Search

Background: Well known for their hemostatic function, platelets are increasingly becoming recognized as important immunomodulators. The purpose of the present study was to assess the impact of platelet depletion on antimicrobial host defense in a mouse model of periprosthetic joint infection (PJI)., Methods: Thrombocytopenia (TCP) was induced in C57BL/6 mice with use of a selective antibody against platelet CD41 (anti-CD41). Whole blood from pre-treated mice was incubated with Staphylococcus aureus to assess antimicrobial efficacy with use of bioluminescent imaging, quantitative histological staining, and colony forming unit (CFU) quantification. In parallel, untreated heterologous platelets were added to TCP blood to assess potential rescue of antimicrobial efficacy. In vivo, TCP and control mice underwent placement of a titanium implant in the femur inoculated with bioluminescent Xen36 S. aureus. Longitudinal bioluminescent imaging was performed postoperatively to quantify the evolution of bacterial burden, which was confirmed via assessment of S. aureus CFUs on the implant and in peri-implant tissue on postoperative day (POD) 28., Results: Anti-CD41 treatment resulted in significant dose-dependent reductions in platelet count. Ex vivo, platelet-depleted whole blood demonstrated significantly less bacterial reduction than control blood. These outcomes were reversed with the addition of untreated rescue platelets. In vivo, infection burden was significantly higher in TCP mice and was inversely correlated with preoperative platelet count (r2 = 0.63, p = 0.037). Likewise, CFU quantification on POD28 was associated with increased bacterial proliferation and severity of periprosthetic infection in TCP mice compared with controls., Conclusions: Thrombocytopenia resulted in an increased bacterial burden both ex vivo and in vivo in a mouse model of PJI., Clinical Relevance: In orthopaedic patients, deficiencies in platelet quantity or function represent an easily modifiable risk factor for PJI., Competing Interests: Disclosure: This study was supported by the H&H Lee Research Program and the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health. On the Disclosure of Potential Conflicts of Interest forms, which are provided with the online version of the article, one or more of the authors checked “yes” to indicate that the author had a relevant financial relationship in the biomedical arena outside the submitted work (http://links.lww.com/JBJS/G396)., (Copyright © 2021 by The Journal of Bone and Joint Surgery, Incorporated.)

Background: Evidence suggests the renin-angiotensin system (RAS) plays key immunomodulatory roles. In particular, angiotensin-converting enzyme (ACE) has been shown to play a role in antimicrobial host defense. ACE inhibitors (ACEi) and angiotensin receptor blockers (ARB) are some of the most commonly prescribed medications, especially in patients undergoing invasive surgery. Thus, the current study assessed the immunomodulatory effect of RAS-modulation in a preclinical model of implant infection. Methods: In vitro antimicrobial effects of ACEi and ARBs were first assessed. C57BL/6J mice subsequently received either an ACEi (lisinopril; 16 mg/kg/day), an ARB (losartan; 30 mg/kg/day), or no treatment. Conditioned mice blood was then utilized to quantify respiratory burst function as well as Staphylococcus aureus Xen36 burden ex vivo in each treatment group. S. aureus infectious burden for each treatment group was then assessed in vivo using a validated mouse model of implant infection. Real-time quantitation of infectious burden via bioluminescent imaging over the course of 28 days post-procedure was assessed. Host response via monocyte and neutrophil infiltration within paraspinal and spleen tissue was quantified by immunohistochemistry for F4/80 and myeloperoxidase, respectively. Results: Blood from mice treated with an ACEi demonstrated a decreased ability to eradicate bacteria when mixed with Xen36 as significantly higher levels of colony forming units (CFU) and biofilm formation was appreciated ex vivo ( p < 0.05). Mice treated with an ACEi showed a higher infection burden in vivo at all times ( p < 0.05) and significantly higher CFUs of bacteria on both implant and paraspinal tissue at the time of sacrifice ( p < 0.05 for each comparison). There was also significantly decreased infiltration and respiratory burst function of immune effector cells in the ACEi group ( p < 0.05). Conclusion: ACEi, but not ARB, treatment resulted in increased S. aureus burden and impaired immune response in a preclinical model of implant infection. These results suggest that perioperative ACEi use may represent a previously unappreciated risk factor for surgical site infection. Given the relative interchangeability of ACEi and ARB from a cardiovascular standpoint, this risk factor may be modifiable., (Copyright © 2020 Trikha, Greig, Kelley, Mamouei, Sekimura, Cevallos, Olson, Chaudry, Magyar, Leisman, Stavrakis, Yeaman and Bernthal.)

T cell cytokines contribute to immunity against Staphylococcus aureus , but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1β, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6 + T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6 + Vδ4 + T cells in immunity against S. aureus skin infections., Competing Interests: Conflict of interest statement: M.R.Y. is a cofounder of NovaDigm Therapeutics, which is developing novel vaccines and immunotherapeutics for infectious diseases, including S. aureus. L.S.M. has received grant support for work unrelated to the work reported in this manuscript from AstraZeneca, Pfizer, Regeneron Pharmaceuticals, Moderna Therapeutics, and Boehringer Ingelheim, is on the scientific advisory board for Integrated Biotherapeutics, and is a shareholder of Noveome Biotherapeutics, which are each developing vaccines and therapeutics against S. aureus and other pathogens.

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous β-defensin (human β-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Many host defense cationic antimicrobial peptides (HDPs) perturb the staphylococcal cell membrane (CM) and alter transmembrane potential (ΔΨ) as key parts of their lethal mechanism. Thus, a sense-response system for detecting and mediating adaptive responses to such stresses could impact organism survival; the Staphylococcus aureus LytSR two-component regulatory system (TCRS) may serve as such a ΔΨ sensor. One well-known target of this system is the lrgAB operon, which, along with the related cidABC operon, has been shown to be a regulator in the control of programmed cell death and lysis. We used an isogenic set of S. aureus strains: (i) UAMS-1, (ii) its isogenic ΔlytS and ΔlrgAB mutants, and (iii) plasmid-complemented ΔlytSR and ΔlrgAB mutants. The ΔlytS strain displayed significantly increased in vitro susceptibilities to all HDPs tested (neutrophil-derived human neutrophil peptide 1 [hNP-1], platelet-derived thrombin-induced platelet microbicidal proteins [tPMPs], and the tPMP-mimetic peptide RP-1), as well as to calcium-daptomycin (DAP), a cationic antimicrobial peptide (CAP). In contrast, the ΔlrgAB strain exhibited no significant changes in susceptibilities to these cationic peptides, indicating that although lytSR positively regulates transcription of lrgAB, increased HDP/CAP susceptibilities in the ΔlytS mutant were lrgAB independent. Further, parental UAMS-1 (but not the ΔlytS mutant) became more resistant to hNP-1 and DAP following pretreatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (a CM-depolarizing agent). Of note, lytSR-dependent survival against CAP/HDP killing was not associated with changes in either surface positive charge, expression of mprF and dlt, or CM fluidity. The ΔlytS strain (but not the ΔlrgAB mutant) displayed a significant reduction in target tissue survival in an endocarditis model during DAP treatment. Collectively, these results suggest that the lytSR TCRS plays an important role in adaptive responses of S. aureus to CM-perturbing HDPs/CAPs, likely by functioning as a sense-response system for detecting subtle changes in ΔΨ.

The investigational vaccine, NDV-3, contains the N-terminal portion of the Candida albicans agglutinin-like sequence 3 protein (Als3p) formulated with an aluminum hydroxide adjuvant in phosphate-buffered saline. Preclinical studies demonstrated that the Als3p vaccine antigen protects mice from oropharyngeal, vaginal and intravenous challenge with C. albicans and other selected species of Candida as well as both intravenous challenge and skin and soft tissue infection with Staphylococcus aureus. The objectives of this first-in-human Phase I clinical trial were to evaluate the safety, tolerability and immunogenicity of NDV-3 at two different antigen levels compared to a saline placebo. Forty healthy, adult subjects were randomized to receive one dose of NDV-3 containing either 30 or 300 μg of Als3p, or placebo. NDV-3 at both dose levels was safe and generally well-tolerated. Anti-Als3p total IgG and IgA1 levels for both doses reached peak levels by day 14 post vaccination, with 100% seroconversion of all vaccinated subjects. On average, NDV-3 stimulated peripheral blood mononuclear cell (PBMC) production of both IFN-γ and IL-17A, which peaked at day 7 for subjects receiving the 300 μg dose and at day 28 for those receiving the 30 μg dose. Six months after receiving the first dose of NDV-3, nineteen subjects received a second dose of NDV-3 identical to their first dose to evaluate memory B- and T-cell immune responses. The second dose resulted in a significant boost of IgG and IgA1 titers in >70% of subjects, with the biggest impact in those receiving the 30 μg dose. A memory T-cell response was also noted for IFN-γ in almost all subjects and for IL-17A in the majority of subjects. These data support the continued investigation of NDV-3 as a vaccine candidate against Candida and S. aureus infections., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The two-component regulatory system, GraRS, appears to be involved in staphylococcal responses to cationic antimicrobial peptides (CAPs). However, the mechanism(s) by which GraRS is induced, regulated, and modulated remain undefined. In this study, we used two well-characterized MRSA strains (Mu50 and COL) and their respective mutants of graR and vraG (encoding the ABC transporter-dependent efflux pump immediately downstream of graRS), and show that (i) the expression of two key determinants of net positive surface charge (mprF and dlt) is dependent on the cotranscription of both graR and vraG, (ii) reduced expression of mprF and dlt in graR mutants was phenotypically associated with reduced surface-positive charge, (iii) this net reduction in surface-positive charge in graR and vraG mutants, in turn, correlated with enhanced killing by a range of CAPs of diverse structure and origin, including those from mammalian platelets (tPMPs) and neutrophils (hNP-1) and from bacteria (polymyxin B), and (iv) the synthesis and translocation of membrane lysyl-phosphatidylglycerol (an mprF-dependent function) was substantially lower in graR and vraG mutants than in parental strains. Importantly, the inducibility of mprF and dlt transcription via the graRS-vraFG pathway was selective, with induction by sublethal exposure to the CAPs, RP-1 (platelets), and polymyxin B, but not by other cationic molecules (hNP-1, vancomycin, gentamicin, or calcium-daptomycin). Although graR regulates expression of vraG, the expression of graR was codependent on an intact downstream vraG locus. Collectively, these data support an important role of the graRS and vraFG loci in the sensing of and response to specific CAPs involved in innate host defenses.

Cell wall thickening is a common feature among daptomycin-resistant Staphylococcus aureus strains. However, the mechanism(s) leading to this phenotype is unknown. We examined a number of cell wall synthesis pathway parameters in an isogenic strain set of S. aureus bloodstream isolates obtained from a patient with recalcitrant endocarditis who failed daptomycin therapy, including the initial daptomycin-susceptible parental strain (strain 616) and two daptomycin-resistant strains (strains 701 and 703) isolated during daptomycin therapy. Transmission electron microscopy demonstrated significantly thicker cell walls in the daptomycin-resistant strains than in the daptomycin-susceptible strain, a finding which was compatible with significant differences in dry cell weight of strain 616 versus strains 701 to 703 (P < 0.05). Results of detailed analysis of cell wall muropeptide composition, the degree of peptide side chain cross-linkage, and the amount of the peptidoglycan precursor, UDP-MurNAc-pentapeptide, were similar in the daptomycin-susceptible and daptomycin-resistant isolates. In contrast, the daptomycin-resistant strains contained less O-acetylated peptidoglycan. Importantly, both daptomycin-resistant strains synthesized significantly more wall teichoic acid (WTA) than the parental strain (P < 0.001). Moreover, the proportion of D-alanylated WTA species was substantially higher in the daptomycin-resistant strains than in the daptomycin-susceptible parental strain (P < 0.05 in comparing strain 616 versus strain 701). The latter phenotypic findings correlated with (i) enhanced tagA and dltA gene expression, respectively, and (ii) an increase in surface positive charge observed in the daptomycin-resistant versus daptomycin-susceptible isolates. Collectively, these data suggest that increases in WTA synthesis and the degree of its D-alanylation may play a major role in the daptomycin-resistant phenotype in some S. aureus strains.

Objectives: The development of daptomycin resistance in Staphylococcus aureus is associated with clinical treatment failures. The mechanism(s) of such resistance have not been clearly defined., Methods: We studied an isogenic daptomycin-susceptible (DAP(S)) and daptomycin-resistant (DAP(R)) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques., Results: Minor differences in the genome content were found between strains by DNA hybridization. Transcriptomic analyses identified a number of genes differentially expressed in important functional categories: cell division; metabolism of bacterial envelopes; and global regulation. Of note, the DAP(R) isolate exhibited reduced expression of the major cell wall autolysis gene coincident with the up-regulation of genes involved in cell wall teichoic acid production. Using quantitative (q)RT-PCR on the gene cadre putatively involved in cationic peptide resistance, we formulated a putative regulatory network compatible with microarray data sets, mainly implicating bacterial envelopes. Of interest, qRT-PCR of this same gene cadre from two distinct isogenic DAP(S)/DAP(R) clinical strain pairs revealed evidence of other strain-dependent networks operative in the DAP(R) phenotype. Comparative proteomics of 616 versus 701 revealed a differential abundance of proteins in various functional categories, including cell wall-associated targets and biofilm formation proteins. Phenotypically, strains 616 and 701 showed major differences in their ability to develop bacterial biofilms in the presence of the antibacterial lipid, oleic acid., Conclusions: Compatible with previous in vitro observations, in vivo-acquired DAP(R) in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelopes.

Carotenoid pigments of Staphylococcus aureus provide integrity to its cell membrane (CM) and limit oxidative host defense mechanisms. However, the role of carotenoids in staphylococcal resistance to nonoxidative host defenses has not been characterized. The current study examined the relationship among CM carotenoid content, membrane order, and in vitro susceptibility to daptomycin or to prototypic neutrophil-derived, platelet-derived, or bacterium-derived cationic antimicrobial peptides (human neutrophil defensin-1 [hNP-1], platelet microbicidal proteins [PMPs], or polymyxin B, respectively). A previously characterized methicillin-susceptible Staphylococcus aureus (MSSA) isogenic clinical strain set was used, including a parental isolate with an intact carotenoid biosynthetic operon (crtOPQMN) containing the crtM gene encoding early steps in staphyloxanthin biosynthesis, a crtM deletion mutant, and a crtMN multicopy plasmid-complemented variant. Compared to the parental and crtM knockout strains, the crtMN-complemented strain exhibited (i) increased carotenoid production, (ii) increased CM rigidity (P < 0.001), and (iii) uniformly reduced susceptibility to killing by the above-mentioned range of cationic peptides (statistically significant for hNP-1 [20 μg/ml]; P = 0.0037). There were no significant differences in phospholipid composition and asymmetry, fatty acid profiles, surface charge, or cell wall thickness among the strain set. Collectively, these data support the concept that carotenoid biosynthesis can contribute to the ability of S. aureus to subvert nonoxidative host defenses mediated by cationic peptides, potentially by increasing target membrane rigidity.

Using isogenic clinical bloodstream Staphylococcus aureus strains from a patient with relapsing endocarditis, we investigated the transcriptional profiles of the mprF and dlt genes in the context of cell-surface charge and daptomycin nonsusceptibility. As in prior studies, a point mutation within mprF was observed in the daptomycin-nonsusceptible strain. However, neither the transcriptional profile of mprF nor the membrane phospholipid analyses were compatible with the anticipated mprF gain-in-function phenotype. In contrast, we demonstrated enhanced dlt expression coincident with increased positive surface charge and reduced daptomycin binding.

Increasingly frequent reports have described the in vivo loss of daptomycin susceptibility in association with clinical treatment failures. The mechanism(s) of daptomycin resistance is not well understood. We studied an isogenic set of Staphylococcus aureus isolates from the bloodstream of a daptomycin-treated patient with recalcitrant endocarditis in which serial strains exhibited decreasing susceptibility to daptomycin. Since daptomycin is a membrane-targeting lipopeptide, we compared a number of membrane parameters in the initial blood isolate (parental) with those in subsequent daptomycin-resistant strains obtained during treatment. In comparison to the parental strain, resistant isolates demonstrated (i) enhanced membrane fluidity, (ii) increased translocation of the positively charged phospholipid lysyl-phosphotidylglycerol to the outer membrane leaflet, (iii) increased net positive surface charge (P < 0.05 versus the parental strain), (iv) reduced susceptibility to daptomycin-induced depolarization, permeabilization, and autolysis (P < 0.05 versus the parental strain), (v) significantly lower surface binding of daptomycin (P < 0.05 versus the parental strain), and (vi) increased cross-resistance to the cationic antimicrobial host defense peptides human neutrophil peptide 1 (hNP-1) and thrombin-induced platelet microbicidal protein 1 (tPMP-1). These data link distinct changes in membrane structure and function with in vivo development of daptomycin resistance in S. aureus. Moreover, the cross-resistance to hNP-1 and tPMP-1 may also impact the capacity of these daptomycin-resistant organisms to be cleared from sites of infection, particularly endovascular foci.

Thrombin-induced platelet microbicidal proteins (e.g. tPMP-1) are small cationic peptides released from mammalian platelets. As the cytoplasmic membrane (CM) is a primary target of tPMPs, distinct CM characteristics are likely to affect the cells' susceptibility profiles. In Staphylococcus aureus, CM surface charge and hydrophobicity are principally determined by the content and distribution of its three major phospholipid (PL) constituents: negatively charged phosphatidylglycerol (PG) and cardiolipin (CL) and positively charged lysyl-PG (LPG). PL composition profiles, and inner vs outer CM leaflet PL distributions, were compared in an isogenic tPMP-susceptible (tPMP(S)) and -resistant (tPMP(R)) S. aureus strain pair (ISP479C vs ISP479R respectively). All PLs were asymmetrically distributed between the outer and inner CM leaflets in both strains. However, in ISP479R, the outer CM leaflet content of LPG was significantly increased vs ISP479C (27.3+/-11.0 % vs 18.6+/-7.0 % respectively; P=0.05). This observation correlated with reduced binding of the cationic proteins cytochrome c, poly-L-lysine, tPMP-1 and the tPMP-1-mimetic peptide, RP1, to tPMP-1(R) whole cells and to model liposomal CMs with LPG content and distribution similar to that of tPMP-1(R) strains. Collectively, selected CM parameters correlated with reduced staphylocidal capacities of tPMP-1 against certain S. aureus strains, including relative increases in outer CM leaflet positive charge and reduced surface binding of cationic molecules. These findings offer new insights into mechanisms of antimicrobial peptide susceptibility and resistance in S. aureus.

Background: The Staphylococcus aureus global regulators--agr, sarA, and sae--coordinately control alpha-toxin gene (hla) expression in vitro. However, their relative in vivo contributions to hla expression, particularly in endovascular infections, have not been defined., Methods: A plasmid-based hla-promoter:green fluorescent protein reporter system was constructed in 2 genetically related S. aureus strains: RN6390 (a natural sigma factor B [sigB]-deficient mutant), SH1000 (a sigB-repaired variant of RN6390 lineage), and their respective agr, sarA, agr/sarA, and sae mutants. These strain sets were used to quantify hla expression in vitro and in an experimental infective endocarditis (IE) model using flow cytometry., Results: In vitro, hla expression was positively modulated by all 3 regulons (sae > agr/sarA > agr and sarA) in both RN6390 and SH1000 backgrounds. In the IE model, hla expression in cardiac vegetations was lower in all single mutants than in the respective parental strains (P<.05 for sae mutant) but was maintained at near-parental levels in the agr/sarA double mutant in both backgrounds. A similar finding was also observed in kidneys and spleens., Conclusions: These results indicate that, although several distinct regulatory circuits can affect hla expression in vitro and in vivo, sae appears to play a crucial role in this context.

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a staphylocidal peptide released by activated platelets. This peptide initiates its microbicidal activity by membrane permeabilization, with ensuing inhibition of intracellular macromolecular synthesis. RP-1 is a synthetic congener modeled on the C-terminal microbicidal alpha-helix of tPMP-1. This study compared the staphylocidal mechanisms of RP-1 with those of tPMP-1, focusing on isogenic tPMP-1-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains for the following quantitative evaluations: staphylocidal efficacy; comparative MIC; membrane permeabilization (MP) and depolarization; and DNA, RNA, and protein synthesis. Although the proteins had similar MICs, RP-1 caused significant killing of ISP479C (<50% survival), correlating with extensive MP (>95%) and inhibition of DNA and RNA synthesis (>90%), versus substantially reduced killing of ISP479R (>80% survival), with less MP (55%) and less inhibition of DNA or RNA synthesis (70 to 80%). Interestingly, RP-1-induced protein synthesis inhibition was equivalent in both strains. RP-1 did not depolarize the cell membrane and caused a relatively short postexposure growth inhibition. These data closely parallel those previously reported for tPMP-1 against this strain set and exemplify how synthetic molecules can be engineered to reflect structure-activity relationships of functional domains in native host defense effector molecules.

Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux.

The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against Staphylococcus aureus. We previously reported that a Tn551 S. aureus transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1(r)) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun. 65:3293-3299, 1997). In the current study, the genetic basis for tPMP-1(r) in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn551 mutant strains showed that the fourth gene in the staphylococcal mnh operon (mnhABCDEFG) was insertionally inactivated. This operon was previously reported to encode a Na(+)/H(+) antiporter involved in pH tolerance and halotolerance. However, the capacity of ISP479R to grow at pH extremes and in high NaCl concentrations (1 to 3 M), coupled with its loss of transmembrane potential (DeltaPsi) during postexponential growth, suggested that the mnh gene products are not functioning as a secondary (i.e., passive) Na(+)/H(+) antiporter. Moreover, we identified protein homologies between mnhD and the nuo genes of Escherichia coli that encode components of a complex I NADH:ubiquinone oxidoreductase. Consistent with these data, exposures of tPMP-1-susceptible (tPMP-1(s)) parental strains (both clinical and laboratory derived) with either CCCP (a proton ionophore which collapses the proton motive force) or pieracidin A (a specific complex I enzyme inhibitor) significantly reduced tPMP-induced killing to levels seen in the tPMP-1(r) mutants. To reflect the energization of the gene products encoded by the mnh operon, we have renamed the locus sno (S. aureus nuo orthologue). These novel findings indicate that disruption of a complex I enzyme locus can confer reduced in vitro susceptibility to tPMP-1 in S. aureus.

The DltABCD and MprF proteins contribute a net positive charge to the Staphylococcus aureus surface envelope by alanylating and lysinylating teichoic acids and membrane phosphatidylglycerol, respectively. These surface charge modifications are associated with increased in vitro resistance profiles of S. aureus to a number of endogenous cationic antimicrobial peptides (CAPs), such as alpha-defensins. The current study investigated the effects of dltA and mprF mutations on the following host factors relevant to endovascular infections: (i) in vitro susceptibility to the CAP thrombin-induced platelet microbicidal protein 1 (tPMP-1), (ii) in vitro adherence to endothelial cells (EC) and matrix proteins, and (iii) in vivo virulence in an endovascular infection model (rabbit endocarditis) in which tPMP-1 is felt to play a role in limiting S. aureus pathogenesis. Both mutations resulted in substantial increases in the in vitro susceptibility to tPMP-1 compared to that of the parental strain. The dltA (but not the mprF) mutation resulted in a significantly reduced capacity to bind to EC in vitro, while neither mutation adversely impacted in vitro binding to fibronectin, fibrinogen, or platelets. In vivo, both mutations significantly attenuated virulence in terms of early colonization of sterile vegetations and subsequent proliferation at this site (versus the parental strain). However, only the dltA mutation significantly reduced metastatic infections in kidneys and spleens compared to those in animals infected with the parental strain. These data underscore the importance of resistance to distinct CAPs and of teichoic acid-dependent EC interactions in the context of endovascular infection pathogenesis.

Perturbation of the Staphylococcus aureus cytoplasmic membrane (CM) is felt to play a key role in the microbicidal mechanism of many antimicrobial peptides (APs). However, it is not established whether membrane permeabilization (MP) alone is sufficient to kill susceptible staphylococci or if the cell wall (CW) and/or intracellular targets contribute to AP-induced lethality. We hypothesized that the relationships between MP and killing may differ for distinct APs. In this study, we investigated the association between AP-induced MP and lethality in S. aureus whole cells versus CW-free protoplasts, and in comparison to the MP of liposomes modeled after whole CMs in terms of phospholipid composition, fluidity and charge. Four APs with different structure-activity relationships were examined: thrombin-induced platelet microbicidal protein 1 (tPMP-1), human neutrophil protein 1 (hNP-1), gramicidin D, and polymyxin B. MP was quantified fluorometrically by calcein release. All APs tested, except polymyxin B, caused concentration-dependent MP and killing of whole cells, but not of protoplasts. The reduced AP susceptibility of protoplasts was associated with increased cardiolipin and lysyl-phosphatidylglycerol content and reduced fluidity of their CMs. However, liposomal MP induced by tPMP-1, hNP-1, and gramicidin D paralleled that of whole cells. Collectively, these results indicate that (i) structurally distinct APs likely exert their staphylocidal effects by differing mechanisms, (ii) MP is not the sole event leading to AP-induced staphylocidal activity, (iii) a complex interrelationship exists between the CM and CW in AP-induced killing, and (iv) liposomes modeled upon whole cell or protoplast CMs can recapitulate the respective susceptibilities to killing by distinct APs.

Loss of agr function, vancomycin exposure, and abnormal autolysis have been linked with both development of the GISA phenotype and low-level resistance in vitro to thrombin-induced platelet microbicidal proteins (tPMPs). We examined the potential in vitro interrelationships among these parameters in well-characterized, isogenic laboratory-derived and clinical Staphylococcus aureus isolates. The laboratory-derived S. aureus strains included RN6607 (agrII-positive parent) and RN6607V (vancomycin-passaged variant; hetero-GISA), RN9120 (RN6607 agr::tetM; agr II knockout parent), RN9120V (vancomycin-passaged variant), and RN9120-GISA (vancomycin passaged, GISA). Two serial isolates from a vancomycin-treated patient with recalcitrant, methicillin-resistant S. aureus (MRSA) endocarditis were also studied: A5937 (agrII-positive initial isolate) and A5940 (agrII-defective/hetero-GISA isolate obtained after prolonged vancomycin administration). In vitro tPMP susceptibility phenotypes were assessed after exposure of strains to either 1 or 2 mug/ml. Triton X-100- and vancomycin-induced lysis profiles were determined spectrophotometrically. For agrII-intact strain RN6607, vancomycin exposure in vitro was associated with modest increases in vancomycin MICs and reduced killing by tPMP, but no change in lysis profiles. In contrast, vancomycin exposure of agrII-negative RN9120 yielded a hetero-GISA phenotype and was associated with defects in lysis and reduced in vitro killing by tPMP. In the clinical isolates, loss of agrII function during prolonged vancomycin therapy was accompanied by emergence of the hetero-GISA phenotype and reduced tPMP killing, with no significant change in lysis profiles. An association was identified between loss of agrII function and the emergence of hetero-GISA phenotype during either in vitro or in vivo vancomycin exposure. In vitro, these events were associated with defective lysis and reduced susceptibility to tPMP. The precise mechanism(s) underlying these findings is the subject of current investigations.

Wall teichoic acids (WTAs) are major surface components of gram-positive bacteria that have recently been shown to play a key role in nasal colonization by Staphylococcus aureus. In the present study, we assessed the impact that WTAs have on endovascular infections by using a WTA-deficient S. aureus mutant ( Delta tagO). There were no significant differences detected between the isogenic parental strain (SA113) and the Delta tagO mutant in polymorphonuclear leukocyte-mediated opsonophagocytosis; killing by a prototypic platelet microbicidal protein; or binding to platelets, fibronectin, or fibrinogen. However, compared with the parental strain, the Delta tagO mutant adhered considerably less well to human endothelial cells, especially under flow conditions (70.3% reduction; P<.05). Beads coated with WTA bound to endothelium in a dose-dependent manner, suggesting that WTA contributes specifically to this interaction. These in vitro data closely paralleled those from a rabbit model of infective endocarditis in which the Delta tagO mutant was compared with the parental strain. Clearances of staphylococcus from the bloodstream were equivalent, but the Delta tagO mutant showed a significantly reduced capacity to both colonize sterile cardiac vegetations (P<.05) and proliferate within these vegetations, the kidneys, and the spleen (P<.001). We conclude that WTA is an important factor in the induction and progression of endovascular S. aureus infection, likely through a specific interaction with endothelial cells.

Background: The causes of persistent bacteremia (PB) due to methicillin-resistant Staphylococcus aureus (MRSA) are poorly understood. This investigation examined potential associations between PB with key clinical features and several in vitro bacterial genotypic and phenotypic characteristics, in isolates from 1 institution., Methods: Pulsed-field gel electrophoresis (PFGE) relatedness, thrombin-induced platelet microbicidal protein (tPMP)-susceptibility phenotype, accessory gene regulator (agr) genotype and functionality (via delta-lysin production), and autolysis phenotypes were assessed in MRSA isolates from the bloodstream of 21 prospectively identified patients with PB (blood cultures positive after > or =7 days of therapy) and of 18 patients with resolving bacteremia (RB) (sterile blood cultures within the first 2-4 days of therapy) due to MRSA., Results: The 2 groups had comparable baseline characteristics but differed in their clinical courses (e.g., endocarditis was more frequent in patients with PB than in those with RB [43% vs. 0%, respectively; P=.0016]); isolates from patients with PB exhibited higher rates of (1) survival in vitro after exposure to tPMP (22.4+/-14.8% vs. 11.6+/-6.5%, respectively; P=.005); (2) defective delta-lysin production (71.4% vs. 38.9%, respectively; P=.057); (3) non-agr genotype II profile (100% vs. 77.8%, respectively; P=.037); and (4) overrepresentation of a specific PFGE genotype (85.7% vs. 44.4%, respectively; P=.015)., Conclusions: Isolates from patients with PB differed from those in patients with RB, in several in vitro characteristics. Further studies will be necessary to define how these factors might affect clinical outcome.

We investigated the impacts of sarA and agr on fnbA expression and fibronectin-binding capacity in Staphylococcus aureus in vitro and in experimental endocarditis. Although sarA up-regulated and agr down-regulated both fnbA expression and fibronectin binding in vitro and in vivo, fnbA expression was positively regulated in the absence of both global regulators. Thus, additional regulatory loci contribute to fnbA regulation and fibronectin-binding capacities in S. aureus.

Aspirin has been previously shown to reduce the in vivo virulence of Staphylococcus aureus in experimental endocarditis, through antiplatelet and antimicrobial mechanisms. In the present study, salicylic acid, the major in vivo metabolite of aspirin, mitigated two important virulence phenotypes in both clinical and laboratory S. aureus strains: alpha-hemolysin secretion and fibronectin binding in vitro. In addition, salicylic acid reduced the expression of the alpha-hemolysin gene promoter, hla, and the fibronectin gene promoter, fnbA. Transcriptional analysis, fluorometry, and flow cytometry revealed evidence of salicylic acid-mediated activation of the stress-response gene sigB. Expression of the sigB-repressible global regulon sarA and the global regulon agr were also mitigated by salicylic acid, corresponding to the reduced expression of the hla and fnbA genes in vitro. Studies in experimental endocarditis confirmed the key roles of both sarA and sigB in mediating the antistaphylococcal effects of salicylic acid in vivo. Therefore, aspirin has the potential to be an adjuvant therapeutic agent against endovascular infections that result from S. aureus, by downmodulating key staphylococcal global regulons and structural genes in vivo, thus abrogating relevant virulence phenotypes.

Small colony variants (SCVs) of Staphylococcus aureus were generated via mutations in menD or hemB, yielding menadione and hemin auxotrophs, respectively, and studied in the rabbit endocarditis model. No differences in the 95% infectious dose occurred between strains with regard to seeding heart valves ( approximately 10(6) cfu) or other target organs. No differences were observed between the response of the hemB mutant to oxacillin therapy and that of the parent strain in any target tissues, and significant reductions in bacterial densities were seen in all tissues (compared with untreated controls). In contrast, oxacillin therapy did not significantly reduce bacterial densities of the menD mutant in either kidney or spleen and significantly reduced densities within vegetations. These data show that SCVs are able to colonize multiple tissues in vivo and that the menD mutation provides the organism with a survival advantage during antimicrobial therapy, compared with its parent strain, in selected target tissues.

This study compared the promoter activation profiles of the 2 major transcripts of the Staphylococcus aureus global regulon, agr (RNAII and RNAIII). In vitro, RNAIII activation temporally followed RNAII activation and was absent in agr mutants. In experimental endocarditis, maximal RNAII activation in vegetations occurred early, followed by progressive increases in RNAIII activation (P<.05; 2 vs. 48 h); this paralleled significant increases in vegetation bacterial densities over time (P<.05; 2 and 6 vs. 48 h). At 48 h of infection, S. aureus densities in vegetations were significantly higher than those in kidney or spleen tissue (P<.05), paralleling a significantly greater RNAIII activation profile in vegetations than in the latter tissues (P<.05). Of importance, RNAIII activation was observed in vegetations in 2 agr mutants. These data demonstrate that RNAIII activation in vivo is time and cell density dependent, may be tissue specific, and can occur through RNAII-dependent and -independent mechanisms.

The expression of antiphagocytic polysaccharide capsules is an important pathogenetic step in establishing Staphylococcus aureus infections. Using a green fluorescent protein reporter gene (gfp) system, we examined the expression and genetic regulation of the cap5 promoter (capsular polysaccharide 5 genes) by two major global regulators of S. aureus (agr and sarA) in vitro and in a rabbit endocarditis model. In vitro, cap5 expression substantially increased during the post-exponential phase in parental, as well assarA mutant constructs. However, cap5 expression was greatly reduced in agr and agr/sarA double mutants. In the endocarditis model, the extent of cap5 expression in vegetations infected with the parental strain was substantially higher than that observed with the agr/sarA double mutants (P<0.05). Similar trends were noted in renal, but not splenic abscesses. Collectively, these data suggest that agr positively regulates cap5 expression both in vitro and in vivo, while the contribution of sarA to cap5 regulation, although modest, is readily discerned in vivo in agr minus background. In addition, the regulation ofcap5 expression by these global regulators may vary in distinct anatomic niches in vivo.

Background: Mammalian platelets contain small, cationic, staphylocidal peptides, termed thrombin-induced platelet-microbicidal proteins (tPMPs). Evidence suggests that tPMPs play a key role in host defense against endovascular infections, such as infective endocarditis (IE). In the present study, we evaluated the influence of differences in staphylococcal tPMP-susceptibility profiles in vitro on disease severity in experimental IE., Methods and Results: Experimental IE was induced in rabbits with either a tPMP-susceptible or an isogenic tPMP-resistant Staphylococcus aureus strain. Vegetation size, left ventricular fractional shortening, and onset of aortic valvular regurgitation were serially assessed by echocardiography over an 11-day postinfection period. In addition, blood cultures were performed daily. Parameters delineated at autopsy included vegetation weights; bacterial densities in vegetations, myocardium, and kidneys; extent of valvular and perivalvular tissue damage; and renal embolization. The following significant differences were observed in animals infected with the tPMP-susceptible versus the tPMP-resistant S aureus strain: substantially lower bacteremia rates (P=0.02); reduced vegetation growth (P<0.001) and weight (P<0.001); a later onset of aortic valvular regurgitation (P=0.0039); increased preservation of left ventricular function (P<0.001); reduced valvular tissue damage (P=0.01) and perivalvular inflammation (P=0.015); and reduced bacterial densities in vegetations (P<0.001) and kidneys (P<0.01)., Conclusions: The in vitro tPMP-susceptibility profile in S aureus substantially affects a number of well-defined cardiac and microbiological parameters related to disease severity and prognosis in IE. These findings underscore the likelihood that platelets mitigate the pathogenesis of endovascular infections via local secretion of antimicrobial peptides.

Thrombin-induced platelet microbicidal proteins (tPMP-1 and tPMP-2) are believed to initiate their staphylocidal effects via cytoplasmic membrane perturbation. The aim of the present study was to investigate the role of subsequent inhibition of macromolecular synthesis in the staphylocidal mechanisms of tPMP-1 and tPMP-2 in an isogenic tPMP-susceptible and -resistant strain pair (ISP479C and ISP479R, respectively). In ISP479C, tPMP-1 and tPMP-2 (2 microg/mL) exerted significant bactericidal effects and significantly reduced DNA and RNA synthesis (P <.05 vs. control). In contrast, tPMP-1 and tPMP-2 exerted reduced staphylocidal effects and significantly reduced inhibition of DNA and RNA synthesis against ISP479R, as compared with ISP479C (P <.05). However, tPMP-1 and tPMP-2 (2 microg/mL) caused equivalent degrees of inhibition of protein synthesis in both ISP479C and ISP479R. Collectively, these observations are consistent with the hypothesis that inhibition of specific macromolecular synthesis pathways is integral to the overall staphylocidal mechanism(s) of tPMPs.

Many antimicrobial peptides permeabilize the bacterial cytoplasmic membrane. However, it is unclear how membrane permeabilization and antimicrobial activity are related for distinct peptides. This study investigated the relationship between Staphylococcus aureus membrane permeabilization and cell death due to the following antistaphylococcal peptides: thrombin-induced platelet microbicidal protein 1 (tPMP-1), gramicidin D, and protamine. Isogenic S. aureus strains ISP479C and ISP479R (tPMP-1 susceptible and resistant, respectively), were loaded with the fluorochrome calcein and exposed to a range of concentrations of each peptide. Flow cytometry was then used to monitor membrane permeabilization by quantifying the release of preloaded calcein. Killing was determined by quantitative culture at time points simultaneous to measurement of membrane permeabilization. Membrane permeabilization and killing caused by tPMP-1 occurred in a time- and concentration-dependent manner, reflecting the intrinsic tPMP-1 susceptibilities of ISP479C and ISP479R. In comparison, gramicidin D killed both S. aureus strains to equivalent extents in a concentration-dependent manner between 0.5 to 50 microg/ml, but cell permeabilization only occurred at the higher peptide concentrations (25 and 50 microg/ml). Protamine permeabilized, but did not kill, either strain at concentrations up to 10 mg/ml. Regression analyses revealed different relationships between membrane permeabilization and staphylocidal activity for the distinct antimicrobial peptides. Taken together, these findings demonstrate that permeabilization, per se, does not invariably result in staphylococcal death due to distinct antimicrobial peptides. Thus, although each of these peptides interacts with the S. aureus cytoplasmic membrane, diversity exists in their mechanisms of action with respect to the relationship between membrane permeabilization and staphylocidal activity.

Platelet microbicidal proteins (PMPs) are small antimicrobial peptides secreted by mammalian platelets. In vitro resistance of Staphylococcus aureus strains to PMPs correlates with more extensive disease in experimental infective endocarditis (IE). To determine whether this same relationship exists in human S. aureus IE, we evaluated the in vitro PMP susceptibility phenotype of isolates from 58 prospectively-identified patients with definite S. aureus IE. On multivariate analyses, patients with S. aureus IE complicating an infected intravascular device were significantly more likely to have IE caused by a PMP-resistant strain (P=.0193). No correlations were detected between in vitro PMP resistance among S. aureus strains and the severity of human IE. This work supports the concept that in vitro PMP resistance in clinical S. aureus strains is associated with important clinical characteristics of S. aureus endovascular infections in vivo.

Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as Staphylococcus aureus. We previously showed that S. aureus strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729-732, 1994; A. S. Bayer et al., Antimicrob. Agents Chemother. 42:3169-3172, 1998; V. K. Dhawan et al., Infect. Immun. 65:3293-3299, 1997). However, the mechanisms mediating tPMP-1 resistance in S. aureus are not fully delineated. The S. aureus cell membrane appears to be a principal target for the action of tPMP-1. To gain insight into the basis of tPMP-1 resistance, we compared several parameters of membrane structure and function in three tPMP-1-resistant (tPMP-1(r)) strains and their genetically related, tPMP-1-susceptible (tPMP-1(s)) counterpart strains. The tPMP-1(r) strains were derived by three distinct methods: transposon mutagenesis, serial passage in the presence of tPMP-1 in vitro, or carriage of a naturally occurring multiresistance plasmid (pSK1). All tPMP-1(r) strains were found to possess elevated levels of longer-chain, unsaturated membrane lipids, in comparison to their tPMP-1(s) counterparts. This was reflected in corresponding differences in cell membrane fluidity in the strain pairs, with tPMP-1(r) strains exhibiting significantly higher degrees of fluidity as assessed by fluorescence polarization. These data provide further support for the concept that specific alterations in the cytoplasmic membrane of S. aureus strains are associated with tPMP-1 resistance in vitro.

Thrombin-induced platelet microbicidal protein-1 (tPMP-1) is a small, cationic staphylocidal peptide from rabbit platelets. In the current study, the outcomes of vancomycin treatment and prophylaxis were compared in experimental infective endocarditis (IE) caused by an isogenic Staphylococcus aureus strain pair differing in tPMP-1 susceptibility (tPMPS) or resistance (tPMPR) in vitro (ISP479C and ISP479R, respectively). Vancomycin therapy (selected for its intrinsically slow bactericidal activity) reduced ISP479C (but not ISP479R) densities in vegetations compared with controls (P<.01). In contrast, prophylactic administration of vancomycin yielded no differences in efficacies for the 2 challenge strains. These data suggest that the tPMPR phenotype in vitro has a negative effect on the antimicrobial therapy (but not the prophylaxis) of experimental S. aureus IE. These disparate results may be explained in part by the requirement for microbicidal effects in the treatment of established IE, whereas prophylactic efficacy depends more on growth inhibitory and antiadhesion effects.

Thrombin-induced platelet microbicidal protein-1 (tPMP-1) and human neutrophil defensin-1 (HNP-1) are small, cationic antimicrobial peptides. These peptides exert potent in vitro microbicidal activity against a broad spectrum of human pathogens, including Staphylococcus aureus. Evidence suggests that tPMP-1 and HNP-1 target and disrupt the bacterial membrane. However, it is not yet clear whether membrane disruption itself is sufficient to kill the bacterium or whether subsequent, presumably intracellular, events are also involved in killing. We investigated the staphylocidal activities of tPMP-1 and HNP-1 in the presence or absence of pretreatment with antibiotics that differ in their mechanisms of action. The staphylocidal effects of tPMP-1 and HNP-1 on control cells (no antibiotic pretreatment) were rapid and concentration dependent. Pretreatment of S. aureus with either penicillin or vancomycin (bacterial cell wall synthesis inhibitors) significantly enhanced the anti-S. aureus effects of tPMP-1 compared with the effects against the respective control cells over the entire tPMP-1 concentration range tested (P < 0.05). Similarly, S. aureus cells pretreated with these antibiotics were more susceptible to HNP-1 than control cells, although the difference in the effects against cells that received penicillin pretreatment did not reach statistical significance (P < 0.05 for cells that received vancomycin pretreatment versus effects against control cells). Studies with isogenic pairs of strains with normal or deficient autolytic enzyme activities demonstrated that enhancement of S. aureus killing by cationic peptides and cell wall-active agents could not be ascribed to a predominant role of autolytic enzyme activation. Pretreatment of S. aureus cells with tetracycline, a 30S ribosomal subunit inhibitor, significantly decreased the staphylocidal effect of tPMP-1 over a wide peptide concentration range (0.16 to 1.25 microgram/ml) (P < 0.05). Furthermore, pretreatment with novobiocin (an inhibitor of bacterial DNA gyrase subunit B) and with azithromycin, quinupristin, or dalfopristin (50S ribosomal subunit protein synthesis inhibitors) essentially blocked the S. aureus killing resulting from exposure to tPMP-1 or HNP-1 at most concentrations compared with the effects against the respective control cells (P < 0.05 for a tPMP-1 concentration range of 0.31 to 1.25 microgram/ml and for an HNP-1 concentration range of 6.25 to 50 microgram/ml). These findings suggest that tPMP-1 and HNP-1 exert anti-S. aureus activities through mechanisms involving both the cell membrane and intracellular targets.

Platelet microbicidal proteins (PMPs), small cationic peptides released at sites of endovascular damage, kill common bloodstream pathogens in vitro. Our group previously showed that in vitro resistance of clinical staphylococcal and viridans group streptococcal bacteremic strains to PMPs correlated with the diagnosis of infective endocarditis (IE) (Wu et al., Antimicrob. Agents Chemother. 38:729-732, 1994). However, that study was limited by (i) the small number of Staphylococcus aureus isolates from IE patients, (ii) the retrospective nature of the case definitions, and (iii) the diverse geographic sources of strains. The present study evaluated the in vitro PMP susceptibility phenotype of a large number of staphylococcemic isolates (n = 60), collected at a single medical center and categorized by defined and validated clinical criteria. A significantly higher proportion of staphylococcemic strains from patients with IE was PMP resistant in vitro than the proportion of strains from patients with soft tissue sepsis (83% and 33%, respectively; P < 0.01). Moreover, the levels of PMP resistance (mean percent survival of strains after 2-h exposure to PMP in vitro) were significantly higher for isolates from patients with IE and with vascular catheter sepsis than for strains from patients with abscess sepsis (P < 0.005 and P < 0.01, respectively). These data further support the concept that bloodstream pathogens that exhibit innate or acquired PMP resistance have a survival advantage with respect to either the induction or progression of endovascular infections.

Platelet microbicidal proteins (PMPs) are hypothesized to exert microbicidal effects via cytoplasmic membrane disruption. Transmission electron microscopy demonstrated a temporal association between PMP exposure, damage of the Staphylococcus aureus cytoplasmic membrane ultrastructure, and subsequent cell death. To investigate the mechanisms of action of PMPs leading to membrane damage, we used flow cytometry to compare the effects of two distinct PMPs (thrombin-induced PMP-1 [tPMP-1] or PMP-2) with human neutrophil defensin-1 (hNP-1) on transmembrane potential (Deltapsi), membrane permeabilization, and killing of S. aureus. Related strains 6850 (Deltapsi -150 mV) and JB-1 (Deltapsi -100 mV; a respiration-deficient menadione auxotroph of 6850) were used to assess the influence of Deltapsi on peptide microbicidal effects. Propidium iodide (PI) uptake was used to detect membrane permeabilization, retention of 3,3'-dipentyloxacarbocyanine (DiOC5) was used to monitor membrane depolarization (Deltapsi), and quantitative culture or acridine orange accumulation was used to measure viability. PMP-2 rapidly depolarized and permeabilized strain 6850, with the extent of permeabilization inversely related to pH. tPMP-1 failed to depolarize strain 6850, but did permeabilize this strain in a manner directly related to pH. Depolarization, permeabilization, and killing of strain JB-1 due to PMPs were significantly less than in strain 6850. Growth in menadione reconstituted Deltapsi of JB-1 to a level equivalent to 6850, and was associated with greater depolarization due to PMP-2, but not tPMP-1. Reconstitution of Deltapsi also enhanced permeabilization and killing of JB-1 due to tPMP-1 or PMP-2. Both PMP-2 and tPMP-1 caused significant reductions in viability of strain 6850. In contrast to tPMP-1 or PMP-2, defensin hNP-1 depolarized, permeabilized, and killed both strains 6850 and JB-1 equally, and in a manner directly related to pH. Collectively, these data indicate that membrane dysfunction and cell death due to tPMP-1, PMP-2, or hNP-1 likely involve different mechanisms. These findings may also reveal new insights into the microbicidal activities versus mammalian cell toxicities of antimicrobial peptides.

Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal densities). Paradoxically, the two variant staphylococcal strains producing alpha-toxin at supraparental levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)] also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the parental strain. The reduced in vivo virulence of the latter variant staphylococcal strains could not be explained by differences in bacteremic clearance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence exhibited by the variant staphylococcal strains in this model was related to pathogenetic events subsequent to bacterial adherence to the damaged endocardium. Excess intravegetation secretion of alpha-toxin, leading to increased PMP release (secondary to either increased platelet secretion or lysis), may well explain the reduced virulence observed in experimental endocarditis.

Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner.

Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic peptide released from rabbit platelets following exposure to thrombin in vitro. This peptide exerts potent in vitro microbicidal activity against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. It is known that the microbicidal actions of other cationic antimicrobial peptides (e.g., neutrophil defensins) are influenced by environmental factors and target cell growth phase. However, whether these parameters affect tPMP microbicidal activity has not been studied. Thus, we assessed the in vitro bactericidal activity of tPMP against two tPMP-susceptible strains, Bacillus subtilis ATCC 6633 and S. aureus 502A, in various target cell growth phases or under various microenvironmental conditions. The conditions studied included differing bacterial growth phase (logarithmic versus stationary), temperature (range, 4 to 42 degrees C), pH (range, 4.5 to 8.5), cationicity (range, 0.1 mM to 2 M), anionicity (range, 0.08 to 5 microM), and neutral carbohydrates ranging in molecular weight (MW) from 180 to 37,700 (range, 50 to 500 mM) as well as rabbit platelet-free plasma and serum. tPMP staphylocidal activity was greater against logarithmic- than stationary-phase cells. tPMP bactericidal activity against both B. subtilis and S. aureus was directly correlated with temperature and pH, with microbicidal activity exhibited near the physiological range (37 to 42 degrees C and pH 7.2 to 8.5, respectively). The presence of cations (Na+, K+, Ca2+, and Mg2+) decreased tPMP bactericidal activity in a time- and concentration-dependent manner, with complete inhibition at monovalent or divalent cation concentrations of > or = 250 or > or = 10 mM, respectively. Staphylocidal activity of tPMP was also inhibited by the polyanions polyanetholsulfonic acid and polyaspartic acid, at 0.1 and 0.4 microM, respectively. Coincident exposure with low-MW carbohydrates (glucose, sucrose, and melezitose) did not affect tPMP staphylocidal activity. However, higher-MW carbohydrates (raffinose and dextrans) decreased tPMP activity in a manner directly proportional to their concentration and MW. Solute-mediated inhibition of tPMP bactericidal activity was independent of solute osmolality but directly related to the duration of tPMP-solute coexposure. tPMP enhanced the staphylocidal activities of platelet-free plasma and heat-inactivated serum, while the activity of normal serum was not affected. These collective observations suggest that tPMP retains antimicrobial activities under physiological conditions which are likely to be relevant to host defense in vivo.

Platelet aggregation by bacteria is felt to play an important role in the pathogenesis of infective endocarditis. However, the mechanisms involved in bacterium-induced platelet aggregation are not well-defined. In the present study, we examined the mechanisms by which Staphylococcus aureus causes rabbit platelet aggregation in vitro. In normal plasma, the kinetics of S. aureus-induced platelet aggregation were rapid and biphasic. The onset and magnitude of aggregation phase 1 varied with the bacterium-platelet ratio, with maximal aggregation observed at a ratio of 5:1. The onset of aggregation phase 2 was delayed in the presence of apyrase (an ADP hydrolase), suggesting that this later aggregation phase may be triggered by secreted ADP. The onset of aggregation phase 2 was delayed in the presence of prostaglandin I2-treated platelets, and this phase was absent when paraformaldehyde-fixed platelets were used, implicating platelet activation in this process. Platelet aggregation phase 2 was dependent on S. aureus viability and an intact bacterial cell wall, and it was mitigated by antibody directed against staphylococcal clumping factor (a fibrinogen-binding protein) and by the cyclooxygenase inhibitor indomethacin. Similarly, aggregation phase 2 was either delayed or absent in three distinct transposon-induced S. aureus mutants with reduced capacities to bind fibrinogen in vitro. In addition, a synthetic pentadecapeptide, corresponding to the staphylococcal binding domain in the C terminus of the fibrinogen delta-chain, blocked aggregation phase 2. However, phase 2 of aggregation was not inhibited by two synthetic peptides (alone or in combination) analogous to the two principal fibrinogen-binding domains on the platelet glycoprotein (GP) IIb/IIIa integrin receptor: (i) a recognition site on the IIIa molecule for the Arg-Gly-Asp (RGD) sequence of the fibrinogen alpha-chain and (ii) a recognition site on the IIb molecule for a dodecapeptide sequence of the fibrinogen delta-chain. This differs from ADP-induced platelet aggregation, which relies on an intact platelet GP IIb/IIIa receptor with an accessible RGD sequence and dodecapeptide recognition site for fibrinogen. Furthermore, a monoclonal antibody directed against the RGD recognition site on rabbit platelet GP IIb/IIIa receptors failed to inhibit rabbit platelet aggregation by S. aureus. Collectively, these data suggest that S. aureus-induced platelet aggregation requires bacterial binding to fibrinogen but is not principally dependent upon the two major fibrinogen-binding domains on the platelet GP IIb/IIIa integrin receptor, the RGD and dodecapeptide recognition sites.

A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this locus is distinct from agr, xpr, and sar, three previously described global regulatory loci. Transduction experiments demonstrated that the genotype associated with mutant 1E3 could be transferred back into the parental strain ISP479C. The transductant 1E3-2 displayed a phenotypic profile similar to that of the original mutant. Northern (RNA) blot studies showed that this locus may be involved in modulating target genes at the mRNA level. In the rabbit endocarditis model, there was a significant decrease in both the infectivity rate and intravegetation bacterial density with mutant 1E3 compared with the parent at an inoculum of 10(3) CFU. Since protein A and the fibrinogen-binding protein(s) are major surface proteins that may mediate bacterial adhesion to host tissues, this locus may be an important genetic element involved in the expression of virulence determinants in S. aureus.

Microbial pathogenicity in Staphylococcus aureus is a complex process involving a number of virulence genes that are regulated by global regulatory systems including sar and agr. To evaluate the roles of these two loci in virulence, we constructed sar-/agr- mutants of strains RN6390 and RN450 and compared their phenotypic profiles to the corresponding single sar- and agr- mutants and parents. The secretion of all hemolysins was absent in the sar-/agr- mutants while residual beta-hemolysin activity remained in single agr- mutants. The fibronectin binding capacity was significantly diminished in both single sar- mutants and double mutants when compared with parents while the reduction in fibrinogen binding capacity in the double mutants was modest. In the rabbit endocarditis model, there was a significant decrease in both infectivity rates and intravegetation bacterial densities with the double mutant as compared to the parent (RN6390) at 10(3)-10(6) CFU inocula despite comparable levels of early bacteremia among various challenge groups. Notably, fewer bacteria in the double mutant group adhered to valvular vegetations at 30 min after challenge (10(6) CFU) than the parent group. These studies suggest that both the sar and agr loci are involved in initial valvular adherence, intravegetation persistence and multiplication of S. aureus in endocarditis.

Bacterial adherence to platelets on the cardiac valve surface is believed to be critical in the induction of infective endocarditis. Recent studies have confirmed that thrombin-activated platelets secrete platelet microbicidal protein (PMP), which can both kill and exert nonlethal antiadherence effects against endovascular pathogens. In the present study, we quantified the influence of antibiotic and/or PMP exposures on in vitro platelet adherence of two Staphylococcus aureus strains, identical by DNA restriction and cell wall protein profiles, that differed in their susceptibility to PMP-induced killing (PMPs or PMPr, respectively). Adherence assays were performed by flow cytometry in the presence of sublethal PMP concentrations (1 to 2.5 micrograms/ml) alone or in combination with ampicillin (AMP) alone, sulbactam (SUL) alone, or AMP plus SUL (AMP-SUL), at levels achievable in serum. Exposure of the PMPs and PMPr S. aureus strains to antibiotics (for 2 h at 37 degrees C) prior to flow cytometry resulted in no substantive changes in the percent adherence to platelets compared with that for S. aureus cells not exposed to antibiotics, except for modestly increased adherence of both PMPs and PMPr cells exposed to AMP-SUL (18.5 and 15.8% increases, respectively). Addition of PMP to antibiotic-S. aureus mixtures (final 30 min) caused a significant decrease in S. aureus adherence to platelets, for both the PMPs and PMPr S. aureus strains, compared with antibiotic exposure alone (e.g., reduction in platelet adherence from 57.9 +/- 8.2% to 12.2 +/- 3.6% for PMPs cells exposed to AMP-SUL and PMP [P = 0.01]). Moreover, addition of PMP following exposure of the PMPs and PMPr strains to AMP-SUL reversed the enhanced bacterium-platelet adherence observed with such antibiotic exposures alone (P < or = 0.005). These data demonstrate that PMP exerts a potent antiplatelet adherence effect which is independent of its microbicidal capacity, rendering S. aureus cells less adherent to platelets in the presence or absence of antibiotics. Reduction of microbial adherence to platelets by PMP alone or with antibiotics provides further insight into the mechanism(s) that may be involved in host defense and antibiotic prophylaxis of infective endocarditis and other endovascular infections.

A regulatory locus on the Staphylococcus aureus chromosome, designated sar, is involved in the expression of cell wall proteins, some of which are potentially important in the pathogenesis of endocarditis. For instance, mutant 11D2 (sar::Tn917LTV1) was found to bind substantially less to matrix proteins (i.e., fibrinogen and fibronectin) than parent strain DB. Remarkably, these two strains did not differ in other phenotypes considered important in the initiation of endocarditis (e.g., binding to platelets and resistance to platelet-derived microbicidal proteins). The isogenic pair were compared for pathogenicity in a rabbit endocarditis model. There were significant differences in infectivity rates between the two strains (71 and 88% for DB versus 17 and 42% for mutant 11D2 at inocula of 10(3) and 10(4) CFU, respectively). In early adherence studies, parent DB adhered substantially better than the mutant to valvular vegetations at an inoculum of 10(6) CFU (P = 0.05). Southern blot analysis of colonies indicated that the location of the Tn917LTV1 insert in mutant 11D2 remained stable after animal passage. In vitro adherence assays revealed that mutant 11D2 was less adherent to cultured human endothelium than parent DB. These studies suggest that the sar locus is involved in the initial adherence of S. aureus to the fibrin-platelet-endothelium matrix on damaged valvular endothelium.

The interaction of bacteria with platelets at the cardiac valve surface represents a critical event in the induction of infective endocarditis. Platelets are thought to modulate induction or propagation of endocarditis via secretion of alpha-granule-derived platelet microbicidal protein (PMP) (a low-molecular-mass, cationic, heat-stable protein distinct from lysozyme). We studied representative PMP-susceptible and PMP-resistant Staphylococcus aureus isolates to determine their in vitro bacteriostatic and bactericidal susceptibilities to combinations of PMP plus antistaphylococcal antibiotics. PMP plus oxacillin exerted a synergistic bactericidal effect, in contrast to either agent alone, regardless of the intrinsic PMP susceptibility of the isolate tested. Exposure of S. aureus to PMP alone resulted in residual postexposure growth-inhibitory effects lasting from 0.9 to 1.8 h. Sequential exposure of S. aureus isolates to PMP for 30 min followed by exposure to either oxacillin or vancomycin (each at 10x the MIC for 120 min) resulted in a significant extension of the postantibiotic-effect duration compared with antibiotic exposure alone (P less than or equal to 0.05). Collectively, these findings indicate that PMP both enhances antibiotic-induced killing of S. aureus and increases the postantibiotic-effect duration in S. aureus.

Quantitative analyses of Staphylococcus aureus binding to platelets were done using flow cytometry after bacterial exposure to the following treatments: proteases (trypsin, protease K), antibiotics (oxacillin, gentamicin), surface carbohydrate modifiers (sodium periodate, anticapsular antibody), or platelet microbicidal protein. In separate studies, platelets were exposed to a monoclonal antibody to their Fc receptor (Fc gamma RII) before binding was quantified. The percentage of bacteria bound to platelets varied significantly among strains (22.1% +/- 3.8% to 76.4 +/- 3.2%). For all isolates, binding to platelets was rapid, saturable, and reversible, suggesting a receptor-ligand interaction. The following modifiers significantly reduced binding: platelet microbicidal protein (by 32.1% +/- 5.2%; P less than .001), homologous (but not heterologous) anticapsular antibody (by 17.7% +/- 1.9%; P less than .05), sodium periodate (by 36.3% +/- 4.3%; P less than .005), and anti-platelet Fc monoclonal antibody (by 41.5% +/- 4.4%; P less than .002). Collectively, these data suggest that the mechanism(s) involved in S. aureus-platelet binding are complex and multimodal, involving carbohydrate-rich and platelet microbicidal protein-susceptible S. aureus surface ligands as well as the platelet Fc receptor.

Bacterium-platelet interactions at the cardiac valve surface represent an important initial step in the induction of infective endocarditis (IE). This cell-cell interaction may play either a protagonistic role in the induction of IE via bacterial adherence to and aggregation of platelets or an antagonistic role via secretion of platelet-derived microbicidal molecules. We examined the spectrum and interrelationship of three aspects of the interaction of 20 clinical Staphylococcus aureus isolates with rabbit platelets in vitro: (i) S. aureus adherence to platelets; (ii) S. aureus-induced platelet aggregation; and (iii) S. aureus resistance to the action of thrombin-induced platelet microbicidal protein (PMP; low-molecular-weight cationic peptides contained in alpha granules). Among the 20 S. aureus isolates (11 bacteremia, 9 endocarditis), there was a heterogeneous distribution profile for each of the bacterium-platelet interaction parameters studied. For S. aureus-platelet adherence and S. aureus-induced platelet aggregation, 3 of 20 and 7 of 20 isolates tested were considered highly active for each respective parameter; 5 of 20 staphylococcal strains were deemed resistant to the bactericidal action of PMP. In addition, more endocarditis isolates (45%) were PMP resistant than strains from patients without endocarditis (19%). When analyzed concomitantly, there was a significant, positive correlation between S. aureus-platelet adherence and S. aureus-induced platelet aggregation among isolates (P = 0.003; r = 0.78). In contrast, there were no statistically significant relationships between either platelet adherence or aggregation and PMP resistance among these 20 S. aureus isolates. These data suggest that platelet adherence and aggregation are related abilities of S. aureus, while resistance to thrombin-induced PMP is an independent phenotypic characteristic and potential virulence factor.

Thrombin-induced platelet microbicidal protein (PMP) is considered to play an important role in preventing an important role in preventing streptococcal endocarditis. However, the structural features and functions of PMPs have not been well characterized, and their antibacterial spectra against other common endocarditis pathogens, such as the staphylococci, are not known. Thrombin stimulation of washed rabbit platelets (10(8)/ml) yielded a PMP-rich preparation with a specific activity of approximately 25 U/mg of protein as determined by Bacillus subtilis bioassay. Twenty-eight clinical and laboratory Staphylococcus aureus isolates, exposed to a standardized PMP preparation (100 U/ml for 2 h at 37 degrees C), exhibited a Poisson-distributed heterogeneity to the bactericidal action of PMP, with approximately one-third designated as PMP resistant. Gel filtration chromatography (Sephadex G-50) identified the bioactive moiety within PMP preparations to be in the major protein elution peak; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) presumptively identified PMP as a low-molecular-weight (MW) (8,500) protein present only in such bioactive protein peaks. Both the bioactivity of PMP preparations and the low-MW protein band were removable by specific anionic membranes (e.g., cellulose-acetate/nitrate), as well as by a variety of anionic resins, further corroborating the suspected cationic charge of PMP. In addition, both PMP bioactivity and the low-MW protein band were recoverable by 1.5 M NaCl elution of the anionic membrane filters post-PMP adsorptive removal. Adsorption of bioactive PMP preparations by highly PMP-susceptible B. subtilis (10(8) CFU/ml, 30 min) resulted in a near-complete loss of residual bioactivity; in contrast, adsorption of bioactive PMP preparations with less PMP-susceptible S. aureus strains failed to reduce bioactivity. Significant lysozyme contamination of PMP-rich preparations was ruled out by determination of differences between bioactive PMP preparations and exogenous lysozyme as regards (i) relative heat stabilities; (ii) differential bactericidal activity versus B. subtilis and Micrococcus luteus; and (iii) SDS-PAGE protein profiles. These data show that the bioactive PMP protein moiety is of low MW, is heat stable, is probably cationic (similar to leukocyte-derived defensins), and possesses potent bactericidal activity against a significant percentage of S. aureus isolates.