An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections

Journal of Infectious Diseases, Nov 2015

High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm–associated, medical device–related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤24 hours on biomaterials conditioned with human plasma under venous shear in iron-free cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin. In contrast, SarA-regulated biofilms formed on uncoated polystyrene in nutrient-rich bacteriological medium were mediated by the previously characterized biofilm factors poly-N-acetyl glucosamine, fibronectin-binding proteins, or autolytic activity and were antibiotic resistant. Coagulase-mediated biofilms exhibited increased antimicrobial resistance over time (>48 hours) but were always susceptible to dispersal by the fibrinolytic enzymes plasmin or nattokinase. Biofilms recovered from infected central venous catheters in a rat model of device-related infection were dispersed by nattokinase, supporting the important role of the biofilm phenotype and identifying a potentially new therapeutic approach with antimicrobials and fibrinolytic drugs, particularly during the early stages of device-related infection.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections

JID An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections Marta Zapotoczna 2 3 Hannah McCarthy 0 2 Justine K. Rudkin 0 2 James P. O'Gara 0 2 Eoghan O'Neill 1 2 3 0 Department of Microbiology, School of Natural Sciences, National University of Ireland , Galway , Ireland 1 Department of Microbiology, Connolly Hospital , Dublin 2 Received 29 April 2015; accepted 30 May 2015; electronically published 4 June 2015. ural Sciences, National University of Ireland , Galway , Ireland 3 Department of Clinical Microbiology, Education and Research Centre, Beaumont Hospital, Royal College of Surgeons in Ireland High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm-associated, medical device-related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤24 hours on biomaterials conditioned with human plasma under venous shear in ironfree cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin. In contrast, SarA-regulated biofilms formed on uncoated polystyrene in nutrient-rich bacteriological medium were mediated by the previously characterized biofilm factors poly-Nacetyl glucosamine, fibronectin-binding proteins, or autolytic activity and were antibiotic resistant. Coagulase-mediated biofilms exhibited increased antimicrobial resistance over time (>48 hours) but were always susceptible to dispersal by the fibrinolytic enzymes plasmin or nattokinase. Biofilms recovered from infected central venous catheters in a rat model of device-related infection were dispersed by nattokinase, supporting the important role of the biofilm phenotype and identifying a potentially new therapeutic approach with antimicrobials and fibrinolytic drugs, particularly during the early stages of device-related infection. Staphylococcus; biofilm; infection; coagulase; antimicrobial; susceptibility - adhesive matrix molecules [2], such as fibrinogenbinding clumping factor A (ClfA) and ClfB and fibronectin-binding and fibrinogen-binding protein A (FnBPA) and FnBPB. S. aureus also interacts with host plasma glycoproteins via secreted proteins associated with the bacterial surface, including the extracellular matrix binding protein (Emp) and the extracellular adherence protein (Eap) [3]. In contrast, bacterial cell attachment to abiotic surfaces is influenced by the charge on teichoic acids and is mediated by a range of surface proteins: poly-N-acetyl glucosamine (PNAG)/polysaccharide intercellular adhesin (PIA), and extracellular DNA (eDNA) or cytoplasmic proteins released following lysis [4, 5]. In methicillin-resistant S. aureus (MRSA), the major cell wall autolysin Atl, eDNA, and FnBPA and FnBPB play fundamental roles in biofilm attachment and accumulation [6–8], whereas methicillin-susceptible S. aureus is more likely to produce PIA/PNAG-mediated biofilm [9]. Other proteins implicated in the biofilm phenotype include biofilm-associated protein, protein A, SasG, SasC, ClfB, and the secreted proteins Eap and Emp [3]. The ability of a number of cell wall and extracellular proteins from S. aureus to recruit and deposit extracellular matrix proteins such as fibrinogen or fibrin on the cell surface has led to the idea that so-called fibrin shields play a role in resistance to opsonophagocytosis and perhaps vaccine failure [2]. Furthermore, a number of cell wall proteins involved in biofilm accumulation (FnBPA, FnBPB, and ClfB) are known to bind fibrinogen, suggesting a relationship between these 2 phenotypes. Consistent with this, Dastgheyb et al recently reported an important role for staphylothrombin-mediated fibrin deposition in the pathogenesis of catheter-related infections caused by S. aureus [10, 11]. Similarly, coagulase was significantly implicated in the formation of biofilm-like aggregates, which play an important role in the pathogenesis of joint infections [12, 13]. The potential contribution of fibrin deposition to biofilm formation under in vivo conditions highlights both the importance of the biofilm phenotype and the possible redundancy among biofilm mechanisms used by S. aureus, given that cells can be embedded in a matrix of bacterial proteins, host glycoproteins, polysaccharide, or extracellular nucleic acids [4, 14, 15]. However, it remains unclear which of these biofilm mechanisms is preferentially deployed under in vivo conditions, and hence the relative contribution of different staphylococcal biofilm matrices to the antimicrobial resistance of biofilms has been largely unexplored. To date, in vitro analysis of staphylococcal biofilm has generally focused on measurements of bacterial cell attachment to and accumulation on abiotic surfaces by using nutrientrich bacteriological growth medium [16] and, more recently, under physiologically relevant shear [17]. In this study, we describe the development of an in vitro biofilm model to more closely reflect the in vivo milieu. Attachment and biofilm accumulation by S. aureus strains grown in cell culture medium on surfaces conditioned with human plasma were measured under venous shear. Antimicrobial susceptibility of biofilms grown under traditional methods and under these more physiologically relevant conditions was compared. Using the Nebraska Transposon Mutant Library (NTML) [18], we studied the relative contribution of known biofilm factors in the community-associated MRSA strain USA300 JE2 and validated this finding in other S. aureus backgrounds. Our data reveal a new biofilm phenotype mediated by coagulase and ClfA and regulated by the SaeRS system. Furthermore, these biofilms exhibit a distinct antimicrobial susceptibility profile and, as demonstrated by in vitro and in vivo findings, can be dispersed by fibrinolysins. MATERIALS AND METHODS Preparation of Platelet-Rich Plasma (PRP), Platelet-Poor Plasma (PPP), and Serum PRP was prepared by collecting human blood specimens in syringes containing heparin at 16 IE/mL. Whole blood was centrifuged at 150 × g for 10 minutes, and the top layer, consisting of PRP, was carefully removed. PPP was obtained by centrifugation of PRP or whole blood at 500 × g for 10 minutes. Serum was obtained by centrifugation of clotted whole blood at 2000 × g for 10 minutes at 4°C. Biofilm Analysis Under Static Conditions Strains used in the study are listed in Table 1. Biofilms were grown in hydrophilic (Nunclon) or hydrophobic (Costar) plates, as indicated. A total of 100 µL of 20% (v/v) plasma in 50 mM carbonate buffer, pH 9.6, was incubated in the wells for 2 hours at 37°C. A range of plasma concentrations (5%– 100% v/v) were tested and showed that a 20% PPP v/v supported optimal biofilm formation (data not shown). S. aureus overnight cultures in Roswell Park Memorial Institute (RPMI) 1640 medium were diluted at a ratio of 1:1 (v/v) in fresh medium and incubated in microtiter wells for 1 hour at 37°C. Unattached bacteria were removed, and the well was refreshed with sterile medium. To grow biofilms in rich medium, overnight cultures were diluted a ratio of 1:200 in either tryptic soy broth (TSB) or brain-heart infusion (BHI) broth, and biofilm assays were performed as described above. Biofilms were grown for 24 hours at 37°C, were stained with 0.5% crystal violet, and underwent absorbance measurement at OD490. To quantify viable bacteria in biofilms, TrpLE (recombinant trypsin; Gibco) was used to disperse biofilms for 5 minutes at 37°C, and the number of colony-forming units (CFU) was determined. Biofilm Treatment With Antibiotics or Enzymes Antibiotics or enzymatic agents ( proteinase K, plasmin, nattokinase, serrapeptase, and TrpLE) were added to biofilms and incubated for 24 hours unless otherwise indicated. Viability after treatment was measured using a resazurin-conversion assay, and biofilm thickness was determined by crystal violet staining. Resazurin-Conversion Assay Conversion of the nonfluorescent redox dye resazurin into the fluorescent resorufin was used to measure viability of bacteria in biofilms. A stock solution of resazurin (Sigma) at 440 µM was diluted to 10% (v/v) in RPMI 1640 medium and added at a ratio of 1:1 (v/v) to microtiter well biofilms before being incubated in the dark at 37°C for 1 hour. Fluorescence at 544-nm excitation and 590-nm emission was read using a Perkin Elmer 2030 Multilabeled Reader Victor X3. Biofilm Analysis Using a Cellix Microfluidic Flow Cell System Vena8 Fluoro+ flow chambers were primed 3 times with 20 µL of PPP at 37°C for 30 minutes. S. aureus overnight cultures were diluted 1:1 in RPMI 1640 medium or 1:200 in TSB. Bacterial suspensions were injected into flow chambers and incubated for 4 hours before the microfluidic pump was engaged to visualize biofilm growth at 37°C for 24 hours under a shear of 6.25 dynes/cm2 (200 µL/min). MRSA; device-related infection isolate MSSA; device-related infection isolate CA-MRSA USA300 derivative; ΔsaeRS USA300 constitutively expressing luciferase (lux) from Photorhabdus luminescens USA300 lacking plasmids p01 and p03; NTML parent Table 1. Staphylococcus aureus Strains Used in This Study Reference Abbreviations: CA, community acquired; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; NTML, Nebraska Transposon Mutant Library. Biofilm Formation Using a Bioflux Microfluidic Flow Cell System The BioFlux 1000z microfluidic system (Fluxion Biosciences, California) was used to assess biofilm formation at 0.6 dynes/cm2 for 18 hours as described previously with the following modification. Each channel of a 48-well plate was coated with 50 µL of undiluted platelet-poor human plasma for 30 minutes at 37°C before biofilm assays were set up. Coagulase Activity Assay S. aureus cultures were grown for 18 hours and adjusted to equal optical densities at 600 nm (OD600). For RPMI 1640 medium, cultures were adjusted to an OD600 of approximately 1, while for TSB, cultures were adjusted to an OD600 approximately 10. Bacterial suspensions were centrifuged to separate culture supernatants, which were collected for coagulase assay. Microtiter wells were inoculated with 100 µL of human plasma. A total of 100 µL of culture supernatant, 1:1 diluted culture supernatant, or medium control was added to the wells. Microtiter plates containing the mixtures were incubated for 4 hours at 37°C following absorbance reading at 600 nm. Peptide Synthesis D- Fg wt γ (1–17) D16A (D-enantiomer of the γ chain of fibrinogen) peptide (NH2-gegqqhhlggakqagac-CONH2) was synthesized by automated solid-phase peptide synthesis on a 433A synthesizer (Applied Biosystems, United Kingdom) from 9-fluorenylmethoxy carbonyl (Fmoc)–protected D-amino acids (Merck, United Kingdom). Rat Jugular Vein Catheter Infection Model Sprague-Dawley rats with preimplanted jugular vein catheters were obtained from Charles River (United Kingdom). The 13.5-cm-long catheters (outside diameter, 1.1 mm; inside diameter, 0.6 mm) were inoculated with a 40-µL suspension of USA300lux containing 106 CFU/mL. The catheters were flushed daily with 150 µL of sodium chloride (0.1% w/v), and the lumen was locked with 40 µL of sodium chloride (0.1% w/v). Intravenous vancomycin (50 mg/kg) was administered twice daily at 10-hour intervals. After 10 days, infected catheters were removed, and luminescence was imaged using a Perkin Elmer IVIS Spectrum instrument (exposure, 20 seconds; binning: 4, f1) before being treated three times with 100 µg/mL nattokinase (2000 FU) for 1 hour at 37°C. Ethics Approval Blood donations were obtained from healthy adult donors. Written, informed consent was obtained from participants at the time of collection. Ethics approval for collection and use of blood was granted by the Ethics Committee of the Royal College of Surgeons in Ireland (RCSI; REC820). Animal experiments were conducted under Irish Government Department of Health guidelines, with ethical approval from the RCSI Ethics Committee (REC931). Statistical Analysis The data presented by this study represent the means ± SD of three experiments unless otherwise stated. Statistical significance was assessed using one-way ANOVA and indicated as * for P < .05, ** for P < .001 and *** for P < .0001. Biofilm Antimicrobial Susceptibility Is Increased on Human Plasma–Conditioned Surfaces To more closely mimic in vivo conditions, PRP, PPP, and serum were used to condition microtiter wells and revealed that plasma proteins but not serum promoted biofilm formation (Supplementary Figure 1). Plasma conditioning contributed significantly to the thickness of biofilms involving of all S. aureus strains, including isolates from device-related infections, such as BH1CC and BH48 (Supplementary Figure 1). To reflect iron limitation encountered by bacteria in vivo, biofilms were also grown in RPMI 1640 medium. All wild-type S. aureus strains, including USA300, MW2, UAMS-1, SH1000, Newman, BH1CC, and BH48, formed biofilms when grown on surfaces conditioned with plasma in RPMI 1640 medium (data not shown). Viable counts revealed that USA300 and SH1000 biofilms grown for 24 hours in RPMI 1640 medium contained 1–2 log fewer cells than biofilms grown in TSB or BHI broth (Supplementary Figure 2A). Similar data were obtained with BH1CC and BH48 (data not shown). Notably the numbers of viable bacteria in RPMI 1640 medium–grown biofilms were similar to the previously reported numbers of bacteria in biofilms recovered from infected catheters in vivo [19]. USA300 and SH1000 biofilms grown for 24 hours on human plasma–coated surfaces in RPMI-1640 medium were susceptible to the antistaphylococcal antibiotics rifampicin and vancomycin at a concentration of <1 mg/mL (Figure 1A), whereas biofilms grown in BHI medium were resistant to 50 mg/mL of either antibiotic (Figure 1B). Resistance of RPMI-1640 medium–grown biofilms to both antibiotics increased significantly beginning at 3 days (Figure 1A), when cell densities were similar to those of biofilms grown in rich laboratory broths (Supplementary Figure 2B). These data suggest that the in vivo biofilm cell density may be an important indicator of susceptibility and identify a therapeutic window for biofilm eradication in the early stages of biofilm-associated infections. The SaeRS System and Coagulase Are Required for Biofilm Formation in an Iron-Free Environment on Human Plasma– Coated Surfaces S. aureus USA300 JE2 mutants from the NTML, which are deficient in known biofilm mediators and regulators, were grown statically in RPMI-1640 medium or TSB on plasma-coated surfaces (Figure 2A). In RPMI-1640 medium only, the saeS:: Tn mutant exhibited a biofilm defect (Figure 2A), whereas in TSB medium, the sarA::Tn, srtA::Tn, and spa::Tn mutants revealed a significant biofilm impairment (Figure 2A). Extending this analysis to a physiologically relevant high venous shear (6.25 dynes/cm2), use of a Cellix microfluidic flow cell system further implicated SaeS in RPMI-1640 medium (Figure 2B) and defibrinated human blood (Figure 2C) and identified a role for ClfA, which was not evident under static conditions. Like the clfA::Tn mutant, the srtA::Tn mutant was also biofilm negative under venous shear in RPMI-1640 medium (Figure 2B). A ΔsaeRS mutation also impaired biofilm formation by UAMS-1, USA300 LAC, and MW2 under venous shear in RPMI-1640 medium but not TSB, and the MW2 ΔsaeRS mutant biofilm defect in RPMI 1640 medium was complemented with a plasmid constitutively expressing the saePQRS genes (Figure 3). When RPMI 1640 medium was replaced with TSB medium, the sarA and srtA mutants exhibited significant biofilm defects, whereas the ClfA and SaeS mutants were unaffected (Supplementary Figure 3). The SaeRS system is repressed by glucose [20], perhaps explaining at least in part the different contributions of this regulatory system to biofilm in minimal RPMI 1640 medium and TSB or BHI medium. Consistent with this, RPMI 1640 medium supplemented with glucose, dose-dependently inhibited biofilm formation by USA300 and SH1000 (Supplementary Figure 4). To elucidate the mechanism of SaeRS-dependent biofilm formation on human plasma-coated surfaces the role of coagulase (coa), which is part of the SaeRS regulon, was investigated. SaeRS positively regulates coagulase [21], which catalyses the conversion of fibrinogen, an abundant protein in human plasma, into fibrin [22]. A JE2 coa::Tn mutant was biofilm negative when grown in RPMI 1640 medium under flow on plasma (Figure 4A). Interestingly, a mutation in the gene encoding von Willebrand factor–binding protein, which produces a second coagulase in S. aureus [23], had no impact on biofilm (Figure 4A). Human plasmin, which degrades fibrin, dispersed USA300 biofilms (Figure 4B), as did proteinase K (Figure 4B). Dispersin B, which degrades PIA/PNAG and nuclease had no effect on biofilms grown on plasma in RPMI 1640 medium (Figure 4B), indicating no role for polysaccharide or eDNA in this biofilm phenotype. Plasmin also dispersed UAMS-1, MW2, and SH1000 biofilms grown on plasma in RPMI 1640 medium (Figure 4C). Consistent with these phenotypes, coagulation of human plasma under iron-limited conditions by MW2 culture supernatants was show to be SaeRS dependent (Supplementary Figure 5). Taken together, these data support previous findings implicating sortase [7] and SarA [24] in the S. aureus biofilm phenotype in rich medium and identify new roles for SaeRS, ClfA, and coagulase in biofilm production on human plasma–coated surfaces under physiologically relevant growth conditions. Critical Role of ClfA in Biofilm Formation on Human Plasma– Coated Surfaces Is Shear Dependent Further investigation of SaeRS/coagulase-mediated biofilm at venous shear (6.25 dynes/cm2) in the Cellix system or 0.6 dynes/cm2 in either Cellix or Bioflux system revealed that the clfA mutation in strains SH1000 and Newman had no effect at the lower shear rate but impaired biofilm at venous shear (Figure 5A). ClfA binds the C-terminal region of the Fg γ chain by using the so-called dock, lock, and latch mechanism [25]. A variant peptide that mimics the γ chain region of Fg and interacts with ClfA with higher affinity has been identified as Fg γ (1–17) D16A [26]. Biofilm formation by SH1000, which was incubated with this peptide prior to inoculation of the microfluidic flow cell chamber, was inhibited in a dose-dependent manner (Figure 5B), indicating that ClfAdependent biofilm production on human plasma is related to its fibrinogen-binding activity, which is then likely to facilitate coagulase-mediated conversion of fibrinogen to fibrin. Dispersal of Biofilms Grown In Vitro and In Vivo With Fibrinolysins Consistent with the effect of plasmin, mature 3-day and 14-day coagulase-mediated US300 and SH1000 biofilms were also effectively dispersed with nattokinase and serrapeptase (Figure 6A). Furthermore, biofilms formed by USA300lux on central venous catheters in a rat model were also dispersed effectively with nattokinase (Figure 6B), supporting the importance of coagulase-mediated biofilm in vivo and the therapeutic potential of targeting this colonization mechanism for the treatment of device-related infections. DISCUSSION The challenge of managing chronic device-associated infections is primarily associated with the inherent drug resistance of biofilms colonizing biomaterial surfaces. Characterization of biofilm susceptibility has generally been performed under in vitro conditions on artificial surfaces. However, the rapid coating of implanted materials by plasma and extracellular matrix proteins and host sequestration of nutrients during infection suggest that in vitro characterization of biofilm antibiotic resistance may not fully reflect the in vivo milieu. To address this, S. aureus biofilms were grown on human plasma–conditioned surfaces under shear flow in chemically defined, iron-limited medium (RPMI 1640 medium). Early biofilms grown for ≤24 hours under these conditions were significantly more sensitive to the antistaphylococcal antibiotics rifampicin and vancomycin than biofilms grown on polystyrene in bacteriological medium. Increased drug susceptibility on plasma-coated surfaces was transient, and older (growth time, >48 hours), denser biofilms exhibited high levels of resistance. The 2-component system SaeRS, which is expressed by all S. aureus strains [27], was required for biofilm production under these conditions. SaeRS has been identified as an important regulator of global gene expression under in vivo conditions [28], including during device-related infections [29]. Furthermore mutation of sae blocks upregulation of proteases, toxins, and surface proteins such as α- and β-hemolysin, FnbA, coagulase, and protein A, which are important for the pathogenesis of S. aureus infection [30–34]. Under iron-limiting conditions, SaeRS was previously shown to increase biofilm production by strain Newman by activating expression of Emp and Eap [20, 35, 36]. However, use of the biofilm model described in this study biofilm production was not dependent on Emp or Eap. Similarly mutations in the hla, hlb, fnbA, or spa genes, which are also regulated by the Sae system [21], had no significant effect on this biofilm phenotype. A subsequent screen of Sae-regulated genes identified a key role for staphylococcal coagulase (Coa) in biofilm production on human plasma–coated surfaces. Coagulase is a secreted enzyme that binds prothrombin to form staphylothrombin, which in turn converts fibrinogen to fibrin, resulting in blood clotting [22]. Our data showed that this process was induced by wildtype S. aureus supernatants but not by supernatants from sae mutant cultures, confirming that coagulase production is controlled by SaeRS in our experimental set up. Moreover, Sae- and Coa-dependent biofilms grown in vitro or in a rat model of central venous catheter infection were dispersed by human plasmin and nattokinase, which both degrade fibrin, further implicating coagulase-catalyzed production of fibrin in the S. aureus biofilm phenotype on human plasma–coated surfaces. Under venous shear in RPMI 1640 medium (and not under static conditions), ClfA, which binds fibrinogen and fibrin [25], was also important for coa-dependent biofilm formation on plasma-coated surfaces. A peptide that binds to ClfA with a higher affinity than the γ chain fragment of fibrinogen blocked biofilm formation, indicating that S. aureus uses this fibrinogen/fibrin receptor to promote attachment via a mechanism that is not critical at lower shear or during static biofilm growth. Our data support a recent report by Vanassche et al demonstrating that an S. aureus mutant lacking both Coa and the second S. aureus coagulase, von Willebrand factor–binding protein, exhibited defective in vitro colonization of polyurethane catheters conditioned with fresh human plasma [10]. Furthermore, chemical inhibition of coagulase activity using dabigatran reduced S. aureus colonization of catheters in a murine model of jugular vein catheter infection [10, 11]. Interestingly, our data showed that mutation of the vwb gene had no significant impact on biofilm formation on plasma-coated surfaces. Consistent with previous reports on the importance of SarA for biofilm formation by S. aureus under in vitro [24, 37–39] and in vivo conditions [40], our data showed that SarAregulated biofilm production on polystyrene (either uncoated or coated with human plasma) in nutrient-rich bacteriological medium was dependent on the previously characterized biofilm mediators PIA/PNAG, on FnBPs or on autolytic activity [14]. The absence of a role for SarA in biofilm production in RPMI 1640 medium underlines the importance of the experimental conditions used for biofilm analysis and the importance of the SaeRS/Coa-mediated biofilm phenotype under physiologically relevant conditions. Taken together, these data suggest that the adhesion and biofilm mechanisms used by staphylococci to colonize implanted biomaterials may, in part, be dependent on the degree of surface conditioning by plasma and extracellular matrix proteins. Insoluble fibrin, produced as a result of coagulase activity, can apparently function as a biofilm scaffold, allowing S. aureus to accumulate on human plasma–coated surfaces. In keeping with studies showing the therapeutic benefit of coagulase inhibitors or plasmin in S. aureus cardiovascular and joint infections [10–12], the data presented in this study reveal the enhanced therapeutic potential of fibrinolytic and antimicrobial drug combinations for S. aureus biofilm eradication, compared with currently used antibiotics, particularly during the early stages of device-related infection. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online ( Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. Acknowledgments. We thank T. J. Foster, J. Geoghegan, J. Voyich, M. Smeltzer, and R. Plaut for kindly providing us with Staphylococcus aureus strains; E. Forde and M. Devocelle, for technical advice regarding peptide synthesis; and J. Kwiecinski, for scientific advice. Financial support. This work was supported by the Irish Health Research Board (grants to E. O. and J. P. O.) and the Healthcare Infection Society (to E. O.). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. 1. Hogan S , Stevens NT , Humphreys H , O'Gara JP , O'Neill E. Current and future approaches to the prevention and treatment of Staphylococcal medical device-related infections . Curr Pharm Des 2014 ; 21 : 100 - 13 . 2. Foster TJ , Geoghegan JA , Ganesh VK , Hook M. Adhesion , invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus . Nat Rev Microbiol 2014 ; 12 : 49 - 62 . 3. Speziale P , Pietrocola G , Foster TJ , Geoghegan JA . Protein-based biofilm matrices in Staphylococci . Front Cell Infect Microbiol 2014 ; 4 : 171 . 4. O'Gara JP . ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus . FEMS Microbiol Lett 2007 ; 270 : 179 - 88 . 5. Foulston L , Elsholz AK , DeFrancesco AS , Losick R. The extracellular matrix of Staphylococcus aureus biofilms comprises cytoplasmic proteins that associate with the cell surface in response to decreasing pH . MBio 2014 ; 5 : e01667 - 14 . 6. Pozzi C , Waters EM , Rudkin JK , et al. Methicillin resistance alters the biofilm phenotype and attenuates virulence in Staphylococcus aureus device-associated infections . PLoS Pathog 2012 ; 8 : e1002626 . 7. O'Neill E , Pozzi C , Houston P , et al. A novel Staphylococcus aureus biofilm phenotype mediated by the fibronectin-binding proteins, FnBPA and FnBPB . J Bacteriol 2008 ; 190 : 3835 - 50 . 8. Geoghegan JA , Monk IR , O'Gara JP , Foster TJ . Subdomains N2N3 of fibronectin binding protein A mediate Staphylococcus aureus biofilm formation and adherence to fibrinogen using distinct mechanisms . J Bacteriol 2013 ; 195 : 2675 - 83 . 9. O'Neill E , Pozzi C , Houston P , et al. Association between methicillin susceptibility and biofilm regulation in Staphylococcus aureus isolates from device-related infections . J Clin Microbiol 2007 ; 45 : 1379 - 88 . 10. Vanassche T , Peetermans M , Van Aelst LN , et al. The role of staphylothrombin-mediated fibrin deposition in catheter-related Staphylococcus aureus infections . J Infect Dis 2013 ; 208 : 92 - 100 . 11. Vanassche T , Verhaegen J , Peetermans WE , Hoylaerts MF , Verhamme P. Dabigatran inhibits Staphylococcus aureus coagulase activity . J Clin Microbiol 2010 ; 48 : 4248 - 50 . 12. Dastgheyb S , Parvizi J , Shapiro IM , Hickok NJ , Otto M. Effect of biofilms on recalcitrance of Staphylococcal joint infection to antibiotic treatment . J Infect Dis 2014 ; 211 : 641 - 50 . 13. Dastgheyb SS , Hammoud S , Ketonis C , et al. Staphylococcal persistence due to biofilm formation in synovial fluid containing prophylactic cefazolin . Antimicrob Agents Chemother 2015 ; 59 : 2122 - 8 . 14. McCarthy H , Rudkin JK , Black NS , Gallagher L , O'Neill E , O'Gara JP . Methicillin resistance and the biofilm phenotype in Staphylococcus aureus . Front Cell Infect Microbiol 2015 ; 5 : 1 . 15. Otto M. Staphylococcal infections: mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity . Annu Rev Med 2013 ; 64 : 175 - 88 . 16. Waters EM , McCarthy H , Hogan S , Zapotoczna M , O'Neill E , O'Gara JP . Rapid quantitative and qualitative analysis of biofilm production by Staphylococcus epidermidis under static growth conditions . Methods Mol Biol 2013 ; 1106 : 157 - 66 . 17. Moormeier DE , Endres JL , Mann EE , et al. Use of microfluidic technology to analyze gene expression during Staphylococcus aureus biofilm formation reveals distinct physiological niches . Appl Environ Microbiol 2013 ; 79 : 3413 - 24 . 18. Fey PD , Endres JL , Yajjala VK , et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes . MBio 2013 ; 4 : e00537 - 12 . 19. Chauhan A , Lebeaux D , Decante B , et al. A rat model of central venous catheter to study establishment of long-term bacterial biofilm and related acute and chronic infections . PLoS One 2012 ; 7 : e37281 . 20. Johnson M , Cockayne A , Morrissey JA . Iron-regulated biofilm formation in Staphylococcus aureus Newman requires ica and the secreted protein Emp . Infect Immun 2008 ; 76 : 1756 - 65 . 21. Mainiero M , Goerke C , Geiger T , Gonser C , Herbert S , Wolz C. Differential target gene activation by the Staphylococcus aureus twocomponent system saeRS . J Bacteriol 2010 ; 192 : 613 - 23 . 22. Cheng AG , McAdow M , Kim HK , Bae T , Missiakas DM , Schneewind O. Contribution of coagulases towards Staphylococcus aureus disease and protective immunity . PLoS Pathog 2010 ; 6 : e1001036 . 23. Thomer L , Schneewind O , Missiakas D. Multiple ligands of von Willebrand factor-binding protein (vWbp) promote Staphylococcus aureus clot formation in human plasma . J Biol Chem 2013 ; 288 : 28283 - 92 . 24. Valle J , Toledo-Arana A , Berasain C , et al. SarA and not sigmaB is essential for biofilm development by Staphylococcus aureus . Mol Microbiol 2003 ; 48 : 1075 - 87 . 25. Geoghegan JA , Ganesh VK , Smeds E , Liang X , Hook M , Foster TJ . Molecular characterization of the interaction of staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMM) ClfA and Fbl with fibrinogen . J Biol Chem 2010 ; 285 : 6208 - 16 . 26. Ganesh VK , Rivera JJ , Smeds E , et al. A structural model of the Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics . PLoS Pathog 2008 ; 4 : e1000226 . 27. Steinhuber A , Goerke C , Bayer MG , Doring G , Wolz C. Molecular architecture of the regulatory Locus sae of Staphylococcus aureus and its impact on expression of virulence factors . J Bacteriol 2003 ; 185 : 6278 - 86 . 28. Voyich JM , Vuong C , DeWald M , et al. The SaeR/S gene regulatory system is essential for innate immune evasion by Staphylococcus aureus . J Infect Dis 2009 ; 199 : 1698 - 706 . 29. Goerke C , Wolz C. Regulatory and genomic plasticity of Staphylococcus aureus during persistent colonization and infection . Int J Med Microbiol 2004 ; 294 : 195 - 202 . 30. Date SV , Modrusan Z , Lawrence M , et al. Global gene expression of methicillin-resistant Staphylococcus aureus USA300 during human and mouse infection . J Infect Dis 2014 ; 209 : 1542 - 50 . 31. Bae T , Banger AK , Wallace A , et al. Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing . Proc Natl Acad Sci U S A 2004 ; 101 : 12312 - 7 . 32. Benton BM , Zhang JP , Bond S , et al. Large-scale identification of genes required for full virulence of Staphylococcus aureus . J Bacteriol 2004 ; 186 : 8478 - 89 . 33. Montgomery CP , Boyle-Vavra S , Daum RS. Importance of the global regulators Agr and SaeRS in the pathogenesis of CA-MRSA USA300 infection . PLoS One 2010 ; 5 : e15177 . 34. Giraudo AT , Raspanti CG , Calzolari A , Nagel R. Characterization of a Tn551-mutant of Staphylococcus aureus defective in the production of several exoproteins . Can J Microbiol 1994 ; 40 : 677 - 81 . 35. Harraghy N , Kormanec J , Wolz C , et al. sae is essential for expression of the staphylococcal adhesins Eap and Emp . Microbiology 2005 ; 151 : 1789 - 800 . 36. Johnson M , Sengupta M , Purves J , et al. Fur is required for the activation of virulence gene expression through the induction of the sae regulatory system in Staphylococcus aureus . Int J Med Microbiol 2011 ; 301 : 44 - 52 . 37. Beenken KE , Blevins JS , Smeltzer MS . Mutation of sarA in Staphylococcus aureus limits biofilm formation . Infect Immun 2003 ; 71 : 4206 - 11 . 38. Mrak LN , Zielinska AK , Beenken KE , et al. saeRS and sarA act synergistically to repress protease production and promote biofilm formation in Staphylococcus aureus . PLoS One 2012 ; 7 : e38453 . 39. Tsang LH , Cassat JE , Shaw LN , Beenken KE , Smeltzer MS . Factors contributing to the biofilm-deficient phenotype of Staphylococcus aureus sarA mutants . PLoS One 2008 ; 3 : e3361 . 40. Snowden JN , Beaver M , Beenken K , Smeltzer M , Horswill AR , Kielian T. Staphylococcus aureus sarA regulates inflammation and colonization during central nervous system biofilm formation . PLoS One 2013 ; 8 : e84089 . 41. Schwartz K , Stephenson R , Hernandez M , Jambang N , Boles BR . The use of drip flow and rotating disk reactors for Staphylococcus aureus biofilm analysis . J Vis Exp 2010 ; doi:10.3791/2470. 42. Kocianova S , Vuong C , Yao Y , et al. Key role of poly-gamma-DLglutamic acid in immune evasion and virulence of Staphylococcus epidermidis . J Clin Invest 2005 ; 115 : 688 - 94 . 43. Plaut RD , Mocca CP , Prabhakara R , Merkel TJ , Stibitz S. Stably luminescent Staphylococcus aureus clinical strains for use in bioluminescent imaging . PLoS One 2013 ; 8 : e59232 . 44. Horsburgh MJ , Aish JL , White IJ , Shaw L , Lithgow JK , Foster SJ. sigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4 . J Bacteriol 2002 ; 184 : 5457 - 67 . 45. Patel AH , Foster TJ , Pattee PA. Physical and genetic mapping of the protein A gene in the chromosome of Staphylococcus aureus 8325-4 . J Gen Microbiol 1989 ; 135 : 1799 - 807 . 46. Fitzgerald JR , Loughman A , Keane F , et al. Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor . Mol Microbiol 2006 ; 59 : 212 - 30 . 47. Baba T , Bae T , Schneewind O , Takeuchi F , Hiramatsu K. Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands . J Bacteriol 2008 ; 190 : 300 - 10 . 48. Burlak C , Hammer CH , Robinson MA , et al. Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection . Cell Microbiol 2007 ; 9 : 1172 - 90 . 49. Cassat JE , Dunman PM , McAleese F , Murphy E , Projan SJ , Smeltzer MS . Comparative genomics of Staphylococcus aureus musculoskeletal isolates . J Bacteriol 2005 ; 187 : 576 - 92 .

This is a preview of a remote PDF:

Marta Zapotoczna, Hannah McCarthy, Justine K. Rudkin, James P. O'Gara, Eoghan O'Neill. An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections, Journal of Infectious Diseases, 2015, 1883-1893, DOI: 10.1093/infdis/jiv319