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Endodontic Topics 2012, 22, 216


All rights reserved

2012 John Wiley & Sons A/S


ENDODONTIC TOPICS 2012
1601-1538

Bacterial biofilm:
structure, function, and
antimicrobial resistance
DELPHINE DUFOUR, VINCENT LEUNG & CLINE M. LVESQUE
Biofilms are microbial communities attached to surfaces and encased in an extracellular matrix of microbial origin.
They represent the predominant form of microbial life. Biofilms are everywhere and can develop on virtually every
natural and man-made surface. Biofilms are also ubiquitous in both normal and pathogenic human processes.
Biofilm 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 biofilm lifestyle helps
bacteria survive and persist within the environment. This review discusses the fundamental biology of microbial
biofilm and how biofilms impact the pathogenesis of human infections. The different mechanisms involved in the
reduced antimicrobial susceptibility of microorganisms in pathogenic biofilms are discussed in detail in this review.
Possible approaches that could be explored in the search for new anti-biofilm strategies to eradicate medically
relevant biofilms are also presented.
Received 20 May 2011; accepted 13 November 2011.

Introduction
A brief history of biofilms
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 first biofilm experimenter.
More than two centuries later, the American bacteriologist 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 find, in


addition to the algae, a thin and uniform coating of
bacteria of various forms, some of unusual morphology. Henrici reported in 1933 that It is quite evident
that for the most part the water bacteria are not free
floating 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 ecologist Claude ZoBell made important contributions to
biofilm microbiology. ZoBell found that when seawater 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).

Bacterial biofilm
What these pioneering scientists were describing was
what we now call biofilm. A biofilm 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 understanding that natural bacterial populations exist mostly
in biofilm communities.

Biofilm: 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 biofilm populations interacting extensively with each other (6). Indeed, most
bacteria in nature do not exist independently but
persist in co-ordinated, spatially organized, and metabolically integrated biofilm communities. Biofilms are
ubiquitous and can develop on virtually every natural
and man-made surface. In natural habitats, biofilms
generally provide homeostasis in the face of fluctuating
and harsh environmental conditions (7). They can
develop under extreme environmental conditions. For
example, a research group has recently reported the
presence of microbial biofilms inhabiting thermal
waters (3550C) more than 60 meters below sea level
in Italy (8). Other biofilms have been found living on
frozen glaciers. In 2009, a glacier sample collection in
the Antarctic coast revealed the presence of a seawater
biofilm composed of a novel bacterial species, Antarcticimonas flava (9). Other than these very hot and cold
environments, biofilms can also be observed in extreme
acidic environments, characterized by a high metal
content and lack of nutrients. Moreover, bacterial biofilms 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, biofilms cause food and drinking water
contamination, metal surface corrosion, and clogging
(12,13). Due to the possibility of water contaminated
with pathogens, biofilms present a serious hazard to
the drinking supply. In the petrochemical industry,
biofilms decrease the efficiency of equipment such as
oil pipelines and platforms. Biofilms can also develop
on ship hulls, fishing traps, and nets, representing a
major economic problem. Although biofilms can be
extremely costly to industry, positive aspects of the
practical application of biofilms exist. For example,

beneficial biofilms are used in the production of industrial chemicals (e.g. ethanol, vinegar) and in the
removal of toxic compounds during the treatment of
wastewater and oily seawater.
Biofilms are also greatly associated with vegetable
life. The role of symbiotic nitrogen fixers and bacterial
biofilms in legumeRhizobium interactions and metaltolerant microbes in the improvement of legumes is of
great interest to professionals working in the field of
agronomy. In contrast, plant pathogens such as
Pseudomonas syringae display detrimental effects on
plant growth, development, and crop yield by infecting a wide range of plant species (e.g. olive, tomato,
rice, soybean) and causing chlorosis, necrosis, or
canker (14).
Biofilms are also ubiquitous in both normal and
pathogenic human processes. Microorganisms comprising the human-associated microbiota are essential
for host development and health. Bacteria play important roles such as in the production of vitamins and
essential amino acids. They can also help prevent colonization by exogenous pathogens. One such example
is the Escherichia coli strain Nissle 1917. This biofilm
organism has been used for many decades as a probiotic against a variety of intestinal disorders (15).
Biofilm 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 (16). Therefore, biofilms provide a new
perspective through which to view infectious diseases.

Biofilms in infectious diseases


Biofilms are a major cause of human infections (17).
The majority of hospital-acquired infections are due to
biofilms because they can be life-threatening colonizers of biomedical devices. Indeed, a large proportion
of nosocomial infections are related to the colonization 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 infections are associated with a urinary catheter, 80% of
pneumonias are associated with mechanical ventilation, and 87% of bloodstream infections are associated
with intravascular devices. Staphylococci and enterococci are responsible for the frequent cases of
hospitalization-acquired infections. These two bacterial genera are commensal inhabitants of the human

Dufour et al.
Table 1: Biofilms 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

Artificial hip prosthesis

CoNS, S. aureus, Enterococcus spp.

Voice prostheses

C. albicans, CoNS

Endotracheal tubes

Enteric Gram-negative species

* CoNS: coagulase-negative staphylococci.

flora of the skin, upper respiratory tract, lower gastrointestinal 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. epidermidis, S. lugdunensis, S. haemolyticus) and Staphylococcus 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. epidermidis nosocomial infections tend to be chronic and
less acute, while S. aureus is involved in acute infections because of its ability to elicit a more acute
immune response from the host (20).
Enterococci have emerged in recent years as pathogens associated with serious nosocomial infections
despite their low level of virulence. They cause infection
almost exclusively in hospitalized patients who have
significantly compromised immune defenses. Two
major bacterial species account for the vast majority of
biofilm 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 flora 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 biofilm infections, including periodontitis, cystic fibrosis 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 (freefloating) growth, in chronic bacterial infections, the
planktonic phenotype generally exists only transiently,
and usually as a minor population. Since chronic infections are fundamentally different from acute infections, different strategies are necessary to treat the
biofilm infections more efficiently. The fact that microbial biofilms represent a protected mode of growth
that allows cells to survive hostile environments presents a challenge for treating chronic biofilm infections
(23,24). Chronic infections are thus important clinically because bacteria in biofilms resist host immune
responses and antibiotic treatment and these characteristics are often cited as the reason bacteria have the
ability to persist for a long time in the human body.

Biofilm lifestyle
Biofilm formation
The development of a microbial biofilm can be
described as a dynamic process involving successive
steps (Fig. 1). The first step is the attachment of the
bacterial cells to the selected abiotic or biotic surface.
Bacteria usually adhere to a conditioning film typically
composed of organic molecules (e.g. nutrients, salivary proteins, large macromolecules) that can promote
the adherence of bacteria to the surface. Initial attachment is mediated through weak reversible van der
Waals interactions between the cell surface and the
substratum, which can lead to stronger adhesionreceptor mediated attachment (25). Bacterial cell
surface structures such as flagella, fimbriae, LPS, and
exopolysaccharides participate in irreversible interactions. 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 first attached cells (primary
colonizers). The micro-colonies progressively enlarge

Bacterial biofilm
Table 2: Diversity of human infections involving biofilms
Anatomic location

Infectious disease

Microorganisms

Eye

Ocular infections

S. aureus

Ear

Otitis media

Non-typeable H. influenzae

Mouth

Dental caries
Endodontic infections
Periodontitis

Acidogenic Gram-positive cocci


Gram-positive anaerobic species, Bacteroides,
Neisseria, Fusobacterium
Gram-negative anaerobic oral bacteria

Respiratory tract

CF pneumonia
Chronic sinusitis

P. aeruginosa, B. cepacia
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 biofilm development. The different stages in biofilm
formation include initial attachment to the surface, formation of a monolayer along the surface with formation of
micro-colonies, biofilm maturation with formation of a three-dimensional structure, and cell dispersion.

Dufour et al.
and coalesce to form the first 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 biofilm characterized 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 biofilm cells can detach individually
or in clumps. In general, biofilm dispersion occurs in
response to environmental changes and is dependent
on growing conditions (26).
The molecular mechanisms that regulate the distinct
developmental steps in biofilm formation vary greatly
between different bacterial species, and even depend
on the environmental conditions for the same given
species. Nevertheless, biofilms display a common
attribute, the biofilm matrix. Contrary to free-floating
planktonic cells, biofilm cells are embedded in a selfproduced extracellular matrix, the extracellular polymeric substance (EPS), that holds them together (27).
Biofilms 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 biofilm and can confer
other beneficial properties. Since the EPS is also highly
hydrated, it can prevent desiccation in some natural
biofilms. 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 biofilms


Most biofilms found in nature are polymicrobials,
where diverse species expressing multiple phenotypes
are involved. The human oral dental plaque biofilm
and the biofilms of periodontal diseases are perhaps
the best described multi-species microbial biofilms
(see next section). Microbial biofilms are thus very
complex and heterogeneous environments characterized by a wide physical, chemical, and biotic diversity.
The most amazing fact is that even in a mono-species
biofilm, phenotypic heterogeneity exists. Cells of the
same bacterial species can exhibit different phenotypes
in a biofilm 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 monospecies microbial biofilm and they are illustrated in
Figure 2.
Mono-species biofilm communities are often composed of phenotypically distinct subpopulations. Cell
differentiation in biofilms may depend on the local
environmental conditions surrounding the cells. Different concentration gradients of oxygen, nutrients,
ions, and chemicals create a wide variety of microhabitats providing conditions suitable for bacterial colonization. Stewart & Franklin (31) provided a nice
picture of phenotypic cell differentiation related to the
oxygen and nutrient gradients found in a monospecies biofilm of a facultative anaerobic bacterial
species. In their model, three different physiological
states are anticipated. Cells located in the upper
biofilm layers consume all available oxygen and grow
aerobically, while an anaerobic micro-niche developed
underneath the aerobic layer. Oxygen- and nutrientdepleted regions are found at the bottom layers of the
biofilm 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 fluctuations in protein abundance among individual cells due
to inherent stochasticity in gene expression (fluctuations in transcription initiation or mRNA degradation). Stochastic gene expression equips cells to face
environmental perturbations, an insurance against
future catastrophe, whether or not it occurs (32).
Genetic mutations in biofilms may also be a direct
cause of phenotypic heterogeneity. Indeed, genetic
variations occurring through point mutation, insertion, or deletion produce genetic variants and contribute to the increased phenotypic variability of the
biofilm. Adaptive mutation is the term for mutations
that originate in a slowly growing or dormant population under environmental stress and counteract the
factors causing the stress (33). Although adaptive
mutations are spontaneous, they result in the phenotypic variations conferring a fitness advantage and promoting survival of the population as a whole under
stressful conditions.

Bacterial biofilm

Fig. 2. Processes leading to intra-species heterogeneity within a mono-species microbial biofilm. 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).

Dental plaque biofilm


Oral microbial communities are some of the most
complex human microbiota. A study done by Zaura
et al. (34), analyzing the microbiomes of several intraoral niches (tooth surface, cheek, hard palate, tongue,
and saliva) from three healthy subjects using pyrosequencing, highlighted the presence of over 3,600
unique nucleotidic sequences per individual. These
sequences corresponded to specific 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, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteria were predominant. The highest diversity was
associated with tooth samples, while the lowest diversity was observed in the cheek samples. Recent estimates of the number of bacterial species using
massively parallel DNA sequencing have suggested that

the oral cavity may contain as many as 19,000 bacterial


phylotypes, while each individual will only have a proportion of these microbes (35).
Several factors may contribute to the microbial
diversity of the oral biofilm, 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
biofilm. Dental plaque is perhaps the best-studied
biofilm and serves as a good model for the study of
other microbial biofilms. 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 flora is responsible for two major
biofilm-related diseases: dental caries and periodontal
diseases (36). The bacteria comprising the dental
plaque biofilm are not randomly distributed. Indeed,
dental plaque shows a high degree of organization and
is formed by sequential and ordered colonization of

Dufour et al.
Table 3: Microbial complexes detected in dental
plaque biofilm
Group
Early colonizers
Yellow complex
Blue complex
Green complex
Purple complex
Late colonizers
Orange complex

Red complex

Species
Streptococcus oralis, S. sanguinis, S. mitis,
S. gordonii, S. intermedius
Actinomyces spp.
Capnocytophaga spp., Eikenella corrodens,
Aggregatibacter actinomycetemcomitans
Veillonella parvula, Actinomyces
odontolyticus
Fusobacterium nucleatum subsp.,
Prevotella intermedia, P. nigrescens,
Peptostreptococcus micros,
Camphylobacter spp., Eubacterium
nodatum, Streptococcus constellatus
Porphyromonas gingivalis, Tannerella
forsythia, Treponema denticola

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 colonizing the tooth surface, studies by Socransky et al.
(38,39) identified six specific microbial groups or bacterial complexes within dental plaque biofilm
(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. actinomycetemcomitans, F. nucleatum, Camphylobacter spp.,
Capnocytophaga spp., E. corrodens, P. gingivalis, P. intermedia, T. forsythia, oral spirochetes).
Under normal circumstances, the dental plaque
remains relatively stable (microbial homeostasis), contributing 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-specific
plaque hypothesis, and the specific plaque hypothesis.
In the non-specific plaque hypothesis, the disease is
considered to be an outcome of the overall activity of
the total oral biofilm community. Indeed, the nonspecific 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 specific plaque hypothesis. This hypothesis states
that only a few species of the dental plaque biofilm are
actively involved in disease. Although distinct differences in the composition of plaque at sites of health
and disease are described, disease occurs in the apparent 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 specific
and non-specific plaque hypotheses. The ecological
plaque hypothesis proposes that potentially pathogenic 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 microflora due to a response to
a change in local environmental conditions (e.g. sugarrich diet, low saliva flow, 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 healthyassociated species (42).

Communication language in biofilm


A few years ago, Watnick & Kolter (43) proposed the
interesting idea that biofilms can be compared to
cities. In these biofilm cities, the microbes are distributed geographically based on their neighbors and environment. In these cities of microbes, microorganisms
are considered social organisms able to communicate with one another. Using different chemical languages, 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

Bacterial biofilm
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 biofilm, 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 fluctuations in the cell population density (44). Differential gene expression results in heterogeneity within the
biofilm. 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
identified. 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 modified before being exported outside the
cells by specific 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-nonspecific 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 specific gene
(45). Nevertheless, the widespread changes in gene
expression induced by AI-2, combined with its apparent activity at extremely low concentrations, are consistent with a role in quorum sensing (46).
The role of a quorum-sensing system in the regulation of biofilm formation was originally reported for
Pseudomonas aeruginosa by Davies et al. (47). Subsequently, other groups have also investigated connections between quorum sensing and biofilms. In some
cases, quorum sensing does not seem to be involved
in biofilm 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 biofilm, or the control
of events leading to the dispersion of cells.
Quorum sensing has been linked to biofilm structure
in many oral streptococcal species. For instance, the

potential role of quorum sensing has been investigated


in the cariogenic biofilm organism Streptococcus
mutans using several quorum-sensing deficient
mutants. In S. mutans, the intra-species quorumsensing system is mediated by a signal peptide molecule known as competence stimulating peptide
(CSP). The CSP of S. mutans is part of the streptococcal 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
deficient mutants were assessed for their ability to
initiate biofilm formation. The results showed that
mutants unable to produce or detect CSP formed
abnormal biofilms (49). Recently, the CSP molecule
has also been shown to trigger autolysis in a fraction of
the S. mutans population at high concentrations, contributing to biofilm formation through the release of
chromosomal DNA into the EPS matrix (50). Work
done by Sztajer et al. (51) showed that biofilm formation was also impaired in an S. mutans DluxS mutant
unable to produce AI-2. Although transcriptome
analysis of DluxS revealed changes in many biofilm
genes, the addition of synthetic AI-2 molecules could
not restore the gene expression profile to the wild-type
level, suggesting that the biofilm-deficient phenotype
may be caused by central metabolic defects rather than
quorum-sensing related signaling.

Multi-drug tolerance of biofilms


A significant characteristic of microbial biofilms is their
high-level drug tolerance. Perhaps the first experiment
showing that biofilm 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 microorganisms were killed if they were first removed from the
teeth and mixed with vinegar in the laboratory. Bacterial biofilms have been shown to have a 100- to 1,000fold increased tolerance toward antibiotics in
comparison to their free-swimming counterparts (52).
Although antibiotics may diminish the amount of bacteria within biofilms, antimicrobial treatment does not
completely eradicate the pathogen, and thus leads to
refractory/relapsing infections. The fact that biofilms

Dufour et al.

Fig. 3. Schematic representation of the different mechanisms involved in the multi-drug tolerance of biofilms. The
EPS matrix acts to restrict the penetration and diffusion of some antimicrobials. Bacteria within the biofilm can also
secrete b-lactamases into their surrounding environment and/or increase the expression of multi-drug resistance
(MDR) efflux 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 biofilm infections.

also represent a protected mode of growth, which


allows microbes to survive the host immune defense,
presents a real challenge in the treatment of biofilm
infections. The tolerance of microbial biofilms to antibiotics does depend on the bacterial species, but nonetheless multiple factors contribute to the increased
tolerance. This renowned tenacity of biofilms may be
due to a diffusion barrier imparted by the EPS matrix,
the physiological state of biofilm-growing cells, the
induction of specific 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 biofilm infections.

Protective role of EPS matrix


A primary function that has been attributed to the
biofilm matrix is protection. Several studies have been
performed to examine the diffusion of antibiotics
through biofilms and they have shown that the EPS
matrix can act as an impermeable barrier to limit antimicrobial penetration, thereby protecting the biofilm
cells (25,29). Such protection can be due either to physical 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
biofilm interface die due to their closer exposure, while
bacteria embedded deep inside the biofilm are able to

10

survive. One such example is pulmonary infection in


cystic fibrosis (CF) patients. During the course of infection, 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 protecting microbial cells from opsonization by host antibodies and by preventing the diffusion of antibiotics to
target cells. The biofilm matrix can also be considered a
chemically active barrier. Anionic EPS matrix can bind
and sequester toxic cationic heavy metals, cationic antimicrobial 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 biofilm involved in antibiotic tolerance.
However, for many antibiotics, the EPS matrix provides little or no barrier to antibiotic penetration.
Therefore, the reduced penetration of antibiotics due
to the biofilm matrix may not be a general protection
mechanism.

Altered microenvironment and


stress responses
Phenotypic heterogeneity occurs within biofilms (as
discussed in the previous section on cell heterogene-

Bacterial biofilm
ity) notably due to different concentration gradients.
Cells localized at the bottom layers of the biofilm are
normally found in a growth stage analogous to
stationary-phase planktonic cells, while the physiology 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 biofilms 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 biofilm layers may thus
contribute to antibiotic tolerance and the persistence
of biofilm 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 biofilm cells,
they alone are not sufficient to explain the recurrence
of biofilm infections. One mechanism that has recently
been explored is the role of drug efflux pumps. There
has been evidence that many membrane-bound drug
efflux pumps found in both Gram-negative and Grampositive 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 efflux out other harmful molecules such as
antimicrobial agents. These efflux pumps can vary
from having specificity for a single antibacterial agent,
to multi-drug pumps with broad substrate specificity
that are able to remove structurally unrelated antimicrobials. In P. aeruginosa, the MexAB-OprM and
MexCD-OprJ efflux pumps are also essential to the
normal development of the biofilm when cells
are challenged by the antibiotic azythromycin, as
mutants defective in both efflux pumps were unable to
form a biofilm when grown under the same stress
conditions (56).
Bacteria in biofilms 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 regulation of multiple mechanisms, but it is also involved
in regulating metabolism. RpoS is induced by high
cell density, and while initially observed in the stationary phase, it has now been linked to biofilms
(58). Mutants that lack rpoS are incapable of forming
biofilms. RpoS has been shown to be up-regulated in
P. aeruginosa biofilms in vitro and from samples
extracted from cystic fibrosis patients (59).
The role of quorum sensing and intracellular signaling molecules in antimicrobial tolerance of biofilms
has also been investigated. Alarmones or pheromones
are synthesized by bacterial cells within the biofilm
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 biofilm formation and in the development of multidrug tolerance (see next section). Studies have shown
that the use of quorum-sensing inhibitors was able to
enhance susceptibility of P. aeruginosa biofilms to
antimicrobial treatments (61). Another study with
P. aeruginosa demonstrated that increased drug tolerance through the formation of non-dividing cells was
induced by quorum sensing-related signaling molecules (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 mechanism of
antibiotic tolerance. Within a given population of bacteria, 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 concentrations of antibiotics. They are not antibiotic-resistant
mutants, but rather phenotypic variants of the wildtype that form stochastically in a clonal population of
genetically identical cells (65). Persisters constitute a

11

Dufour et al.
small fraction of stationary phase planktonic cultures
and biofilm populations of numerous species. The
number of persisters in a growing population of bacteria rises at the mid-logarithmic phase and reaches a
maximum of approximately 1% at the stationary phase.
Similarly, slow-growing biofilms produce substantial
numbers of persister cells. Persisters resuscitate from
their dormant state when a stationary culture is inoculated into fresh medium. A model of antibiotic tolerance of bacterial biofilms based on persister survival has
been proposed (66). An initial treatment with antibiotic kills planktonic cells and the majority of biofilm
cells, leaving persisters intact. The host immune system
targets and kills planktonic persisters, but the biofilm
persisters are protected from host defenses by the
biofilm matrix. After the antibiotic treatment is
stopped, persister cells repopulate the biofilm and the
infection relapses. Based on this model, persisters have
a much broader clinical significance than only in the
context of biofilm 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 immunocompromised patients (e.g. HIV-positive, cancer
chemotherapy) or immunocompetent patients in cases
where the pathogen is located at bodily sites inaccessible 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-

12

tic expression of a broad variety of genes, some of them


encoding products that may become toxic.

New weapons in the battle against


biofilm infections
The presence of microbial biofilms is the main cause of
inefficient eradication by antibiotic therapy. Biofilm
bacteria are often tolerant to doses of antibiotics
several hundred-fold over the minimum lethal dose for
bacteria living outside the biofilm. The concentrations
that would be expected to eradicate biofilm infections
often exceed the highest deliverable doses of antibiotics. Furthermore, the widespread use of antibiotics
(both inside and outside the medical field) is playing a
significant role in the emergence of antibiotic-resistant
bacteria. Consequently, more work is needed to fully
understand the mechanisms involved in antibiotic tolerance and to develop new therapeutic strategies to
combat biofilm infections. This section will briefly
introduce some possible approaches that could be
explored in the search for new anti-biofilm strategies
to eradicate medically relevant biofilms.

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 biofilm formation. There is growing evidence that quorum sensing
constitutes a global regulatory system in many different bacterial species (68). Consequently, interfering
with the action of a global regulator would necessarily
produce pleiotropic phenotypes. For example, anything that affects cell motility or cell surface chemistry
might translate into a biofilm-deficient phenotype.
The idea to use anti-quorum sensing molecules originated from the discovery of quorum-sensing inhibition by halogenated furanones from the Australian red
macro alga Delisea pulchra (69). This alga had developed chemical defense mechanisms to inhibit bacterial
colonization by producing natural products (halogenated furanone compounds) that have been shown
to interfere with the quorum-sensing systems of
several bacteria (70). One of the major problems associated with antibiotics is the development of resistance. Conventional antibiotics inherently favor the
evolution of resistance because they pose a strong

Bacterial biofilm
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. Quorumsensing inhibitors could then be viewed as blockers of
pathogenicity rather than as antimicrobials.

Specifically targeted antimicrobial peptide


(STAMP) technology
Broad-spectrum antibiotics may disrupt the patients
normal bacterial flora. Antimicrobial treatment
specifically targeting each undesirable pathogen represents an attractive strategy. In 2006, a research group
from the University of California, Los Angeles,
reported the design of narrow spectrum molecules
known as specifically targeted antimicrobial peptides
(STAMPs) (71,72). Construction of these molecules
requires the fusion of a species-specific targeting
peptide domain that provides specific binding to a
selected pathogen with a wide-spectrum antimicrobial
peptide domain. The killing peptide domain is usually
a membrane-active antimicrobial peptide either synthesized chemically or a natural component of the
innate immunity (e.g. defensins, integrins, cathelicidins). STAMPs have been shown to effectively eliminate the cariogenic biofilm organism S. mutans from a
multi-species biofilm without affecting closely related
non-cariogenic organisms. These STAMPs were constructed with peptides derived from the quorumsensing 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 associated with a healthy flora.

Persister eradication
Dormant persister cells are likely to be largely responsible for multi-drug tolerance and for many recurrent
biofilm infections. With their ability to survive even
high concentrations of antibiotics, the need for novel
therapeutics capable of killing persister cells is important for treating microbial biofilm infections. Further
studies into the elucidation of the mechanistic basis for
the formation of persisters could lead to the development 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 difficult. However, antimicrobials that target the
bacterial membrane organization may be promising.
These agents would be similar to antimicrobial peptides 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 production (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 biofilms
through the permeabilization of the bacterial membrane. Resistance toward these antibiotics is likely to be
rare, but is still possible. Alternatively, the identification
of factors specifically 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 resuscitationpromoting factors might be promising candidates.

Probiotics
A promising alternative strategy to treat chronic
biofilm 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
modified) 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 flora in children with recurrent
otitis media. Numerous studies have reported the preventive effect of long-term probiotic consumption on
dental caries in children (78,79). The probiotic bacterium 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 lactococci probiotics (L. rhamnosus, L. reuteri, L. plan-

13

Dufour et al.
tarum) were able to inhibit the formation of biofilm
by different clinical isolates of S. mutans, and even to
significantly reduce their viability (81).

Concluding remarks
Microbiologists have traditionally focused on freeswimming bacteria growing in laboratory flasks; yet
they have recently come to realize that in the natural
world more than 99% of all bacteria live in biofilm
communities. As a result, biofilm research is now one of
the hottest topics in microbiology. The cost to society
associated with biofilms (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 biofilm as an integral part of
their pathogenesis. The fact that microbial biofilms
represent a protected mode of growth that allows cells
to survive hostile environments presents a challenge in
the treatment of biofilm infections. Of importance with
respect to medicine, biofilms compromise quality of life
and may be associated with mortality. The existence of
microbial biofilms suggests an important conceptual
change in our understanding of the bacteria involved in
mild or life-threatening illnesses. Antibiotics have not
been specifically developed to target microbial biofilm
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 biofilm formation within
the body and the genetic basis of how pathogenic
microorganisms persist in biofilms and how this relates
to tolerance mechanisms is essential in the development
of novel antibiofilm strategies.

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