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1.  Alternative Mutational Pathways to Intermediate Resistance to Vancomycin in Methicillin-Resistant Staphylococcus aureus 
Background. We used 2 in vitro experimental systems to compare phenotypic and genotypic changes that accompany selection of mutants of methicillin-resistant Staphylococcus aureus (MRSA) strain JH1 with low-level vancomycin resistance similar to the type found in vancomycin-intermediate S. aureus (VISA).
Methods. The previously described MRSA strain JH1 and its vancomycin-intermediate mutant derivative JH2, both of which were recovered from a patient undergoing vancomycin chemotherapy, were used in this study. Mutants of JH1 were selected in vitro by means of a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs) and by exposure to vancomycin in laboratory growth medium. Phenotypic abnormalities of JH1 mutants generated by each in vitro experimental system were compared to those of JH2, and whole genomes of 2 in vitro JH1 mutants were sequenced to identify mutations that may be associated with an increased vancomycin minimum inhibitory concentration.
Results. JH1R1 was selected from the PK/PD model, and JH1R2 was selected in laboratory growth medium. Both mutants displayed reduced vancomycin and daptomycin susceptibility and phenotypic alterations (eg, thicker cell walls and abnormal autolysis) that are typical of in vivo VISA mutants. Genome sequencing of JH1R1 identified point mutations in 4 genes, all of which were different from the mutations described in JH2, including 1 mutation in yycG, a component of the WalKR sensory regulatory system. Sequencing of the JH1R2 genome identified mutations in 7 genes, including 2 in rpoB.
Conclusion. Our findings indicate that JH1 is able to develop VISA-type resistance through several alternative genetic pathways.
doi:10.1093/infdis/jit127
PMCID: PMC3666135  PMID: 23539745
PK/PD model; JH1; VISA; genetic alterations; phenotypic alteration; genome sequencing; cell wall thickness; pharmacokinetics/pharmacodynamics; vancomycin resistance
2.  β-Lactam Antibiotics Targeting PBP1 Selectively Enhance Daptomycin Activity against Methicillin-Resistant Staphylococcus aureus 
Antimicrobial Agents and Chemotherapy  2013;57(10):5005-5012.
The activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA) is enhanced in the presence of subinhibitory concentrations of antistaphylococcal β-lactam antibiotics by an undefined mechanism. Given the variability in the penicillin-binding protein (PBP)-binding profiles of different β-lactam antibiotics, the purpose of this study was to examine the relative enhancement of DAP activity against MRSA by different β-lactam antibiotics to determine if a specific PBP-binding profile is associated with the ability to enhance the anti-MRSA activity of DAP. We determined that both broad- and narrow-spectrum β-lactam antibiotics known to exhibit PBP1 binding demonstrated potent enhancement of DAP anti-MRSA activity, whereas β-lactam antibiotics with minimal PBP1 binding (cefoxitin, ceftriaxone, cefaclor, and cefotaxime) were less effective. We suspect that PBP1 disruption by β-lactam antibiotics affects pathways of cell division in S. aureus that may be a compensatory response to DAP membrane insertion, resulting in DAP hypersusceptibility.
doi:10.1128/AAC.00594-13
PMCID: PMC3811465  PMID: 23896478
3.  Multicenter Study of High-Dose Daptomycin for Treatment of Enterococcal Infections 
Enterococci are among the leading pathogens isolated in hospital-acquired infections. Current antimicrobial options for vancomycin-resistant enterococci (VRE) are limited. Prior data suggest that daptomycin at >6 mg/kg of body weight/day may be used to treat enterococcal infections. We retrospectively evaluated the effectiveness and safety of high-dose daptomycin (HD-daptomycin) therapy (>6 mg/kg) in a multicenter cohort of adult patients with enterococcal infections to describe the characteristics and outcomes. Two hundred forty-five patients were evaluated. Enterococcus faecium was identified in 175 (71%), followed by Enterococcus faecalis in 49 (20%) and Enterococcus spp. in 21 (9%); overall, 204 (83%) isolates were VRE. Enterococcal infections included bacteremia (173, 71%) and intra-abdominal (35, 14%) and bone and joint (25, 10%) infections. The median dosage and duration of HD-daptomycin were 8.2 mg/kg/day (interquartile range [IQR], 7.7 to 9.7) and 10 days (IQR, 6 to 15), respectively. The overall clinical success rate was 89% (193/218), and microbiological eradication was observed in 93% (177/191) of patients. The median time to clearance of blood cultures on HD-daptomycin was 3 days (IQR, 2 to 5). The 30-day all-cause mortality rate was 27%, and 5 (2%) patients developed daptomycin-nonsusceptible enterococcal strains while on HD-daptomycin. Seven patients (3%) had creatine phosphokinase (CPK) elevation, yet no HD-daptomycin regimen was discontinued due to an elevated CPK and all patients were asymptomatic. Overall, there was a high frequency of clinical success and microbiological eradication in patients treated with HD-daptomycin for enterococcal infections, even in patients with complicated and difficult-to-treat infections. No adverse event-related discontinuation of HD-daptomycin was noted. HD-daptomycin may be an option for the treatment of enterococcal infections.
doi:10.1128/AAC.00526-13
PMCID: PMC3754344  PMID: 23774437
4.  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.
doi:10.1128/AAC.02481-12
PMCID: PMC3719745  PMID: 23689728
5.  Reduced Susceptibility to Host-Defense Cationic Peptides and Daptomycin Coemerge in Methicillin-Resistant Staphylococcus aureus From Daptomycin-Naive Bacteremic Patients 
The Journal of Infectious Diseases  2012;206(8):1160-1167.
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 neutrophil-derived 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.
doi:10.1093/infdis/jis482
PMCID: PMC3448966  PMID: 22904338
7.  Multicenter Evaluation of the Clinical Outcomes of Daptomycin with and without Concomitant β-Lactams in Patients with Staphylococcus aureus Bacteremia and Mild to Moderate Renal Impairment 
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.
doi:10.1128/AAC.02192-12
PMCID: PMC3591880  PMID: 23254428
8.  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 [fCmax], 15.2 mg/liter; half-life [t1/2], 2.3 h), daptomycin at 10 mg/kg/day (fCmax, 11.3 mg/liter; t1/2, 8 h), vancomycin at 2 g q12h (fCmax, 30 mg/liter; t1/2, 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 log10 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.
doi:10.1128/AAC.01586-12
PMCID: PMC3535972  PMID: 23070161
9.  Activity of the thiopeptide antibiotic nosiheptide against contemporary strains of methicillin-resistant Staphylococcus aureus 
The Journal of antibiotics  2012;65(12):593-598.
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. Nosiheptide was also highly active against Enterococcus spp and the contemporary hypervirulent BI 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 hours at 10X MIC. Furthermore, nosiheptide was found to be non-cytotoxic against mammalian cells at >> 100X MIC, and its anti-MRSA activity was not inhibited by 20% human serum. Notably, nosiheptide exhibited a significantly prolonged post-antibiotic effect (PAE) against both healthcare- and community-associated MRSA compared to 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.
doi:10.1038/ja.2012.77
PMCID: PMC3528839  PMID: 23047246
contemporary MRSA; marine actinomycete; nosiheptide; thiopeptide
10.  Genotypic and Phenotypic Evaluation of the Evolution of High-Level Daptomycin Nonsusceptibility in Vancomycin-Resistant Enterococcus faecium 
Antimicrobial Agents and Chemotherapy  2012;56(11):6051-6053.
Whole-genome sequencing and cell membrane studies of three clonal Enterococcus faecium strains with daptomycin MICs of 4, 32, and 192 μg/ml were performed, revealing nonsynonymous single nucleotide variants in eight open reading frames, including those predicted to encode a phosphoenolpyruvate-dependent, mannose-specific phosphotransferase system, cardiolipin synthetase, and EzrA. Membrane studies revealed a higher net surface charge among the daptomycin-nonsusceptible isolates and increased septum formation in the isolate with a daptomycin MIC of 192 μg/ml.
doi:10.1128/AAC.01318-12
PMCID: PMC3486580  PMID: 22948885
11.  Altering the Proclivity towards Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus Using Combinations with Other Antibiotics 
Antimicrobial Agents and Chemotherapy  2012;56(10):5046-5053.
Daptomycin (DAP) is increasingly used as a part of combination therapy, particularly in complex methicillin-resistant Staphylococcus aureus (MRSA) infections. While multiple studies have reported the potential for synergy between DAP and adjunctive anti-infectives, few have examined the influence of adjunctive therapy on the emergence of DAP resistance. This study examined eight adjunctive antimicrobial combinations with DAP in vitro and the emergence of DAP resistance over time (up to 4 weeks) using clinical isolates of DAP-susceptible MRSA (MIC, 0.5 μg/ml) in which DAP resistance subsequently developed during patient therapy (MIC, 3 μg/ml). In addition to DAP susceptibility testing, selected strains were examined for phenotypic changes associated with DAP resistance, including changes to cell wall thickness (CWT) and cell membrane alterations. The addition of either oxacillin or clarithromycin in medium containing DAP significantly inhibited the development of DAP resistance through the entirety of the 4-week exposure (10- to 32-fold MIC reduction from that of DAP alone). Combinations with rifampin or fosfomycin were effective in delaying the emergence of DAP resistance through the end of week one only (week one MIC, 0.5 μg/ml; week four MIC, 24 μg/ml). Cell wall thickening was observed for all antibiotic combinations regardless of their effect on the DAP MIC (14 to 70% increase in CWT), while changes in cell membrane fluidity were variable and treatment dependent. DAP showed reduced activity against strains with DAP MICs of 1 to 12 μg/ml, but cell membrane integrity was still disrupted at concentrations achieved with doses greater than 10 mg/kg of body weight. The emergence of DAP resistance in MRSA is strongly influenced by the presence of subinhibitory concentrations of adjunctive antimicrobials. These data suggest that combining DAP with oxacillin or clarithromycin may delay the development of DAP resistance in cases requiring prolonged antibiotic therapy.
doi:10.1128/AAC.00502-12
PMCID: PMC3457407  PMID: 22802248
12.  Microbiological Profile of Organisms Causing Bloodstream Infection in Critically Ill Patients 
Background
Bloodstream infection (BSI) is the most frequent infection in critically ill patients. As BSI’s among patients in intensive care units (ICU’s) are usually secondary to intravascular catheters, they can be caused by both Gram-positive and Gram-negative microorganisms as well as fungi. Infection with multidrug-resistant (MDR) organisms is becoming more common, making the choice of empirical antimicrobial therapy challenging. The objective of this study is to evaluate the spectrum of microorganisms causing BSI’s in a Medical-Surgical Intensive Care Unit (MSICU) and their antimicrobial resistance patterns.
Methods
A prospective observational study among all adult patients with clinical signs of sepsis was conducted in a MSICU of an inner-city hospital in New York City between May 1, 2010 and May 30, 2011.
Results
A total of 722 adult patients with clinical signs of systemic inflammatory response syndrome (SIRS) and/or sepsis were admitted to the MSICU between May 1, 2010 and May 30, 2011. From those patients, 91 (12.6%) had one or more positive blood culture. A 122 isolates were identified: 72 (59%) were Gram-positive bacteria, 38 (31.1%) were Gram-negative organisms, and 12 (9.8%) were fungi. Thirteen (34.2%) Gram-negative organisms and 14 (19.4%) Gram-positive bacteria were classified as MDR.
Conclusions
Antimicrobial resistance, particularly among Gram-negative organisms, continues to increase at a rapid rate, especially in the ICU’s. Coordinated infection control interventions and antimicrobial stewardship policies are warranted in order to slow the emergence of resistance.
doi:10.4021/jocmr1099w
PMCID: PMC3513418  PMID: 23226169
Bloodstream infection (BSI); Multidrug-resistant (MDR); Extended-spectrum β-lactamase (ESBL); Carbapenem-resistant Enterobacteriaceae (CRE); Intensive care units (ICU’s)
13.  Pharmacological Inhibition of the ClpXP Protease Increases Bacterial Susceptibility to Host Cathelicidin Antimicrobial Peptides and Cell Envelope-Active Antibiotics 
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.
doi:10.1128/AAC.05131-11
PMCID: PMC3318395  PMID: 22252821
14.  Daptomycin resistance mechanisms in clinically derived Staphylococcus aureus strains assessed by a combined transcriptomics and proteomics approach 
Objectives
The development of daptomycin resistance in Staphylococcus aureus is associated with clinical treatment failures. The mechanism(s) of such resistance have not been clearly defined.
Methods
We studied an isogenic daptomycin-susceptible (DAPS) and daptomycin-resistant (DAPR) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques.
Results
Minor differences in the genome content were found between strains by DNA hybridization. Transcriptomic analyses identified a number of genes differentially expressed in important functional categories: cell division; metabolism of bacterial envelopes; and global regulation. Of note, the DAPR isolate exhibited reduced expression of the major cell wall autolysis gene coincident with the up-regulation of genes involved in cell wall teichoic acid production. Using quantitative (q)RT–PCR on the gene cadre putatively involved in cationic peptide resistance, we formulated a putative regulatory network compatible with microarray data sets, mainly implicating bacterial envelopes. Of interest, qRT–PCR of this same gene cadre from two distinct isogenic DAPS/DAPR clinical strain pairs revealed evidence of other strain–dependent networks operative in the DAPR phenotype. Comparative proteomics of 616 versus 701 revealed a differential abundance of proteins in various functional categories, including cell wall-associated targets and biofilm formation proteins. Phenotypically, strains 616 and 701 showed major differences in their ability to develop bacterial biofilms in the presence of the antibacterial lipid, oleic acid.
Conclusions
Compatible with previous in vitro observations, in vivo-acquired DAPR in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelopes.
doi:10.1093/jac/dkr195
PMCID: PMC3133485  PMID: 21622973
cell wall metabolism; antibiotic resistance; biofilms; δ-haemolysis; oleic acid; microarrays; virulence; quantitative proteomics
15.  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 β-lactam-mediated shifts in surface charge remains to be defined, these finding suggest a potential for β-lactam-mediated enhancement of activity of both daptomycin and innate host defense peptides against antibiotic-resistant bacteria.
doi:10.1128/AAC.05551-11
PMCID: PMC3264218  PMID: 22123698
16.  Use of Antistaphylococcal β-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 β-lactams (ASBL) to clear refractory MRSA bacteremia. In vitro studies showed enhanced daptomycin bactericidal activity, increased membrane daptomycin binding, and decrease in positive surface charge induced by ASBLs against daptomycin nonsusceptible MRSA. Addition of ASBLs to daptomycin may be of benefit in refractory MRSA bacteremia. (Although the official designation is “daptomycin nonsusceptiblity,” we will use the term “daptomycin-resistance” in this paper for facility of presentation.)
doi:10.1093/cid/cir340
PMCID: PMC3697476  PMID: 21690622
17.  Reduced vancomycin susceptibility among clinical Staphylococcus aureus isolates (‘the MIC Creep’): implications for therapy 
Methicillin-resistant S. aureus (MRSA) has emerged as the most common hospital-acquired pathogen and is associated with increased morbidity and mortality compared with other strains. Vancomycin has been the cornerstone of treatment of patients with serious MRSA infections for some decades and while more than 99% of clinical S. aureus isolates remain susceptible to vancomycin, we are beginning to see strains of MRSA with reduced susceptibility. This review discusses this phenomenon, the predictors of infection with such forms of MRSA, and current and future management options.
doi:10.3410/M4-4
PMCID: PMC3270590  PMID: 22312414
18.  Novel Bacterial Metabolite Merochlorin A Demonstrates in vitro Activity against Multi-Drug Resistant Methicillin-Resistant Staphylococcus aureus 
PLoS ONE  2012;7(1):e29439.
Background
We evaluated the in vitro activity of a merochlorin A, a novel compound with a unique carbon skeleton, against a spectrum of clinically relevant bacterial pathogens and against previously characterized clinical and laboratory Staphylococcus aureus isolates with resistance to numerous antibiotics.
Methods
Merochlorin A was isolated and purified from a marine-derived actinomycete strain CNH189. Susceptibility testing for merochlorin A was performed against previously characterized human pathogens using broth microdilution and agar dilution methods. Cytotoxicity was assayed in tissue culture assays at 24 and 72 hours against human HeLa and mouse sarcoma L929 cell lines.
Results
The structure of as new antibiotic, merochlorin A, was assigned by comprehensive spectroscopic analysis. Merochlorin A demonstrated in vitro activity against Gram-positive bacteria, including Clostridium dificile, but not against Gram negative bacteria. In S. aureus, susceptibility was not affected by ribosomal mutations conferring linezolid resistance, mutations in dlt or mprF conferring resistance to daptomycin, accessory gene regulator knockout mutations, or the development of the vancomycin-intermediate resistant phenotype. Merochlorin A demonstrated rapid bactericidal activity against MRSA. Activity was lost in the presence of 20% serum.
Conclusions
The unique meroterpenoid, merochlorin A demonstrated excellent in vitro activity against S. aureus and C. dificile and did not show cross-resistance to contemporary antibiotics against Gram positive organisms. The activity was, however, markedly reduced in 20% human serum. Future directions for this compound may include evaluation for topical use, coating biomedical devices, or the pursuit of chemically modified derivatives of this compound that retain activity in the presence of serum.
doi:10.1371/journal.pone.0029439
PMCID: PMC3261149  PMID: 22279537
19.  Comparative effectiveness of nafcillin or cefazolin versus vancomycin in methicillin-susceptible Staphylococcus aureus bacteremia 
BMC Infectious Diseases  2011;11:279.
Background
The high prevalence of methicillin-resistant S. aureus (MRSA) has led clinicians to select antibiotics that have coverage against MRSA, usually vancomycin, for empiric therapy for suspected staphylococcal infections. Clinicians often continue vancomycin started empirically even when methicillin-susceptible S. aureus (MSSA) strains are identified by culture. However, vancomycin has been associated with poor outcomes such as nephrotoxicity, persistent bacteremia and treatment failure. The objective of this study was to compare the effectiveness of vancomycin versus the beta-lactam antibiotics nafcillin and cefazolin among patients with MSSA bacteremia. The outcome of interest for this study was 30-day in-hospital mortality.
Methods
This retrospective cohort study included all adult in-patients admitted to a tertiary-care facility between January 1, 2003 and June 30, 2007 who had a positive blood culture for MSSA and received nafcillin, cefazolin or vancomycin. Cox proportional hazard models were used to assess independent mortality hazards comparing nafcillin or cefazolin versus vancomycin. Similar methods were used to estimate the survival benefits of switching from vancomycin to nafcillin or cefazolin versus leaving patients on vancomycin. Each model included statistical adjustment using propensity scores which contained variables associated with an increased propensity to receive vancomycin.
Results
267 patients were included; 14% (38/267) received nafcillin or cefazolin, 51% (135/267) received both vancomycin and either nafcillin or cefazolin, and 35% (94/267) received vancomycin. Thirty (11%) died within 30 days. Those receiving nafcillin or cefazolin had 79% lower mortality hazards compared with those who received vancomycin alone (adjusted hazard ratio (HR): 0.21; 95% confidence interval (CI): 0.09, 0.47). Among the 122 patients who initially received vancomycin empirically, those who were switched to nafcillin or cefazolin (66/122) had 69% lower mortality hazards (adjusted HR: 0.31; 95% CI: 0.10, 0.95) compared to those who remained on vancomycin.
Conclusions
Receipt of nafcillin or cefazolin was protective against mortality compared to vancomycin even when therapy was altered after culture results identified MSSA. Convenience of vancomycin dosing may not outweigh the potential benefits of nafcillin or cefazolin in the treatment of MSSA bacteremia.
doi:10.1186/1471-2334-11-279
PMCID: PMC3206863  PMID: 22011388
20.  Increased Mortality with Accessory Gene Regulator (agr) Dysfunction in Staphylococcus aureus among Bacteremic Patients ▿ †  
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.
doi:10.1128/AAC.00918-10
PMCID: PMC3067101  PMID: 21173172
21.  Combinatorial Phenotypic Signatures Distinguish Persistent from Resolving Methicillin-Resistant Staphylococcus aureus Bacteremia Isolates ▿  
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.
doi:10.1128/AAC.01028-10
PMCID: PMC3028773  PMID: 21098242
22.  Enhanced expression of dltABCD is associated with development of daptomycin nonsusceptibility in a clinical endocarditis isolate of Staphylococcus aureus 
The Journal of infectious diseases  2009;200(12):1916-1920.
Using isogenic clinical bloodstream Staphylococcus aureus strains from a patient with relapsing endocarditis, we investigated transcriptional profiles of mprF and dlt genes in the context of cell surface charge and daptomycin nonsusceptibility. As in prior studies, a point mutation within mprF was observed in the daptomycin-nonsusceptible strain. However, neither the transcriptional profile of mprF, nor membrane phospholipid analyses were compatible with the anticipated mprF gain-in-function phenotype. In contrast, we demonstrated enhanced dlt expression coincident with increased positive surface charge and reduced daptomycin binding.
doi:10.1086/648473
PMCID: PMC2779839  PMID: 19919306
Staphylococcus aureus; daptomycin; cationic antimicrobial peptide (CAP); mprF; dlt
23.  Polyphyletic Emergence of Linezolid-Resistant Staphylococci in the United States ▿  
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 (Linr) 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 Linr 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.
doi:10.1128/AAC.00621-09
PMCID: PMC2812165  PMID: 19933808
24.  Empiric Antibiotic Therapy for Staphylococcus aureus Bacteremia May Not Reduce In-Hospital Mortality: A Retrospective Cohort Study 
PLoS ONE  2010;5(7):e11432.
Background
Appropriate empiric therapy, antibiotic therapy with in vitro activity to the infecting organism given prior to confirmed culture results, may improve Staphylococcus aureus outcomes. We aimed to measure the clinical impact of appropriate empiric antibiotic therapy on mortality, while statistically adjusting for comorbidities, severity of illness and presence of virulence factors in the infecting strain.
Methodology
We conducted a retrospective cohort study of adult patients admitted to a tertiary-care facility from January 1, 2003 to June 30, 2007, who had S. aureus bacteremia. Time to appropriate therapy was measured from blood culture collection to the receipt of antibiotics with in vitro activity to the infecting organism. Cox proportional hazard models were used to measure the association between receipt of appropriate empiric therapy and in-hospital mortality, statistically adjusting for patient and pathogen characteristics.
Principal Findings
Among 814 admissions, 537 (66%) received appropriate empiric therapy. Those who received appropriate empiric therapy had a higher hazard of 30-day in-hospital mortality (Hazard Ratio (HR): 1.52; 95% confidence interval (CI): 0.99, 2.34). A longer time to appropriate therapy was protective against mortality (HR: 0.79; 95% CI: 0.60, 1.03) except among the healthiest quartile of patients (HR: 1.44; 95% CI: 0.66, 3.15).
Conclusions/Significance
Appropriate empiric therapy was not associated with decreased mortality in patients with S. aureus bacteremia except in the least ill patients. Initial broad antibiotic selection may not be widely beneficial.
doi:10.1371/journal.pone.0011432
PMCID: PMC2896397  PMID: 20625395
25.  Evaluating Aztreonam and Ceftazidime Pharmacodynamics with Escherichia coli in Combination with Daptomycin, Linezolid, or Vancomycin in an In Vitro Pharmacodynamic Model ▿  
Antimicrobial Agents and Chemotherapy  2009;53(10):4549-4555.
In an in vitro pharmacodynamic model, linezolid attenuated the activity of aztreonam and ceftazidime against Escherichia coli. Conversely, synergy was detected at 24 and 48 h when daptomycin or vancomycin was added to aztreonam and ceftazidime. We conclude that significant yet underappreciated interactions may occur between gram-positive-spectrum and gram-negative-spectrum antibacterials.
doi:10.1128/AAC.00180-09
PMCID: PMC2764192  PMID: 19620335

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