SCH79797 improves outcomes in experimental bacterial pneumonia by boosting neutrophil killing and direct antibiotic activity.

Objectives: The role of protease-activated receptor 1 (PAR1) in the pathogenesis of pneumonia and sepsis is ambiguous given the existing literature. As PAR1 is classically activated by the coagulation-based protease thrombin and leads to vascular leakage, our hypothesis was that PAR1 blockade with SCH79797 would be therapeutically beneficial in an experimental model of murine Gram-negative pneumonia. Methods: In this study, we administered SCH79797 via the intrapulmonary route 6 h after the establishment of Escherichia coli pneumonia and observed a significant improvement in survival, lung injury, bacterial clearance and inflammation. We focused on neutrophils as a potential target of the PAR1 antagonist, since they are the predominant inflammatory cell type in the infected lung. Results: Neutrophils appear to express PAR1 at low levels and the PAR1 antagonist SCH79797 enhanced neutrophil killing. Part of this effect may be explained by alterations in the generation of reactive oxygen species (ROS). SCH79797 also led to robust neutrophil extracellular trap (NET) generation and cathelicidin-related antimicrobial peptide (CRAMP) release by neutrophils. Surprisingly, SCH79797 also had a potent, direct antibiotic effect with disruption of the E. coli cell membrane. However, the newer-generation PAR1 antagonist, vorapaxar (SCH530348), had no appreciable effect on neutrophil activity or direct bacterial killing, which suggests the effects seen with SCH79797 may be PAR1 independent. Conclusions: In summary, we observed that intrapulmonary treatment with SCH79797 has significant therapeutic effects in a model of E. coli pneumonia that appear to be due, in part, to both neutrophil-stimulating and direct antibacterial effects of SCH79797.

Classical ß-Lactamase Inhibitors Potentiate the Activity of Daptomycin against Methicillin-Resistant Staphylococcus aureus and Colistin against Acinetobacter baumannii.

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-log10 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.

Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for ß-Lactam-Daptomycin Synergy.

The activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA) is enhanced in the presence of ß-lactam antibiotics. This effect is more pronounced with ß-lactam antibiotics that exhibit avid binding to penicillin binding protein 1 (PBP1). Here, we present evidence that PBP1 has a significant role in responding to DAP-induced stress on the cell. Expression of the pbpA transcript, encoding PBP1, was specifically induced by DAP exposure whereas expression of pbpB, pbpC, and pbpD, encoding PBP2, PBP3, and PBP4, respectively, remained unchanged. Using a MRSA COL strain with pbpA under an inducible promoter, increased pbpA transcription was accompanied by reduced susceptibility to, and killing by, DAP in vitro. Exposure to ß-lactams that preferentially inactivate PBP1 was not associated with increased DAP binding, suggesting that synergy in the setting of anti-PBP1 pharmacotherapy results from increased DAP potency on a per-molecule basis. Combination exposure in an in vitro pharmacokinetic/pharmacodynamic model system with ß-lactams that preferentially inactivate PBP1 (DAP-meropenem [MEM] or DAP-imipenem [IPM]) resulted in more-rapid killing than did combination exposure with DAP-nafcillin (NAF) (nonselective), DAP-ceftriaxone (CRO) or DAP-cefotaxime (CTX) (PBP2 selective), DAP-cefaclor (CEC) (PBP3 selective), or DAP-cefoxitin (FOX) (PBP4 selective). Compared to ß-lactams with poor PBP1 binding specificity, exposure of S. aureus to DAP plus PBP1-selective ß-lactams resulted in an increased frequency of septation and cell wall abnormalities. These data suggest that PBP1 activity may contribute to survival during DAP-induced metabolic stress. Therefore, targeted inactivation of PBP1 may enhance the antimicrobial efficiency of DAP, supporting the use of DAP-ß-lactam combination therapy for serious MRSA infections, particularly when the ß-lactam undermines the PBP1-mediated compensatory response.

Cefazolin and Ertapenem, a Synergistic Combination Used To Clear Persistent Staphylococcus aureus Bacteremia.

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 log10-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.

Examining the use of ceftaroline in the treatment of Streptococcus pneumoniae meningitis with reference to human cathelicidin LL-37.

Five cases of bacterial meningitis treated with ceftaroline (4 Streptococcus pneumoniae and 1 Staphylococcus aureus) are summarized here. The pharmacodynamics of human cathelicidin LL-37 and ceftaroline were evaluated against S. pneumoniae. Patients who received ceftaroline 600 mg every 8 h (q8h) (1 S. aureus and 3 S. pneumoniae) were successfully treated; treatment failed in 1 patient with S. pneumoniae who received 600 mg q12h. Ceftaroline increased the negative surface charge and sensitized S. pneumoniae to killing by LL-37, a peptide implicated in blood-brain barrier defense.

Differential Effects of Penicillin Binding Protein Deletion on the Susceptibility of Enterococcus faecium to Cationic Peptide Antibiotics.

Beta-lactam antibiotics sensitize Enterococcus faecium to killing by endogenous antimicrobial peptides (AMPs) of the innate immune system and daptomycin through mechanisms yet to be elucidated. It has been speculated that beta-lactam inactivation of select E. faecium penicillin binding proteins (PBPs) may play a pivotal role in this sensitization process. To characterize the specific PBP inactivation that may be responsible for these phenotypes, we utilized a previously characterized set of E. faecium PBP knockout mutants to determine the effects of such mutations on the activity of daptomycin and the AMP human cathelicidin (LL-37). Enhanced susceptibility to daptomycin was dependent more on a cumulative effect of multiple PBP deletions than on inactivation of any single specific PBP. Selective knockout of PBPZ rendered E. faecium more vulnerable to killing by both recombinant LL-37 and human neutrophils, which produce the antimicrobial peptide in high quantities. Pharmacotherapy targeting multiple PBPs may be used as adjunctive therapy with daptomycin to treat difficult E. faecium infections.

In vitro activity of daptomycin in combination with ß-lactams, gentamicin, rifampin, and tigecycline against daptomycin-nonsusceptible enterococci.

Enterococci that are nonsusceptible (NS; MIC > 4 µg/ml) to daptomycin are an emerging clinical concern. The synergistic combination of daptomycin plus beta-lactams has been shown to be effective against vancomycin-resistant Enterococcus (VRE) species in vitro. This study systematically evaluated by in vitro time-kill studies the effect of daptomycin in combination with ampicillin, cefazolin, ceftriaxone, ceftaroline, ertapenem, gentamicin, tigecycline, and rifampin, for a collection of 9 daptomycin-NS enterococci that exhibited a broad range of MICs and different resistance-conferring mutations. We found that ampicillin plus daptomycin yielded the most consistent synergy but did so only for isolates with mutations to the liaFSR system. Daptomycin binding was found to be enhanced by ampicillin in a representative isolate with such mutations but not for an isolate with mutation to the yycFGHIJ system. In contrast, ampicillin enhanced the killing of the LL-37 human antimicrobial peptide against daptomycin-NS E. faecium with either the liaFSR or yycFGHIJ mutation. Antagonism was noted only for rifampin and tigecycline and only for 2 or 3 isolates. These data add support to the growing body of evidence indicating that therapy combining daptomycin and ampicillin may be helpful in eradicating refractory VRE infections.

Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles.

Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions - such as virion proteins and repressors - cannot always be recognized. Here we describe a functional genomic dissection of mycobacteriophage Giles, in which the virion proteins are identified, genes required for lytic growth are determined, the repressor is identified, and the transcription patterns determined. We find that although all of the predicted phage genes are expressed either in lysogeny or in lytic growth, 45% of the predicted genes are non-essential for lytic growth. We also describe genes required for DNA replication, show that recombination is required for lytic growth, and that Giles encodes a novel repressor. RNAseq analysis reveals abundant expression of a small non-coding RNA in a lysogen and in late lytic growth, although it is non-essential for lytic growth and does not alter lysogeny.

Ceftaroline restores daptomycin activity against daptomycin-nonsusceptible vancomycin-resistant Enterococcus faecium.

Daptomycin-nonsusceptible vancomycin-resistant Enterococcus faecium (VRE) strains are a formidable emerging threat to patients with comorbidities, leaving few therapeutic options in cases of severe invasive infections. Using a previously characterized isogenic pair of VRE strains from the same patient differing in their daptomycin susceptibilities (Etest MICs of 0.38 mg/liter and 10 mg/liter), we examined the effect of ceftaroline, ceftriaxone, and ampicillin on membrane fluidity and susceptibility of VRE to surface binding and killing by daptomycin and human cathelicidin antimicrobial peptide LL37. Synergy was noted in vitro between daptomycin, ampicillin, and ceftaroline for the daptomycin-susceptible VRE strain, but only ceftaroline showed synergy against the daptomycin-nonsusceptible VRE strain (∼2 log10 CFU reduction at 24 h). Ceftaroline cotreatment increased daptomycin surface binding with an associated increase in membrane fluidity and an increase in the net negative surface charge of the bacteria as evidenced by increased poly-l-lysine binding. Consistent with the observed biophysical changes, ceftaroline resulted in increased binding and killing of daptomycin-nonsusceptible VRE by human cathelicidin LL37. Using a pair of daptomycin-susceptible/nonsusceptible VRE strains, we noted that VRE is ceftaroline resistant, yet ceftaroline confers significant effects on growth rate as well as biophysical changes on the cell surface of VRE that can potentiate the activity of daptomycin and innate cationic host defense peptides, such as cathelicidin. Although limited to just 2 strains, these finding suggest that additional in vivo and in vitro studies need to be done to explore the possibility of using ceftaroline as adjunctive anti-VRE therapy.

Heterogeneity of mprF sequences in methicillin-resistant Staphylococcus aureus clinical isolates: role in cross-resistance between daptomycin and host defense antimicrobial peptides.

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.

Potent synergy of ceftobiprole plus daptomycin against multiple strains of Staphylococcus aureus with various resistance phenotypes.

OBJECTIVES: Ceftobiprole is a broad-spectrum cephalosporin that demonstrates activity against Staphylococcus aureus resistant to methicillin, including strains with reduced susceptibility to glycopeptides and lipopeptides. The addition of this agent provides a potential therapeutic option for difficult-to-treat infections. Synergy has been demonstrated between ß-lactams combined with glycopeptides and lipopeptides against S. aureus. This study sought to determine whether ceftobiprole was synergistic with daptomycin, vancomycin or standard-of-care combination agents (gentamicin or rifampicin) against methicillin-resistant S. aureus (MRSA) strains with varying degrees of vancomycin susceptibility. METHODS: Broth microdilution MICs of ceftobiprole, daptomycin, vancomycin, rifampicin and gentamicin were evaluated for 20 MRSA isolates. Combination MICs were additionally evaluated in the presence of subinhibitory concentrations of ceftobiprole to assess synergism. Time-kill curves for five representative isolates were performed utilizing combinations of ceftobiprole plus daptomycin, vancomycin, rifampicin and gentamicin to further quantify the degree of synergy for each regimen. RESULTS: Ceftobiprole plus daptomycin represented the most potent combination with a 4-fold decrease in MIC and synergy against all strains evaluated in time-kill evaluations. Additionally, binding studies demonstrated enhanced daptomycin binding in the presence of subinhibitory concentrations of ceftobiprole. CONCLUSIONS: The use of combination therapy with ceftobiprole may provide a needed addition for the treatment of Gram-positive infections resistant to daptomycin or vancomycin. Consistent with what has been observed with other ß-lactams, ceftobiprole increased bodipy-tagged daptomycin binding on the surface of S. aureus, potentially explaining this potent synergy observed in time-kill evaluations. More detailed evaluation of ceftobiprole is warranted to better characterize observed synergy.

Ceftaroline increases membrane binding and enhances the activity of daptomycin against daptomycin-nonsusceptible vancomycin-intermediate Staphylococcus aureus in a pharmacokinetic/pharmacodynamic model.

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 (fC(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.

Treatment of high-level gentamicin-resistant Enterococcus faecalis endocarditis with daptomycin plus ceftaroline.

A recurrent case of left-sided endocarditis caused by high-level aminoglycoside-resistant Enterococcus faecalis was successfully treated with ceftaroline and daptomycin. This combination demonstrated excellent synergy in vitro. Mechanistically, ceftaroline enhanced binding of daptomycin to the cell membrane and sensitized E. faecalis to killing by human cathelicidin LL-37, a cationic innate host defense peptide. Daptomycin plus ceftaroline may be considered in salvage therapy in E. faecalis endovascular infections and requires further study.

Nafcillin Augmentation of Daptomycin and Cathelicidin LL-37 Killing of Methicillin-resistant Staphylococcus epidermidis: Foundations of Successful Therapy of Endocarditis.

Methicillin-resistant Staphylococcus epidermidis (MRSE) endocarditis failing conventional therapy has been successfully treated with nafcillin plus daptomycin in the clinic. In vitro studies showed that nafcillin enhanced daptomycin killing of MRSE in both planktonic cells and biofilm. Nafcillin exposure also sensitized MRSE to killing by human neutrophils and cathelicidin antimicrobial peptide LL-37. Fluorescent microscopy showed increased daptomycin and LL-37 binding to the MRSE bacterial surface upon nafcillin treatment. Ceftaroline also increased MRSE killing by daptomycin in planktonic cultures and biofilms, as well as daptomycin and LL-37 binding on the bacterial surface. Nafcillin, ceftaroline, and possibly other ß-lactams, may serve an important role in the therapy of MRSE endocarditis through augmentation of cationic peptide, the innate immune system, and daptomycin killing. Clinical studies will be needed to determine how early these regimens should be deployed to optimize clinical outcome.

Ampicillin enhances daptomycin- and cationic host defense peptide-mediated killing of ampicillin- and vancomycin-resistant Enterococcus faecium.

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 of body weight) plus linezolid. Blood cultures cleared within 24 h 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. MICs and time-kill curves with daptomycin were assessed in the presence and absence of ampicillin. The impact of ampicillin on surface charge was assessed by flow cytometry and a poly-l-lysine binding assay. The effects of ampicillin preexposures upon VRE killing by five distinct cationic peptides of different structure, charge, origin, and mechanism 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 (bacteria derived). Fluoroscein-Bodipy-labeled daptomycin was used to evaluate daptomycin binding to VRE membranes in the presence or absence of ampicillin. In media containing ampicillin (25 to 100 mg/liter), daptomycin MICs decreased from 1.0 to 0.38 mg/liter. Based on time-kill analysis 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 to 150 mg/liter) demonstrated an incremental reduction in its relative net positive surface charge. When grown in the presence (versus absence) of ampicillin (25 and 100 mg/liter), 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. We conclude that ampicillin induces 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 ß-lactam-mediated shifts in surface charge remains to be defined, these finding suggest a potential for ß-lactam-mediated enhancement of activity of both daptomycin and innate host defense peptides against antibiotic-resistant bacteria.

Use of antistaphylococcal beta-lactams to increase daptomycin activity in eradicating persistent bacteremia due to methicillin-resistant Staphylococcus aureus: role of enhanced daptomycin binding.

We used daptomycin plus antistaphylococcal ß-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.).

Genetic Determinants Enabling Medium-Dependent Adaptation to Nafcillin in Methicillin-Resistant Staphylococcus aureus.

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.

Characterization of CA-MRSA TCH1516 exposed to nafcillin in bacteriological and physiological media.

Cation adjusted-Mueller Hinton Broth (CA-MHB) is the standard bacteriological medium utilized in the clinic for the determination of antibiotic susceptibility. However, a growing number of literature has demonstrated that media conditions can cause a substantial difference in the efficacy of antibiotics and antimicrobials. Recent studies have also shown that minimum inhibitory concentration (MIC) tests performed in standard cell culture media (e.g. RPMI and DMEM) are more indicative of in vivo antibiotic efficacy, presumably because they are a better proxy for the human host's physiological conditions. The basis for the bacterial media dependent susceptibility to antibiotics remains undefined. To address this question, we characterized the physiological response of methicillin-resistant Staphylococcus aureus (MRSA) during exposure to sub-inhibitory concentrations of the beta-lactam antibiotic nafcillin in either CA-MHB or RPMI + 10% LB (R10LB). Here, we present high quality transcriptomic, exo-metabolomic and morphological data paired with growth and susceptibility results for MRSA cultured in either standard bacteriologic or more physiologic relevant medium.

The Mla pathway is critical for Pseudomonas aeruginosa resistance to outer membrane permeabilization and host innate immune clearance.

Pseudomonas aeruginosa is an important opportunistic pathogen that has become a serious problem due to increased rates of antibiotic resistance. Due to this along with a dearth in novel antibiotic development, especially against Gram-negative pathogens, new therapeutic strategies are needed to prevent a post-antibiotic era. Here, we describe the importance of the vacJ/Mla pathway in resisting bactericidal actions of the host innate immune response. P. aeruginosa tn5 transposon mutants in genes from the VacJ/Mla pathway showed increased susceptibility to killing by the host cathelicidin antimicrobial peptide, LL-37, when compared to the wild-type parent strain. The P. aeruginosa vacJ - mutant demonstrated increased membrane permeability upon damage as well as sensitivity to killing in the presence of the detergent sodium dodecyl sulfate and the divalent cation chelator EDTA. When exposed to human whole blood and serum complement, the vacJ - mutant was killed more rapidly when compared to the wild-type parent strain and complemented mutant. Finally, in an in vivo mouse lung infection model, infection with the vacJ - mutant resulted in reduced mortality, lower bacterial burden, and reduced lung damage when compared to the wild-type strain. This study highlights the potential in therapeutically targeting the VacJ/Mla pathway in sensitizing P. aeruginosa to killing by the host innate immune response. KEY MESSAGES: • The Mla pathway regulates outer membrane dynamics in human pathogen Pseudomonas aeruginosa (PA). • Disruption of Mla pathway gene vacJ sensitizes PA to host cathelicidin antimicrobial peptide LL-37. • Loss of vacJ expression renders PA more sensitive to human whole blood and serum killing. • Loss of vacJ expression reduces PA survival and virulence in a murine lung infection model. • The Mla pathway merits exploration as a pharmacologic target to sensitize PA to host innate immunity.

Nafcillin enhances innate immune-mediated killing of methicillin-resistant Staphylococcus aureus.

UNLABELLED: 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 ß-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. KEY MESSAGES: 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.