel, at the expense of healthy-
associated species (42).
Communication language in biolm
A few years ago, Watnick & Kolter (43) proposed the
interesting idea that biolms can be compared to
cities. In these biolm cities, the microbes are distrib-
uted geographically based on their neighbors and envi-
ronment. In these cities of microbes, microorganisms
are considered social organisms able to communi-
cate with one another. Using different chemical lan-
guages, bacteria learn about their current cell
population and determine when they have reached a
critical mass. Using that information, bacteria can thus
modify their behavior to carry out processes that
Table 3: Microbial complexes detected in dental
plaque biolm
Group Species
Early colonizers
Yellow complex Streptococcus oralis, S. sanguinis, S. mitis,
S. gordonii, S. intermedius
Blue complex Actinomyces spp.
Green complex Capnocytophaga spp., Eikenella corrodens,
Aggregatibacter actinomycetemcomitans
Purple complex Veillonella parvula, Actinomyces
odontolyticus
Late colonizers
Orange complex Fusobacterium nucleatum subsp.,
Prevotella intermedia, P. nigrescens,
Peptostreptococcus micros,
Camphylobacter spp., Eubacterium
nodatum, Streptococcus constellatus
Red complex Porphyromonas gingivalis, Tannerella
forsythia, Treponema denticola
Dufour et al.
8
would require many cells acting in conjunction to be
effective. Cell-to-cell communication is generally
carried out by diffusible signal molecules produced
and released by bacteria.
When bacteria are growing within a biolm, they
secrete signaling molecules (autoinducers) that
increase in concentration as a function of bacterial cell
density. In a process called quorum sensing, bacteria
communicate with one another by using autoinducers
to regulate their gene expression in response to uc-
tuations in the cell population density (44). Differen-
tial gene expression results in heterogeneity within the
biolm. Two types of quorum-sensing systems are
recognized in bacteria: intra-species communication
and inter-species communication. During intra-species
communication, several autoinducers have been
identied. Gram-negative bacteria usually use acyl
homoserine lactone (AHL) as signal molecules, while
Gram-positive bacteria utilize small peptides. The
small peptides used by Gram-positive bacteria are
usually the products of oligopeptides that are cleaved
and/or modied before being exported outside the
cells by specic transporters. During inter-species
communication, bacteria use autoinducer-2 (AI-2), a
furanosyl borate diester. The AI-2 molecule and the
luxS gene required for its synthesis occur in a wide
variety of Gram-negative and Gram-positive bacteria;
it is the only species-nonspecic autoinducer. For this
reason, AI-2 has been proposed as the universal signal
for inter-species communication. Despite AI-2 being
produced by many genera, there is very little evidence
linking AI-2 with direct activation of any specic gene
(45). Nevertheless, the widespread changes in gene
expression induced by AI-2, combined with its appar-
ent activity at extremely low concentrations, are con-
sistent with a role in quorum sensing (46).
The role of a quorum-sensing system in the regula-
tion of biolm formation was originally reported for
Pseudomonas aeruginosa by Davies et al. (47). Subse-
quently, other groups have also investigated connec-
tions between quorum sensing and biolms. In some
cases, quorum sensing does not seem to be involved
in biolm structural development, while for other
species, there is evidence that quorum sensing is
important for the attachment of bacteria to the
surface, the maturation of the biolm, or the control
of events leading to the dispersion of cells.
Quorum sensing has been linked to biolm structure
in many oral streptococcal species. For instance, the
potential role of quorum sensing has been investigated
in the cariogenic biolm organism Streptococcus
mutans using several quorum-sensing decient
mutants. In S. mutans, the intra-species quorum-
sensing system is mediated by a signal peptide mol-
ecule known as competence stimulating peptide
(CSP). The CSP of S. mutans is part of the strepto-
coccal competence stimulating peptide family. This
family of autoinducers consists exclusively of signal
peptide precursors that are up to 50 amino acids long.
In all members of this family, the precursor is cleaved
after two conserved glycine residues to produce the
mature CSP signal molecule used in quorum-sensing
regulation (48). Several quorum-sensing S. mutans
decient mutants were assessed for their ability to
initiate biolm formation. The results showed that
mutants unable to produce or detect CSP formed
abnormal biolms (49). Recently, the CSP molecule
has also been shown to trigger autolysis in a fraction of
the S. mutans population at high concentrations, con-
tributing to biolm formation through the release of
chromosomal DNA into the EPS matrix (50). Work
done by Sztajer et al. (51) showed that biolm forma-
tion was also impaired in an S. mutans DluxS mutant
unable to produce AI-2. Although transcriptome
analysis of DluxS revealed changes in many biolm
genes, the addition of synthetic AI-2 molecules could
not restore the gene expression prole to the wild-type
level, suggesting that the biolm-decient phenotype
may be caused by central metabolic defects rather than
quorum-sensing related signaling.
Multi-drug tolerance of biolms
A signicant characteristic of microbial biolms is their
high-level drug tolerance. Perhaps the rst experiment
showing that biolm cells were more tolerant to drugs
than planktonic cells was done by Leeuwenhoek when
he failed to kill plaque bacteria in situ on his teeth by
prolonged rinsing with vinegar, while the microorgan-
isms were killed if they were rst removed from the
teeth and mixed with vinegar in the laboratory. Bacte-
rial biolms have been shown to have a 100- to 1,000-
fold increased tolerance toward antibiotics in
comparison to their free-swimming counterparts (52).
Although antibiotics may diminish the amount of bac-
teria within biolms, antimicrobial treatment does not
completely eradicate the pathogen, and thus leads to
refractory/relapsing infections. The fact that biolms
Bacterial biolm
9
also represent a protected mode of growth, which
allows microbes to survive the host immune defense,
presents a real challenge in the treatment of biolm
infections. The tolerance of microbial biolms to anti-
biotics does depend on the bacterial species, but none-
theless multiple factors contribute to the increased
tolerance. This renowned tenacity of biolms may be
due to a diffusion barrier imparted by the EPS matrix,
the physiological state of biolm-growing cells, the
induction of specic resistance mechanisms, and/or the
development of dormant persister cells (Fig. 3). It is
thus essential to understand the mechanisms that
promote tolerance to antimicrobials in order to develop
novel strategies to treat biolm infections.
Protective role of EPS matrix
A primary function that has been attributed to the
biolm matrix is protection. Several studies have been
performed to examine the diffusion of antibiotics
through biolms and they have shown that the EPS
matrix can act as an impermeable barrier to limit anti-
microbial penetration, thereby protecting the biolm
cells (25,29). Suchprotectioncanbe due either to phys-
ical hindrance in antimicrobial diffusion or to direct
binding of the antibiotics by the EPS matrix. Upon
antibiotic treatment, cells at the top of the liquid
biolm interface die due to their closer exposure, while
bacteria embedded deep inside the biolm are able to
survive. One such example is pulmonary infection in
cystic brosis (CF) patients. During the course of infec-
tion, P. aeruginosa usually undergoes a phenotypic
switch to a mucoid colony, which is characterized by the
overproduction of the EPS alginate (52). The alginate
produced by P. aeruginosa directly contributes to the
tenacity of the bacterial infections in the lung by pro-
tecting microbial cells from opsonization by host anti-
bodies and by preventing the diffusion of antibiotics to
target cells. The biolmmatrix can also be considered a
chemically active barrier. Anionic EPS matrix can bind
and sequester toxic cationic heavy metals, cationic anti-
microbial peptides, and positively-charged antibiotics
(e.g. aminoglycosides) (53). In contrast, for relatively
uncharged antibiotics such as b-lactams, such binding
to the EPS matrix is unlikely to occur (54).
The EPS matrix is one of the distinguishing features
of a microbial biolm involved in antibiotic tolerance.
However, for many antibiotics, the EPS matrix pro-
vides little or no barrier to antibiotic penetration.
Therefore, the reduced penetration of antibiotics due
to the biolm matrix may not be a general protection
mechanism.
Altered microenvironment and
stress responses
Phenotypic heterogeneity occurs within biolms (as
discussed in the previous section on cell heterogene-
Fig. 3. Schematic representation of the different mechanisms involved in the multi-drug tolerance of biolms. The
EPS matrix acts to restrict the penetration and diffusion of some antimicrobials. Bacteria within the biolm can also
secrete b-lactamases into their surrounding environment and/or increase the expression of multi-drug resistance
(MDR) efux pumps. The activation of quorum-sensing systems along with different concentration gradients of
nutrients, oxygen, and metabolic waste products also make important contributions to antibiotic tolerance and
resistance to the host immune system. The presence of persisters that are resistant to killing by all antibiotics may also
be responsible for recalcitrant biolm infections.
Dufour et al.
10
ity) notably due to different concentration gradients.
Cells localized at the bottom layers of the biolm are
normally found in a growth stage analogous to
stationary-phase planktonic cells, while the physiol-
ogy of cells localized at the top surface layers is
similar to exponentially growing planktonic cells
(31). Microbial cells, especially those in the deeper
layers of the biolms where nutrients and oxygen are
limited, are associated with a lower growth rate. This
reduced metabolic activity might account for the
enhanced tolerance toward antibiotics that target
bacterial cellular processes such as DNA replication
or translation. Conventional antibiotics used to treat
infections are mostly effective at killing rapidly
growing cells. The decreased metabolic activity of
cells found within the deeper biolm layers may thus
contribute to antibiotic tolerance and the persistence
of biolm infections.
Many bacterial species encode antibiotic resistance
mechanisms in the planktonic phase that aid in cell
survival. Although these resistance mechanisms do
contribute to the antibiotic tolerance of biolm cells,
they alone are not sufcient to explain the recurrence
of biolm infections. One mechanism that has recently
been explored is the role of drug efux pumps. There
has been evidence that many membrane-bound drug
efux pumps found in both Gram-negative and Gram-
positive bacteria are induced by exposure to sub-lethal
concentrations of various antibiotics (55). The origin
of these transporters was to remove metabolites and
by-products within bacterial cells and, over time, they
evolved to efux out other harmful molecules such as
antimicrobial agents. These efux pumps can vary
from having specicity for a single antibacterial agent,
to multi-drug pumps with broad substrate specicity
that are able to remove structurally unrelated antimi-
crobials. In P. aeruginosa, the MexAB-OprM and
MexCD-OprJ efux pumps are also essential to the
normal development of the biolm when cells
are challenged by the antibiotic azythromycin, as
mutants defective in both efux pumps were unable to
form a biolm when grown under the same stress
conditions (56).
Bacteria in biolms and planktonic cultures can also
express stress-responsive genes and switch to more
tolerant phenotypes upon environmental stressors
(e.g. starvation, heat or cold shock, cell density, pH,
osmolarity). Sigma factors are key regulators of
general stress responses in bacteria. In Salmonella,
cells lacking sigmaE had an increased susceptibility to
oxidative stress during the stationary phase (57). In
E. coli, the main transcriptional regulator induced by
environmental stresses is the alternate sigma factor,
RpoS. This sigma factor is involved in preventing
DNA damage from stress factors through the regu-
lation of multiple mechanisms, but it is also involved
in regulating metabolism. RpoS is induced by high
cell density, and while initially observed in the sta-
tionary phase, it has now been linked to biolms
(58). Mutants that lack rpoS are incapable of forming
biolms. RpoS has been shown to be up-regulated in
P. aeruginosa biolms in vitro and from samples
extracted from cystic brosis patients (59).
The role of quorum sensing and intracellular signal-
ing molecules in antimicrobial tolerance of biolms
has also been investigated. Alarmones or pheromones
are synthesized by bacterial cells within the biolm
community due to certain types of stresses (60). An
example is the synthesis of ppGpp in response to
amino acid starvation that leads to the generation of
non-dividing cells. Such dormant cells are important
in biolm formation and in the development of multi-
drug tolerance (see next section). Studies have shown
that the use of quorum-sensing inhibitors was able to
enhance susceptibility of P. aeruginosa biolms to
antimicrobial treatments (61). Another study with
P. aeruginosa demonstrated that increased drug toler-
ance through the formation of non-dividing cells was
induced by quorum sensing-related signaling mol-
ecules (62). Because they are not yet fully understood,
signaling molecules and cell-to-cell communication
in relation to multi-drug tolerance require further
investigation.
Persister cells
The formation of persister cells is another mechanismof
antibiotic tolerance. Within a given population of bac-
teria, a small subpopulation known as non-growing
persisters exists (63,64). It has been suggested that
these specialized cells enter into a state of dormancy,
which allows them to survive stress conditions and
prevents death because antibiotics target cell growth.
Persister cells are extremely tolerant to high concentra-
tions of antibiotics. They are not antibiotic-resistant
mutants, but rather phenotypic variants of the wild-
type that form stochastically in a clonal population of
genetically identical cells (65). Persisters constitute a
Bacterial biolm
11
small fraction of stationary phase planktonic cultures
and biolm populations of numerous species. The
number of persisters in a growing population of bacte-
ria rises at the mid-logarithmic phase and reaches a
maximum of approximately 1% at the stationary phase.
Similarly, slow-growing biolms produce substantial
numbers of persister cells. Persisters resuscitate from
their dormant state when a stationary culture is inocu-
lated into fresh medium. A model of antibiotic toler-
ance of bacterial biolms based on persister survival has
been proposed (66). An initial treatment with antibi-
otic kills planktonic cells and the majority of biolm
cells, leaving persisters intact. The host immune system
targets and kills planktonic persisters, but the biolm
persisters are protected from host defenses by the
biolm matrix. After the antibiotic treatment is
stopped, persister cells repopulate the biolm and the
infection relapses. Based on this model, persisters have
a much broader clinical signicance than only in the
context of biolm infections. Indeed, these specialized
cells are likely to play a critical role in recalcitrance to
therapy whenever the immune response is limited. Such
cases would include disseminated infections in immu-
nocompromised patients (e.g. HIV-positive, cancer
chemotherapy) or immunocompetent patients in cases
where the pathogen is located at bodily sites inacces-
sible to many of the host defense mechanisms (63).
All antibiotic resistance mechanisms do essentially the
same thing: they prevent an antibiotic from binding to
the target. By contrast, persister tolerance works by
shutting down these targets. One of the main questions
about bacterial persistence is whether bacteria have
evolved a dedicated mechanism that allows them to
survive exposure to bactericidal antibiotics. Such a
mechanism may potentially be based on a deliberate or
sporadic expression of toxic proteins in a small fraction
of cells to maintain them in a dormant or non-growing
state. Single-cell analysis revealed that several bacterial
toxinantitoxin systems are over-expressed in persister
cells. Toxinantitoxin systems are small genetic
modules consisting in general of two components: a
stable toxin and its labile antitoxin. Typically, the toxin
inhibits an essential microbial cell function such as
translation or DNA replication (67). Under conditions
that preclude the continuous synthesis of the antitoxin,
the toxin can exert its toxic effect to inhibit cell growth.
It has been shown that ectopic mild over-expression of
unrelated proteins also induces persistence (63). This
indicates that persistence might be the result of stochas-
tic expression of a broad variety of genes, some of them
encoding products that may become toxic.
New weapons in the battle against
biolm infections
The presence of microbial biolms is the main cause of
inefcient eradication by antibiotic therapy. Biolm
bacteria are often tolerant to doses of antibiotics
several hundred-fold over the minimum lethal dose for
bacteria living outside the biolm. The concentrations
that would be expected to eradicate biolm infections
often exceed the highest deliverable doses of antibiot-
ics. Furthermore, the widespread use of antibiotics
(both inside and outside the medical eld) is playing a
signicant role in the emergence of antibiotic-resistant
bacteria. Consequently, more work is needed to fully
understand the mechanisms involved in antibiotic tol-
erance and to develop new therapeutic strategies to
combat biolm infections. This section will briey
introduce some possible approaches that could be
explored in the search for new anti-biolm strategies
to eradicate medically relevant biolms.
Interference with bacterial
communication systems
One strategy might be to develop analogs of microbial
signaling molecules that interfere with the cell-to-cell
communication required for normal biolm forma-
tion. There is growing evidence that quorum sensing
constitutes a global regulatory system in many differ-
ent bacterial species (68). Consequently, interfering
with the action of a global regulator would necessarily
produce pleiotropic phenotypes. For example, any-
thing that affects cell motility or cell surface chemistry
might translate into a biolm-decient phenotype.
The idea to use anti-quorum sensing molecules origi-
nated from the discovery of quorum-sensing inhibi-
tion by halogenated furanones from the Australian red
macro alga Delisea pulchra (69). This alga had devel-
oped chemical defense mechanisms to inhibit bacterial
colonization by producing natural products (halo-
genated furanone compounds) that have been shown
to interfere with the quorum-sensing systems of
several bacteria (70). One of the major problems asso-
ciated with antibiotics is the development of resis-
tance. Conventional antibiotics inherently favor the
evolution of resistance because they pose a strong
Dufour et al.
12
selective pressure on bacteria. Because quorum sensing
is not involved in bacterial growth, inhibitors of
quorum sensing should not yield a strong selective
pressure for the development of resistance. Quorum-
sensing inhibitors could then be viewed as blockers of
pathogenicity rather than as antimicrobials.
Specically targeted antimicrobial peptide
(STAMP) technology
Broad-spectrum antibiotics may disrupt the patients
normal bacterial ora. Antimicrobial treatment
specically targeting each undesirable pathogen repre-
sents an attractive strategy. In 2006, a research group
from the University of California, Los Angeles,
reported the design of narrow spectrum molecules
known as specically targeted antimicrobial peptides
(STAMPs) (71,72). Construction of these molecules
requires the fusion of a species-specic targeting
peptide domain that provides specic binding to a
selected pathogen with a wide-spectrum antimicrobial
peptide domain. The killing peptide domain is usually
a membrane-active antimicrobial peptide either syn-
thesized chemically or a natural component of the
innate immunity (e.g. defensins, integrins, cathelici-
dins). STAMPs have been shown to effectively elimi-
nate the cariogenic biolm organism S. mutans from a
multi-species biolm without affecting closely related
non-cariogenic organisms. These STAMPs were con-
structed with peptides derived from the quorum-
sensing CSP signal molecule for selective S. mutans
binding. STAMPs represent a promising technology
for the elimination of pathogens in human microbiota
in disease while preserving the microorganisms asso-
ciated with a healthy ora.
Persister eradication
Dormant persister cells are likely to be largely respon-
sible for multi-drug tolerance and for many recurrent
biolm infections. With their ability to survive even
high concentrations of antibiotics, the need for novel
therapeutics capable of killing persister cells is impor-
tant for treating microbial biolm infections. Further
studies into the elucidation of the mechanistic basis for
the formation of persisters could lead to the develop-
ment of drugs that prevent the formation of persisters.
However, due to the multiple genetic and redundant
mechanisms that appear to be involved in persister
formation, ascertaining a drug for target selection may
be difcult. However, antimicrobials that target the
bacterial membrane organization may be promising.
These agents would be similar to antimicrobial pep-
tides synthesized by the host innate immune system,
where these molecules act directly on the membrane to
kill cells. Such membrane-acting antimicrobials would
be lipophilic to directly bind and permeabilize the
bacterial membrane bilayer to disrupt the physical
integrity and numerous cellular functions, such as
membrane-bound enzymes involved in energy produc-
tion (73). Two recently approved membrane-acting
antibiotics, daptomycin and telavancin, have been in
clinical use for treating S. aureus infections (74). In
addition, both of these antimicrobial agents, as well as
other membrane-acting antibiotics used in clinical
trials, have shown activity against S. aureus biolms
through the permeabilization of the bacterial mem-
brane. Resistance toward these antibiotics is likely to be
rare, but is still possible. Alternatively, the identication
of factors specically enhancing the switching rate from
dormant to normal growing cells could also suggest
different drug targets (75). Bacterial growth factors,
quorum sensing-related factors, or resuscitation-
promoting factors might be promising candidates.
Probiotics
A promising alternative strategy to treat chronic
biolm infections is bacteriotherapy or the use of
selected harmless bacteria to displace pathogenic
organisms. The introduced probiotic strains can be
either wild-type commensals or avirulent (genetically
modied) bacterial strains. Probiotic microorganisms
can be used to prevent or to combat diseases caused by
pathogens (76). Species-replacement studies have
been found to be effective in the prevention of dental
caries and in preventing recurrent otitis media. Roos
et al. (77) showed that nasal spray bacteriotherapy
with commensal streptococci were used to replace the
normal nasopharyngeal ora in children with recurrent
otitis media. Numerous studies have reported the pre-
ventive effect of long-term probiotic consumption on
dental caries in children (78,79). The probiotic bacte-
rium Streptococcus salivarius TOVE-R was successfully
used to competitively displace the cariogenic strains
S. mutans and S. sobrinus on rat teeth (80). More
recently, an in vitro study showed that different lacto-
cocci probiotics (L. rhamnosus, L. reuteri, L. plan-
Bacterial biolm
13
tarum) were able to inhibit the formation of biolm
by different clinical isolates of S. mutans, and even to
signicantly reduce their viability (81).
Concluding remarks
Microbiologists have traditionally focused on free-
swimming bacteria growing in laboratory asks; yet
they have recently come to realize that in the natural
world more than 99% of all bacteria live in biolm
communities. As a result, biolmresearch is nowone of
the hottest topics in microbiology. The cost to society
associated with biolms (e.g. infections, equipment
damage, product contamination) is estimated to be in
the billions of dollars annually. At least 65% of all
bacterial infections have biolm as an integral part of
their pathogenesis. The fact that microbial biolms
represent a protected mode of growth that allows cells
to survive hostile environments presents a challenge in
the treatment of biolminfections. Of importance with
respect to medicine, biolms compromise quality of life
and may be associated with mortality. The existence of
microbial biolms suggests an important conceptual
change in our understanding of the bacteria involved in
mild or life-threatening illnesses. Antibiotics have not
been specically developed to target microbial biolm
infections. Despite extensive efforts, no antimicrobial
drug has yet been found that completely eradicates
adherent microbial populations. New information on
the physical factors that affect biolm formation within
the body and the genetic basis of how pathogenic
microorganisms persist in biolms and how this relates
to tolerance mechanisms is essential in the development
of novel antibiolm strategies.
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