Staphylococcus aureus biofilms

Abstract

Increasing attention has been focused on understanding bacterial biofilms and this growth modality's 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 regulated 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 community, typified by cells that are attached to a substratum, interface, or to each other, are embedded in a matrix of extracellular polymeric substance, and exhibit an altered phenotype with regard to growth, gene expression and protein production.¹ Biofilm thickness 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 depending on the environmental conditions.² Intricate channel networks 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.

Although a biofilm can arise from a single cell, differential environmental conditions throughout the community can potentiate 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 identified: 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 demonstrated 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 growing cells that are tolerant to high levels of antibiotics but also a proportion of persister cells (no more than ∼1% of total population).⁷ These cells are found in biofilm communities but also exist in planktonic cultures.⁸ In addition, persister cells demonstrate 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 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 antimicrobial 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 antimicrobial 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 surface 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 antimicrobial agents and the host immune response, while simultaneously enabling continuous shedding to encourage bacterial spread.

Staphylococcus aureus Biofilms

Staphylococcus aureus is a Gram-positive, ubiquitous bacterial species. The ecological niche of S. aureus in humans is the anterior nares. In the human population, approximately 20–25% have become persistently colonized and 75–80% 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 cellular 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 predilection to transform from an acute infection to one that is persistent, 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 heterogeneous 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 (80–85%) and an anionic fraction with a lower content of non-N-acetylated D-glucosaminyl residues that contains phosphate and ester-linked succinate (15–20%).²⁰

Figure 1.

IgGs against recombinant forms of cell wall-associated biofilm proteins bind to intact S. aureus biofilms. IgG against each selected candidate protein was applied followed by the secondary goat anti-rabbit F(ab′)2 antibody (upper right in A–D). After a washing step, SYTO 9 was applied to stain all bacterial cells (upper left in A–D). 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 (A–D). 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.

Figure 2.

SEM micrograph from an implant recovered at five days showing massive numbers of cocci partially occluded by dehydrated material. Bar = 5 µm.

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 virulence and are upregulated in response to anaerobic growth, such as the conditions seen in the biofilm environment.²² The staphylococcal respiratory response regulator, SrrAB, is responsible for PIA induction under anaerobic environments via binding of a 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 antibiotics such as tetracycline.¹⁸ In the homologous Staphylococcus epidermidis locus, regulation of ica can occur via reversible inactivation by insertion sequence (IS256) phase variation in 25–33% 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 transcriptional 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 modulating IcaR.²⁹

PIA-independent biofilm formation.

Despite the importance of the ica gene locus in biofilm development, biofilms can occur in an ica-independent fashion. The arlRS two-component 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 deletion of the icaADBC operon, suggesting that in this double deletion 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, deletion of ica and thus, lack of PIA production caused no decrease in virulence.³¹ In addition, Fitzpatrick et al.³² showed that biofilm 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, protein A (SpA) production was found essential for biofilm formation.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 arbitrate biofilm formation through an essential role by the major autolysin (Atl) and sigB regulation,³⁵ and in Staphylococcus epidermidis, 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 elements.³⁸ These reports suggest that proteinaceous cell-to-cell 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 biofilms 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 successful 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 effector 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 formation, 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 regulator 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 environment 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 recovered 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 µm.

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 µm.

Biofilm regulation by agr/sarA.

The major global regulators, staphylococcal accessory regulator (sarA) and accessory gene regulator (agr), have also been implicated in biofilm formation. A two component regulatory gene locus encoded by arlRS, a member of the OmpR-PhoB family of response regulators, is regulated 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 recovered 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 pathway.^49,50,52,53 Despite this, there is evidence that the agr locus is associated with biofilm development. The agr quorum sensing system has been shown to downregulate genes of cell wall-associated 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 essential component of the quorum sensing system, results in seeding dispersal in mature biofilms.⁵⁵ This mechanism occurs in a protease-dependent manner and requires an active agr system since both protease inhibitors and inhibitory AIP block seeding dispersal.^55,56 As well, agr has been shown to upregulate the expression of detergent-like peptides and nucleases that seem to increase biofilm detachment.^53,57 Therefore, the staphylococcal global regulators 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 seeding dispersal in a nuclease, protease and surfactant-dependent manner.

Biofilm regulation by sigB.

Additional levels of control are accomplished through the sigB operon product σ^B in Staphylococcus aureus (regulated by operon genes rsbU, rsbW and rsbV).⁵⁸ Factors necessary for the early stages of biofilm formation, including clumping factor, fibronectin binding protein A (FnbpA) and coagulase, are all upregulated by σ^B.^59,60 In addition, factors that correlate with seeding dispersal and a planktonic phenotype, including β-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 σ^B and is deficient in biofilm formation.⁶³ Additional deletion 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 hypothesize that its role is to sterically hinder the binding of σ^B, thus preventing activation of the ica locus. In contrast, Valle et al.⁴⁹ found that a sigB deletion mutant still effectively developed biofilm. 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 biofilms.

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 microbial 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 damage to the host.⁶⁵ In a fully mature biofilm, staphylococci are slow growing and nutrient deprived resulting in the downregulation 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 discussion 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.

Figure 5.

Flowchart of regulatory factors involved in S. aureus biofilm formation, maintenance and detachment. (A) PIA-dependent biofilm formation—expression 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 expression, 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 formation—eDNA 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 regulation—expression 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.

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 antimicrobial 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 biofilm 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 mimicked in many other diseases and host sites.

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 inoculation during surgery, trauma or an overlying infection.⁷⁰ While in the blood in the planktonic mode of growth, S. aureus upregulates the production of adhesins for many host adhesive matrix molecules (e.g., fibrinogen, fibrin, osteopontin, fibronectin, collagen, elastin, etc.).⁶⁴ Once in the bone, S. aureus attaches to localized 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, resulting in the production of toxins, immunoavoidance factors and other agr-controlled virulence factors with a concomitant downregulation in microbial adhesins.⁶⁵ Simultaneously, a number of microbial inflammatory mediators such as formylmethionyl peptides, low level hemolysins, lipotechoic acids, peptidoglycan, 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 immunoavoidance factors resulting in host cell lysis and frustrated phagocytosis.⁷¹ The end result is a maturing S. aureus biofilm population with detaching flocs and tissue damage due to PMN-induced collateral host tissue damage, thereby providing the essential devitalized surface for further biofilm development (see Fig. 7 and Movie S1).^70,72 In adults, the resulting tissue destruction means that the biofilm and devitalized tissue must be surgically debrided in order resolve the infection.⁶⁹ However, in cases of juvenile osteomyelitis, children are often able to resorb the dead bone and tissue during antimicrobial therapy, resulting in the elimination of the bacterial attachment surface. Therefore, this type of biofilm infection in juveniles is one of the few types of biofilm infections 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.

Figure 6.

SEM micrograph demonstrating cocci adherent to a bone sequestra. Collapsed glycocalyx material is apparent associated with the bacterial biofilm (arrow). Bar = 5 µm.

Figure 7.

(A) Nasal colonization confers Staphylococcus aureus a reservoir for dissemination onto skin and various other host epithelial surfaces. Damage 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, fibronectin binding protein A; Fib, fibrinogen binding protein; FbpA, fibronectin binding protein; CflA, clumping factor A; EbpS, elastin binding protein. (C) Initial 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 polysaccharide 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 formation of a nascent biofilm can occur to repeat the cycle.

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 biofilm 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 dental 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 the oral cavity.⁷⁷ S. aureus has a high affinity to titanium surfaces and is recovered at high levels from infected peri-implant pockets. Although this pathogen is not strongly associated with 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 diabetic 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 88–93.5% of infections.^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 biofilm 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 molecular 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 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, surgically recalcitrant infection and unfavorable post-operative evolution.^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 carries 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 clinically 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 endophthalmitis.^88,89 Staphylococci, specifically S. aureus, had the highest isolation 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-compromised patients. Candida albicans and S. aureus have been co-cultured from oral and vaginal mucosal surfaces, both exhibiting 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 biofilm infections and delineating which confer clearance or foster persistence.

In a mouse tibial implant model of S. aureus biofilm osteomyelitis, 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, TNFα and IL-1β (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.

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 complement inhibitor (SCIN) that binds to and stabilizes C3 convertases, 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 system's primary defense against extracellular pathogens, S. aureus has evolved a number of different strategies to disarm this early inflammatory 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 biofilms.⁹⁶ Neutrophils co-cultured with planktonic S. aureus cultures, 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 phagocytosis, 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 activation 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 resulting in concomitant host tissue damage. Interestingly, S. aureus biofilms, and not planktonic cultures, were conferred a growth advantage in the presence of IL-1β.¹⁰⁰ This insinuates that induction of inflammatory mediators, such as IL-1β, may bolster biofilm formation and therefore mitigate the immune system's ability to clear S. aureus infection. In addition, recent studies in our laboratory have shown that the development of chronic infection 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 prevention 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 infection, are not activated until persistent infection has already developed.¹⁰¹ However, even though the host produces these IgG₁ antibodies, they may also be less effective due to the heterogeneous 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 bacterial 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 factors.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 complicated by the fact that S. aureus—during biofilm formation and maturation—releases 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., vancomycin) or difficulties with absorption from the gastrointestinal tract.¹⁰⁷ However, the evidence suggesting that parenteral administration 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 criteria.¹⁰⁷ The majority of patients with surgically managed biofilm infections can be treated orally with one or more of several antibiotics, 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.

β-lactam agents are often used in the treatment of biofilm infections such as osteomyelitis. They demonstrate time-dependent killing, maximal when the serum concentration is ca. four times the MIC.¹⁰⁹ β-lactams do not show a post-antibiotic effect; i.e., antimicrobial activity after antibiotic concentrations have decreased below therapeutic levels. Penicillin G (12–20 million U every 24 h IV) and oxacillin (1–2 g IV every 4–6 h) are the current recommended first-line therapy for osteomyelitis caused by penicillin-sensitive and −resistant S. aureus, respectively.¹¹⁰

Unlike β-lactams, fluoroquinolones and aminoglycosides demonstrate both concentration-dependent activity and a postantibiotic effect.¹¹¹ Dosing of such agents is limited by their toxicity (aminoglycoside nephrotoxicity). Therefore, systemic aminoglycosides are usually recommended for prolonged therapy 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-dependent mode of action. Vancomycin is the principal agent used for treatment of methicillin resistant biofilm infection; the recommended 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 daptomycin may be effective alternatives for treatment of S. aureus biofilm infection.¹¹⁴

Rifampin has the capacity to kill metabolically dormant sessile bacteria, making it highly useful for musculoskeletal infections 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 management 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 concentrations of antibiotics and may be useful for situations such as infection prophylaxis following open fractures.¹¹⁶ Calcium sulfate beads also dissolve and so do not require surgical removal. Additionally, they do not generate heat during formation, facilitating 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 leaching 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 antimicrobial 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 antigens. The sequencing of the whole genome of multiple S. aureus strains,¹²³ followed by those of methicillin-resistant^124,125 and vancomycin-intermediate¹²⁶ and epidemic¹²⁷ strains undoubtedly marked a significant step forward. The subsequent development of bioinformatics to manipulate and analyze these data has facilitated high-throughput genomic, transcriptomic and proteomic analyses of microbial growth and pathogenesis.^128,129

The first question that must be answered is which component of the biofilm should be targeted. Broadly speaking, two alternatives exist: bacterial cells within the biofilm and the biofilm matrix itself. The biofilm matrix may be composed of polysaccharides, protein or extracellular DNA, in proportions that vary between bacterial genera, species and strains. Most antibiofilm 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-β-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 preventing 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 analyzing individual virulence factors from pathogens cultured in vitro.¹⁴⁰ Identification of putative antigens—principally surface proteins—by systematic searching of the pathogen's 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 selection 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 problematic. 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 vivo expression technology (RIVET) and signature-tagged mutagenesis.^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 techniques do not require selective pressure on the bacteria and therefore allow for a natural progression of infection.¹⁴¹

Proteomics and its derivatives.

Proteomic profiling identifies 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) solubility 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-associated proteins, use of extraction protocols that solubilize membrane 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 immunoproteomics, 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 identify immunogenic anchorless S. aureus surface proteins. Proteins reacting with an intravenous immunoglobulin (IVIG) preparation (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 anchorless 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 anchor-less 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 effective 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 gradients 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 rabbit 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 quadrivalent vaccine administered to rabbits that were then infected with S. aureus M2 in a biofilm model of infection of osteomyelitis. Vaccination, in combination with post-infection vancomycin 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 treatment 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 interactions with the immune system.

Conclusion

Staphylococcus aureus is a clinically relevant pathogen due to its antimicrobial resistance and evasion of the host immune system. In conjunction with the multitude and redundancy of its virulence factors in avoiding host responses and influencing disease, S. aureus is able to form intricate micro-colonies termed biofilms. Although neutrophils are capable of invading the biofilm, the bacterial community is able to thwart this attack and may also skew the immune response to survive attack. Staphylococcus aureus is the etiological agent to a myriad of human acute infections, however, its ability to form biofilm in host emanates into chronic and recalcitrant disease. Current therapies for treating and preventing chronic biofilm-mediated infections are limited to surgical intervention and prolonged antibiotic regiments or addition of antimicrobial compounds to indwelling-medical devices. Vaccination studies have begun to take biofilm development into consideration, and with the combination of genomic and proteomic-based techniques have identified numerous potential vaccination candidates. However, the physiological heterogeneity and subsequent multifarious protein expression throughout the biofilm must be carefully examined for development of an efficacious S. aureus vaccine. Alternatively, modulating the host immune response may prove advantageous in resolving chronic S. aureus infections and warrants further investigation.