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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
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.
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.
Dufour et al.
Table 1: Biofilms of indwelling medical devices
Medical device
Principal microorganisms
Contact lens
Denture
Candida spp.
Urinary catheter
CoNS*, S. aureus
CoNS, S. aureus
Voice prostheses
C. albicans, CoNS
Endotracheal tubes
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
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
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
Skin
Necrotizing fasciitis
Group A streptococci
Foot
Prostate gland
Bacterial prostatis
Vagina
S. aureus
Biliary system
Gallstone
Enteric bacteria
Urinary tract
Large intestine
Persistent diarrhea
E. coli
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).
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).
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
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
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.
10
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,
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
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.
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
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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