Background:We studied an ampicillin- and vancomycin-resistant Enterococcus faecium (VRE) isolate from a patient with endocarditis and bacteremia refractory to treatment with daptomycin (6 mg/kg) plus linezolid. Blood cultures cleared within 24 hours of changing therapy to daptomycin (12 mg/kg) plus ampicillin. We examined the effects of ampicillin on daptomycin-induced growth inhibition and killing, surface charge, and susceptibility to several prototypical host defense cationic antimicrobial peptides.Methods:Minimum inhibitory concentrations (MICs) and time-kill curves with daptomycin were assessed in the presence or absence of ampicillin. The impact of ampicillin on surface charge was assessed by flow cytometry using a poly-L-lysine binding assay. The effects of ampicillin pre-exposures upon VRE killing by five distinct cationic peptides of different structure, charge, origin and mechanisms of action were analyzed using the epidermal cathelicidin, LL-37, thrombin-induced platelet microbicidal proteins (tPMPs), and a synthetic congener modeled after tPMP microbicidal domains (RP-1), human neutrophil peptide-1 (hNP-1), and polymyxin B (bacterial-derived). Fluoroscein-bodipy-labeled daptomycin was used to evaluate daptomycin binding to VRE membranes in the presence or absence of ampicillin.Results:In media containing ampicillin (25-100 mg/L), daptomycin MICs decreased from 1.0 to 0.38 mg/L. By time-kill and an in vitro pharmacodynamic model, ampicillin enhanced daptomycin activity against the study VRE from a bacteriostatic to a bactericidal profile. VRE grown in ampicillin (25-150 mg/L) demonstrated an incremental reduction in its relative net positive surface charge. When grown in the presence (vs. absence) of ampicillin (25 and 100 mg/L), the VRE strain i) was more susceptible to killing by LL-37, tPMPs, hNP-1, and RP-1, but not to polymyxin B, and ii) exhibited greater binding to Bodipy-labeled daptomycin.Conclusion:Ampicillin induced reductions in net positive bacterial surface charge of VRE, correlating with enhanced bactericidal effects of cationic calcium-daptomycin and a diverse range of other cationic peptides in vitro. While the mechanism(s) of such β-lactam-mediated shifts in surface charge remain to be defined, these finding suggest a potential for β-lactam-mediated enhancement of activity of both daptomycin and innate host defense peptides against antibiotic-resistant bacteria.

The two-component regulatory system, GraRS, appears to be involved in staphylococcal responses to cationic antimicrobial peptides (CAPs). However, 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 showed that: i) expression of two key determinants of net positive surface charge (mprF and dlt) were dependent on the co-transcription 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 bacteria (polymyxin B); and iv) synthesis and translocation of membrane lysyl-phosphotidyl glycerol (an mprF-dependent function) was substantially lower in graR and vraG mutants than parental strains. Importantly, 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 co-dependent 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.

The accessory gene regulator (agr) locus has been shown to be important for virulence in several animal models of S. aureus infection. However, the role of agr in human infections, and specifically in antibiotic treatment is controversial. Interestingly, agr dysfunction has been associated with reduced vancomycin responses. To systematically investigate the role of agr in virulence and treatment outcome in the context of endovascular infection, ten well-characterized vancomycin-susceptible MRSA bloodstream isolates (5 agr-I [CC45] and 5 agr-II [CC5]) were studied for: i) agr function; ii) RNAIII transcriptional profiles; iii) agr locus sequences; iv) intrinsic virulence and responses to vancomycin therapy in an experimental endocarditis (IE) model, and v) in vivo RNAIII expression. Significant differences in agr function (determined by δ-hemolysin activity) correlated with the time-point of RNAIII transcription (earlier RNAIII onset = increased agr function). Unexpectedly, four MRSA strains with strong δ-hemolysin activity exhibited significant resistance to vancomycin treatment in experimental IE. In contrast, five of six MRSA strains with weak or no δ-hemolysin activity were highly susceptible to vancomycin therapy in the IE model. agr sequence analyses showed no common single nucleotide polymorphism predictive of agr functionality. In vivo RNAIII expression in cardiac vegetations did not correlate with virulence or vancomycin treatment outcomes in the IE model. Inactivation of agr in two strains with strong δ-hemolysin activity did not affect virulence or in vivo efficacy of vancomycin. Our findings suggest that agr-dysfunction does not correlate with vancomycin treatment failures in experimental IE model in two distinct MRSA genetic backgrounds.

Cationic antimicrobial peptides (CAPs) play important roles in host immune defenses. Plectasin is a defensin-like CAP isolated from the saprophytic fungus, Pseudoplectania nigrella. NZ2114 is a novel variant of plectasin with potent activity against Gram-positive bacteria. In this study, we investigated: i) the in vivo pharmacokinetic and pharmacodynamic (PK/PD) characteristics of NZ2114; and ii) the in vivo efficacy of NZ2114 in comparison with two conventional antibiotics, vancomycin or daptomycin, in a rabbit experimental endocarditis model (IE) due to a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC33591). All NZ2114 regimens (5, 10 and 20 mg/kg, iv, twice daily for three days), significantly decreased MRSA densities in cardiac vegetations, kidneys and spleen vs. untreated controls, except in one scenario (5 mg/kg vs. splenic MRSA counts). The efficacy of NZ2114 was clearly dose-dependent in all target tissues. At 20 mg/kg, NZ2114 showed a significantly greater efficacy vs. vancomycin (P < 0.001), and similar efficacy to daptomycin. Of importance, only NZ2114 (at 10 and 20 mg/kg regimen) prevented post-therapy relapse in cardiac vegetations, kidneys and spleen, while bacterial counts in these target tissues continued to increase in vancomycin- and daptomycin-treated animals. These in vivo efficacies were equivalent and significantly correlated with three PK indices investigated: fC(max)/MIC, fAUC/MIC and f%T>MIC, as analyzed by a sigmoid E(max) model (R(2)> 0.69). The superior efficacy of NZ2114 in this MRSA IE model suggest the potential for further development of this compound for treating serious MRSA infections.

The pathogenesis of Staphylococcus aureus infective endocarditis (IE) is postulated to involve invasion and damage of endothelial cells (ECs). However, the precise relationships between S. aureus-EC interactions in vitro and IE virulence and treatment outcomes in vivo are poorly defined. Ten methicillin-resistant S. aureus (MRSA) clinical isolates previously tested for their virulence and vancomycin responsiveness in an experimental IE model were assessed in vitro for their haemolytic activity, protease production, and capacity to invade and damage ECs. There was a significant positive correlation between the in vitro EC damage caused by these MRSA strains and their virulence during experimental IE (in terms of bacterial densities in target tissues; P < 0.02). Importantly, higher EC damage was also significantly correlated with poor microbiological response to vancomycin in the IE model (P < 0.001). Interestingly, the extent of EC damage was unrelated to a strain's ability to invade ECs, haemolytic activity and protease production, or β-toxin gene transcription. Inactivation of the agr locus in two MRSA strains caused ∼20% less damage as compared with the corresponding parental strains, indicating that a functional agr is required for maximal EC damage induction. Thus, MRSA-induced EC damage in vitro is a unique virulence phenotype that is independent of many other prototypical MRSA virulence factors, and may be a key biomarker for predicting MRSA virulence potential and antibiotic outcomes during endovascular infections.

We investigated the hypothesis that methicillin-resistant Staphylococcus aureus (MRSA) isolates developing reduced susceptibilities to daptomycin (DAP; a calcium-dependent molecule acting as a cationic antimicrobial peptide [CAP]) may also coevolve reduced in vitro susceptibilities to host defense cationic antimicrobial peptides (HDPs). Ten isogenic pairs of clinical MRSA DAP-susceptible/DAP-resistant (DAP(s)/DAP(r)) strains were tested against two distinct HDPs differing in structure, mechanism of action, and origin (thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]) and one bacterium-derived CAP, polymyxin B (PMB). Seven of 10 DAP(r) strains had point mutations in the mprF locus (with or without yyc operon mutations), while three DAP(r) strains had neither mutation. Several phenotypic parameters previously associated with DAP(r) were also examined: cell membrane order (fluidity), surface charge, and cell wall thickness profiles. Compared to the 10 DAP(s) parental strains, their respective DAP(r) strains exhibited (i) significantly reduced susceptibility to killing by all three peptides (P < 0.05),(ii) increased cell membrane fluidity, and (iii) significantly thicker cell walls (P < 0.0001). There was no consistent pattern of surface charge profiles distinguishing DAP(s) and DAP(r) strain pairs. Reduced in vitro susceptibility to two HDPs and one bacterium-derived CAP tracked closely with DAP(r) in these 10 recent MRSA clinical isolates. These results suggest that adaptive mechanisms involved in the evolution of DAP(r) also provide MRSA with enhanced survivability against HDPs. Such adaptations appear to correlate with MRSA variations in cell membrane order and cell wall structure. DAP(r) strains with or without mutations in the mprF locus demonstrated significant cross-resistance profiles to these unrelated CAPs.

We used daptomycin plus antistaphylococcal β-lactams (ASBL) to clear refractory MRSA bacteremia. In vitro studies showed enhanced daptomycin bactericidal activity, increased membrane daptomycin binding, and decrease in positive surface charge induced by ASBLs against daptomycin nonsusceptible MRSA. Addition of ASBLs to daptomycin may be of benefit in refractory MRSA bacteremia.(Although the official designation is "daptomycin nonsusceptiblity," we will use the term "daptomycin-resistance" in this paper for facility of presentation.).

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.

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.

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.