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Background Staphylococcus aureus is the most common cause of life-threatening endovascular infections, including infective endocarditis (IE). These infections, especially when caused by methicillin-resistant strains (MRSA), feature limited therapeutic options and high morbidity and mortality rates. Methods Herein, we investigated the role of the purine biosynthesis repressor, PurR, in virulence factor expression and vancomycin (VAN) treatment outcomes in experimental IE due to MRSA. Results The PurR-mediated repression of purine biosynthesis was confirmed by enhanced purF expression and production of an intermediate purine metabolite in purR mutant strain. In addition, enhanced expression of the transcriptional regulators, sigB and sarA , and their key downstream virulence genes (eg, fnbA , and hla) was demonstrated in the purR mutant in vitro and within infected cardiac vegetations. Furthermore, purR deficiency enhanced fnbA / fnbB transcription, translating to increased fibronectin adhesion versus the wild type and purR- complemented strains. Notably, the purR mutant was refractory to significant reduction in target tissues MRSA burden following VAN treatment in the IE model. Conclusions These findings suggest that the purine biosynthetic pathway intersects the coordination of virulence factor expression and in vivo persistence during VAN treatment, and may represent an avenue for novel antimicrobial development targeting MRSA. [ABSTRACT FROM AUTHOR]

Background Clinical outcomes in bacterial bloodstream infections (BSIs) are influenced by bacterial species, host immunity, and antibiotic therapy. The mechanisms by which such factors influence outcomes are poorly understood. We aimed to identify bacterial- and antibiotic-specific host transcriptional signatures in patients with bacterial BSI. Methods RNA sequencing was performed on blood samples from patients with BSI due to gram-negative (GN) versus gram-positive (GP) pathogens: Escherichia coli (n = 30) or Klebsiella pneumoniae (n = 28) versus methicillin-susceptible Staphylococcus aureus (MSSA) (n = 24) or methicillin-resistant S. aureus (MRSA) (n = 58). Patients were matched by age, sex, and race. Results No significant host transcriptome differences were detected in patients with E. coli versus K. pneumoniae BSI, so these were considered together as GN BSI. Relative to S. aureus BSI, patients with GN BSI had increased activation of the classic complement system. However, the most significant signal was a reduction in host transcriptional signatures involving mitochondrial energy transduction and oxidative burst in MRSA versus MSSA. This attenuated host transcriptional signature remained after controlling for antibiotic therapy. Conclusions Given the importance of immune cellular energetics and reactive oxygen species in eliminating hematogenous or intracellular MRSA, these findings may offer insights into its persistence relative to other bacterial BSIs. [ABSTRACT FROM AUTHOR]

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinical-therapeutic challenge. Of particular concern is antibiotic treatment failure in infections caused by MRSA that are "susceptible" to antibiotic in vitro. In the current study, we investigate specific purine biosynthetic pathways and stringent response mechanism(s) related to this life-threatening syndrome using genetic matched persistent and resolving MRSA clinical bacteremia isolates (PB and RB, respectively), and isogenic MRSA strain sets. We demonstrate that PB isolates (vs RB isolates) have significantly higher (p)ppGpp production, phenol-soluble-modulin expression, polymorphonuclear leukocyte lysis and survival, fibronectin/endothelial cell (EC) adherence, and EC damage. Importantly, an isogenic strain set, including JE2 parental, relP-mutant and relP-complemented strains, translated the above findings into significant outcome differences in an experimental endocarditis model. These observations indicate a significant regulation of purine biosynthesis on stringent response, and suggest the existence of a previously unknown adaptive genetic mechanism in persistent MRSA infection. [ABSTRACT FROM AUTHOR]

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (PB) represents an important subset of S. aureus endovascular infections. In this study, we investigated potential genetic mechanisms underlying the persistent outcomes. Compared with resolving bacteremia (RB) isolates (defined as isolates associated with negative results of blood cultures 2-4 days after initiation of therapy), PB strains (defined as isolates associated with positive results of blood cultures ≥7 days after initiation of therapy) had significantly earlier onset activation of key virulence regulons and structural genes (eg, sigB, sarA, sae, and cap5), higher expression of purine biosynthesis genes (eg, purF), and faster growth rates, with earlier entrance into stationary phase. Importantly, an isogenic strain set featuring a wild-type MRSA isolate, a purF mutant strain, and a purF-complemented strain and use of strategic purine biosynthesis inhibitors implicated a causal relationship between purine biosynthesis and the in vivo persistent outcomes. These observations suggest that purine biosynthesis plays a key role in the outcome of PB and may represent a new target for enhanced efficacy in treating life-threatening MRSA infections. [ABSTRACT FROM AUTHOR]

Background:  The global regulator sarA modulates virulence of methicillin-resistant Staphylococcus aureus (MRSA) via regulation of principal virulence factors (eg, adhesins and toxins) and biofilm formation. Resistance of S. aureus strains to β-lactam antibiotics (eg, oxacillin) depends on the production of penicillin-binding protein 2a (PBP2a), encoded by mecA METHODS:  In the present study, we investigated the impact of sarA on the phenotypic and genotypic characteristics of oxacillin resistance both in vitro and in an experimental endocarditis model, using prototypic healthcare- and community-associated MRSA parental and their respective sarA mutant strain sets.Results:  All sarA mutants (vs respective MRSA parental controls) displayed significant reductions in oxacillin resistance and biofilm formation in vitro and oxacillin persistence in an experimental endocarditis model in vivo. These phenotypes corresponded to reduced mecA expression and PBP2a production and an interdependency of sarA and sigB regulators. Moreover, RNA sequencing analyses showed that sarA mutants exhibited significantly increased levels of primary extracellular proteases and suppressed pyrimidine biosynthetic pathway, argininosuccinate lyase-encoding, and ABC transporter-related genes as compared to the parental strain.Conclusions:  These results suggested that sarA regulates oxacillin resistance in mecA-positive MRSA. Thus, abrogation of this regulator represents an attractive and novel drug target to potentiate efficacy of existing antibiotic for MRSA therapy. [ABSTRACT FROM AUTHOR]

Daptomycin is a lipopeptide antibiotic that is used clinically against many gram-positive bacterial pathogens and is considered a key frontline bactericidal antibiotic to treat multidrug-resistant enterococci. Emergence of daptomycin resistance during therapy of serious enterococcal infections is a major clinical issue. In this work, we show that deletion of the gene encoding the response regulator, LiaR (a member of the LiaFSR system that controls cell envelope homeostasis), from daptomycin-resistant Enterococcus faecalis not only reversed resistance to 2 clinically available cell membrane-targeting antimicrobials (daptomycin and telavancin), but also resulted in hypersusceptibility to these antibiotics and to a variety of antimicrobial peptides of diverse origin and with different mechanisms of action. The changes in susceptibility to these antibiotics and antimicrobial peptides correlated with in vivo attenuation in a Caenorhabditis elegans model. Mechanistically, deletion of liaR altered the localization of cardiolipin microdomains in the cell membrane. Our findings suggest that LiaR is a master regulator of the enterococcal cell membrane response to diverse antimicrobial agents and peptides; as such, LiaR represents a novel target to restore the activity of clinically useful antimicrobials against these organisms and, potentially, increase susceptibility to endogenous antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Background. Staphylococcus aureus is the most common cause of endovascular infections. The staphylococcal accessory regulator A locus (sarA) is a major virulence determinant that may potentially impact methicillin-resistant S. aureus (MRSA) persistence in such infections via its influence on biofilm formation.Methods. Two healthcare-associated MRSA isolates from patients with persistent bacteremia and 2 prototypical community-acquired MRSA strains, as well as their respective isogenic sarA mutants, were studied for in vitro biofilm formation, fibronectin-binding capacity, autolysis, and protease and nuclease activities. These assays were done in the presence or absence of sub–minimum inhibitory concentrations (MICs) of vancomycin. In addition, these strain pairs were compared for intrinsic virulence and responses to vancomycin therapy in experimental infective endocarditis, a prototypical biofilm model.Results. All sarA mutants displayed significantly reduced biofilm formation and binding to fibronectin but increased protease production in vitro, compared with their respective parental strains. Interestingly, exposure to sub-MICs of vancomycin significantly promoted biofilm formation and fibronectin-binding in parental strains but not in sarA mutants. In addition, all sarA mutants became exquisitely susceptible to vancomycin therapy, compared with their respective parental strains, in the infective endocarditis model.Conclusions. These observations suggest that sarA activation is important in persistent MRSA endovascular infection, potentially in the setting of biofilm formation. [ABSTRACT FROM AUTHOR]

The occurrence of mutations in methicillin-resistant Staphylococcus aureus (MRSA) during persistent infection leads to antimicrobial resistance but may also impact host-pathogen interactions. Here, we investigate the host-pathogen consequences of 2 mutations arising in clinical MRSA during persistent infection: RpoB H481Y, which is linked to rifampicin resistance, and RelA F128Y, which is associated with an active stringent response. Allelic exchange experiments showed that both mutations cause global transcriptional changes, leading to upregulation of capsule production, with attenuated virulence in a murine bacteremia model and reduced susceptibility to both antimicrobial peptides and whole-blood killing. Disruption of capsule biosynthesis reversed these impacts on innate immune function. These data clearly link MRSA persistence and reduced virulence to the same mechanisms that alter antimicrobial susceptibility. Our study highlights the wider consequences of suboptimal antimicrobial use, where drug resistance and immune escape mechanisms coevolve, thus increasing the likelihood of treatment failure. [ABSTRACT FROM AUTHOR]

Background: Persistent MRSA bacteremia (PB) represents an important subset of Staphylococcus aureus infections and correlates with poor clinical outcomes.Methods: We profiled relevant in vitro phenotypic and genotypic characteristics of MRSA isolates from 39 persons with bacteremia (21 had PB and 18 had resolving bacteremia [RB]). We also compared the intrinsic virulence and responsiveness to vancomycin of selected PB and RB strains in an experimental endocarditis model (IE).Results: PB and RB isolates differed significantly with regard to several in vitro characteristics that are believed to impact endovascular infections. PB isolates exhibited significantly more resistance to the cationic defensin hNP-1, enhanced membrane fluidity, and substantially greater adhesion to fibronectin, fibrinogen, and endothelial cells. Genotypically, PB isolates had higher frequency of SCCmec II, CC30, and spa 16; and higher rates of agr type III, cap8, tst-1, and cna carriage. Finally, a prototypic PB strain was more resistant to vancomycin treatment in the infective endocarditis model than a RB comparator strain, despite equivalent virulence profiles.Conclusions: Our findings indicate that PB isolates may have specific virulence signatures that distinguish them from RB isolates. These data suggest that methods might be developed to identify patients at higher risk for PB in real-time, thereby optimizing the effectiveness of anti-MRSA therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background. The Staphylococcus aureus global regulators—agr, sarA, and sae—coordinately control α-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 staphylo-cidal 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 mg/mL) exerted significant bactericidal effects and significantly reduced DNAand RNAsynthesis (P ,.05 vs. con-trol). 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 mg/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. [ABSTRACT FROM AUTHOR]

Studies the in vitro resistance to thrombin-induced platelet microbicidal protein in isolates of Stapylococcus aureus from endocarditis patients. Data supporting 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.