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1.  MgrA Activates Expression of Capsule Genes, but Not the α-Toxin Gene in Experimental Staphylococcus aureus Endocarditis 
The Journal of Infectious Diseases  2013;208(11):1841-1848.
Background. Staphylococcus aureus produces numerous virulence factors but little is known about their in vivo regulation during an infection.
Methods. The production of capsule and α-toxin, and the expression of their respective genes, cap5 and hla, were analyzed by comparing CYL11481 (derivative of Newman) and its isogenic regulatory mutants in vitro. The temporal expression of cap5 and hla and the regulatory genes in vivo was carried out using a rat infective endocarditis model.
Results. In vitro analyses showed that capsule was positively regulated by MgrA, Agr, Sae, ArlR, and ClpC, and negatively by CodY and SbcDC. The α-toxin was positively regulated by MgrA, Agr, Sae, ArlR, and SbcDC but negatively by ClpC and CodY. In vivo analyses showed that cap5 expression correlated best with mgrA expression, whereas hla expression correlated best with sae expression. Mutation in mgrA drastically reduced cap5 expression in vivo.
Conclusions. Our results suggest that, in vitro, Agr is the most important regulator for capsule and α-toxin production, as well as for cap5 transcription, but SaeR is the most critical for hla transcription. However, in vivo, MgrA is the major transcriptional regulator of capsule, but not α-toxin, whereas saeR expression correlates best with hla expression.
doi:10.1093/infdis/jit367
PMCID: PMC3814835  PMID: 23901087
Staphylococcus aureus; virulence; infective endocarditis model; mgrA; capsule; α-toxin
2.  Phenotypic and Genotypic Characterization of Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus Strains: Relative Roles of mprF and dlt Operons 
PLoS ONE  2014;9(9):e107426.
Development of in vivo daptomycin resistance (DAP-R) among Staphylococcus aureus clinical isolates, in association with clinical treatment failures, has become a major therapeutic problem. This issue is especially relevant to methicillin-resistant S. aureus (MRSA) strains in the context of invasive endovascular infections. In the current study, we used three well-characterized and clinically-derived DAP-susceptible (DAP-S) vs. resistant (DAP-R) MRSA strain-pairs to elucidate potential genotypic mechanisms of the DAP-R phenotype. In comparison to the DAP-S parental strains, DAP-R isolates demonstrated (i) altered expression of two key determinants of net positive surface charge, either during exponential or stationary growth phases (i.e., dysregulation of dltA and mprF), (ii) a significant increase in the D-alanylated wall teichoic acid (WTA) content in DAP-R strains, reflecting DltA gain-in-function; (iii) heightened elaboration of lysinylated-phosphatidylglyderol (L-PG) in DAP-R strains, reflecting MprF gain-in-function; (iv) increased cell membrane (CM) fluidity, and (v) significantly reduced susceptibility to prototypic cationic host defense peptides of platelet and leukocyte origins. In the tested DAP-R strains, genes conferring positive surface charge were dysregulated, and their functionality altered. However, there were no correlations between relative surface positive charge or cell wall thickness and the observed DAP-R phenotype. Thus, charge repulsion mechanisms via altered surface charge may not be sufficient to explain the DAP-R outcome. Instead, changes in the compositional or biophysical order of the DAP CM target of such DAP-R strains (i.e., increased fluidity) may be essential to this phenotype. Taken together, DAP-R in S. aureus appears to involve multi-factorial and strain-specific adaptive mechanisms.
doi:10.1371/journal.pone.0107426
PMCID: PMC4166420  PMID: 25226591
3.  Virulence of Endemic Nonpigmented Northern Australian Staphylococcus aureus Clone (Clonal Complex 75, S. argenteus) Is Not Augmented by Staphyloxanthin 
The Journal of Infectious Diseases  2013;208(3):520-527.
Staphylococcus aureus clonal complex 75 (herein referred to as S. argenteus) lacks the carotenoid pigment operon, crtOPQMN, responsible for production of the putative virulence factor, staphyloxanthin. Although a common cause of community-onset skin infections among Indigenous populations in northern Australia, this clone is infrequently isolated from hospital-based patients with either bacteremic or nonbacteremic infections. We hypothesized that S. argenteus would have attenuated virulence compared to other S. aureus strains due to its staphyloxanthin “deficiency.” Compared to prototypical S. aureus strains, S. argenteus was more susceptible to oxidative stress and neutrophil killing in vitro and had reduced virulence in murine sepsis and skin infection models. Transformation with pTX-crtOPQMN resulted in staphyloxanthin expression and increased resistance to oxidative stress in vitro. However, neither resistance to neutrophil killing nor in vivo virulence was increased. Thus, reduced virulence of S. argenteus in these models is due to mechanisms unrelated to lack of staphyloxanthin production.
doi:10.1093/infdis/jit173
PMCID: PMC3699000  PMID: 23599317
Staphylococcus aureus; staphyloxanthin; virulence; Australia; carotenoid pigment
4.  High-Dose Daptomycin Therapy for Left-Sided Infective Endocarditis: a Prospective Study from the International Collaboration on Endocarditis 
Antimicrobial Agents and Chemotherapy  2013;57(12):6213-6222.
The use of daptomycin in Gram-positive left-sided infective endocarditis (IE) has significantly increased. The purpose of this study was to assess the influence of high-dose daptomycin on the outcome of left-sided IE due to Gram-positive pathogens. This was a prospective cohort study based on 1,112 cases from the International Collaboration on Endocarditis (ICE)-Plus database and the ICE-Daptomycin Substudy database from 2008 to 2010. Among patients with left-sided IE due to Staphylococcus aureus, coagulase-negative staphylococci, and Enterococcus faecalis, we compared those treated with daptomycin (cohort A) to those treated with standard-of-care (SOC) antibiotics (cohort B). The primary outcome was in-hospital mortality. Time to clearance of bacteremia, 6-month mortality, and adverse events (AEs) ascribable to daptomycin were also assessed. There were 29 and 149 patients included in cohort A and cohort B, respectively. Baseline comorbidities did not differ between the two cohorts, except for a significantly higher prevalence of diabetes and previous episodes of IE among patients treated with daptomycin. The median daptomycin dose was 9.2 mg/kg of body weight/day. Two-thirds of the patients treated with daptomycin had failed a previous antibiotic regimen. In-hospital and 6-month mortalities were similar in the two cohorts. In cohort A, median time to clearance of methicillin-resistant S. aureus (MRSA) bacteremia was 1.0 day, irrespective of daptomycin dose, representing a significantly faster bacteremia clearance compared to SOC (1.0 versus 5.0 days; P < 0.01). Regimens with higher daptomycin doses were not associated with increased incidence of AEs. In conclusion, higher-dose daptomycin may be an effective and safe alternative to SOC in the treatment of left-sided IE due to common Gram-positive pathogens.
doi:10.1128/AAC.01563-13
PMCID: PMC3837915  PMID: 24080644
5.  Causal Role of Single Nucleotide Polymorphisms within the mprF Gene of Staphylococcus aureus in Daptomycin Resistance 
Antimicrobial Agents and Chemotherapy  2013;57(11):5658-5664.
Single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr) Staphylococcus aureus strains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if such mprF SNPs are causal in DAPr strains or are merely a biomarker for this phenotype. In this study, we used an isogenic set of S. aureus strains: (i) Newman, (ii) its isogenic ΔmprF mutant, and (iii) several in trans plasmid complementation constructs, expressing either a wild-type or point-mutated form of the mprF ORF cloned from two isogenic DAP-susceptible (DAPs)-DAPr strain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprF strain with singly point-mutated mprF genes (mprFS295L or mprFT345A) revealed that (i) individual and distinct point mutations within the mprF ORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding to S. aureus by a charge repulsion mechanism. Thus, for these two DAPr strains, the defined mprF SNPs appear to be causally related to this phenotype.
doi:10.1128/AAC.01184-13
PMCID: PMC3811309  PMID: 24002096
6.  Genome Sequences of Sequence Type 45 (ST45) Persistent Methicillin-Resistant Staphylococcus aureus (MRSA) Bacteremia Strain 300-169 and ST45 Resolving MRSA Bacteremia Strain 301-188 
Genome Announcements  2014;2(2):e00174-14.
Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (positive blood cultures after ≥7 days) represents a challenging subset of invasive MRSA infections. The comparison of genome sequences of persistent (300-169) and resolving (301-188) MRSA bacteremia isolates with similar genetic background (sequence type 45 [ST45]) will help us to better understand underlying mechanisms of persistent MRSA bacteremia.
doi:10.1128/genomeA.00174-14
PMCID: PMC3953194  PMID: 24625873
7.  Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat 
Endovascular infections, including endocarditis, are life-threatening infectious syndromes1–3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4–6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.
doi:10.3791/3863
PMCID: PMC3471301  PMID: 22711072
Infection; Issue 64; Immunology; Staphylococcus aureus; endocarditis; animal model; methicillin resistance; MRSA; rat
8.  Role of the LytSR Two-Component Regulatory System in Adaptation to Cationic Antimicrobial Peptides in Staphylococcus aureus 
Many host defense cationic antimicrobial peptides (HDPs) perturb the staphylococcal cell membrane (CM) and alter transmembrane potential (ΔΨ) as key parts of their lethal mechanism. Thus, a sense-response system for detecting and mediating adaptive responses to such stresses could impact organism survival; the Staphylococcus aureus LytSR two-component regulatory system (TCRS) may serve as such a ΔΨ sensor. One well-known target of this system is the lrgAB operon, which, along with the related cidABC operon, has been shown to be a regulator in the control of programmed cell death and lysis. We used an isogenic set of S. aureus strains: (i) UAMS-1, (ii) its isogenic ΔlytS and ΔlrgAB mutants, and (iii) plasmid-complemented ΔlytSR and ΔlrgAB mutants. The ΔlytS strain displayed significantly increased in vitro susceptibilities to all HDPs tested (neutrophil-derived human neutrophil peptide 1 [hNP-1], platelet-derived thrombin-induced platelet microbicidal proteins [tPMPs], and the tPMP-mimetic peptide RP-1), as well as to calcium-daptomycin (DAP), a cationic antimicrobial peptide (CAP). In contrast, the ΔlrgAB strain exhibited no significant changes in susceptibilities to these cationic peptides, indicating that although lytSR positively regulates transcription of lrgAB, increased HDP/CAP susceptibilities in the ΔlytS mutant were lrgAB independent. Further, parental UAMS-1 (but not the ΔlytS mutant) became more resistant to hNP-1 and DAP following pretreatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (a CM-depolarizing agent). Of note, lytSR-dependent survival against CAP/HDP killing was not associated with changes in either surface positive charge, expression of mprF and dlt, or CM fluidity. The ΔlytS strain (but not the ΔlrgAB mutant) displayed a significant reduction in target tissue survival in an endocarditis model during DAP treatment. Collectively, these results suggest that the lytSR TCRS plays an important role in adaptive responses of S. aureus to CM-perturbing HDPs/CAPs, likely by functioning as a sense-response system for detecting subtle changes in ΔΨ.
doi:10.1128/AAC.00412-13
PMCID: PMC3719743  PMID: 23733465
9.  Mechanisms of daptomycin resistance in Staphylococcus aureus: role of the cell membrane and cell wall 
The bactericidal, cell membrane-targeting lipopeptide antibiotic daptomycin (DAP) is an important agent in treating invasive Staphylococcus aureus infections. However, there have been numerous recent reports of development of daptomycin-resistance (DAP-R) during therapy with this agent. The mechanisms of DAP-R in S. aureus appear to be quite diverse. DAP-R strains often exhibit progressive accumulation of single nucleotide polymorphisms in the multipeptide resistance factor gene (mprF) and the yycFG components of the yycFGHI operon. Both loci are involved in key cell membrane (CM) events, with mprF being responsible for the synthesis and outer CM translocation of the positively-charged phospholipid, lysyl-phosphotidylglycerol (L-PG), while the yyc operon is involved in the generalized response to stressors such as antimicrobials. In addition, other perturbations of the CM have been identified in DAP-R strains including: extremes in CM order; resistance to CM depolarization and permeabilization; and reduced surface binding of DAP. Moreover, modifications of the cell wall (CW) appear to also contribute to DAP-R, including enhanced expression of the dlt operon (involved in D-alanylation of CW teichoic acids) and progressive CW thickening.
doi:10.1111/j.1749-6632.2012.06819.x
PMCID: PMC3556211  PMID: 23215859
Staphylococcus aureus; daptomycin; antibiotic resistance; endocarditis
10.  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
11.  Reduced Vancomycin Susceptibility in an In Vitro Catheter-Related Biofilm Model Correlates with Poor Therapeutic Outcomes in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus 
Staphylococcus aureus is the most common cause of endovascular infections, including catheter sepsis and infective endocarditis (IE). Vancomycin (VAN) is the primary choice for treatment of methicillin-resistant S. aureus (MRSA) infections. However, high rates of VAN treatment failure in MRSA infections caused by VAN-susceptible strains have been increasingly reported. Biofilm-associated MRSA infections are especially prone to clinical antibiotic failure. The present studies examined potential relationships between MRSA susceptibility to VAN in biofilms in vitro and nonsusceptibility to VAN in endovascular infection in vivo. Using 10 “VAN-susceptible” MRSA bloodstream isolates previously investigated for VAN responsiveness in experimental IE, we studied the mechanism(s) of such in vivo VAN resistance, including: (i) VAN binding to MRSA organisms; (ii) the impact of VAN on biofilm formation and biofilm composition; (iii) VAN efficacy in an in vitro catheter-related biofilm model; (iv) effects on cell wall thickness. As a group, the five strains previously categorized as VAN nonresponders (non-Rsp) in the experimental IE model differed from the five responders (Rsp) in terms of lower VAN binding, increased biofilm formation, higher survival in the presence of VAN within biofilms in the presence or absence of catheters, and greater biofilm reduction upon proteinase K treatment. Interestingly, sub-MICs of VAN significantly promoted biofilm formation only in the non-Rsp isolates. Cell wall thickness was similar among all MRSA strains. These results suggest that sublethal VAN levels that induce biofilm formation and reduce efficacy of VAN in the in vitro catheter-associated biofilms may contribute to suboptimal treatment outcomes for endovascular infections caused by “VAN-susceptible” MRSA strains.
doi:10.1128/AAC.02073-12
PMCID: PMC3591927  PMID: 23295925
12.  Emergence of Daptomycin Resistance in Daptomycin-Naïve Rabbits with Methicillin-Resistant Staphylococcus aureus Prosthetic Joint Infection Is Associated with Resistance to Host Defense Cationic Peptides and mprF Polymorphisms 
PLoS ONE  2013;8(8):e71151.
Background
Previous studies of both clinically-derived and in vitro passage-derived daptomycin–resistant (DAP-R) Staphylococcus aureus strains demonstrated the coincident emergence of increased DAP MICs and resistance to host defense cationic peptides (HDP-R).
Methods
In the present investigation, we studied a parental DAP-susceptible (DAP-S) methicillin-resistant Staphylococcus aureus (MRSA) strain and three isogenic variants with increased DAP MICs which were isolated from both DAP-treated and DAP-untreated rabbits with prosthetic joint infections. These strains were compared for: in vitro susceptibility to distinct HDPs differing in size, structure, and origin; i.e.; thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]; cell membrane (CM) phospholipid and fatty acid content; CM order; envelope surface charge; cell wall thickness; and mprF single nucleotide polymorphisms (SNPs) and expression profiles.
Results
In comparison with the parental strain, both DAP-exposed and DAP-naive strains exhibited: (i) significantly reduced susceptibility to each HDP (P<0.05); (ii) thicker cell walls (P<0.05); (iii) increased synthesis of CM lysyl-phosphatidylglycerol (L-PG); (iv) reduced content of CM phosphatidylglycerol (PG); and (v) SNPs within the mprF locus No significant differences were observed between parental or variant strains in outer CM content of L-PG, CM fluidity, CM fatty acid contents, surface charge, mprF expression profiles or MprF protein content. An isolate which underwent identical in vivo passage, but without evolving increased DAP MICs, retained parental phenotypes and genotype.
Conclusions
These results suggest: i) DAP MIC increases may occur in the absence of DAP exposures in vivo and may be triggered by organism exposure to endogenous HDPs: and ii) gain-in-function SNPs in mprF may contribute to such HDP-DAP cross-resistance phenotypes, although the mechanism of this relationship remains to be defined.
doi:10.1371/journal.pone.0071151
PMCID: PMC3747195  PMID: 23990934
13.  Correlation of Cell Membrane Lipid Profiles with Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus 
We compared the cell membrane (CM) lipid composition among nine well-characterized daptomycin-susceptible (Daps)/Dap-resistant (Dapr) methicillin-resistant Staphylococcus aureus (MRSA) strain pairs. Compared to the 9 Daps parental strains, Dapr strains (with or without mprF-yycFG mutations) exhibited significantly reduced phosphatidylglycerol (PG) content (P < 0.01), significantly increased total synthesis of lysyl-PG (LPG) (P < 0.01), and reduced carotenoid content (P < 0.05 for 5/9 strains). There were no significant changes in LPG flipping, cardiolipin content, or fatty acid composition among strain pairs.
doi:10.1128/AAC.02182-12
PMCID: PMC3553710  PMID: 23254419
14.  Daptomycin-Resistant Enterococcus faecalis Diverts the Antibiotic Molecule from the Division Septum and Remodels Cell Membrane Phospholipids 
mBio  2013;4(4):e00281-13.
ABSTRACT
Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides.
IMPORTANCE  The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.
IMPORTANCE 
The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.
doi:10.1128/mBio.00281-13
PMCID: PMC3735187  PMID: 23882013
15.  Whole-Genome Analysis of a Daptomycin-Susceptible Enterococcus faecium Strain and Its Daptomycin-Resistant Variant Arising during Therapy 
Development of daptomycin (DAP) resistance in Enterococcus faecalis has recently been associated with mutations in genes encoding proteins with two main functions: (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase [cls]). However, the genetic bases for DAP resistance in Enterococcus faecium are unclear. We performed whole-genome comparative analysis of a clinical strain pair, DAP-susceptible E. faecium S447 and its DAP-resistant derivative R446, which was recovered from a single patient during DAP therapy. By comparative whole-genome sequencing, DAP resistance in R446 was associated with changes in 8 genes. Two of these genes encoded proteins involved in phospholipid metabolism: (i) an R218Q substitution in Cls and (ii) an A292G reversion in a putative cyclopropane fatty acid synthase enzyme. The DAP-resistant derivative R446 also exhibited an S333L substitution in the putative histidine kinase YycG, a member of the YycFG system, which, similar to LiaFSR, has been involved in cell envelope homeostasis and DAP resistance in other Gram-positive cocci. Additional changes identified in E. faecium R446 (DAP resistant) included two putative proteins involved in transport (one for carbohydrate and one for sulfate) and three enzymes predicted to play a role in general metabolism. Exchange of the “susceptible” cls allele from S447 for the “resistant” one belonging to R446 did not affect DAP susceptibility. Our results suggest that, apart from the LiaFSR system, the essential YycFG system is likely to be an important mediator of DAP resistance in some E. faecium strains.
doi:10.1128/AAC.01454-12
PMCID: PMC3535923  PMID: 23114757
16.  Increased Cell Wall Teichoic Acid Production and D-alanylation Are Common Phenotypes among Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus (MRSA) Clinical Isolates 
PLoS ONE  2013;8(6):e67398.
Multiple mechanisms have been correlated with daptomycin-resistance (DAP-R) in Staphylococcus aureus. However, one common phenotype observed in many DAP-R S. Aureus strains is a thickened cell wall (CW). The first evidence for an impact of CW-linked glycopolymers on this phenotype was recently demonstrated in a single, well-characterized DAP-R methicillin-susceptible S. aureus (MSSA) strain. In this isolate the thickened CW phenotype was linked to an increased production and D-alanylation of wall teichoic acids (WTA). In the current report, we extended these observations to methicillin-resistant daptomycin-sensitive/daptomyin-resistant (DAP-S/DAP-R) strain-pairs. These pairs included methicillin-resistant S. aureus (MRSA) isolates with and without single nucleotide polymorphisms (SNPs) in mprF (a genetic locus linked to DAP-R phenotype). We found increased CW dry mass in all DAP-R vs DAP-S isolates. This correlated with an increased expression of the WTA biosynthesis gene tagA, as well as an increased amount of WTA in the DAP-R vs DAP-S isolates. In addition, all DAP-R isolates showed a higher proportion of WTA D-alanylation vs their corresponding DAP-S isolate. We also detected an increased positive surface charge amongst the DAP-R strains (presumably related to the enhanced D-alanylation). In comparing the detailed CW composition of all isolate pairs, substantive differences were only detected in one DAP-S/DAP-R pair. The thickened CW phenotype, together with an increased surface charge most likely contributes to either: i) a charge-dependent repulsion of calcium complexed-DAP; and/or ii) steric-limited access of DAP to the bacterial cell envelope target. Taken together well-defined perturbations of CW structural and functional metrics contribute to the DAP-R phenotype and are common phenotypes in DAP-R S. Aureus isolates, both MSSA and MRSA.
Note: Although “daptomycin-nonsusceptibility” is the generally accepted terminology, we have utilized the term “daptomycin resistance” for ease of presentation in this manuscript
doi:10.1371/journal.pone.0067398
PMCID: PMC3681945  PMID: 23785522
17.  Telavancin in Therapy of Experimental Aortic Valve Endocarditis in Rabbits Due to Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus 
Antimicrobial Agents and Chemotherapy  2012;56(11):5528-5533.
A number of cases of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains that have developed daptomycin resistance (DAP-R) have been reported. Telavancin (TLV) is a lipoglycopeptide agent with a dual mechanism of activity (cell wall synthesis inhibition plus depolarization of the bacterial cell membrane). Five recent daptomycin-susceptible (DAP-S)/DAP-R MRSA isogenic strain pairs were evaluated for in vitro TLV susceptibility. All five DAP-R strains (DAP MICs ranging from 2 to 4 μg/ml) were susceptible to TLV (MICs of ≤0.38 μg/ml). In vitro time-kill analyses also revealed that several TLV concentrations (1-, 2-, and 4-fold MICs) caused rapid killing against the DAP-R strains. Moreover, for 3 of 5 DAP-R strains (REF2145, A215, and B2.0), supra-MICs of TLV were effective at preventing regrowth at 24 h of incubation. Further, the combination of TLV plus oxacillin (at 0.25× or 0.50× MIC for each agent) increased killing of DAP-R MRSA strains REF2145 and A215 at 24 h (∼2-log and 5-log reductions versus TLV and oxacillin alone, respectively). Finally, using a rabbit model of aortic valve endocarditis caused by DAP-R strain REF2145, TLV therapy produced a mean reduction of >4.5 log10 CFU/g in vegetations, kidneys, and spleen compared to untreated or DAP-treated rabbits. Moreover, TLV-treated rabbits had a significantly higher percentage of sterile tissue cultures (87% in vegetations and 100% in kidney and spleen) than all other treatment groups (P < 0.0001). Together, these results demonstrate that TLV has potent bactericidal activity in vitro and in vivo against DAP-R MRSA isolates.
doi:10.1128/AAC.00922-12
PMCID: PMC3486568  PMID: 22890759
18.  Clinical Presentation, Etiology and Outcome of Infective Endocarditis in the 21st Century: The International Collaboration on Endocarditis-Prospective Cohort Study 
Archives of internal medicine  2009;169(5):463-473.
Background
The aim of this study was to provide a contemporary picture of the presentation, etiology and outcome of infective endocarditis (IE) in a large patient cohort from multiple locations worldwide.
Methods
Prospective cohort study of 2781 adults with definite IE admitted to 58 hospitals in 25 countries between June 2000 and September 2005.
Results
The median age of the cohort was 57.9 (IQR 43.2–71.8) years and 72% had native valve IE. Most (77%) patients presented early in the disease (<30 days) with few of the classic clinical hallmarks of IE. Recent health-care exposure was found in one quarter of patients. Staphylococcus aureus was the most common pathogen (31%). Mitral (41%) and aortic (38%) valves were infected most commonly. Complications were common: stroke (17%); embolization other than stroke (23%); heart failure (32%) and intracardiac abscess (14%). Surgical therapy was common (48%) and in-hospital mortality remained high (18%). Prosthetic valve involvement (OR 1.47, 95%CI 1.13–1.90), increasing age (OR 1.30, 95%CI 1.17–1.46 per 10-year interval), pulmonary edema (OR 1.79, 95%CI 1.39–2.30), S. aureus infection (OR 1.54, 95%CI 1.14–2.08), coagulase-negative staphylococcal infection (OR 1.50, 95%CI 1.07–2.10), mitral valve vegetation (OR 1.34, 95%CI 1.06–1.68), and paravalvular complications (OR 2.25, 95%CI 1.64–3.09) were associated with increased risk of in-hospital death, while viridans streptococcal infection (OR 0.52, 95%CI 0.33–0.81) and surgery (OR 0.61, 95%CI 0.44–0.83) were associated with decreased risk.
Conclusions
In the early 21st century, IE is more often an acute disease, characterized by a high rate of S. aureus infection. Mortality remains relatively high.
doi:10.1001/archinternmed.2008.603
PMCID: PMC3625651  PMID: 19273776
19.  In vitro Endothelial Cell Damage is Positively Correlated with Enhanced Virulence and Poor Vancomycin Responsiveness in Experimental Endocarditis due to Methicillin Resistant Staphylococcus aureus 
Cellular microbiology  2011;13(10):1530-1541.
Summary
The pathogenesis of Staphylococcus aureus infective endocarditis (IE) is postulated to involve invasion and damage of endothelial cells (ECs). However, the precise relationships between S. aureus – EC interactions in vitro and IE virulence and treatment outcomes in vivo are poorly defined. Ten methicillin-resistant S. aureus (MRSA) clinical isolates previously tested for their virulence and vancomycin responsiveness in an experimental IE model were assessed in vitro for their hemolytic activity, protease production, and capacity to invade and damage ECs. There was a significant positive correlation between the in vitro EC damage caused by these MRSA strains and their virulence during experimental IE (in terms of bacterial densities in target tissues; P < 0.02). Importantly, higher EC damage was also significantly correlated with poor microbiologic response to vancomycin in the IE model (P < 0.001). Interestingly, the extent of EC damage was unrelated to a strain's ability to invade ECs, hemolytic activity and protease production, or β-toxin gene transcription. Inactivation of the agr locus in two MRSA strains caused ∼20% less damage as compared to the corresponding parental strains, indicating that a functional agr is required for maximal EC damage induction. Thus, MRSA-induced EC damage in vitro is a unique virulence phenotype that is independent of many other prototypical MRSA virulence factors, and may be a key biomarker for predicting MRSA virulence potential and antibiotic outcomes during endovascular infections.
doi:10.1111/j.1462-5822.2011.01639.x
PMCID: PMC3173605  PMID: 21777408
20.  Daptomycin Resistance in Enterococci Is Associated with Distinct Alterations of Cell Membrane Phospholipid Content 
PLoS ONE  2012;7(8):e43958.
Background
The lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains.
Methodology
Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs.
Principal Findings
Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i) phosphatidic acid; and ii) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM.
Conclusion
Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.
doi:10.1371/journal.pone.0043958
PMCID: PMC3428275  PMID: 22952824
21.  Differential Adaptations of Methicillin-Resistant Staphylococcus aureus to Serial In Vitro Passage in Daptomycin: Evolution of Daptomycin Resistance and Role of Membrane Carotenoid Content and Fluidity 
Previous studies showed serial 20 d in vitro passage of MRSA strain MW2 in sublethal daptomycin (DAP) resulted in diverse perturbations in both cell membrane (CM) and cell wall (CW) characteristics, including increased CM rigidity; increased CW thickness; “gain-in-function” single nucleotide polymorphisms (SNPs) in the mprF locus (i.e., increased synthesis and translocation of lysyl-phosphatidylglycerol (L-PG)); progressive accumulation of SNPs in yyc and rpo locus genes; reduced carotenoid production; cross-resistance to innate host defense peptides. The current study was designed to characterize the reproducibility of these phenotypic and genotypic modifications following in vitro serial passages of the same parental strain. After a second 20d serial in vitro passage of parental MW2, emergence of DAP-R was associated with evolution of several phenotypes closely mirroring previous passage outcomes. However, in contrast to the initial serial passage strain set, we observed (i) only modest increase in L-PG synthesis and no increase in L-PG outer CM translocation; (ii) significantly increased carotenoid synthesis (P < 0.05); (iii) a different order of SNP accumulations (mprF ≫ rpoB ≫ yycG); (iv) a different cadre and locations of such SNPs. Thus, MRSA strains are not “pre-programmed” to phenotypically and/or genotypically adapt in an identical manner during induction of DAP resistance.
doi:10.1155/2012/683450
PMCID: PMC3431083  PMID: 22956961
22.  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
23.  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
24.  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
25.  The Staphylococcus aureus Two-Component Regulatory System, GraRS, Senses and Confers Resistance to Selected Cationic Antimicrobial Peptides 
Infection and Immunity  2012;80(1):74-81.
The two-component regulatory system, GraRS, appears to be involved in staphylococcal responses to cationic antimicrobial peptides (CAPs). However, the mechanism(s) by which GraRS is induced, regulated, and modulated remain undefined. In this study, we used two well-characterized MRSA strains (Mu50 and COL) and their respective mutants of graR and vraG (encoding the ABC transporter-dependent efflux pump immediately downstream of graRS), and show that (i) the expression of two key determinants of net positive surface charge (mprF and dlt) is dependent on the cotranscription of both graR and vraG, (ii) reduced expression of mprF and dlt in graR mutants was phenotypically associated with reduced surface-positive charge, (iii) this net reduction in surface-positive charge in graR and vraG mutants, in turn, correlated with enhanced killing by a range of CAPs of diverse structure and origin, including those from mammalian platelets (tPMPs) and neutrophils (hNP-1) and from bacteria (polymyxin B), and (iv) the synthesis and translocation of membrane lysyl-phosphatidylglycerol (an mprF-dependent function) was substantially lower in graR and vraG mutants than in parental strains. Importantly, the inducibility of mprF and dlt transcription via the graRS-vraFG pathway was selective, with induction by sublethal exposure to the CAPs, RP-1 (platelets), and polymyxin B, but not by other cationic molecules (hNP-1, vancomycin, gentamicin, or calcium-daptomycin). Although graR regulates expression of vraG, the expression of graR was codependent on an intact downstream vraG locus. Collectively, these data support an important role of the graRS and vraFG loci in the sensing of and response to specific CAPs involved in innate host defenses.
doi:10.1128/IAI.05669-11
PMCID: PMC3255649  PMID: 21986630

Results 1-25 (86)