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Platelets are a critical immune defense against Staphylococcus aureus bloodstream infections. Staphylococcus aureus α-toxin is a virulence factor that decreases platelet viability and accelerates platelet clearance. It has been shown that ticagrelor blocks α-toxin–mediated platelet injury and resulting thrombocytopenia, protecting mice in a lethal S. aureus sepsis model. We now present the use of ticagrelor as adjunctive therapy in a patient with a S. aureus endovascular infection and thrombocytopenia, associated with restoration of platelet count and bacteremia clearance. Ticagrelor enhanced platelet killing of the S. aureus bloodstream isolate from the treated patient in vitro.

Background Within the last decade, methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a frequent cause of purulent skin and soft tissue infections. New therapeutic options are being investigated for these infections. Methods We report an integrated analysis of 2 randomized, controlled studies involving omadacycline, a novel aminomethylcycline, and linezolid for the treatment of acute bacterial skin and skin structure infections (ABSSSI). Omadacycline in Acute Skin and Skin Structure Infections Study 1 (OASIS-1) initiated patients on intravenous omadacycline or linezolid, with the option to transition to an oral formulation after day 3. OASIS-2 was an oral-only study of omadacycline versus linezolid. Results In total, 691 patients received omadacycline and 689 patients received linezolid. Infection types included wound infection in 46.8% of patients, cellulitis/erysipelas in 30.5%, and major abscess in 22.7%. Pathogens were identified in 73.2% of patients. S. aureus was detected in 74.7% and MRSA in 32.4% of patients in whom a pathogen was identified. Omadacycline was noninferior to linezolid using the Food and Drug Administration primary endpoint of early clinical response (86.2% vs 83.9%; difference 2.3, 95% confidence interval –1.5 to 6.2) and using the European Medicines Agency primary endpoint of investigator-assessed clinical response at the posttreatment evaluation. Clinical responses were similar across different infection types and infections caused by different pathogens. Treatment-emergent adverse events, mostly described as mild or moderate, were reported by 51.1% of patients receiving omadacycline and 41.2% of those receiving linezolid. Conclusions Omadacycline was effective and safe in ABSSSI. Clinical Trials Registration NCT02378480 and NCT02877927.

While microbiological resistance to vancomycin in Staphylococcus aureus is rare, clinical vancomycin treatment failures are common, and methicillin-resistant S. aureus (MRSA) strains isolated from patients after prolonged vancomycin treatment failure remain susceptible. Adaptive laboratory evolution was utilized to uncover mutational mechanisms associated with MRSA vancomycin resistance in a physiological medium as well as a bacteriological medium used in clinical susceptibility testing. Sequencing of resistant clones revealed shared and media-specific mutational outcomes, with an overlap in cell wall regulons (walKRyycHI, vraSRT). Evolved strains displayed similar properties to resistant clinical isolates in their genetic and phenotypic traits. Importantly, resistant phenotypes that developed in physiological media did not translate into resistance in bacteriological media. Further, a bacteriological media-specific mechanism for vancomycin resistance associated with a mutated mprF was confirmed. This study bridges the gap between the understanding of clinical and microbiological vancomycin resistance in S. aureus and expands the number of allelic variants (18 ± 4 mutations for the top 5 mutated genes) that result in vancomycin resistance phenotypes., Henrique Machado et al. describe mutational mechanisms associated with MRSA vancomycin resistance in Staphylococcus aureus using adaptive laboratory evolution experiments focused on tolerance. Their results reveal environment-dependent mutational strategies to vancomycin tolerization and the impact of mutations in regulatory genes, providing insight into the development of antibiotic resistance under multiple conditions.

Staphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20 to 30%) despite modern supportive care. In a human bacteremia cohort, we found that development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta), a commonly prescribed P2Y12 receptor inhibitor used after myocardial infarction, blocked α-toxin-mediated platelet injury and resulting thrombocytopenia, thereby providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu) provided similar therapeutic benefit. Thus, a "toxin-platelet-AMR" regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof of concept for repurposing two commonly prescribed drugs as adjunctive therapies to improve patient outcomes.

Given that it is unlikely that randomized clinical trials will yield answers for treating the most challenging bacteremic infections caused by methicillin-resistant Staphylococcus aureus , clinicians, microbiologists, and pharmacists will have to cooperate to discover novel ways to select successful individualized antimicrobial therapy for these patients. An example of such a strategy was demonstrated in the identification and utilization of imipenem/cilastatin plus fosfomycin to treat a particularly recalcitrant MRSA bacteremia and spinal abscess.

BACKGROUND: Prosthetic joint infections (PJI) are difficult to treat complications of joint arthroplasty. Debridement with implant retention is a common treatment strategy and frequently involves the use of pulsed lavage (PL). However, PL effects on biofilms and antibiotic activity have been scarcely studied in-vitro. We report the effects of PL, vancomycin or flucloxacillin used independently or in combination against Staphylococcus aureus biofilms. METHODS: Biofilms of 3 methicillin-susceptible (MSSA) and of 3 methicillin-resistant (MRSA) S. aureus were grown on Ti6Al4V coupons in TGN (TSB + 1%glucose + 2%NaCl). After 24 h, PL was applied to half of the samples (50 mL saline from 5 cm). Samples were either reincubated for 24 h in TGN or TGN + flucloxacillin or vancomycin. Analyses included CFUs counts, biomass assays or fluorescence microscopy. RESULTS: PL transiently reduced bacterial counts by 3–4 Log10 CFU/coupon, but bacterial regrowth to baseline levels was seen after 24 h. At 20 mg/L, flucloxacillin reduced both the CFU counts (3 Log10 CFU/coupon) and biomass (−70%) in one MSSA only, while vancomycin had no effects against MRSA. PL combined with a 24 h reincubation with vancomycin or flucloxacillin at 20 mg/L was synergistic (−5 to 6.5 Log10 CFU/coupon; 81–100% biomass reduction). Fluorescence microscopy confirmed that PL removed most of the biofilm and that subsequent antibiotic treatment partially killed bacteria. CONCLUSIONS: While PL only transiently reduces the bacterial load and antibiotics at clinically relevant concentrations show no or limited activity on biofilms, their combination is synergistic against MRSA and MSSA biofilms. These results highlight the need for thorough PL before antibiotic administration in PJI.

Staphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20-30%) despite modern supportive care. In a human bacteremia cohort, development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta®), a commonly prescribed P2Y12 receptor inhibitor used post-myocardial infarction, blocked α-toxin-mediated platelet injury and resulting thrombocytopenia, thus providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu®) provided similar therapeutic benefit. Thus a “toxin-platelet-AMR” regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof-of-concept for repurposing two FDA-approved drugs as adjunctive therapies to improve patient outcomes.

Staphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20–30%) despite modern supportive care. In a human bacteremia cohort, development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta), a commonly prescribed P2Y12 receptor inhibitor used post-myocardial infarction, blocked α-toxin-mediated platelet injury and resulting thrombocytopenia, thus providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu) provided similar therapeutic benefit. Thus a “toxin-platelet-AMR” regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof-of-concept for repurposing two commonly prescribed drugs as adjunctive therapies to improve patient outcomes.

While microbiological resistance to vancomycin in Staphylococcus aureus is rare, clinical vancomycin treatment failures are common, and methicillin-resistant S. aureus (MRSA) strains isolated from patients after prolonged vancomycin treatment failure remain susceptible. Adaptive laboratory evolution was utilized to uncover mutational mechanisms associated with MRSA vancomycin resistance in a bacteriological medium used in clinical susceptibility testing and a physiological medium. Sequencing of resistant clones revealed shared and media-specific mutational outcomes, with an overlap in cell wall regulons (walKRyycHI, vraSRT). Evolved strains displayed similar genetic and phenotypic traits to resistant clinical isolates. Importantly, resistant phenotypes that developed in physiological media did not translate into resistance in bacteriological media. Further, a bacteriological media-specific mechanism for vancomycin resistance enabled by a mutated mprF was confirmed. This study bridges the gap of understanding between clinical and microbiological vancomycin resistance in S. aureus and expands the number of allelic variants that result in vancomycin resistance phenotypes.

Staphylococcus aureus strain TX0117 is a methicillin-susceptible bacterium with type A beta-lactamase exhibiting a high cefazolin inoculum effect. TX0117 was cured of blaZ, yielding TX0117c with increased antimicrobial peptide resistance. The sequencing and genome assembly of TX0117 elucidate six mutations between TX0117 and TX0117c, including relA truncation and mnA_1 substitution.

Staphylococcus aureus bacteremia (SaB) causes significant disease in humans, carrying mortality rates of ~25%. The ability to rapidly predict SaB patient responses and guide personalized treatment regimens could reduce mortality. Here, we present a resource of SaB prognostic biomarkers. Integrating proteomic and metabolomic techniques enabled the identification of >10,000 features from >200 serum samples collected upon clinical presentation. We interrogated the complexity of serum using multiple computational strategies, which provided a comprehensive view of the early host response to infection. Our biomarkers exceed the predictive capabilities of those previously reported, particularly when used in combination. Lastly, we validated the biological contribution of mortality-associated pathways using a murine model of SaB. Our findings represent a starting point for the development of a prognostic test for identifying high-risk patients at a time early enough to trigger intensive monitoring and interventions.

Objective Development of antimicrobial resistance during monotherapy of complicated methicillin-resistant Staphylococcus aureus bacteremia is problematic due to cross-resistance between vancomycin (VAN) and daptomycin, the only approved agents for this condition. Our objective was to demonstrate that development of resistance under conditions of suboptimal VAN (200 mg q 12 h) exposure in S. aureus can be attenuated by addition of cefazolin (CFZ). Methods Two strains of S. aureus, 1 methicillin-susceptible Staphylococcus aureus (MSSA) (RN9120) and 1 methicillin-resistant S. aureus (MRSA) (JH1), were evaluated. The organisms were exposed to subtherapeutic VAN concentrations in a 1-compartment pharmacokinetic/pharmacodynamic model combined with recycling in the presence and absence of CFZ. Changes in MIC to glyco/lipopeptides and β-lactams along with susceptibility to human cathelicidin LL-37 killing were studied. Population analysis profiles (PAPs) were performed to detect changes in VAN heteroresistance. Results VAN MIC of both organisms increased from 1 to 4 mg/L within 144 h under subtherapeutic VAN exposure. Increase in VAN MIC was associated with increased glyco/lipopeptides MICs. Additionally, increased survival in LL-37 killing assays from 40% to >90% accompanied the increase in VAN MIC. Addition of CFZ prevented the emergence of VAN-intermediate S. aureus. PAPs demonstrated an attenuation of VAN area under the curve shift (reduced organism selection with higher MICs values) when suboptimal VAN exposure was accompanied with CFZ compared to VAN alone (MSSA 17.81 versus 36.027, MRSA −0.35 versus 17.92, respectively). Given the emerging data on the clinical benefits of β-lactam adjunctive therapy in refractory MRSA bacteremia, additional studies on a larger collection of clinical isolates are needed to establish the utility of VAN plus CFZ for treatment of MRSA bacteremia.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved numerous antimicrobial resistance mechanisms and is identified as a serious public health threat by the World Health Organization and U.S. Centers for Disease Control and Prevention. The glycopeptide vancomycin (VAN) remains a cornerstone of therapy for severe MRSA infections despite increasing reports of therapeutic failure in hospitalized patients with bacteremia or pneumonia. Recently, the role of released bacterial-derived membrane vesicles (MVs) in antibiotic resistance has garnered attention. Here we examined the effect of exogenous MRSA-derived MVs on VAN activity against MRSA in vitro, using minimum inhibitory concentration and checkerboard assays, and ex vivo, incorporating components of host innate immunity such as neutrophils and serum complement present in blood. Additionally, the proteome of MVs from VAN-exposed MRSA was characterized to determine if protein expression was altered. The presence of MVs increased the VAN MIC against MRSA to values where clinical failure is commonly observed. Furthermore, the presence of MVs increased survival of MRSA pre-treated with sub-MIC concentrations of VAN in whole blood and upon exposure to human neutrophils but not human serum. Unbiased proteomic analysis also showed an elevated expression of MV proteins associated with antibiotic resistance (e.g., marR) or proteins that are functionally linked to cell membrane/wall metabolism. Together, our findings indicate MRSA-derived MVs are capable of lowering susceptibility of the pathogen to VAN, whole-blood- and neutrophil-mediated killing, a new pharmacodynamic consideration for a drug increasingly linked to clinical treatment failures.

Cell wall peptidoglycan stimulates interleukin 10 (IL-10) production in Staphylococcus aureus bacteremia (SaB) animal models, but clinical data are not available. This study evaluates the impact of intravascular bacterial cell numbers (ie, the level of bacteremia), in patients at the time of clinical presentation on IL-10 production and its association with S. aureus bacteremia (SaB) mortality. Blood and isolates were collected in 133 consecutive SaB patients. Serum IL-10 was quantified by an electrochemoluminescence assay. Bacterial inoculum was measured in patient sera with elevated (n = 8) or low (n = 8) IL-10 using a magnetic bacterial capture assay. Staphylococcus aureus from these 2 groups were introduced into whole blood ex vivo to determine IL-10 production with variable inocula. IL-10 serum concentration was higher in SaB patient mortality (n = 27) vs survival (n = 106) (median, 36.0 pg/mL vs 10.4 pg/mL, respectively, P Elevated IL-10 serum concentrations at clinical presentation of SaB were highly associated with mortality. High intravascular peptidoglycan concentration, driven by a higher level of bacteremia, is a key mediator of IL-10 anti-inflammatory response that portends poor clinical outcome. Using IL-10 as an initial biomarker, clinicians may consider more aggressive antimicrobials for rapid bacterial load reduction in high-risk SaB patients.

Background The pore-forming alpha-toxin produced by Staphylococcus aureus (SA) decreases the viability and increases the clearance of platelets, a critical element of innate immune defense in endovascular infection. Our group recently identified that ticagrelor (TICA) blocks alpha-toxin-induced platelet clearance, protecting mice in a lethal systemic SA infection model. Here, we describe a case report in which TICA, added to antimicrobial therapy of a persistent methicillin-susceptible SA (MSSA) bacteremia associated with a septic aortic thrombus, resulted in immediate bacteremia clearance. We further explore TICA synergy with antibiotics and human platelets in vitro. Methods Antibiotic susceptibility of an MSSA strain from a patient treated with TICA for refractory bacteremia was tested by MIC and checkerboard assays in MHB or RPMI at standard (105 CFU/mL) or high (107 CFU/mL) inocula using TICA, ertapenem (ETP), cefazolin (CZ), or nafcillin (NAF) alone vs. ETP+CZ ± TICA. Killing assays with human platelets ± TICA against SA were also performed. Results SA bacteremia secondary to a septic aortic thrombus (>4 mm) with multiple secondary pyogenic foci refractory to standard CZ and subsequent salvage CZ+ETP for 5 days rapidly cleared within 24 h after the addition of TICA. Thrombocytopenia resolved concurrently. Discontinuation of TICA on day 12 led to rebound thrombocytopenia, and TICA was restarted, once again resulting in resolution of thrombocytopenia. TICA alone lacked in vitro activity against SA, nor was TICA synergistic with ETP+CZ. In contrast, addition of a physiological achievable concentration of TICA dramatically sensitized SA to human platelet killing (p< 0.001) in vitro. Figure 1 Figure 2 Figure 3 Conclusion In a complex case of aortic plaque rupture with septic thrombus, multiple septic emboli, and refractory MSSA bacteremia, addition of TICA to antimicrobial therapy yielded unanticipated immediate clinical and microbiological success. The profound therapeutic effect of TICA in vivo was corroborated by the enhanced staphylocidal activity of human platelets in vitro in the presence of physiological concentrations of the antiplatelet agent. TICA warrants further study as adjunctive treatment of refractory SA bacteremia due to a primary endovascular focus when thrombocytopenia is present. Disclosures Victor Nizet, MD, Centauri Therapeutics (Advisor or Review Panel member)Cidara Therapeutics (Advisor or Review Panel member)InhibRx (Advisor or Review Panel member)Roche Pharmaceutical (Advisor or Review Panel member)

The ability of pathogens such as Staphylococcus aureus to evolve resistance to antibiotics used in the treatment of infections has been an important concern in the last decades. Resistant acquisition usually translates into treatment failure and puts patients at risk of unfavorable outcomes. Furthermore, the laboratory testing of antibiotic resistance does not account for the different environment the bacteria experiences within the human body, leading to results that do not translate into the clinic. In this study, we forced methicillin-resistant S. aureus to develop nafcillin resistance in two different environments, a laboratory environment and a physiologically more relevant environment. This allowed us to identify genetic changes that led to nafcillin resistance under both conditions. We concluded that not only does the environment dictate the evolutionary strategy of S. aureus to nafcillin but also that the evolutionary strategy is specific to that given environment., Antimicrobial susceptibility testing standards driving clinical decision-making have centered around the use of cation-adjusted Mueller-Hinton broth (CA-MHB) as the medium with the notion of supporting bacterial growth, without consideration of recapitulating the in vivo environment. However, it is increasingly recognized that various medium conditions have tremendous influence on antimicrobial activity, which in turn may have major implications on the ability of in vitro susceptibility assays to predict antibiotic activity in vivo. To elucidate differential growth optimization and antibiotic resistance mechanisms, adaptive laboratory evolution was performed in the presence or absence of the antibiotic nafcillin with methicillin-resistant Staphylococcus aureus (MRSA) TCH1516 in either (i) CA-MHB, a traditional bacteriological nutritionally rich medium, or (ii) Roswell Park Memorial Institute (RPMI), a medium more reflective of the in vivo host environment. Medium adaptation analysis showed an increase in growth rate in RPMI, but not CA-MHB, with mutations in apt, adenine phosphoribosyltransferase, and the manganese transporter subunit, mntA, occurring reproducibly in parallel replicate evolutions. The medium-adapted strains showed no virulence attenuation. Continuous exposure of medium-adapted strains to increasing concentrations of nafcillin led to medium-specific evolutionary strategies. Key reproducibly occurring mutations were specific for nafcillin adaptation in each medium type and did not confer resistance in the other medium environment. Only the vraRST operon, a regulator of membrane- and cell wall-related genes, showed mutations in both CA-MHB- and RPMI-evolved strains. Collectively, these results demonstrate the medium-specific genetic adaptive responses of MRSA and establish adaptive laboratory evolution as a platform to study clinically relevant resistance mechanisms. IMPORTANCE The ability of pathogens such as Staphylococcus aureus to evolve resistance to antibiotics used in the treatment of infections has been an important concern in the last decades. Resistant acquisition usually translates into treatment failure and puts patients at risk of unfavorable outcomes. Furthermore, the laboratory testing of antibiotic resistance does not account for the different environment the bacteria experiences within the human body, leading to results that do not translate into the clinic. In this study, we forced methicillin-resistant S. aureus to develop nafcillin resistance in two different environments, a laboratory environment and a physiologically more relevant environment. This allowed us to identify genetic changes that led to nafcillin resistance under both conditions. We concluded that not only does the environment dictate the evolutionary strategy of S. aureus to nafcillin but also that the evolutionary strategy is specific to that given environment.

Cefazolin and ertapenem combination therapy was used successfully to salvage 11 cases (6 endocarditis) of persistent methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia, including immediate clearance (≤24 hours) in 8 cases. While in vitro synergy was modest, cefazolin plus ertapenem exhibited synergistic action in a rat model of MSSA endocarditis. The combination of cefazolin and ertapenem provides potent in vivo activity against MSSA beyond what is predicted in vitro and warrants further clinical study in the treatment of refractory MSSA bacteremia and endocarditis.

Background Patient interleukin (IL)-1β and IL-10 responses early in Staphylococcus aureus bacteremia (SaB) are associated with bacteremia duration and mortality. We hypothesized that these responses vary depending on antimicrobial therapy, with particular interest in whether the superiority of β-lactams links to key cytokine pathways. Methods Three medical centers included 59 patients with SaB (47 methicillin-resistant S. aureus [MRSA], 12 methicillin-sensitive S. aureus [MSSA]) from 2015–2017. In the first 48 hours, patients were treated with either a β-lactam (n = 24), including oxacillin, cefazolin, or ceftaroline, or a glyco-/lipopeptide (n = 35), that is, vancomycin or daptomycin. Patient sera from days 1, 3, and 7 were assayed for IL-1β and IL-10 by enzyme-linked immunosorbent assay and compared using the Mann-Whitney U test. Results On presentation, IL-10 was elevated in mortality (P = .008) and persistent bacteremia (P = .034), while no difference occurred in IL-1β. Regarding treatment groups, IL-1β and IL-10 were similar prior to receiving antibiotic. Patients treated with β-lactam had higher IL-1β on days 3 (median +5.6 pg/mL; P = .007) and 7 (+10.9 pg/mL; P = .016). Ex vivo, addition of the IL-1 receptor antagonist anakinra to whole blood reduced staphylococcal killing, supporting an IL-1β functional significance in SaB clearance. β-lactam–treated patients had sharper declines in IL-10 than vancomycin or daptomycin –treated patients over 7 days. Conclusions These data underscore the importance of β-lactams for SaB, including consideration that the adjunctive role of β-lactams for MRSA in select patients helps elicit favorable host cytokine responses.

Recent studies have demonstrated that anti-staphylococcal beta-lactam antibiotics, like nafcillin, render methicillin-resistant Staphylococcus aureus (MRSA) more susceptible to killing by innate host defense peptides (HDPs), such as cathelicidin LL-37. We compared the effects of growth in 1/4 minimum inhibitory concentration (MIC) of nafcillin or vancomycin on LL-37 killing of 92 methicillin-susceptible S. aureus (MSSA) isolates. For three randomly selected strains among these, we examined the effects of nafcillin, vancomycin, daptomycin, or linezolid on LL-37 killing and autolysis. Growth in the presence of sub-inhibitory nafcillin significantly enhanced LL-37 killing of MSSA compared to vancomycin and antibiotic-free controls. Nafcillin also reduced MSSA production of the golden staphylococcal pigment staphyloxanthin in 39% of pigmented strains vs. 14% for vancomycin. Among antibiotics tested, only nafcillin resulted in significantly increased MSSA autolysis. These studies point to additional mechanisms of anti-staphylococcal activity of nafcillin beyond direct bactericidal activity, properties that vancomycin and other antibiotic classes do not exhibit. The ability of nafcillin to enhance sensitivity to innate host defense peptides may contribute to its superior effectiveness against MSSA as suggested by studies comparing clinical outcomes to vancomycin treatment.

Successful treatment of bacterial infections requires the timely administration of appropriate antimicrobial therapy. The failure to initiate the correct therapy in a timely fashion results in poor clinical outcomes, longer hospital stays, and higher medical costs. Current approaches to antibiotic susceptibility testing of cultured pathogens have key limitations ranging from long run times to dependence on prior knowledge of genetic mechanisms of resistance. We have developed a rapid antimicrobial susceptibility assay for Staphylococcus aureus based on bacterial cytological profiling (BCP), which uses quantitative fluorescence microscopy to measure antibiotic induced changes in cellular architecture. BCP discriminated between methicillin-susceptible (MSSA) and -resistant (MRSA) clinical isolates of S. aureus (n = 71) within 1–2 h with 100% accuracy. Similarly, BCP correctly distinguished daptomycin susceptible (DS) from daptomycin non-susceptible (DNS) S. aureus strains (n = 20) within 30 min. Among MRSA isolates, BCP further identified two classes of strains that differ in their susceptibility to specific combinations of beta-lactam antibiotics. BCP provides a rapid and flexible alternative to gene-based susceptibility testing methods for S. aureus, and should be readily adaptable to different antibiotics and bacterial species as new mechanisms of resistance or multidrug-resistant pathogens evolve and appear in mainstream clinical practice., Highlights • Bacterial cytological profiling identifies antibiotic resistant S. aureus. • BCP predicts best treatment options for multidrug resistant MRSA. • Resistant strains are correctly identified within 1 h. • BCP does not require prior knowledge of resistance mechanism. There is a great need for rapid antimicrobial susceptibility testing (AST) as it can dramatically improve clinical outcome for bacterial infections. Most currently proposed ASTs are dependent on knowledge of known resistance genes or based solely on growth/lysis. We have developed a new diagnostic method for rapidly determining antibiotic susceptibility of Staphylococcus aureus using quantitative fluorescence microscopy to measure antibiotic induced changes in cellular architecture. Our test has the potential to change the way antibiotic susceptibility testing is done in the future and is readily adaptable to different antibiotics and bacterial species regardless of the mechanisms of resistance.

Background Low serum concentrations of tetracycline antibiotics may raise concerns on their efficacy in patients with secondary bacteremia, especially in comorbid patients and in those with higher acuity of illness. Omadacycline (OMC), an aminomethylcycline antibiotic, showed noninferiority to linezolid (LZD) in two acute bacterial skin and skin structure infections (ABSSSI) studies (Omadacycline in Acute Skin and skin structure Infections Study [OASIS]-1 and -2), or to moxifloxacin (MOX) in a community-acquired bacterial pneumonia (CABP) study (Omadacycline for Pneumonia Treatment In the Community study [OPTIC]). This analysis considers clinical outcomes in patients with secondary bacteremia identified in these studies. Methods Baseline blood cultures were taken from adult patients enrolled in Phase 3 randomized, double-blind clinical studies of OMC. Patients received ≥1 treatment dose: N = 1,347 ABSSSI (OMC n = 676; LZD n = 671); N = 774 CABP (OMC n = 386; MOX n = 388). Efficacy was evaluated by investigator assessment of clinical response at a post-treatment evaluation (PTE) 7–14 days (ABSSSI) or 5–10 days (CABP) after the last dose. Results Bacteremia was confirmed in 63 patients (3%) across the three studies. Staphylococcus aureus was the most common pathogen in patients with ABSSSI (N = 30: OMC n/N = 7/13; LZD n/N = 9/17), with a median treatment duration of median 9 days (OMC) and median 10 days (LZD). Streptococcus pneumoniae was the most common pathogen in patients with CABP (N = 33; OMC n/N = 11/15; MOX n/N = 11/18), with a median treatment duration of 11 days (OMC) and 14 days (MOX). Clinical success was numerically comparable between treatment groups within each study (figure) after completion of therapy. Conclusion The clinical successes of patients receiving OMC for ABSSSI or CABP with secondary bacteremia were comparable to results for LZD and MOX, respectively. Although limited by the small sample size, data from ABSSSI and CABP study patients with secondary bacteremia provide reassurance for the use of OMC. Real-world OMC data in patients with ABSSSI and CABP will be needed to assess outcomes in patients with additional comorbidities and/or higher acuity of illness. Disclosures All authors: No reported disclosures.

Background Imbalance among innate mediators such as IL-1β, IL-10, and TNF-α portends poor outcomes of persistence and death in patients with SAB. Previous studies did not consider the role of antibiotic treatment in this important host–pathogen relationship. In this study of SAB, we determined cytokine signatures that correlate with the composite endpoint of clinical failure (bacteremia duration >4 days or 30-day mortality) in Australian patients treated with FLU or VAN. Methods Sera from 86 patients with SAB (24.4% MRSA) were obtained from a clinical study of patients treated with FLU or VAN. All of the patient samples were collected at clinical presentation (day 0 or day 1 of infection) and were treated with FLU or VAN throughout. Patients were classified into either clinical success (CS = 68) or clinical failure (CF = 18), defined as death or prolonged bacteremia >4 days. Patient demographic and infection characteristics were collected. A 10-multiplex TH1/TH2 cytokine analysis was performed using electrochemoluminescence with the Meso-Scale Discovery platform analyzed by Mann–Whitney U. Results Patients’ median values were significantly elevated and above the normal range in CF for IL-1β (P = 0.029), IL-10 (P = 0.018), TNF-α (P = 0.042), and IL-6 (P = 0.006) (figure). Epidural abscess source was associated with CF, but no other host or pathogen characteristics correlated to outcome. Patients infected with isolates with VAN MIC = 2 mg/L (by Etest and broth dilution) had lower concentrations of IL-1β and IL-10 (P = 1.5 mg/L. In ROC analysis, IL-1β, IL-10, TNF, and IL-6 were higher sensitivity and specificity predictors of CF (AUC 0.65–0.71; P = 0.05). Conclusion A suboptimal host immune response to SAB at presentation predicts adverse clinical outcomes. IL-10, TNF-α, and IL-6 serum concentrations appear to reflect immunopathology in patients with SAB. These predictive markers may be considered in therapeutic clinical decision-making, such as escalation of alternative therapies in high-risk patients and/or de-escalation treatment in low-risk patients. These data offer steps toward further refining therapeutic precision for patients with SAB beyond the standard clinical or microbiological metrics that are employed in current practice. Disclosures All authors: No reported disclosures.

Daptomycin is increasingly used in combination with other antibiotics to enhance antimicrobial efficacy and/or to mitigate the emergence of daptomycin nonsusceptibility (DNS). This study used a clinical methicillin-resistant Staphylococcus aureus (MRSA) strain in which DNS emerged upon therapy to examine the influence of antibiotic combinations on the development of mutations in specific genes ( mprF , rpoBC , dltA , cls2 , and yycFG ) previously associated with DNS. Whole genomes of bacteria obtained following 28 days of in vitro exposure to daptomycin with or without adjunctive clarithromycin, linezolid, oxacillin, or trimethoprim-sulfamethoxazole were sequenced, and the sequences were compared to that of the progenitor isolate. The addition of oxacillin to medium containing daptomycin prevented the emergence of mprF mutation but did not prevent rpoBC mutation ( P < 0.01). These isolates maintained susceptibility to daptomycin during the combined exposure (median MIC, 1 mg/liter). Daptomycin plus clarithromycin or linezolid resulted in low-level (1.5 to 8 mg/liter) and high-level (12 to 96 mg/liter) DNS, respectively, and did not prevent mprF mutation. However, these same combinations prevented rpoBC mutation. Daptomycin alone or combined with linezolid or trimethoprim-sulfamethoxazole resulted in high-level DNS and mutations in mprF plus rpoBC , cls2 , and yycFG . Combining daptomycin with different antimicrobials alters the mutational space available for DNS development, thereby favoring the development of predictable collateral susceptibilities.

The extensive use of daptomycin (DAP) for treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections in the last decade has led to the emergence of DAP non-susceptible (DNS) Staphylococcus aureus strains. A better understanding of the molecular changes underlying DAP-non-susceptibility is required for early diagnosis and intervention with alternate combination therapies. The phenotypic changes associated with DNS strains have been well established. However, the genotypic changes-especially the kinetics of expression of the genes responsible for DAP-non-susceptibility are not well understood. In this study, we used three clinically derived isogenic pairs of DAP-susceptible (DAP-S) and DNS S. aureus strains to study gene expression profiles with the objective of identifying the potential genotypic changes associated with DAP-nonsusceptibility. We determined the expression profiles of genes involved in cell membrane (CM) charge, autolysis, cell wall (CW) synthesis, and penicillin binding proteins in DAP-S and DNS isogenic pairs. Our results demonstrate characteristic expression profiles for mprF, dltABCD, vraS, femB, and pbp2a genes, which are common to all the DNS S. aureus strains tested. Whole genome sequencing of DAP-S and DNS clinical isolates of S. aureus showed non-synonymous mutations in all DNS strains in genes involved in CM charge, CM composition, CW thickness and CW composition. To conclude, this study unravels some of the complex molecular changes involved in the development of DAP-nonsusceptibility by demonstrating distinct differences in gene expression profiles and mutations in the DNS S. aureus strains. This knowledge will aid in rapid identification of DNS S. aureus in clinical settings.

We asked whether beta-lactamase inhibitors (BLIs) increased the activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA), the peptide antibiotic colistin (COL) against the emerging Gram-negative nosocomial pathogen Acinetobacter baumannii , and the human host defense peptide cathelicidin LL37 against either pathogen. DAP and LL37 kill curves were performed with or without BLIs against MRSA, vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA). COL and LL37 kill curves were performed against A. baumannii . Boron-dipyrromethene (BODIPY)-labeled DAP binding to MRSA grown with the BLI tazobactam (TAZ) was assessed microscopically. The combination of COL plus TAZ was studied in a murine model of A. baumannii pneumonia. TAZ alone lacked in vitro activity against MRSA or A. baumannii . The addition of TAZ to DAP resulted in a 2- to 5-log 10 reduction in recoverable MRSA CFU at 24 h compared to the recoverable CFU with DAP alone. TAZ plus COL showed synergy by kill curves for 4 of 5 strains of A. baumannii tested. Growth with 20 mg/liter TAZ resulted in 2- to 2.5-fold increases in the intensity of BODIPY-DAP binding to MRSA and hVISA strains. TAZ significantly increased the killing of MRSA and A. baumannii by LL37 in vitro . TAZ increased the activity of COL in a murine model of A. baumannii pneumonia. Classical BLIs demonstrate synergy with peptide antibiotics. Since BLIs have scant antimicrobial activity on their own and are thus not expected to increase selective pressure toward antibiotic resistance, their use in combination with peptide antibiotics warrants further study.

Over the past several years, single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been proposed to be associated with a gain-of-function phenotype in terms of daptomycin (DAP) nonsusceptibility (referred to as daptomycin resistance [DAP-R] herein for ease of presentation) in Staphylococcus aureus . We investigated the frequencies of SNPs within the mprF ORF and the relationships of such SNPs to cross-resistance between DAP and cationic host defense peptides (HDPs). Thirty-five well-characterized, unique DAP-susceptible (DAP-S) and DAP-R methicillin-resistant S. aureus (MRSA) isolates of the clonal complex 5 genotype were used. In addition to mprF SNPs and DAP-HDP cross-resistance, several other key genotypic and phenotypic metrics often associated with DAP-R were delineated, as follows: (i) mprF expression, (ii) membrane phospholipid content, (iii) positive surface charge, (iv) DAP binding, and (v) cell wall thickness profiles. A number of DAP-S strains (MICs of ≤1 μg/ml) exhibited mprF SNPs, occasionally with high-level mprF sequence variation from the genotype reference strain. However, none of these SNPs were localized to well-chronicled mprF hot spot locations associated with DAP-R in S. aureus . In contrast, all 8 DAP-R isolates demonstrated SNPs within such known mprF hot spots. Moreover, only the DAP-R strains showed MprF gain-of-function phenotypes, enhanced mprF expression, higher survival against two prototypical HDPs, and reduced DAP binding. Although a heterogenous array of mprF SNPs were often found in DAP-S strains, only selected hot spot SNPs, combined with concurrent mprF dysregulation, were associated with the DAP-R phenotype.

Purpose Guidelines recommend daptomycin combination therapy as an option for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia after vancomycin failure. Recent data suggest that combining daptomycin with a β-lactam may have unique benefits; however, there are very limited clinical data regarding the use of ceftaroline with daptomycin. Methods All 26 cases from the 10 medical centers in which ceftaroline plus daptomycin was used for treatment of documented refractory staphylococcal bacteremia from March 2011 to November 2012 were included. In vitro (synergy studies, binding assays, cathelicidin LL-37 killing assays), and in vivo (virulence assays using a murine subcutaneous infection model) studies examining the effects of ceftaroline with daptomycin were also performed. Findings Daptomycin plus ceftaroline was used in 26 cases of staphylococcal bacteremia (20 MRSA, 2 vancomycin-intermediate S aureus , 2 methicillin-susceptible S aureus [MSSA], 2 methicillin-resistant S epidermidis ). Bacteremia persisted for a median of 10 days (range, 3–23 days) on previous antimicrobial therapy. After daptomycin plus ceftaroline was started, the median time to bacteremia clearance was 2 days (range, 1–6 days). In vitro studies showed ceftaroline synergy against MRSA and enhanced MRSA killing by cathelicidin LL-37 and neutrophils. Ceftaroline also induced daptomycin binding in MSSA and MRSA to a comparable degree as nafcillin. MRSA grown in subinhibitory concentrations of ceftaroline showed attenuated virulence in a murine subcutaneous infection model. Implications Ceftaroline plus daptomycin may be an option to hasten clearance of refractory staphylococcal bacteremia. Ceftaroline offers dual benefit via synergy with both daptomycin and sensitization to innate host defense peptide cathelicidin LL37, which could attenuate virulence of the pathogen.

Purpose Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important nosocomial pathogens. Resistance to antibiotic therapy has been known to emerge especially in clinically complex scenarios, resulting in challenges in determining optimal treatment of serious MRSA. Daptomycin, in combination with other antibiotics, has been successfully used in the treatment of these infections, with the aims of resulting in reducing the prevention of antimicrobial resistance and increased killing compared with daptomycin monotherapy. Methods This article reviews all the published studies that used daptomycin combination therapy for the treatment of bacteremia and associated complicated infections caused by gram-positive organisms, including MRSA. We discuss the rationale of combination antibiotics and the mechanisms that enhance the activity of daptomycin, with special focus on the role of β-lactam antibiotics. Findings There are limited clinical data on the use of daptomycin in combination with other antibiotics. Most of this use was as successful salvage therapy in the setting of failing primary, secondary, or tertiary therapy and/or relapsing infection. Synergy between β-lactams and daptomycin is associated with several characteristics, including increased daptomycin binding and β-lactam–mediated potentiation of innate immunity, but the precise molecular mechanism is unknown. Implications Use of daptomycin in combination with other antibiotics, especially β-lactams, offers a promising treatment option for complicated MRSA bacteremia in which emergence of resistance during treatment may be anticipated. Because it is currently not possible to differentiate complicated from uncomplicated bacteremia at the time of presentation, combination therapy may be considered as first-line therapy, with de-escalation to monotherapy in uncomplicated cases and cases with stable pharmacologic and surgical source control.

Based on in vitro synergy studies, the addition of nafcillin to daptomycin was used to treat refractory methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Daptomycin is a de facto cationic antimicrobial peptide in vivo, with antistaphylococcal mechanisms reminiscent of innate host defense peptides (HDPs). In this study, the effects of nafcillin on HDP activity against MRSA were examined in vitro and in vivo. Exposures to β-lactam antimicrobials in general, and nafcillin in particular, significantly increased killing of S. aureus by selected HDPs from keratinocytes, neutrophils, and platelets. This finding correlated with enhanced killing of MRSA by whole blood, neutrophils, and keratinocytes after growth in nafcillin. Finally, nafcillin pretreatment ex vivo reduced MRSA virulence in a murine subcutaneous infection model. Despite the lack of direct activity against MRSA, these studies show potent, consistent, and generalized nafcillin-mediated "sensitization" to increased killing of MRSA by various components of the innate host response. The use of nafcillin as adjunctive therapy in MRSA bacteremia merits further study and should be considered in cases refractory to standard therapy.Nafcillin has been used as adjunctive therapy to clear persistent MRSA bacteremia. Nafcillin enhances killing of MRSA by a cadre of innate host defense peptides. Nafcillin increases binding of human cathelicidin LL-37 to the MRSA membrane. Nafcillin enhances killing of MRSA by neutrophils. Nafcillin reduces virulence of MRSA in a murine subcutaneous infection model.

SUMMARY Daptomycin is a lipopeptide antimicrobial with in vitro bactericidal activity against Gram-positive bacteria that was first approved for clinical use in 2004 in the United States. Since this time, significant data have emerged regarding the use of daptomycin for the treatment of serious infections, such as bacteremia and endocarditis, caused by Gram-positive pathogens. However, there are also increasing reports of daptomycin nonsusceptibility, in Staphylococcus aureus and, in particular, Enterococcus faecium and Enterococcus faecalis . Such nonsusceptibility is largely in the context of prolonged treatment courses and infections with high bacterial burdens, but it may occur in the absence of prior daptomycin exposure. Nonsusceptibility in both S. aureus and Enterococcus is mediated by adaptations to cell wall homeostasis and membrane phospholipid metabolism. This review summarizes the data on daptomycin, including daptomycin's unique mode of action and spectrum of activity and mechanisms for nonsusceptibility in key pathogens, including S. aureus , E. faecium , and E. faecalis . The challenges faced by the clinical laboratory in obtaining accurate susceptibility results and reporting daptomycin MICs are also discussed.

We demonstrated a significant inverse correlation between vancomycin and beta-lactam susceptibilities in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates. Using time-kill assays, vancomycin plus oxacillin or ceftaroline was synergistic against 3 of 5 VISA and 1 of 5 hVISA isolates or 5 of 5 VISA and 4 of 5 hVISA isolates, respectively. Beta-lactam exposure reduced overall vancomycin-Bodipy ( dipy rromethene bo ron difluoride [4,4-difluoro-4-bora-3a,4a-diaza- s -indacene] fluorescent dye) binding but may have improved vancomycin-cell wall interactions to improve vancomycin activity. Further research is warranted to elucidate the mechanism behind vancomycin and beta-lactam synergy.

Patients with underlying renal disease may be vulnerable to vancomycin-mediated nephrotoxicity and Staphylococcus aureus bacteremia treatment failure. In light of recent data demonstrating the successful use of β-lactam plus daptomycin in very difficult cases of S. aureus bacteremia, we examined safety and clinical outcomes for patients who received daptomycin with or without concomitant β-lactams. We identified 106 patients who received daptomycin for S. aureus bacteremia, had mild or moderate renal insufficiency according to FDA criteria, and enrolled in the Cubicin Outcomes Registry and Experience (CORE), a multicenter registry, from 2005 to 2009. Daptomycin treatment success was 81%. Overall treatment efficacy was slightly enhanced with the addition of a β-lactam (87% versus 78%; P = 0.336), but this trend was most pronounced for bacteremia associated with endocarditis or bone/joint infection or bacteremia from an unknown source (90% versus 57%; P = 0.061). Factors associated with reduced daptomycin efficacy (by logistic regression) were an unknown source of bacteremia (odds ratio [OR] = 7.59; 95% confidence interval [CI] = 1.55 to 37.2), moderate renal impairment (OR = 9.11; 95% CI = 1.46 to 56.8), and prior vancomycin failure (OR = 11.2; 95% CI = 1.95 to 64.5). Two patients experienced an increase in creatine phosphokinase (CPK) that resolved after stopping daptomycin. No patients developed worsening renal insufficiency related to daptomycin. In conclusion, daptomycin appeared to be effective and well tolerated in patients with S. aureus bacteremia and mild to moderate renal insufficiency. Daptomycin treatment efficacy might be enhanced with β-lactam combination therapy in primary endovascular and bone/joint infections. Additional studies will be necessary to confirm these findings.

The evidence for using combination therapy for the treatment of serious methicillin-resistant Staphylococcus aureus (MRSA) infections is growing. In this study, we investigated the synergistic effect of daptomycin (DAP) combined with piperacillin-tazobactam and ampicillin-sulbactam against MRSA in time-kill experiments. Six of eight strains demonstrated synergy between DAP and the β-lactam–β-lactamase inhibitor (BLI) combination. In 5/8 strains, the synergy occurred only in the presence of the BLI, highlighting a role for BLIs in peptide–β-lactam synergy.

New antimicrobial agents and novel combination therapies are needed to treat serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to daptomycin and vancomycin. The purpose of this study was to evaluate the combination of ceftaroline plus daptomycin or vancomycin in an in vitro pharmacokinetic/pharmacodynamic model. Simulations of ceftaroline-fosamil at 600 mg per kg of body weight every 8 h (q8h) (maximum free-drug concentration in serum [ fC max ], 15.2 mg/liter; half-life [ t 1/2 ], 2.3 h), daptomycin at 10 mg/kg/day ( f C max , 11.3 mg/liter; t 1/2 , 8 h), vancomycin at 2 g q12h ( fC max , 30 mg/liter; t 1/2 , 6 h), ceftaroline plus daptomycin, and ceftaroline plus vancomycin were evaluated against a clinical, isogenic MRSA strain pair: D592 (daptomycin susceptible and heterogeneous vancomycin intermediate) and D712 (daptomycin nonsusceptible and vancomycin intermediate) in a one-compartment in vitro pharmacokinetic/pharmacodynamic model over 96 h. Therapeutic enhancement of combinations was defined as ≥2 log 10 CFU/ml reduction over the most active single agent. The effect of ceftaroline on the membrane charge, cell wall thickness, susceptibility to killing by the human cathelicidin LL37, and daptomycin binding were evaluated. Therapeutic enhancement was observed with daptomycin plus ceftaroline in both strains and vancomycin plus ceftaroline against D592. Ceftaroline exposure enhanced daptomycin-induced depolarization (81.7% versus 72.3%; P = 0.03) and killing by cathelicidin LL37 ( P < 0.01) and reduced cell wall thickness ( P < 0.001). Fluorescence-labeled daptomycin was bound over 7-fold more in ceftaroline-exposed cells. Whole-genome sequencing and mutation analysis of these strains indicated that change in daptomycin susceptibility is related to an fmtC ( mprF ) mutation. The combination of daptomycin plus ceftaroline appears to be potent, with rapid and sustained bactericidal activity against both daptomycin-susceptible and -nonsusceptible strains of MRSA.

Ertapenem and cefazolin were used in combination to successfully clear refractory methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. In addition, recent work has demonstrated activity of combination therapy with beta-lactams from different classes against methicillin-resistant S. aureus (MRSA). The ertapenem-plus-cefazolin combination was evaluated for synergy in vitro and in vivo in a murine skin infection model using an index MSSA bloodstream isolate from a patient in whom persistent bacteremia was cleared with this combination and against a cadre of well-described research strains and clinical strains of MSSA and MRSA. Against the index MSSA bloodstream isolate, ertapenem and cefazolin showed synergy using both checkerboard (fractional inhibitory concentration [FIC] index = 0.375) and time-kill assays. Using a disk diffusion ertapenem potentiation assay, the MSSA isolate showed a cefazolin disk zone increased from 34 to 40 mm. In vitro pharmacokinetic/pharmacodynamic modeling at clinically relevant drug concentrations demonstrated bactericidal activity (>3 log 10 -CFU/ml reduction) of the combination but bacteriostatic activity of ether drug alone at 48 h. A disk diffusion potentiation assay showed that ertapenem increased the cefazolin zone of inhibition by >3 mm for 34/35 (97%) MSSA and 10/15 (67%) MRSA strains. A murine skin infection model of MSSA showed enhanced activity of cefazolin plus ertapenem compared to monotherapy with these agents. After successful use in clearance of MSSA bacteremia, the combination of ertapenem and cefazolin showed synergy against MSSA in vitro and in vivo . This combination may warrant consideration for future clinical study in MSSA bacteremia.

The rapid rise in antimicrobial resistance in bacteria has generated an increased demand for the development of novel therapies to treat contemporary infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). However, antimicrobial development has been largely abandoned by the pharmaceutical industry. We recently isolated the previously described thiopeptide antibiotic nosiheptide from a marine actinomycete strain and evaluated its activity against contemporary clinically relevant bacterial pathogens. Nosiheptide exhibited extremely potent activity against all contemporary MRSA strains tested including multiple drug-resistant clinical isolates, with MIC values 0.25 mg l(-1). Nosiheptide was also highly active against Enterococcus spp. and the contemporary hypervirulent BI/NAP1/027 strain of Clostridium difficile but was inactive against most Gram-negative strains tested. Time-kill analysis revealed nosiheptide to be rapidly bactericidal against MRSA in a concentration- and time-dependent manner, with a nearly 2-log kill noted at 6 h at 10 × MIC. Furthermore, nosiheptide was found to be non-cytotoxic against mammalian cells at100 × MIC, and its anti-MRSA activity was not inhibited by 20% human serum. Notably, nosiheptide exhibited a significantly prolonged post-antibiotic effect against both healthcare- and community-associated MRSA compared with vancomycin. Nosiheptide also demonstrated in vivo activity in a murine model of MRSA infection, and therefore represents a promising antibiotic for the treatment of serious infections caused by contemporary strains of MRSA.

Background. We hypothesized that, for methicillin-resistant Staphylococcus aureus (MRSA), in vitro daptomycin susceptibility could be influenced by exposures to endogenous host defense peptides (HDPs) prior to clinical exposure to daptomycin. Methods. Two endovascular HDPs were used: thrombin-induced platelet microbicidal protein (tPMP) and human neutrophil defensin-1 (hNP-1) from neutrophils. Forty-seven unique MRSA isolates obtained from bacteremic patients in multicenter prospective clinical trials were studied. Clinical characteristics, microbiologic parameters, prior vancomycin therapy, and susceptibilities to tPMP, hNP-1, and daptomycin were compared using univariate and multivariate analyses. Results. All strains were daptomycin susceptible. Daptomycin minimum inhibitory concentrations (MICs) were inversely related to in vitro tPMP (but not hNP-1) killing. Strains with a daptomycin MIC of 1 mg/L exhibited significantly less killing by tPMP, compared with strains with an MIC of ≤ 0.5 mg/L. Prior vancomycin therapy did not influence this relationship. Regression tree modeling confirmed that reduced tPMP-induced killing in vitro was the strongest predictor of higher daptomycin MICs within the daptomycin-susceptible range. Conclusions. Among daptomycin-susceptible MRSA isolates from patients who had never received daptomycin, higher daptomycin MICs tracked with increased resistance to killing by platelet-derived but not neutrophilderived HDPs. These findings support the notion that endogenous exposure of MRSA to specific HDPs may play a role in selecting strains with an intrinsically higher daptomycin MIC phenotype.

The ClpXP protease is a critical bacterial intracellular protease that regulates protein turnover in many bacterial species. Here we identified a pharmacological inhibitor of the ClpXP protease, F2, and evaluated its action in Bacillus anthracis and Staphylococcus aureus . We found that F2 exhibited synergistic antimicrobial activity with cathelicidin antimicrobial peptides and antibiotics that target the cell well and/or cell membrane, such as penicillin and daptomycin, in B. anthracis and drug-resistant strains of S. aureus . ClpXP inhibition represents a novel therapeutic strategy to simultaneously sensitize pathogenic bacteria to host defenses and pharmaceutical antibiotics.

Background Toxins contribute to the pathogenicity of Staphylococcus aureus infections by inducing a dysregulated inflammatory response. This study evaluated the impact of anti-staphylococcal antibiotic exposures over an increasing concentration range on cytokine production from peripheral blood mononuclear cells (PBMCs) after S. aureus toxin exposures. Methods Human PBMCs were suspended in complete Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum at 10(6) cells/mL with 100 ng/mL S. aureus toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin A (SEA), α-toxin or Panton-Valentine leucocidin (PVL). Vancomycin, trimethoprim/sulfamethoxazole, tigecycline, daptomycin, linezolid, clindamycin and azithromycin were added at a concentration range of 0.5-100 mg/L. Cytokine [interleukin-1β (IL-1β), IL-6, IL-8, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α)] concentrations were measured in duplicate by ELISA following exposure and were compared with response with toxin alone. Results At concentrations approximating serum C(max), tigecycline decreased IL-6 by 52%-57% and IFN-γ production by 43%-53% compared with toxin alone (P ≤ 0.05) and linezolid inhibited TNF-α by 12%-35% and IL-8 by 25%-42% (P ≤ 0.02). However, trimethoprim/sulfamethoxazole increased TNF-α and IL-8 production (P = 0.002). Clindamycin, daptomycin, vancomycin and azithromycin had no consistent significant effect at approximate serum C(max) concentrations. All antibiotics had a concentration-dependent effect on cytokine production, with tigecycline, clindamycin and trimethoprim/sulfamethoxazole being the most potent inhibitors of cytokine production at concentrations exceeding 25 mg/L. Conclusions S. aureus toxins stimulate production of inflammatory cytokines in PBMCs. Antimicrobials with high tissue penetration, including tigecycline, clindamycin, trimethoprim/sulfamethoxazole and linezolid, reduced cytokine production, which, along with their antimicrobial effects, may have importance in the therapeutic outcome of severe infections.

Background: Therapeutic use of vancomycin is characterized by decreased susceptibilities and increasing reports of clinical failures. Few studies have examined the clinical outcomes of patients with methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia treated with vancomycin. The primary objective was to compare clinical outcomes of patients with MRSA bacteraemia treated according to standard of care practices. Methods: Patients were included if: (i) admitted to University of New Mexico Hospital between 2002 and 2009; (ii) ≥18 years of age; (iii) had one blood culture positive for MRSA; and (iv) received vancomycin. Clinical outcomes were defined as cure, failure (relapse of infection 30 days after completion of therapy, death or change in therapy) or unevaluable. Patient demographics, source of bacteraemia, treatment regimen, and microbiological characteristics were determined. Results: Two hundred patients with MRSA bacteraemia were included. Sixty-one patients were unevaluable, leaving 139 patients for the final analysis. Seventy-two (51.8%) patients were cured and 67 (48.2%) experienced vancomycin failure. Vancomycin MIC90 was 2 mg/L for both groups by Etest. Patients with endocarditis (P ¼0.02) or pneumonia (P ¼0.02) were more likely to fail therapy. Panton‐Valentine leucocidin, loss of agr functionality and strain type were not predictors of outcomes in this study. Conclusions: High failure rates were observed in patients with MRSA bacteraemia treated with vancomycin, despite high vancomycin troughs and low rates of nephrotoxicity. Predictors of vancomycin failure included endocarditis and pneumonia. In these situations, vancomycin provides suboptimal therapy.

Accessory gene regulator ( agr ) dysfunction in Staphylococcus aureus has been associated with a longer duration of bacteremia. We aimed to assess the independent association between agr dysfunction in S. aureus bacteremia and 30-day in-hospital mortality. This retrospective cohort study included all adult inpatients with S. aureus bacteremia admitted between 1 January 2003 and 30 June 2007. Severity of illness prior to culture collection was measured using the modified acute physiology score (APS). agr dysfunction in S. aureus was identified semiquantitatively by using a δ-hemolysin production assay. Cox proportional hazard models were used to measure the association between agr dysfunction and 30-day in-hospital mortality, statistically adjusting for patient and pathogen characteristics. Among 814 patient admissions complicated by S. aureus bacteremia, 181 (22%) patients were infected with S. aureus isolates with agr dysfunction. Overall, 18% of patients with agr dysfunction in S. aureus died, compared to 12% of those with functional agr in S. aureus ( P = 0.03). There was a trend toward higher mortality among patients with S. aureus with agr dysfunction (adjusted hazard ratio [HR], 1.34; 95% confidence interval [CI], 0.87 to 2.06). Among patients with the highest APS (scores of >28), agr dysfunction in S. aureus was significantly associated with mortality (adjusted HR, 1.82; 95% CI, 1.03 to 3.21). This is the first study to demonstrate an independent association between agr dysfunction and mortality among severely ill patients. The δ-hemolysin assay examining agr function may be a simple and inexpensive approach to predicting patient outcomes and potentially optimizing antibiotic therapy.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (PB) (positive blood cultures after ≥7 days of therapy) represents a clinically challenging subset of invasive MRSA infections. In this investigation, we examined the potential correlation of specific virulence signatures with PB versus resolving MRSA bacteremia (RB) (negative blood cultures within 2 to 4 days of therapy) strains. Thirty-six MRSA isolates from patients enrolled in a recent multinational clinical trial were studied for (i) susceptibility to host defense cationic peptides (HDPs) (i.e., thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide 1 [hNP-1]); (ii) adherence to host endovascular ligands (fibronectin) and cells (endothelial cells); and (iii) biofilm formation. We found that PB isolates exhibited significantly reduced susceptibilities to tPMPs and hNP-1 ( P < 0.001 and P = 0.023, respectively). There was no significant association between the PB outcome and fibronectin binding, endothelial cell binding, or biofilm formation ( P = 0.25, 0.97, and 0.064 versus RB strains, respectively). However, multiple logistic regression analysis revealed that the PB outcome was significantly associated with the combination of reduced susceptibilities to HDPs and extent of biofilm formation ( P < 0.0001). Similar results were obtained in a second analysis using days of bacteremia as a continuous outcome, showing that reduced HDP susceptibilities and increased biofilm formation cocontributed to predict the duration of bacteremia. Our data indicate that PB isolates have specific pathogenic signatures independent of conventional antimicrobial susceptibility. These combinatorial mosaics can be defined and used to prospectively distinguish PB from RB strains in advance and potentially to predict ultimate clinical outcomes.

Background: Proton-pump inhibitors (PPIs) are commonly used in clinical practice for gastric acid suppression. However, these agents have also been associated with certain negative clinical outcomes. We evaluated the real-world effects of incident PPI use on clinical outcomes in patients with Staphylococcus aureus bacteremia. Methods: This retrospective cohort study included patients admitted to Veterans Affairs hospitals with positive S. aureus blood cultures collected between 2002 and 2013 that received appropriate antibiotics within 48 hours of culture collection. Clinical outcomes among three PPI exposure groups, each compared to nonusers, were assessed with propensity-score-matched Cox proportional-hazard regression models: pretreated PPI users initiating therapy in the 30 days prior to culture and either (a) continuing PPI therapy after culture, or (b) not continuing after culture, and (c) de novo users initiating at culture. Results: Clinical outcomes, including inpatient mortality, intensive care discharge, 30-day mortality, 30-day readmission, and 30-day Clostridium difficile infection (CDI) were similar among PPI users and nonusers. Though length of stay was longer in pretreated, continuing PPI users [time-to-discharge hazard ratio (HR) 0.78, 95% confidence interval (CI) 0.65–0.93], 14-day mortality was significantly lower than in nonusers (HR 0.66, 95% CI 0.50–0.87). Conclusions: In our large national cohort study, PPIs were not associated with an increased risk of negative clinical outcomes, including mortality and CDI, in patients with S. aureus bacteremia.

Since the year 2000, linezolid has been used in the United States to treat infections caused by antimicrobial-resistant Gram-positive cocci. At present, linezolid-resistant (Lin r ) Staphylococcus aureus and Staphylococcus epidermidis strains are rare and the diversity of their genetic backgrounds is unknown. We performed sequence-based strain typing and resistance gene characterization of 46 Lin r isolates that were collected from local and national sources between the years 2004 and 2007. Resistance was found to occur in at least three clonal complexes (CCs; lineages) of S. aureus and in at least four subclusters of a predominant, phylogenetically unstable CC of S. epidermidis . New candidate resistance mutations in 23S rRNA and the L4 riboprotein were identified among the S. epidermidis isolates. These findings suggest that linezolid resistance has emerged independently in multiple clones of S. aureus and with a variety of ribosomal mutations in multiple clones of S. epidermidis .

The decrease in vancomycin treatment efficacy that is accompanying increases in vancomycin minimum inhibitory concentration (MIC) within the susceptible range (so-called MIC creep) has led to the suggestion that vancomycin is losing its potency in treating serious Staphylococcus aureus infections. Understanding the clinical importance of the microbiological effects of glycopeptides on bacterial lipopeptides and lipoglycopeptides will be crucial in treating serious meticillin-resistant S aureus infections. We review the observed effects of reduced glycopeptide susceptibility on the activities of daptomycin in S aureus in vitro and in vivo. Factors associated with loss of susceptibility and ways to reduce the risk of resistance to daptomycin are reviewed, including the importance of prompt mechanical reduction of bacterial inoculum through surgery or through potent or combination antibiotic therapy, as well as optimisation of daptomycin pharmacodynamic exposure.

Background: Daptomycin is most commonly used as a second-line treatment. Previous studies have not differentiated the effect of prior antibiotic therapy on daptomycin clinical outcomes. Objectives: The primary objective of this study was to compare clinical outcomes of patients treated with daptomycin as first-line therapy versus after prior antibiotic therapy (specifically, vancomycin). A secondary objective was to identify other factors associated with the clinical failure of daptomycin therapy. Methods: This was a retrospective cohort study using data from a postlabeling registry database. The effects of relevant patient characteristics on the clinical outcome of individuals treated for Staphylococcus aureus infections with daptomycin were examined in an unblinded approach using univariate and multivariate analyses. Only patients with an evaluable clinical outcome (ie, cure, improvement, failure) and culture-confirmed S aureus infection were included in the analysis cohort. Results: Of 1227 clinically evaluable patients, 250 (20%) received daptomycin as first-line therapy and 977 (80%) received daptomycin after other prior antibiotic therapy. Overall, 53% of patients were male; 64% were aged 31 to 65 years and 26% were aged ≥66 years. Race information was collected beginning in 2007; of the patients studied, 71% were white and 18% were black. The initial daptomycin dose (mean [SD]) overall was 5.1 (1.1) mg/kg and was highest for patients with endocarditis (5.9 [1.2] mg/kg) and lowest for those with uncomplicated skin and skin structure infections (4.4 [0.9] mg/kg). Clinical success, defined as an outcome of cured or improved at the end of daptomycin therapy, was reported for 1140 (93%) of the 1227 evaluable patients. The clinical success rates for first-line therapy with daptomycin and after prior antibiotics were both 93%. Using univariate analysis, 8 variables were associated with clinical failure (receipt of daptomycin in an intensive care unit setting, severe renal dysfunction [creatinine clearance