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Keywords:

  • Biofilms;
  • genetics;
  • ica operon;
  • Staphylococcus;
  • resistance;
  • review

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The ica operon, biofilm and pathogenesis
  5. Regulation of ica operon expression and biofilm formation
  6. Other factors involved in staphylococcal biofilm formation
  7. Implications for the management of device-related infections
  8. Anti-biofilm antibiotics
  9. Conclusions
  10. Acknowledgements
  11. References

Staphylococcus epidermidis and Staphylococcus aureus are common causes of biofilm-mediated prosthetic device-related infection. The polysaccharide adhesion mechanism encoded by the ica operon is currently the best understood mediator of biofilm development, and represents an important virulence determinant. More recently, the contributions of other virulence regulators, including the global regulators agr, sarA and σB, to the biofilm phenotype have also been investigated. Nevertheless, little has changed at the bedside; the clinical and laboratory diagnosis of device-related infection can be difficult, and biofilm resistance frequently results in failure of therapy. This review assesses the way in which advances in the understanding of biofilm genetics may impact on the clinical management of device-related infection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The ica operon, biofilm and pathogenesis
  5. Regulation of ica operon expression and biofilm formation
  6. Other factors involved in staphylococcal biofilm formation
  7. Implications for the management of device-related infections
  8. Anti-biofilm antibiotics
  9. Conclusions
  10. Acknowledgements
  11. References

Staphylococcus aureus and Staphylococcus epidermidis have evolved to become highly adaptable human pathogens. Colonisation by either species does not usually lead to adverse events; however, once the epithelial and mucosal surfaces have been breached, serious disease can result, ranging from minor skin infections to systemic life-threatening infection. While the clinical presentation of staphylococcal infection is not unique, treatment of these infections is increasingly problematic because of the resistance of clinical isolates to an increasing number of antimicrobial agents. Most staphylococcal infections result in acute disease; however, bacterial persistence and recurrent infections are also observed commonly, particularly among patients with indwelling medical devices, and the increasing use of such devices has resulted in an increase in staphylococcal device-related infection.

The pathogenesis of these infections depends on the ability of staphylococci to first adhere and then form a mucoid biofilm, previously referred to as ‘slime’. Biofilms are sophisticated communities of matrix-encased, surface-attached bacteria that exhibit a distinct phenotype. They are highly hydrated structures, and contain water channels that allow the inward diffusion of nutrients and oxygen; they also contain non-microbial host-derived components such as platelets [1]. Cell-to-cell signalling (quorum-sensing) within a biofilm plays an important role in the coordinated regulation of multiple genes involved in both attachment and biofilm formation, and also in cell detachment in response to changing environmental conditions. Staphylococcal cells embedded in dense polysaccharide biofilms are inherently resistant to host immune responses and antimicrobial chemotherapy. Importantly, device-associated biofilms represent a focus of infection from which individual cells or clusters of cells may detach, resulting in bloodstream infection, emboli and metastatic spread.

During the past decade, there have been numerous published studies concerning the pathogenesis of staphylococcal device-related infection. The optimal approach for treatment of these infections involves removal of the device. However, this is frequently difficult in patients with bleeding tendencies (e.g., because of thrombocytopenia or tunnelled devices) or in unstable patients in the intensive care unit. This review discusses how developments in understanding of the pathogenesis of staphylococcal biofilm infection may lead to improved clinical management.

The ica operon, biofilm and pathogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. The ica operon, biofilm and pathogenesis
  5. Regulation of ica operon expression and biofilm formation
  6. Other factors involved in staphylococcal biofilm formation
  7. Implications for the management of device-related infections
  8. Anti-biofilm antibiotics
  9. Conclusions
  10. Acknowledgements
  11. References

Production of the extracellular polysaccharide, termed ‘poly-N-acetylglucosamine (PNAG)’ in S. aureus and ‘polysaccharide intercellular adhesin (PIA)’ in S. epidermidis, is currently the best understood mechanism of biofilm development. PNAG/PIA is synthesised by enzymes encoded by the ica (intercellular adhesin) operon [2–4]. The ica operon is associated more commonly with invasive isolates of S. epidermidis than with carriage strains [5,6]. In animal models of foreign body infection, inactivation of ica has been reported to be associated with decreased virulence in S. epidermidis[7,8]. Given that S. epidermidis is frequently a contaminant of sterile sites, the development of a discriminatory test to distinguish pathogenic from non-pathogenic isolates would assist greatly in the diagnosis of significant infections. PCR-based detection of the ica locus has been proposed as such a test [5,9]. However, high rates (50%) of S. epidermidis ica positivity have been found in intensive care unit isolates representative of specimen contamination [9], suggesting that detection of ica alone should not guide clinical decision-making [6].

In contrast to the situation in S. epidermidis, the ica operon is found in up to 100% of clinical isolates of S. aureus[4,10–12]. However, in animal models, the relationship between PNAG/PIA production and S. aureus virulence is uncertain. PNAG/PIA has been shown to be a virulence factor in a rat model of endocarditis [13]; however, in guinea-pig tissue cage infection, deletion of ica and absence of PNAG/PIA production had no effect on virulence [14]. Deletion of the ica locus in S. aureus isolate UAMS-1 did not impact on biofilm formation in vitro or in an in-vivo murine model of catheter-related infection [15]. Consistent with this finding, four clinical isolates of methicillin-resistant S. aureus in which glucose-mediated biofilm development was independent of the ica operon have been reported [16] In addition, the rbf (regulator of biofilm formation) gene in S. aureus encodes a putative AraC-type transcription factor required for biofilm development in media supplemented with glucose or NaCl, but does not regulate ica operon expression [17]. Clearly ica-independent mechanisms of biofilm formation exist in S. aureus, and detection of the ica operon in clinical isolates of S. aureus has no useful role in diagnosing isolates associated with biofilm-mediated device-related infection.

Congo red agar (CRA) has been used in the past to detect biofilm production by S. epidermidis[18], and this approach correlates well with a biofilm-positive phenotype observed in vitro[5]. Among clinical isolates, a correlation appears to exist between the phenotype on CRA and the presence of the ica locus [12,19]. However, there is a poor correlation between the phenotype on CRA and biofilm formation among hospital isolates of S. epidermidis[9]. Similarly, the phenotype on CRA was found to be an unreliable indicator of biofilm-forming capacity among clinical isolates of S. aureus[11]. Therefore, while screening on CRA may be easier to perform than a molecular analysis of the genes implicated in biofilm production, and could be performed easily in a diagnostic laboratory, it may be a poor method for determining the biofilm-forming capacity of clinical isolates in the diagnostic laboratory.

Regulation of ica operon expression and biofilm formation

  1. Top of page
  2. Abstract
  3. Introduction
  4. The ica operon, biofilm and pathogenesis
  5. Regulation of ica operon expression and biofilm formation
  6. Other factors involved in staphylococcal biofilm formation
  7. Implications for the management of device-related infections
  8. Anti-biofilm antibiotics
  9. Conclusions
  10. Acknowledgements
  11. References

Regulation of ica operon expression and biofilm development is negatively controlled by the ica operon regulator, IcaR [20–22], and the teicoplanin-associated locus regulator, TcaR [22], and is influenced by environmental conditions, including glucose [17,23], ethanol [24,25], high osmolarity and high temperature [25], anaerobiosis [26], and sub-inhibitory concentrations of tetracycline or quinupristin–dalfopristin [27]. In S. epidermidis, the alternative sigma factor σB positively influences ica operon expression by negatively regulating icaR expression [28]. Mutations affecting σB activity therefore result in a biofilm-negative phenotype [25,28,29]. Unlike S. epidermidis, mutations in the S. aureus sigB locus do not result in a biofilm-negative phenotype [30], thus highlighting an important difference between the species in the regulation of ica operon expression and biofilm development.

Mutations in another global regulator, the staphylococcal accessory regulator (SarA), result in a biofilm-negative phenotype in both S. aureus[30,31] and S. epidermidis[29]. SarA positively regulates ica transcription and PNAG/PIA production [30,31]. A family of sarA homologues, including sarR, sarT, sarU, sarS and rot, have been identified in the S. aureus genome; many of these are involved in the SarA regulatory cascade, and therefore may have an effect on biofilm formation [32]. Given that SarA affects the expression of over 100 genes [33], including the staphylococcal accessory gene regulator (Agr) system, which in turn controls expression of many secreted virulence factors [34], it may prove to be an attractive target for antibacterial agents, because inhibition of its activity would simultaneously control extracellular toxin production and biofilm formation. However, disruption of agr, which encodes the only known quorum-sensing system in staphylococci, is associated with increased biofilm development [35–38]. Activation of the Agr system may therefore contribute to biofilm detachment and metastatic spread to secondary infection sites. Thus, the complexities of the SarA family network and their interactions with the Agr system must be fully elucidated before therapeutic applications are considered.

Other factors involved in staphylococcal biofilm formation

  1. Top of page
  2. Abstract
  3. Introduction
  4. The ica operon, biofilm and pathogenesis
  5. Regulation of ica operon expression and biofilm formation
  6. Other factors involved in staphylococcal biofilm formation
  7. Implications for the management of device-related infections
  8. Anti-biofilm antibiotics
  9. Conclusions
  10. Acknowledgements
  11. References

Atl/AtlE

Bacterial autolysins are peptidoglycan hydrolases that are involved in many processes, such as cell division and separation, cell wall turnover and antibiotic-induced lysis of bacterial cells. Many are considered to be virulence factors, and some are involved in adhesion. The atl gene product (Atl) is the major cell wall autolysin in S. aureus[39]. The atl equivalent in S. epidermidis is atlE, and its product (AtlE) is thought to play a role in the early stages of S. epidermidis adherence by interacting directly with hydrophobic surfaces (such as medical devices) [40]. It may also contribute to later stages of adherence, when polymer surfaces become coated with extracellular matrix proteins, by virtue of its vitronectin-binding ability [40]. It has therefore been speculated that Atl of S. aureus also has adhesion-like functions. In addition, AtlE is thought to play a role in the pathogenesis of biofilm-mediated infection in vivo, as atlE mutants are significantly less virulent than wild-type strains in animal models of intravenous catheter infection [41].

Teichoic acid

The electrical charge of S. aureus teichoic acids is thought to play a role in the initial steps of biofilm formation. Isolates with mutations in the dlt operon (which is responsible for incorporation of D-alanine into teichoic acids) have increased negative charge on the cell surface, are no longer able to colonise glass or polystyrene, despite unaltered PNAG/PIA production, and exhibit a biofilm-negative phenotype [42]. The stronger net negative charge of the dlt mutant has been proposed to increase repulsive forces, thereby preventing adherence. In S. epidermidis, cell wall teichoic acids have also been shown to have fibronectin-binding activity [43] and, given that most medical devices become coated rapidly with a conditioning film of host-derived extracellular matrix components, the fibronectin-binding ability of S. epidermidis teichoic acids may play an important role in the initial steps of biofilm formation.

MSCRAMMs (microbial surface components recognising adhesive matrix molecules)

Staphylococcal protein adhesins also play an important role in adherence, particularly to biomaterials coated with a host-derived conditioning film. Some of the host proteins in the conditioning film can serve as receptors for bacterial attachment. The binding of S. aureus to a wide range of matrix proteins is mediated by surface proteins termed ‘MSCRAMMs’[44]. S. epidermidis does not possess the wide variety of MSCRAMMs present in S. aureus; however, S. epidermidis surface proteins with similarity to the S. aureus clumping factors ClfA and ClfB have been identified [45] and may play a role in S. epidermidis adherence to implanted biomaterials.

Accumulation-associated protein

Accumulation-associated protein (AAP), a homologue of the S. aureus SasG protein [46], is a cell wall-associated protein, expressed predominantly under sessile growth conditions, that is implicated in biofilm accumulation on polymer surfaces [47]. AAP is proposed to play a role in anchoring PNAG/PIA to cell surfaces, because mutants produce PNAG/PIA that is only loosely attached to the cell surface [48]. Consistent with this, a novel domain with five glycine residues, termed the ‘G5 domain’, which is present in AAP, is shared with other proteins that have been implicated in N-acetylglucosamine binding in bacteria with a low GC content [49].

Implications for the management of device-related infections

  1. Top of page
  2. Abstract
  3. Introduction
  4. The ica operon, biofilm and pathogenesis
  5. Regulation of ica operon expression and biofilm formation
  6. Other factors involved in staphylococcal biofilm formation
  7. Implications for the management of device-related infections
  8. Anti-biofilm antibiotics
  9. Conclusions
  10. Acknowledgements
  11. References

Will advances in the understanding of staphylococcal biofilm genetics make a difference in the day-to-day management of device-related infection and, in particular, is treatment likely to be significantly different in the near future? The findings of various investigators in the field of staphylococcal biofilm genetics have certainly expanded our understanding of the complexities of biofilm formation, and have also pointed to potential new therapies for device-related infection. However, as more is discovered, more gaps in knowledge are uncovered and, in the absence of good in-vivo models of device-related infection, it remains to be seen whether this knowledge can be translated directly into clinically significant therapeutic interventions. Furthermore, current antimicrobial susceptibility tests do not assess activity against sessile cells, which are major components of a biofilm.

Anti-biofilm antibiotics

  1. Top of page
  2. Abstract
  3. Introduction
  4. The ica operon, biofilm and pathogenesis
  5. Regulation of ica operon expression and biofilm formation
  6. Other factors involved in staphylococcal biofilm formation
  7. Implications for the management of device-related infections
  8. Anti-biofilm antibiotics
  9. Conclusions
  10. Acknowledgements
  11. References

The major problem in the treatment of device-related infection is that all currently available antibiotics are selected for their ability to kill planktonic cells. There is an obvious need to identify new biofilm-specific antimicrobial targets. Bacteria within biofilms are intrinsically more resistant to antimicrobial agents than planktonic cells, for a wide variety of reasons, including diffusion limitation, altered metabolic activity, the phenotypic and genotypic states of biofilm cells [15,33], and the extreme microenvironmental conditions found in biofilms [50–52]. Antimicrobial resistance in biofilms can also be viewed as a multicellular strategy, in which the biofilm cells collectively withstand antimicrobial agents that would kill a lone cell. In addition, methicillin- and glycopeptide-resistant planktonic staphylococci, which are being isolated increasingly from clinical specimens, are making these ‘difficult-to-treat’ biofilm-associated infections even more difficult to treat with currently available antimicrobial agents.

One potential therapeutic approach would be to interfere with the bacterial cell-to-cell communication that leads to the virulence phenotype. If antimicrobial resistance in the biofilm is a multicellular strategy, then disruption of cell-to-cell communication, together with the use of conventional antimicrobial agents, could potentially be used to treat these infections. The central role of the agr-encoded quorum-sensing system in the regulation of virulence makes it an attractive target. However, mutations in agr, or interfering with agr activity with a cross-inhibiting agr pheromone, promotes the production of colonisation factors such as MSCRAMMs, as well as biofilm development [35,36]. Other approaches have involved the development of anti-PNAG/PIA antibodies to prevent the formation of PNAG/PIA [3,53], or have targeted surface-binding proteins with specific antibodies to prevent adhesion [54]. Recently, a biofilm-releasing enzyme, produced by the Gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans, that removed S. epidermidis biofilms rapidly and efficiently from plastic surfaces was described [55]. This enzyme, termed ‘DspB’ or ‘dispersin B’, which might potentially be used to coat susceptible medical devices, specifically cleaves PIA/PNAG [56]. However, recent evidence of ica-independent biofilm formation [15,16] suggests that the effectiveness of anti-PNAG/PIA therapeutics against diverse clinical isolates must be considered further.

Another promising strategy involves the use of enzymes such as varidase (streptokinase) [57] or lysostaphin [58] to disrupt the staphylococcal biofilm matrix. The use of aspirin and related drugs to inhibit or eradicate staphylococcal biofilms has also been reported [59–61]. More recently, using an endocarditis model, Kupferwasser et al.[62,63] found that aspirin has potential as an adjuvant therapeutic agent and can attenuate virulence phenotypes by downregulating the activity of the global regulator sarA and the agr-encoded quorum-sensing system. In addition, Sun et al.[64] reported that monoclonal antibodies targeting AAP can inhibit S. epidermidis biofilm development significantly. These approaches appear attractive, as they could be used in combination with conventional antimicrobial agents to prevent or treat established biofilm infections, and could also be used to coat catheters used in antibiotic lock therapy to prevent biofilm-mediated device-related infection. Other novel technologies that enhance the susceptibility of biofilm bacteria to conventional antibiotics, such as the use of direct current electric fields [65,66] or ultrasonic radiation [67], may prove useful in the future.

In the meantime, in cases of biofilm-mediated device-associated infection, early removal of the device, while avoiding replacement of devices over guidewires, in conjunction with aggressive antimicrobial therapy to prevent recolonisation of replacement devices, is strongly recommended. Future research concerning the effects of anti-PNAG/PIA antibodies (specific antibodies to prevent adhesion), enzymes such as varidase, or quorum-sensing blockers on medical devices in relevant animal models, should help to improve the management of biofilm-mediated device-associated infections.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. The ica operon, biofilm and pathogenesis
  5. Regulation of ica operon expression and biofilm formation
  6. Other factors involved in staphylococcal biofilm formation
  7. Implications for the management of device-related infections
  8. Anti-biofilm antibiotics
  9. Conclusions
  10. Acknowledgements
  11. References

In summary, staphylococcal biofilm formation is multifactorial and influenced profoundly by the infection milieu. While ica operon expression and PNAG/PIA production are of central importance in generating biofilm, the role of other factors and co-factors has yet to be fully elucidated. In addition, knowledge regarding the role of regulatory pathways in controlling the biofilm phenotype, and in coordinating the various stages of biofilm formation and cellular detachment from the mature biofilm, is still relatively limited. Because of the genetic variability between clinical staphylococcal isolates, it is important that future research characterises biofilm formation in both well-recognised laboratory strains and clinical isolates. In addition, investigators should attempt to realistically mimic the in-vivo environmental conditions of device-related infection. Finally, as the genetic basis for biofilm development emerges, it should become possible to design more ways of preventing and treating staphylococcal device-related infections that are biofilm-specific.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. The ica operon, biofilm and pathogenesis
  5. Regulation of ica operon expression and biofilm formation
  6. Other factors involved in staphylococcal biofilm formation
  7. Implications for the management of device-related infections
  8. Anti-biofilm antibiotics
  9. Conclusions
  10. Acknowledgements
  11. References
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