review special focus REVIEW: biofilms

Virulence 2:5, 445-459; September/October 2011; 2011 Landes Bioscience

Staphylococcus aureus biofilms

Properties, regulation and roles in human disease
Nathan K. Archer,1,2 Mark J. Mazaitis,3 J. William Costerton,4 Jeff G. Leid,5 Mary Elizabeth Powers6 and Mark E. Shirtliff1,7,*
1
Department of Microbial Pathogenesis; Dental School; 2Graduate Program in Life Sciences, Microbiology and Immunology Program; 3Institute for Genome Sciences;
University of Maryland; Baltimore, MD USA 4Center for Genomic Sciences; Allegheny-Singer Research Institute; Pittsburgh, PA USA; 5Department of Biological Sciences;
Northern Arizona University; Flagstaff, AZ USA; 6University of Waterloo; Waterloo, ON Canada; 7Department of Microbiology and Immunology; Medical School;
University of Maryland; Baltimore, MD USA

Key words: Staphylococcus aureus, biofilms, MRSA, therapy, vaccine, host response, pathogenesis

Increasing attention has been focused on understanding
bacterial biofilms and this growth modalitys relation
to human disease. In this review we explore the genetic
regulation and molecular components involved in biofilm
formation and maturation in the context of the Gram-positive
cocci, Staphylococcus aureus. In addition, we discuss diseases
and host immune responses, along with current therapies
associated with S. aureus biofilm infections and prevention
strategies.
Staphylococcus aureus biofilm mode of growth is tightly regu-
lated by complex genetic factors. Host immune responses against
persistent biofilm infections are largely ineffective and lead to
chronic disease. However, current research has taken biofilm
formation into account in terms of elucidating host immunity
toward infection, and may lead to the development of efficacious
anti-biofilm S. aureus therapies.
Biofilms
A biofilm can be defined as a microbially-derived sessile commu-
nity, typified by cells that are attached to a substratum, interface,
or to each other, are embedded in a matrix of extracellular poly-
meric substance, and exhibit an altered phenotype with regard to
growth, gene expression and protein production.1 Biofilm thick-
ness can range from a single cell layer to a substantial community
encased by a viscous polymeric milieu.2 Structural analyses have
shown that in some cases unique pillar or mushroom-shaped
structures can be formed by the micro-colony architecture of
these dense biofilms; however, other structures do form depend-
ing on the environmental conditions.2 Intricate channel net-
works flow through these complex structures and provide some
accessibility to essential nutrients even in the deepest regions of
the biofilm. Although biofilm formation is not a prerequisite
for persistent infection,3 biofilm eradication is arduous, usually
requiring surgical intervention and therefore warrants further
investigation.
*Correspondence to: Mark E. Shirtliff; Email: mshirtliff@umaryland.edu
Submitted: 06/04/11; Revised: 08/10/11; Accepted: 08/11/11
http://dx.doi.org/10.4161/viru.2.5.17724
Although a biofilm can arise from a single cell, differential
environmental conditions throughout the community can poten-
tiate the development of distinct subpopulations. Gradients in
oxygen, nutrients and electron acceptors can cause heterogeneous
gene expression throughout a biofilm.4 In a staphylococci in vitro
colony biofilm model, four distinct metabolic states were identi-
fied: cells growing aerobically, fermentatively, dormant (including
very slow growing cells and persisters), or dead. The cells exposed
to the upper air oxygen-rich interface and lower liquid-nutrient-
rich interface were metabolically active.4 However, the majority
of cells were dormant and located in an anoxic environment.4 In
addition, heterogeneity of biofilm protein expression was demon-
strated with multiple cell wall-associated proteins. Expression was
shown to vary in cell clusters throughout the biofilm, and in one
case, differential expression was visualized on a cell-cell basis.5
By adopting this sessile mode of life, biofilm-embedded
microorganisms benefit from a number of advantages over their
planktonic counterparts. The extracellular matrix is capable of
sequestering and concentrating environmental nutrients such as
carbon, nitrogen and phosphate.6 An additional benefit to the
biofilm growth modality is the ability to evade multiple clearance
mechanisms produced by host and synthetic sources. Examples
of ineffective clearance strategies include antimicrobial and anti-
fouling agents, shear stress, host phagocytic elimination and host
radical and protease defenses. Resistance to antimicrobial factors
is mediated through a dormant phenotype caused by adaptation
to an anoxic environment and nutrient deprivation, and results
in low metabolic levels and radically downregulated rates of cell
division.7 This stressed environment produces many slow grow-
ing cells that are tolerant to high levels of antibiotics but also a
proportion of persister cells (no more than ~1% of total popu-
lation).7 These cells are found in biofilm communities but also
exist in planktonic cultures.8 In addition, persister cells dem-
onstrate multidrug tolerance that is inherently different from
resistance, which is prevention of the antibiotic from hitting its
microbial target.7,8 Instead, persister tolerance is accomplished by
shutting down the microbial targets or the cellular need for those
targets by maintaining a metabolically quiescent state, thereby
protecting the cell from even bactericidal antibiotics.7 Although
the mechanism for achieving this metabolic state in S. aureus
is not completely understood, in E. coli it has been implicated
that it is accomplished through a certain proportion of cells in

www.landesbioscience.com Virulence 445

 Figure 1. IgGs against recombinant forms of cell wall-associated biofilm proteins bind to intact S. aureus biofilms. IgG against each selected candi-
date protein was applied followed by the secondary goat anti-rabbit F(ab)2 antibody (upper right in AD). After a washing step, SYTO 9 was applied
to stain all bacterial cells (upper left in AD). Biofilms were probed with (A) anti-SA0486 (autolysin) IgG and secondary antibody, (B) anti-SA0486
(lipoprotein) IgG and secondary antibody, (C) anti-SA0688 (lipoprotein) IgG and secondary antibody and (D) anti-SA0037 (conserved hypothetical) IgG
and secondary antibody. The merged image is shown in the lower part in (AD). The base of the glass is located at the bottom of each image, and each
image is a cross-sectional view of the biofilm from the base into the lumen.

the total microbial population having drastically downregulated
biosynthetic pathways and toxin/antitoxin production, thereby
producing a persister phenotype in this population subset.7 By
protecting these cells within the biofilm, effectors of the immune
system are prevented from clearing out these populations.9
Therefore, once antibiotic regimens are halted, these persisters
are able to spontaneously shift out of their quiescent state and
produce a reactivation of infection.9
While low metabolic rates may explain a great deal of the anti-
microbial resistance properties of biofilms, other factors may play
a role. One such feature may be the capability of biofilms to act
as a diffusion barrier to slow down the infiltration of some anti-
microbial agents.10 For example, reactive chlorine species (such
as hypochlorite, chloramines or chlorine dioxide) in a number of
antimicrobial/antifouling agents may be deactivated in the sur-
face layers of the biofilm before they are able to disseminate into
the lower layers.11 In addition, a recent study showed that several
antibiotics (oxacillin, cefotaxime and vancomycin) had reduced
penetration throughout S. aureus and S. epidermidis biofilms.12
The final benefit to the biofilm development is the potential
for seeding dispersal or cellular detachment. Micro-colonies may
detach under the direction of fluid mechanical shear forces or
through a genetically programmed response that mediates the
seeding dispersal process.13 In a similar fashion to a metastatic
cancer cell, detached micro-colonies migrate from the original
biofilm community to uninfected regions of the host, attach and
promote nascent biofilm formation. In addition, while not the
case for non-motile bacteria like S. aureus, seeding dispersal can
also be mediated by the movement of single, motile cells from
the adherent micro-colony.14 Therefore, this advantage allows an
enduring bacterial source population that is resilient against anti-
microbial agents and the host immune response, while simultane-
ously enabling continuous shedding to encourage bacterial spread.
Staphylococcus aureus Biofilms
Staphylococcus aureus is a Gram-positive, ubiquitous bacterial spe-
cies. The ecological niche of S. aureus in humans is the anterior
nares. In the human population, approximately 2025% have
become persistently colonized and 7580% intermittently or
never colonized.15,16 Previous studies have shown that there is a
strong causal connection between S. aureus nasal carriage and
increased risk of nosocomial infection. Nasal carriage provides a
staging ground for S. aureus to disseminate to other areas of the
body where, once transmitted to the circulatory system through
an epithelial breach, planktonic growth and upregulation of
adherence factors occurs.17,18 Invading staphylococci are then
either removed by the host innate immune response or attach to
host extracellular matrix proteins and form a biofilm. The cel-
lular physiology is then quickly transformed into one reflective of
a biofilm. Owing to the escalating involvement of Staphylococcus
aureus in foreign body-related infections, the swift development
and exhibition of multiple-antibiotic resistance, and their predi-
lection to transform from an acute infection to one that is per-
sistent, chronic and recurrent, this pathogen continues to receive
considerable attention.
Staphylococcus aureus can produce a multilayered biofilm
embedded within a glycocalyx or slime layer with heteroge-
neous protein expression throughout (see Fig. 1). Early studies
described the solid component of the glycocalyx as primarily
composed of teichoic acids (80%) and staphylococcal and host
proteins (see Fig. 2).19 In later studies, a specific polysaccharide
antigen named polysaccharide intercellular antigen (PIA) was
isolated. PIA is composed of b-1,6-linked N-acetylglucosamine
residues (8085%) and an anionic fraction with a lower con-
tent of non-N-acetylated D-glucosaminyl residues that con-
tains phosphate and ester-linked succinate (1520%).20
PIA-dependent biofilm formation. PIA is produced in vitro
from UDP-N-acetylglucosamine via products of the intercellular
adhesion (ica) locus.21 The genes and products of the ica locus
[icaR (regulatory) and icaADBC (biosynthetic) genes] have been
demonstrated to be necessary for biofilm formation and viru-
lence and are upregulated in response to anaerobic growth, such
as the conditions seen in the biofilm environment.22 The staphy-
lococcal respiratory response regulator, SrrAB, is responsible for
PIA induction under anaerobic environments via binding of a

446 Virulence Volume 2 Issue 5

100 bp DNA sequence upstream of the icaADBC operon.23
Other environmental factors can also play a role in regulation of
ica, including glucose, ethanol, osmolarity, temperature and anti-
biotics such as tetracycline.18 In the homologous Staphylococcus
epidermidis locus, regulation of ica can occur via reversible inacti-
vation by insertion sequence (IS256) phase variation in 2533%
of variants,24 and this has been observed in some S. aureus strains
as well.25 In addition, PIA expression is repressed by TcaR, a tran-
scriptional regulator of the teicoplanin-associated locus; however,
deletion of the tcaR gene had no effect on PIA synthesis.26 Strong
negative regulation is conferred by IcaR, through binding of
the ica cluster promoter and deletion of the icaR gene results in
enhanced ica cluster gene expression.26,27 The protein regulator of
biofilm formation, Rbf, however, represses transcription of IcaR,
albeit indirectly, leading to augmented ica gene expression, PIA
production and biofilm formation.28 In addition, Spx, a global
regulator of stress response genes, was shown to have a negative
regulatory impact on biofilm formation, seemingly by modulat-
ing IcaR.29
PIA-independent biofilm formation. Despite the impor-
tance of the ica gene locus in biofilm development, biofilms
can occur in an ica-independent fashion. The arlRS two-com-
ponent system was shown to repress biofilm development, and
when deleted led to enhanced attachment and PIA production.30
However, biofilm synthesis was unaffected by additional dele-
tion of the icaADBC operon, suggesting that in this double dele-
tion mutant, PIA was not essential for biofilm development.30
The S. aureus clinical isolate, UAMS-1 (University of Arkansas
Medical System-1), had unabated biofilm formation in vitro and
in vivo in a catheter infection model even with mutation of the
ica cluster.17 In a guinea pig model of biofilm infection, dele-
tion of ica and thus, lack of PIA production caused no decrease
in virulence.31 In addition, Fitzpatrick et al.32 showed that bio-
film formation in MRSA strain BH1CC was unaffected by ica
locus deletion. However, other mutant strains lost the ability to
form biofilm. Interestingly, when S. aureus icaADBC operon-
deletion mutants are categorized by methicillin susceptibility,
MRSA strains are capable of biofilm development, whereas
MSSA strains are impaired in biofilm formation.33 These data
propose that biofilm formation in an ica-independent manner
is strain specific. In an ica-deletion mutant S. aureus strain, pro-
tein A (SpA) production was found essential for biofilm forma-
tion.34 Furthermore, biofilm development could be recovered in
spa mutants by addition of exogenous SpA, indicating that it
is not necessary for SpA to be covalently anchored to the cell
wall.34 The fibronectin-binding proteins (FnBPs) can also arbi-
trate biofilm formation through an essential role by the major
autolysin (Atl) and sigB regulation,35 and in Staphylococcus epi-
dermidis, PIA-independent biofilms were mediated through the
accumulation-associated protein (Aap).36,37 In addition, biofilm-
associated protein (Bap) and Bap-related proteins of S. aureus
can confer biofilm development independent of PIA production
through cell-to-cell aggregation, and are characterized by their
high-molecular weight, presence on the bacterial surface, role
as a virulence factor and occasional containment in mobile ele-
ments.38 These reports suggest that proteinaceous cell-to-cell
www.landesbioscience.com Virulence 447
Figure 2. SEM micrograph from an implant recovered at five days
showing massive numbers of cocci partially occluded by dehydrated
material. Bar = 5 m.
adhesion can substitute PIA-mediated biofilm development in
ica-independent biofilms.
eDNA and biofilm formation. Another important component
of the staphylococcal biofilm is extracellular DNA (eDNA). The
discovery that this substance is an important component of bio-
films was previously made in P. aeruginosa.39-41 This is supported
by the in vitro finding that early, immature biofilm clearance
could be attained by DNase treatment.39 In addition, support of
the importance of eDNA in biofilms in vivo includes the success-
ful clinical usage of DNase concurrently with antibiotic therapy
in the treatment of cystic fibrosis patients,42 and the finding that
DNase found on skin cells can lessen biofilm formation.43 Rice et
al.44 discovered that mutation of cidA, a gene encoding an effec-
tor of murein hydrolase activity and regulating cell death, led to
a less adherent biofilm that contained lower levels of genomic
DNA. In addition, treatment with DNase I minimally affected
biofilm formation in the cidA mutant, whereas the parental strain
had a dramatic inhibition. The cidA gene has been shown to be a
holin homolog involved in cell lysis, and these data suggest that
this cell lysis activity is necessary for DNA release and biofilm
development.44 Cell lysis and subsequent genomic DNA release
must also occur early in cell attachment for proper biofilm for-
mation, as shown by Mann et al.45 This cell lysis and the biofilm
matrix interspersed between whole intact bacterial cells can be
readily seen in Figures 3 and 4. The lrg gene, another regula-
tor of murein hydrolase and cell death, counteracts the effects of
cidA DNA release as revealed by enhanced biofilm attachment
and matrix-associated eDNA in lrgAB mutants.45 Other globally
upregulated genes in S. aureus biofilms included autolysin, an
enzyme that hydrolyzes staphylococcal cells, and phage genes due
to the change from a lysogenic to lytic form in the stressed envi-
ronment of the biofilm.46,47 Therefore, S. aureus has a number of
modalities to add eDNA into the early biofilm matrix, thereby
providing the structural support for biofilm formation. Control
of cell lysis and eDNA release is another branch in the complex
regulation of biofilm formation by S. aureus.

Figure 3. TEM micrograph of a cross-section through a biofilm recov-
ered from an indwelling medical device implanted in the peritoneum
of a rat for five days. Bacteria are apparent on the surface of the biofilm
and interspersed with host material and lysed bacteria within the
biofilm. Bar = 5 m.
Biofilm regulation by agr/sarA. The major global regulators,
staphylococcal accessory regulator (sarA) and accessory gene reg-
ulator (agr), have also been implicated in biofilm formation. A
two component regulatory gene locus encoded by arlRS, a mem-
ber of the OmpR-PhoB family of response regulators, is regu-
lated by the agr and sarA loci.48 When upregulated, the product
of arlS prevents biofilm formation and may mediate attachment
to polymer surfaces by affecting peptidoglycan hydrolase activity.
Transcripts of sarA are upregulated in biofilms when compared
with planktonic cultures.49 In addition, capacity to form biofilm
is reduced in sarA mutants.50 Biofilm formation can be recov-
ered in sarA mutants by concurrently mutating the gene encoding
the S. aureus thermostable nuclease, nuc or addition of protease
inhibitors, as nuc and extracellular proteases were found to be
transcriptionally upregulated in sarA mutants.51 By inhibiting
nuc expression or protease activity, sarA expression may prevent
degradation of extracellular DNA and protein, important biofilm
structural components. Although SarA affects and is affected by
agr, the effect of agr in vitro mutants on biofilm development
is minimal and has been shown to be an agr-independent path-
way.49,50,52,53 Despite this, there is evidence that the agr locus is
associated with biofilm development. The agr quorum sensing
448 Virulence Volume 2 Issue 5
system has been shown to downregulate genes of cell wall-associ-
ated adherence factors.54 This would lead to lesser adherence and
thus, indirectly, decreased initial biofilm formation. Repression
of agr has been shown to be necessary for biofilm formation and
induction of agr through auto-inducing peptides (AIP), an essen-
tial component of the quorum sensing system, results in seeding
dispersal in mature biofilms.55 This mechanism occurs in a pro-
tease-dependent manner and requires an active agr system since
both protease inhibitors and inhibitory AIP block seeding disper-
sal.55,56 As well, agr has been shown to upregulate the expression
of detergent-like peptides and nucleases that seem to increase bio-
film detachment.53,57 Therefore, the staphylococcal global regula-
tors function in a complementary and opposing fashion where
sarA induces attachment and early biofilm formation conceivably
by repressing nucleolytic and proteolytic extracellular enzymes,
and once biofilms have attained a significant quorum population,
agr expression leads to upregulation of a number of virulence
factors to thwart the host immune response and supports seed-
ing dispersal in a nuclease, protease and surfactant-dependent
manner.
Biofilm regulation by sigB. Additional levels of con-
trol are accomplished through the sigB operon product B in
Staphylococcus aureus (regulated by operon genes rsbU, rsbW and
rsbV ).58 Factors necessary for the early stages of biofilm forma-
tion, including clumping factor, fibronectin binding protein A
(FnbpA) and coagulase, are all upregulated by B.59,60 In addi-
tion, factors that correlate with seeding dispersal and a plank-
tonic phenotype, including -hemolysin, enterotoxin B, serine
protease (SplA), cysteine protease (SplB), the metalloprotease
Aur, staphopain and leukotoxin D are all negatively controlled.61
Rachid et al. revealed that a sigB-deficient S. aureus could not
form biofilm and resulted in upregulation of RNAIII, a vital
component of the anti-biofilm promoting agr system.62 This is
exemplified in the case of the laboratory strain RN6390, which
lacks B and is deficient in biofilm formation.63 Additional dele-
tion of agr or use of protease inhibitors reverted S. aureus to a
biofilm-positive phenotype.63 Jefferson et al.27 discovered that
IcaR binds to a 42-bp region just upstream of icaA and hypoth-
esize that its role is to sterically hinder the binding of B, thus
preventing activation of the ica locus. In contrast, Valle et al.49
found that a sigB deletion mutant still effectively developed bio-
film. These conflicting reports suggest that the sigB operon may
act in a strain-specific manner in controlling biofilm formation.
Transcriptomic and proteomic expression results in bio-
films. Based on studies from other researchers and our own
laboratory, we have found a staged progression of virulence gene
expression in the biofilm under control of the sigB, agr, sarA and
sae regulons.17,46,64 In the planktonic mode of growth, the micro-
bial surface components recognizing adhesive matrix molecules
(MSCRAMMS) are upregulated while the staphylococci are in
a low concentration such as that seen in a case of early sepsis.64
Once an early biofilm develops, transient upregulation of sarA
and agr systems downregulate the adhesins and upregulates a
number of immunoavoidance factors and toxins that cause dam-
age to the host.65 In a fully mature biofilm, staphylococci are slow
growing and nutrient deprived resulting in the downregulation
 Figure 4. TEM micrograph depicts bacteria within a medical implant biofilm. The bacteria are associated with large amounts of host extracellular
matrix materials and lysed bacteria. Fibrils are apparent radiating from the bacterial cell surface in a clear halo surrounding the bacteria (arrow).
Bar = 1 m.
in virulence factor production and the upregulation of stress
response genes, phages genes and systems for maintaining pH
homeostasis.64 The implications of these various gene expression
changes can only be understood in the context of the human
host and will be explored more thoroughly below in the dis-
cussion of one of the well studied diseases caused by S. aureus:
osteomyelitis.
For a summary diagram of the regulatory factors involved in
biofilm formation, maintenance and detachment, please refer to
Figure 5.
S. aureus Biofilm-Related Diseases
Staphylococcus aureus has re-emerged as a clinically relevant
pathogen due to its resistance to antibiotics and the increased
use of indwelling medical devices. Infections associated with
S. aureus in the US have a crude mortality rate of 25% along
with hospitalizations resulting in approximately twice the length
of stay, deaths and medical costs of typical hospitalizations.66,67
S. aureus biofilms, once established, are recalcitrant to antimi-
crobial treatment and the host response, and therefore are the
etiological agent of many recurrent infections.68 Following is an
incomplete list of S. aureus diseases that have a demonstrated bio-
film component. Osteomyelitis will be first discussed in detail
in the context of S. aureus stages of colonization, infection and
maturation of the biofilm as this progression of disease is mim-
icked in many other diseases and host sites.
www.landesbioscience.com Virulence 449
Osteomyelitis. Osteomyelitis is defined as an infection of
the bone, and can be caused by a variety of bacteria or fungi.
However, S. aureus provides the majority of cases.69 Bacteria can
be introduced through a hematogenous route or direct inocula-
tion during surgery, trauma or an overlying infection.70 While in
the blood in the planktonic mode of growth, S. aureus upregu-
lates the production of adhesins for many host adhesive matrix
molecules (e.g., fibrinogen, fibrin, osteopontin, fibronectin, colla-
gen, elastin, etc.).64 Once in the bone, S. aureus attaches to local-
ized areas of trauma and divides by binary fission to form an early
biofilm (see Fig. 6) while constitutively expressing a low level of
the auto-inducing peptide (AIP) of the quorum sensing system.70
As the population expands and the AIP levels increase, a quorum
is attained and the agr quorum sensing system is activated, result-
ing in the production of toxins, immunoavoidance factors and
other agr-controlled virulence factors with a concomitant down-
regulation in microbial adhesins.65 Simultaneously, a number
of microbial inflammatory mediators such as formylmethionyl
peptides, low level hemolysins, lipotechoic acids, peptidogly-
can, phenol soluble modulins and staphylococcal DNA (due to
unmethylated cytosine-phosphate-guanosine [CpG] motifs) call
the innate inflammatory response effectors (e.g., PMNs) to bear
on the invading pathogen.71 However, PMNs attempt to attack
the virulent early biofilm in a non-specific way and are met with
an armamentarium of staphylococcal toxins and immunoavoid-
ance factors resulting in host cell lysis and frustrated phagocyto-
sis.71 The end result is a maturing S. aureus biofilm population
 Figure 5. For figure legend, see page 451.

with detaching flocs and tissue damage due to PMN-induced col- that the biofilm and devitalized tissue must be surgically debrided
lateral host tissue damage, thereby providing the essential devi- in order resolve the infection.69 However, in cases of juvenile
talized surface for further biofilm development (see Fig. 7 and osteomyelitis, children are often able to resorb the dead bone and
Movie S1).70,72 In adults, the resulting tissue destruction means tissue during antimicrobial therapy, resulting in the elimination

450 Virulence Volume 2 Issue 5

 Figure 5 (See opposite page). Flowchart of regulatory factors involved in S. aureus biofilm formation, maintenance and detachment. (A) PIA-de-
pendent biofilm formationexpression of the icaADBC gene cluster results in PIA expression and biofilm formation.21 Expression of icaADBC can be
suppressed by production of tcaR and icaR, resulting in downregulation of PIA and thus biofilm formation. 26,27 In the case of the icaR gene, expression
can be up or downregulated by the proteins Spx and Rbf, respectively. 28,29 Consequently, Spx induction of icaR expression results in downregulation
of icaADBC expression, PIA production and biofilm formation.29 Conversely, Rbf inhibits icaR expression leading to upregulation of icaADBC expres-
sion, PIA production and biofilm formation.28 Additionally, anaerobic conditions induce production of SrrAB, causing expression of the icaADBC gene
cluster, PIA production and biofilm formation.23 PIA-independent biofilm formation*in ica-deletion mutants, PIA-independent biofilm formation can
be mediated through cell wall-associated protein cell-to-cell adhesion (MRSA-specific). 33 Examples of proteins arbitrating biofilm formation include
SpA, FnBPs and Bap. 34,35,38 icaADBC, intercellular adhesion biosynthetic genes; PIA, polysaccharide intercellular antigen; tcaR, transcriptional regulator
of the teicoplanin-associated locus; icaR, intercellular adhesion regulatory gene; Spx, global regulator of stress response genes; Rbf, protein regulator
of biofilm formation; SrrAB, staphylococcal respiratory response regulator; SpA, S. aureus protein A; FnBPs, fibronectin-binding proteins; Bap, biofilm-
associated protein. (B) eDNA and biofilm formationeDNA leads to enhanced biofilm formation.39-41 DNase treatment degrades eDNA and inhibits
eDNA-mediated biofilm formation. 39,42,43 DNA release is arbitrated through cell lysis and controlled by lrg and cidA gene expression.44,45 Upregulation
of the lrg gene results in inhibition of cellular lysis, DNA release and biofilm formation.45 Conversely, cidA gene expression enhances cellular lysis, DNA
release and biofilm formation.44 eDNA, extracellular DNA; lrg, regulator of murein hydrolase and cell death; cidA, regulator of murein hydrolase and
cell death. (C) agr/sarA/sigB regulationexpression of the sarA gene results in downregulation of proteases and the thermostable nuclease, allowing
for development of an immature biofilm. 51 Expression of sigB similarly downregulates protease production, but additionally promotes expression of
adherence factors that aid in initial biofilm formation.59-61 The immature biofilm increases in cell density until a mature biofilm develops. At this stage,
the density of AIPs throughout the bacterial community reaches a quorum sensing threshold and induces expression of the agr gene. 55 Induction of
agr results in upregulation of detergent-like peptide, protease and thermostable nuclease expression; leading to release of bacterial cells from the
mature biofilm, termed seeding dispersal. 53,55-57 sarA, staphylococcal accessory regulator; sigB, sigmaB; AIP, auto-inducing peptides; agr, accessory gene
regulator.

of the bacterial attachment surface. Therefore, this type of bio-

film infection in juveniles is one of the few types of biofilm infec-
tions that can be successfully resolved with antibiotic therapy
alone.69 This mode of infection is much like other modes of
S. aureus chronic infection such as those discussed below.
Indwelling medical device infection. Millions of indwelling
medical devices are implanted into hosts every year and S. aureus
is the major culprit for their infection and failure.73 S. aureus
has been known to infect and form a chronic biofilm infection
most often on orthopedic implants including prosthetic joints,
wires, pins, external fixators, plates, screws, nails and mini-large
fragment implants.74 However, other indwelling medical devices
that are prone to staphylococcal biofilm infection include stents,
ventilators, intravenous catheters, invasive blood pressure units,
infusion pumps, cardiac defibrillators, mechanical heart valves,
aspirators, pacemakers, stitch materials, ear and central nervous
system shunts, cosmetic surgical implants, penile implants and
orthopedic devices.74 During cases of implant infection, the
implant becomes coated with host derived extracellular matrix
proteins, providing a rich surface for bacterial attachment.75
Whether infected during implantation, during subsequent
trauma or hematogenously, the result is the same. In order to
resolve the infection, the device must be removed to completely
resolve the infection since antibiotics cannot eliminate this bio-
film population.
Periodontitis and peri-implantitis. Periodontitis is defined
as inflammation and infection of the ligaments and bones
that support the teeth and peri-implantitis as destructive
inflammation of soft and hard tissues surrounding a den-
tal implant.76 S. aureus biofilm formation in the oral cavity
can act as a reservoir for oral infections, and the prevalence of
S. aureus in the periodontal pocket is 13.4% and 15.8% in Figure 6. SEM micrograph demonstrating cocci adherent to a bone
the oral cavity.77 S. aureus has a high affinity to titanium sur- sequestra. Collapsed glycocalyx material is apparent associated with
faces and is recovered at high levels from infected peri-implant the bacterial biofilm (arrow). Bar = 5 m.
pockets. Although this pathogen is not strongly associated with

www.landesbioscience.com Virulence 451

 Figure 7. (A) Nasal colonization confers Staphylococcus aureus a reservoir for dissemination onto skin and various other host epithelial surfaces. Dam-
age to the epithelia can occur from extraneous sources. (B) Damage allows S. aureus to breach the epithelial layer and bind to host matrix via surface
expressed colonization factors. Map, MHC class II analog protein; Cna, collagen-binding adhesin; FnbB, fibronectin binding protein B; FnbA, fibronec-
tin binding protein A; Fib, fibrinogen binding protein; FbpA, fibronectin binding protein; CflA, clumping factor A; EbpS, elastin binding protein. (C) Ini-
tial attachment and cell division produces an early S. aureus biofilm. The quorum sensing compound of S. aureus, termed auto-inducing peptide (AIP),
is secreted and taken up by the microcolony. SarA expression is upregulated, leading to production of virulence and immunoavoidance factors (CHIPS,
chemotaxis inhibitory protein of staphylococci; Eap, extracellular adherence protein; SCIN, staphylococcal complement inhibitor). Additionally, AIP has
reached quorum threshold levels, resulting in agr activation and a downregulation of adhesins and an upregulation of virulence factor expression that
cause damage to the host and evade the immune response. The nascent S. aureus biofilm avoids immunological destruction by initiating leukocyte
apoptosis and/or developing a protective environment via inflammation and tissue damage. (D) Mature S. aureus biofilm is encapsulated by polysac-
charide intercellular antigen (PIA), protein and extracellular DNA (eDNA). AIP has reached quorum threshold levels, resulting in agr gene expression.
Subsequently, protease and detergent-like peptides are secreted into the biofilm and promote seeding dispersal. Large biofilm aggregates detach
forming flocs and planktonic bacterial cells migrate from the mature biofilm into the circulatory system, where adherence to distal tissue and forma-
tion of a nascent biofilm can occur to repeat the cycle.

periodontitis, it is associated with the more difficult-to-treat
refractory cases.76
Chronic wound infection. Staphylococcus aureus biofilms have
been implicated in cases of chronic wound infections such as dia-
betic foot ulcers, venous stasis ulcers and pressure sores. In fact,
S. aureus is the most commonly isolated bacteria from such wound
infections and studies involving patients with chronic venous leg
ulcers found S. aureus positive cultures in 8893.5% of infec-
tions.78,79 Davis et al.80 observed S. aureus biofilm structures in
a porcine wound colonization model using multiple microscopic
techniques. Diabetic foot wound patients with S. aureus wound
colonization have a 2-fold increase in healing time.81 Similarly, a
murine cutaneous wound model demonstrated that S. aureus bio-
film delays re-epithelialization and healing.82 In addition, delayed
re-epithelialization was shown to be specifically dependent on
S. aureus biofilm development.82 However, with modern molec-
ular and culture independent techniques, this prevalence may
change in the future.
Chronic rhinosinusitis. Chronic rhinosinusitis (CRS) is an
inflammatory disorder with an unknown etiopathogenesis.83

452 Virulence Volume 2 Issue 5

Bacterial biofilms have been repeatedly demonstrated on the
mucosal surface of CRS patients and correlate with more severe
disease.84,85 Staphylococcus aureus is recovered from 50% of CRS
cases, and more interestingly, is associated with more severe, sur-
gically recalcitrant infection and unfavorable post-operative evo-
lution.83,86 Despite the connections between biofilm and CRS,
little is known of the pathogenesis conferred by S. aureus biofilms.
Endocarditis. Infection of the heart valve with S. aureus car-
ries a high rate of morbidity and mortality. The left heart valve is
most often infected and while the mortality rate is low in persons
under the age of 50, it easily surpass 50% in older patients clini-
cally diagnosed with S. aureus endocarditis.87 The treatment is
most often monitoring and antimicrobial therapy since biofilms
are often sloughed and limited in size due to the high shear forces.
Ocular infection. Ocular infections associated with S. aureus
biofilms include conjunctivitis, keratitis and endophthalmi-
tis.88,89 Staphylococci, specifically S. aureus, had the highest isola-
tion rate from patients with conjunctivitis, and it is predicted that
MRSA cultures are more common in serious ocular infections
than methicillin-susceptible strains.88 In addition, S. aureus is the
second most common cause of post-operative endophthalmitis
and biofilm formation may play a large role in such infection.89
Polymicrobial biofilm infections. Microorganisms rarely
live in isolated environments, but rather exist in polymicrobial
communities. Recent interest has been formed in understanding
the relationships between multi-species biofilms and infection.
A mixed microorganism interaction with medical importance
exists between S. aureus and Candida albicans, a pathogenic
fungus associated with nosocomial infections in immuno-com-
promised patients. Candida albicans and S. aureus have been
co-cultured from oral and vaginal mucosal surfaces, both exhib-
iting biofilm modes of growth.90 A recent study by Peters et al.90
demonstrates the ability of S. aureus to favorably bind the hyphal
form of C. albicans and develop biofilm. Interestingly, C. albicans
hyphae can penetrate host epithelial cells, potentially providing
an entrance for S. aureus local and systemic infections.91
Host Response to S. aureus Biofilm Infection
Biofilm-associated infections result in chronic disease that
are resistant to the host immune response. Although a myriad
of information exists on biofilm development, little is known
how the immune system reacts to infection. Recent research
has focused on elucidating the host responses to S. aureus bio-
film infections and delineating which confer clearance or foster
persistence.
In a mouse tibial implant model of S. aureus biofilm osteo-
myelitis, a pro-inflammatory Th1/Th17 and anti-inflammatory
Th2/Treg paradigm was discovered. C57BL/6J mice were shown
to hold a chronic infection and resulted in upregulation of IL-2,
IL-12p70, TNF and IL-1 (Th1-associated cytokines) and IL-6
and IL-17 (Th17-associated cytokines).92 In addition, IgG2a and
IgG2b (Th1-associated antibodies) levels were increased early in
infection, and IgG1 (Th2-associated Ab) and suppressive Tregs
were not upregulated until late in infection.92 This data suggests
that pro-inflammatory Th1/Th17 responses occur early.
www.landesbioscience.com Virulence 453
This early inflammatory response may be ineffective at
microbial clearance since the host produces IgG subclasses of
IgG2a and IgG2b that are very effective at fixing complement to
which Gram positive bacteria like staphylococci are inherently
resistant.93 Also, S. aureus possesses a staphylococcal comple-
ment inhibitor (SCIN) that binds to and stabilizes C3 conver-
tases, interfering with additional C3b deposition through the
classical, lectin and alternative complement pathways.94 This
leads to a substantial decrease in phagocytosis and killing of
S. aureus by human neutrophils [polymorphonuclear leukocytes
(PMN)].
While neutrophils comprise the immune systems primary
defense against extracellular pathogens, S. aureus has evolved
a number of different strategies to disarm this early inflamma-
tory response.95 Some of these mechanisms include the release of
chemotaxis inhibitory protein of staphylococci (CHIPS), SCIN,
clumping factor A (ClfA) and extracellular adherence protein
(Eap),71 not to mention the vast array of staphylococcal toxins.
The ability to develop biofilms may also be an effective means of
evading PMN phagocytosis. Neutrophils were able to penetrate,
but not phagocytose biofilm when cultured with S. aureus bio-
films.96 Neutrophils co-cultured with planktonic S. aureus cul-
tures, however, were able to internalize S. aureus bacterial cells.96
A study by Guenther et al. contradicts this by showing that
S. aureus biofilms are not protected by the effects of PMN phago-
cytosis, and in fact, PMNs release lactoferrin and elastase, along
with DNA.98 This difference may be due to the strain of S. aureus
used, duration of biofilm development, growth conditions or acti-
vation state of co-cultured neutrophils. The opsonization activity
of IgG can also influence PMN action. For example, addition
of serum IgG to S. aureus/neutrophil co-cultures resulted in
enhanced biofilm clearance, not by increased de-granulation or
adherence, but rather by upregulated oxygen radical production
in PMNs.99
Early activation of the inflammatory response may not only
be ineffective but may also even be detrimental to the host due
to the PMN activation and inflammatory mediator influx result-
ing in concomitant host tissue damage. Interestingly, S. aureus
biofilms, and not planktonic cultures, were conferred a growth
advantage in the presence of IL-1.100 This insinuates that induc-
tion of inflammatory mediators, such as IL-1, may bolster
biofilm formation and therefore mitigate the immune systems
ability to clear S. aureus infection. In addition, recent studies in
our laboratory have shown that the development of chronic infec-
tion was prevented by the early inhibition of Th1/Th17 through
the administration of monoclonal antibodies against the IL12p40
antigen common to Th1/Th17 activation through the preven-
tion of the early and non-specific host damage elicited by the
inflammatory response. Th2/Treg anti-inflammatory responses
and effective IgG1 antibodies, which may inhibit chronic infec-
tion, are not activated until persistent infection has already devel-
oped.101 However, even though the host produces these IgG1
antibodies, they may also be less effective due to the heteroge-
neous antigenic character of the biofilm where an antigen may
be produced in certain areas of the biofilm but completely absent
from other areas (see Fig. 1).
 Therapy and Prophylaxis of S. aureus Biofilm
Infections
In terms of the annual burden of morbidity and mortality it
imposes on society, S. aureus is arguably the most significant bac-
terial pathogen in the western world. However, no concomitant
advances in therapy or prevention have been forthcoming. This is
particularly so in the case of chronic, S. aureus biofilm infections.
The biofilm mode of growth is now recognized as a major
mediator of infection, with an estimated 80% of all infections
caused by biofilms.102 Resolution of S. aureus biofilm infections
continues to require surgical removal of the nidus of infection
due to innate resistance to antimicrobial and host response fac-
tors.103 However, attempts to develop an effective anti-S. aureus
vaccine have often ignored this mode of growth in favor of using
planktonic cells and infection models.104-106 This is further com-
plicated by the fact that S. aureusduring biofilm formation
and maturationreleases a number of extracellular factors that
induce an inflammatory, Th1-type immune response rather than
the more effective Th2-type as mentioned before.92
Antimicrobial therapy. It is important to note that the vast
majority of S. aureus biofilm infections must be either prevented
from forming or be surgically removed once formed in order to
resolve the infection. In general, antimicrobial therapy alone is
not effective. In conjunction with surgical intervention such as
debridement, incision and drainage, indwelling medical device
removal, antimicrobial therapy is often prolonged and often takes
place in the outpatient setting. Some individuals require extended
parenteral therapy due to either of the agent itself (e.g., vanco-
mycin) or difficulties with absorption from the gastrointestinal
tract.107 However, the evidence suggesting that parenteral admin-
istration of vancomycin results in better outcomes than oral is
limited. Parenteral antimicrobial therapy as an outpatient is both
convenient and cost-effective, but is appropriate only for those
patients who meet the Infectious Disease Society of America cri-
teria.107 The majority of patients with surgically managed biofilm
infections can be treated orally with one or more of several antibi-
otics, so long as the causative organism is not resistant to them.108
The requirement for prolonged treatment regimens means it is
vital to take into consideration toxicities and drug interactions
when selecting a regimen.
-lactam agents are often used in the treatment of biofilm
infections such as osteomyelitis. They demonstrate time-depen-
dent killing, maximal when the serum concentration is ca. four
times the MIC.109 -lactams do not show a post-antibiotic effect;
i.e., antimicrobial activity after antibiotic concentrations have
decreased below therapeutic levels. Penicillin G (1220 million
U every 24 h IV) and oxacillin (12 g IV every 46 h) are the
current recommended first-line therapy for osteomyelitis caused
by penicillin-sensitive and -resistant S. aureus, respectively.110
Unlike -lactams, fluoroquinolones and aminoglycosides
demonstrate both concentration-dependent activity and a post-
antibiotic effect.111 Dosing of such agents is limited by their
toxicity (aminoglycoside nephrotoxicity). Therefore, systemic
aminoglycosides are usually recommended for prolonged therapy
454 Virulence Volume 2 Issue 5
only when equally efficacious and less toxic alternative agents are
unavailable. Aminoglycosides can safely achieve high levels at the
site of infection when implanted close to the infectious nidus.112
Glycopeptides are usually thought of as having a time-depen-
dent mode of action. Vancomycin is the principal agent used
for treatment of methicillin resistant biofilm infection; the rec-
ommended regimen in osteomyelitis being 15 mg/kg IV every
24 h.110 The MICs to vancomycin of S. aureus strains have risen
over the most recent ten years, but aiming for slightly higher
minimum serum concentrations most commonly overcomes
this. Resistance to vancomycin among staphylococci remains
rare; less than 15 cases have been reported worldwide.113 One
study reported that a use of continuous infusion vancomycin
at a high dose was linked to an increased risk of occurrence of
antibiotic-associated diarrhea caused by Clostridium difficile.
Should this become a significant problem, linezolid or dapto-
mycin may be effective alternatives for treatment of S. aureus
biofilm infection.114
Rifampin has the capacity to kill metabolically dormant ses-
sile bacteria, making it highly useful for musculoskeletal infec-
tions of which a biofilm is the focus.115 Furthermore, it exhibits
high bioavailability and has very few known side-effects. In
order to prevent emergence of resistance to rifampin, it should
be only used in combination with another antibiotic agent that
is active against S. aureus, such as vancomycin or one of the
fluroquinolones.
Inclusion of antimicrobial agents at the site of infection.
A more preventative alternative to post-infection surgical man-
agement and antibiotic treatment is inclusion of the agent in an
implanted medical device. This was initially performed with
permanent, antibiotic-containing bone cement. Use of cement or
calcium sulfate116 beads has shown some efficacy in preventing
infection.117 These provide a more rapid release of high concen-
trations of antibiotics and may be useful for situations such as
infection prophylaxis following open fractures.116 Calcium sul-
fate beads also dissolve and so do not require surgical removal.
Additionally, they do not generate heat during formation, facili-
tating use of a greater variety of antibiotics.
The antibiotic selected for inclusion in beads must be (1) active
against the causative microbes, (2) available as a powder that
will harden properly and (3) able to maintain activity despite the
heat generated during the polymerization process.110 The most
commonly used agents are gentamicin in mainland Europe, and
in the United States and United Kingdom, tobramycin. Release
of antibiotic agents from cement or beads of any type shows a
biphasic profile. The majority of the agent is released in the first
few hours to days after implantation, with the remainder leach-
ing out slowly over a matter of weeks, months or in some cases,
years.118
While antibiotics and novel derivatives have been the mainstay
of therapeutic alternatives, modern advances using other antimi-
crobial agents such as nanosilver,119 cytokines such as IL-12,120
nitric oxide,121 or anti-staphylococcal phages122 are being tested.
However, the widespread clinical utility of these other agents
have yet to be demonstrated.
 Vaccines
The greatest obstacle to development of a vaccine effective against
S. aureus biofilm infection remains selection of appropriate anti-
gens. The sequencing of the whole genome of multiple S. aureus
strains,123 followed by those of methicillin-resistant124,125 and
vancomycin-intermediate126 and epidemic127 strains undoubtedly
marked a significant step forward. The subsequent development
of bioinformatics to manipulate and analyze these data has facili-
tated high-throughput genomic, transcriptomic and proteomic
analyses of microbial growth and pathogenesis.128,129
The first question that must be answered is which compo-
nent of the biofilm should be targeted. Broadly speaking, two
alternatives exist: bacterial cells within the biofilm and the bio-
film matrix itself. The biofilm matrix may be composed of poly-
saccharides, protein or extracellular DNA, in proportions that
vary between bacterial genera, species and strains. Most anti-
biofilm vaccine efforts to-date have been directed toward the
extracellular matrix.130 Perhaps the best example of this is the
staphylococcal polysaccharide inter-cellular adhesin (PIA), or
poly-N-acetyl--1,6-glucosamine (PNAG), production of which
is encoded by the icaADBC locus.131 PIA is produced by both
S. epidermidis132 and S. aureus21 and is involved in adherence of
S. epidermidis to host tissues133 and biomaterials.134 However,
only 57% of icaADBC-positive strains135 produced a biofilm in
vitro,136 suggesting distinct strain differences in any correlation
of PIA and biofilm formation. The proportion of ica-positive
S. aureus clinical isolates varies according to the clinical origin
and even between infection sites that are both biofilm-mediated.
For example, the proportion of icaADBC-positive S. aureus strains
was higher in orthopedic prosthesis-associated infections (92%)
than in those that were catheter-associated (63%).137 There is also
some evidence that PIA expression undergoes phase variation.138
Although it has been tested against planktonic-type infection in
animal models,139 the efficacy of vaccination with PIA in prevent-
ing biofilm-type infections remains to be determined.
Genomics and its derivatives. Genome-based technologies
facilitated rapid identification of vaccine candidates compared
with the more conventional approach of identifying and ana-
lyzing individual virulence factors from pathogens cultured in
vitro.140 Identification of putative antigensprincipally surface
proteinsby systematic searching of the pathogens genome
using bioinformatics is termed reverse vaccinology.140 This has a
number of advantages compared with traditional methodologies:
(1) there is no need for in vitro culture and (2) antigen selec-
tion can proceed independent of in vivo expression levels and/or
immunogenicity. As a result, many antigens that would have been
passed over in conventional studies may be detected. However,
such analyses yield only limited information regarding levels of
expression of vaccine candidate antigens during pathogenesis.
Transcriptomic analysis of bacterial pathogens in vivo using
microarrays has the potential to generate such data; however, the
large quantity of host RNA in any sample makes this problem-
atic. This need for information concerning in vivo expression of
potential vaccine antigens led to development of positive- and
negative-selection technologies such as recombination-based in
www.landesbioscience.com Virulence 455
vivo expression technology (RIVET) and signature-tagged muta-
genesis.141,142 RIVET allows for identification of in vivo expressed
antigens during infection whereas signature-tagged mutagenesis
identifies antigens required for pathogenesis and survival. These
techniques have the advantage of discovering vaccine candidates
that in vitro methods may have missed. Additionally, these tech-
niques do not require selective pressure on the bacteria and there-
fore allow for a natural progression of infection.141
Proteomics and its derivatives. Proteomic profiling identi-
fies the spectrum of proteins expressed by bacteria under varying
growth conditions using a combination of two-dimensional gel
electrophoresis (2DGE) and mass spectrometry (MS). Detection
of membrane and cell wall proteins is a limitation of proteomic
profiling due to (1) their relatively low abundance and (2) solu-
bility constraints due to hydrophobicity (the presence of varying
numbers of transmembrane domains), and an isoelectric point
at pH 8 +.143 Since vaccine development focuses on surface-asso-
ciated proteins, use of extraction protocols that solubilize mem-
brane proteins or isoelectric focusing performed in the alkaline
pH range are essential. Reference maps of the surface proteomes
of S. aureus strains Phillips and VISA generated by lysostaphin
extraction are available.144,145 Another novel antigen discovery
strategy involves identification of surface proteins shaved from
group A Streptococcus cells by trypsin digestion.146,147 Use of this
technique has led to discovery of 42 S. aureus surface proteins
that may have potential as vaccine antigens.148
Serological probing of proteomic samples, known as immu-
noproteomics, followed by peptide identification using matrix-
assisted laser desorption ionization time-of-flight MS is a direct
method for defining antigenic proteins. An initial 2DGE
immunoproteomic study of S. aureus identified 15 known and
novel proteins that were immunoreactive with patient sera.149
Additionally, subtractive proteome analysis was used to iden-
tify immunogenic anchorless S. aureus surface proteins. Proteins
reacting with an intravenous immunoglobulin (IVIG) prepara-
tion (pooled IgG extracted from sera of multiple donors) but not
with IVIG depleted of S. aureus-specific opsonizing antibodies
were considered vaccine candidates.150 Of a total of ~40 anchor-
less cell wall proteins identified in this manner, three were cloned.
Although not tested in a biofilm model of infection, recombinant
versions thereof provided partial protection in a mouse sepsis
model.150 Such anchorless wall proteins lack either a conserved
signal peptide or LPXTG motif, and so may have been omitted
from classical reverse vaccinology screens.151
An additional consideration when developing vaccines effec-
tive against chronic, biofilm-mediated S. aureus infection is the
physiological heterogeneity of these communities (see Fig. 1).
Biofilm communities are inherently complex systems, usually
existing in close proximity to a surface. This complexity arises
from a number of factors. First, distinct physicochemical gradi-
ents are found within microbial biofilm communities.4 In most
cases, organic compounds, oxygen, or water enter the biofilm
from the surrounding bulk fluid and diffuse through the matrix
to the depths closer to the surface.152 Bacteria resident within a
biofilm consume these compounds at varying rates, resulting in
differential availability of nutrients, dependent on the location
of a particular cell within the community. This effect has been
observed experimentally in the case of oxygen tension.152 The
situation is further complicated by very low metabolic levels
and radically downregulated rates of cell division of the deeply
entrenched microorganisms.153
Brady and coworkers used immunoproteomics to elucidate
S. aureus surface proteins that were immunogenic in the rab-
bit model154 of osteomyelitis.46 In subsequent work, the same
group demonstrated that expression of these antigens during
S. aureus biofilm growth in vitro was spatially heterogeneous.5
Based on these data, four antigens were included in a quadri-
valent vaccine administered to rabbits that were then infected
with S. aureus M2 in a biofilm model of infection of osteomy-
elitis. Vaccination, in combination with post-infection vancomy-
cin therapy, was effective at preventing development of chronic
S. aureus osteomyelitis.155
Despite the considerable human and fiscal resources that have
been expended, the search for a vaccine effective against S. aureus
biofilm infections continues. A caveat to consider in developing
anti-S. aureus biofilm therapies are the potential losses of treat-
ment efficacy when given to immunocompromised patients at
risk of infection, specifically in the case of vaccination. It seems
likely that the answer lies in an increased understanding of
S. aureus biofilm development in vivo, and particularly its inter-
actions with the immune system.
Conclusion
Staphylococcus aureus is a clinically relevant pathogen due to its
antimicrobial resistance and evasion of the host immune system.
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In conjunction with the multitude and redundancy of its viru-
lence factors in avoiding host responses and influencing disease,
S. aureus is able to form intricate micro-colonies termed bio-
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Acknowledgments
Completion of this article was funded by the National Institute
of Allergy and Infectious Diseases, National Institutes of Health
(grant R01 AI69568-01A2).
Note
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