Daptomycin Nonsusceptibility Research Articles

Failure of daptomycin β-Lactam combination therapy to prevent resistance emergence in Enterococcus faecium

Daptomycin β-Lactam combination therapy offers “protection” against daptomycin non-susceptibility (DNS) development in Enterococcus faecium. We report failure of this strategy and the importance of source control. Mutations were detected in the LiaF and cls genes in DNS isolates. A single DNS isolate contained an unrecognized mutation, which requires confirmation.

Emergence of Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus Clinical Isolates Among Daptomycin-Naive Patients in Korea.

This study was conducted to assess emergence of daptomycin-nonsusceptible (DAP-NS) phenotype in DAP-naive patients with invasive Staphylococcus aureus (ISA) infections in Korea. A total of 208 S. aureus clinical isolates were selected from a previous prospective study on ISA infections and evaluated for DAP-NS. Although DAP has never been introduced in Korea, five DAP-NS S. aureus strains (2.4%) were identified among 208 S. aureus strains collected from ISA infections. The DAP-NS phenotype was observed only in methicillin-resistant S. aureus (MRSA) strains, but not in methicillin-susceptible S. aureus strains. One DAP-NS MRSA strain belonged to sequence type 72 (ST72) and four were ST5 MRSA strains, three of which were heteroresistant vancomycin (VAN)-intermediate S. aureus. All these five DAP-NS MRSA strains were from healthcare-associated infections without prior exposure to VAN within 30 days. While the ST72 MRSA strain exhibited DAP-NS phenotype via charge repulsion mechanism, four ST5 DAP-NS S. aureus strains had charge-independent DAP-NS mechanism. None of the five DAP-NS strains displayed significant increase in cell wall thickness, indicating that altered cell wall thickness was not associated with the observed DAP-NS phenotype.

Early in vitro development of daptomycin non-susceptibility in high-level aminoglycoside-resistant Enterococcus faecalis predicts the efficacy of the combination of high-dose daptomycin plus ampicillin in an in vivo model of experimental endocarditis

Previous studies showed development of daptomycin non-susceptibility (DNS: MIC >4 mg/L) in Enterococcus faecalis infections. However, no studies have assessed the efficacy of the combination of daptomycin/ampicillin against E. faecalis strains developing DNS in the experimental endocarditis (EE) model. To assess the in vitro and in vivo efficacy of daptomycin at 10 mg/kg/day, daptomycin/ampicillin and ampicillin/ceftriaxone against two high-level aminoglycoside-resistant E. faecalis strains, one developing DNS after in vitro exposure to daptomycin and another that did not (DS). Subculture of 82 E. faecalis strains from patients with endocarditis with daptomycin MICs, time-kill and in vivo experiments using the EE model. 33% of the strains (27 of 82) displayed DNS after subculture with daptomycin. Daptomycin MIC rose from 0.5-2 to 8-16 mg/L. In time-kill experiments, when using a high inoculum (10 8 cfu/mL), daptomycin/ampicillin was synergistic for one-third of DS strains and none of DNS strains, while ampicillin/ceftriaxone retained synergy in all cases. In the EE model, daptomycin did not significantly reduce cfu/g from vegetations compared with control against either strain, while daptomycin/ampicillin reduced significantly more cfu/g than daptomycin against the DS strain, but not against the DNS strain [2.9 (2.0-4.1) versus 6.1 (4.5-8.0); P = 0.002]. Ampicillin/ceftriaxone was synergistic and bactericidal against both strains, displaying the same activity as daptomycin/ampicillin against the DS strain. Performance of an Etest for daptomycin MIC after subculture with daptomycin inhibitory doses on strains of high-level aminoglycoside-resistant E. faecalis endocarditis may be an easy test to predict the in vivo efficacy of daptomycin/ampicillin.

Increasing rate of daptomycin non-susceptible strains of Staphylococcus aureus in patients with atopic dermatitis.

IntroductionDaptomycin is a cyclic lipopeptide that is bactericidal against Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) strains. Daptomycin exerts its antimicrobial effect by a calcium-dependent interaction with the cytoplasmic membrane resulting in depolarization, ion loss and rapid cell death. Unfortunately, loss of daptomycin susceptibility in S. aureus in the clinical setting has been noted.AimTo evaluate the susceptibility profile to daptomycin among S. aureus strains isloted from patients with atopic dermatitis (AD). Another point was to correlate the results obtained by broth microdilution method and Etest, which is commonly applied in clinical setting.Material and methodsOne hundred patients with the diagnosis of atopic dermatitis were microbiologically assessed for the carriage of S. aureus. Antimicrobial susceptibility tests were performed using broth-microdilution (BMD) and Etests for daptomycin.Results Staphylococcus aureus strains were isolated from the majority of our patients, either from the skin (73%) or the anterior nares (75%). Six of the 100 nasal swabs (6%) and 5 of the 100 skin swabs (5%) were positive for methicillin-resistant Staphylococcus aureus (MRSA). A total of 81 of 148 (54.7%) daptomycin non-susceptible isolates of S. aureus were identified by BMD. Only 19 of 81 were also classified as non-susceptible by Etest.ConclusionsClinicians and microbiologists should be aware of the possibility of the emergence of daptomycin non-susceptibility (or increase in minimal inhibitory concentration) during prolonged therapy and closely monitor the susceptibility of persisting isolates that might be recovered during therapy.

Characteristics of Tedizolid Non-susceptible Enterococcal Clinical Isolates

BackgroundTedizolid is a novel oxazolidinone antibiotic with activity across a broad range of gram-positive pathogens. The aim of this study was to describe the characteristics of tedizolid (TZD) resistant enterococcal clinical isolates.MethodsTedizolid resistant isolates were recovered from patients at Westchester Medical Center, New York from 2012 to 2016. In vitrosusceptibility of tedizolid isolates was performed by broth microdilution using the Sensititre™ panel in accordance with the guidelines of the Clinical and Laboratory Standards Institute. The sequence type (ST) of enterococci was determined based on multilocus sequence typing (MLST) data derived from assembled next-generation sequencing.ResultsDuring the study period, we identified 8 clinical isolates which were resistant to Tedizolid. Clinical characteristics, genomic, treatment and outcome data are summarized in Table 1. 7/8 isolates were E. faecium belonging to a unique ST736 which predominates in our hospital. 7/8 isolates showed G2567T mutation of 23S rRNA. Only one patient had prior reciept of Linezolid. None of the patients had recieved Tedizolid in past.ConclusionThere is de-novo emergence of Tedizolid resistant enterococcal isolates. This along with higher incidence daptomycin non-susceptibility in our institution is of significant concern. This highlights the need for further improvement in infection control practices and newer options for the treatment of enterococcal infections.Disclosures A. Dhand, Merck, Pfizer, Allergan: Speaker’s Bureau, Speaker honorarium; Astellas: Consultant, Consulting feeDateSiteSequence typePrior Linezolid useMIC mg/LTreatmentOutcomeLINTZDDAPVAN62/F2012Urine736No81161NA-59/F2013Peritoneum736Yes826>16DAP x 14 dCure61/F2013BloodNANo>8242Tigecycline x 4 d + Doxycycline x 10 dCure80/M2015Urine736No1612>16DAP x 7 dCure47/F2015Urine736No814>16DAP x 21 dCure72/F2015Urine736No818>16NA-75/M2015Urine736No824>16NA-60/F2016Peritoneum736No1623>16DAP x 21 dCure

A single-center experience with infections due to daptomycin-nonsusceptible Enterococcus faecium in liver transplant recipients.

Infections caused by vancomycin-resistant Enterococcus faecium (VRE) are a major cause of morbidity and mortality in the liver transplant population. Daptomycin (DAP) is often used to treat infections caused by VRE, but DAP nonsusceptibility in Enterococcus is increasing. Patients with DAP-nonsusceptible Enterococcus (DNSE) infections who had undergone liver transplantation between January 1, 2010 and July 31, 2014 were retrospectively reviewed. A convenience sample of DNSE isolates was analyzed by pulsed-field gel electrophoresis (PFGE). We identified 14 liver transplant recipients (LTRs) who developed DNSE infections post transplantation. Postoperative complications were common, and most patients required repeat abdominal surgery within 90 days of transplantation. The initial DNSE culture was taken a median of 74.5 days post transplant and was secondary to an intra-abdominal infection in all but 1 patient. Half of patients were VRE colonized before or at the time of organ transplantation, and all those who were not VRE colonized at the time of transplantation later became colonized, a median of 27 days post transplant. Overall mortality in this cohort was 71%. PFGE did not demonstrate genetic relatedness among DNSE isolates. This study, the largest published series to our knowledge of DNSE infections in LTRs, demonstrates that these infections occur in patients with serious surgical complications and are associated with high morbidity and mortality. Established risk factors for VRE infection were common, as was DAP exposure. Although many risk factors for DNSE infection cannot be changed, this case series identifies several potentially modifiable variables. Further work is needed to identify interventions to decrease the risk of developing DNSE infections in this complex patient population.

Oritavancin Combinations with β-Lactams against Multidrug-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococci.

Oritavancin possesses activity against vancomycin-resistant enterococci (VRE) and methicillin-resistantStaphylococcus aureus(MRSA).In vitrodata suggest synergy between beta-lactams (BLs) and vancomycin or daptomycin, agents similar to oritavancin. We evaluated the activities of BLs combined with oritavancin against MRSA and VRE. Oritavancin MICs were determined for 30 strains, 5 each of MRSA, daptomycin-nonsusceptible (DNS) MRSA, vancomycin-intermediate MRSA (VISA), heteroresistant VISA (hVISA), vancomycin-resistantEnterococcus faecalis, and vancomycin-resistantEnterococcus faecium Oritavancin MICs were determined in the presence of subinhibitory concentrations of BLs. Oritavancin combined with ceftaroline, cefazolin, or nafcillin was evaluated for lethal synergy against MRSA, and oritavancin combined with ceftaroline, ampicillin, or ertapenem was evaluated for lethal synergy against VRE in 24-h time-kill assays. Oritavancin at 0.5× the MIC was combined with BLs at 0.5× the MIC or the biological free peak concentration, whichever one was lower. Synergy was defined as a ≥2-log10-CFU/ml difference between the killing achieved with the combination and that achieved with the most active single agent at 24 h. Oritavancin MICs were ≤0.125 μg/ml for all MRSA isolates except three VISA isolates with MICs of 0.25 μg/ml. Oritavancin MICs for VRE ranged from 0.03 to 0.125 μg/ml. Oritavancin in combination with ceftaroline was synergistic against all MRSA phenotypes and statistically superior to all other combinations against DNS MRSA, hVISA, and MRSA isolates (P< 0.02). Oritavancin in combination with cefazolin and oritavancin in combination with nafcillin were also synergistic against all MRSA strains. Synergy between oritavancin and all BLs was revealed against VRE strain 8019, while synergy between oritavancin and ampicillin or ertapenem but not ceftaroline was demonstrated against VRE strain R7164. The data support the potential use of oritavancin in combination with BLs, especially oritavancin in combination with ceftaroline, for the treatment of infections caused by MRSA. The data from the present study are not as strong for oritavancin in combination with BLs for VRE. Further study of both MRSA and VRE in more complex models is warranted.

Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci.

Chlorhexidine is a bisbiguanide antiseptic used for infection control. Vancomycin-resistantE. faecium(VREfm) is among the leading causes of hospital-acquired infections. VREfm may be exposed to chlorhexidine at supra- and subinhibitory concentrations as a result of chlorhexidine bathing and chlorhexidine-impregnated central venous catheter use. We used RNA sequencing to investigate how VREfm responds to chlorhexidine gluconate exposure. Among the 35 genes upregulated ≥10-fold after 15 min of exposure to the MIC of chlorhexidine gluconate were those encoding VanA-type vancomycin resistance (vanHAX) and those associated with reduced daptomycin susceptibility (liaXYZ). We confirmed thatvanAupregulation was not strain or species specific by querying other VanA-type VRE. VanB-type genes were not induced. ThevanHpromoter was found to be responsive to subinhibitory chlorhexidine gluconate in VREfm, as was production of the VanX protein. UsingvanHreporter experiments withBacillus subtilisand deletion analysis in VREfm, we found that this phenomenon is VanR dependent. Deletion ofvanRdid not result in increased chlorhexidine susceptibility, demonstrating thatvanHAXinduction is not protective against chlorhexidine. As expected, VanA-type VRE is more susceptible to ceftriaxone in the presence of sub-MIC chlorhexidine. Unexpectedly, VREfm is also more susceptible to vancomycin in the presence of subinhibitory chlorhexidine, suggesting that chlorhexidine-induced gene expression changes lead to additional alterations in cell wall synthesis. We conclude that chlorhexidine induces expression of VanA-type vancomycin resistance genes and genes associated with daptomycin nonsusceptibility. Overall, our results indicate that the impacts of subinhibitory chlorhexidine exposure on hospital-associated pathogens should be further investigated in laboratory studies.

Bacterial Cytological Profiling (BCP) as a Rapid and Accurate Antimicrobial Susceptibility Testing Method for Staphylococcus aureus

Successful treatment of bacterial infections requires the timely administration of appropriate antimicrobial therapy. The failure to initiate the correct therapy in a timely fashion results in poor clinical outcomes, longer hospital stays, and higher medical costs. Current approaches to antibiotic susceptibility testing of cultured pathogens have key limitations ranging from long run times to dependence on prior knowledge of genetic mechanisms of resistance. We have developed a rapid antimicrobial susceptibility assay for Staphylococcus aureus based on bacterial cytological profiling (BCP), which uses quantitative fluorescence microscopy to measure antibiotic induced changes in cellular architecture. BCP discriminated between methicillin-susceptible (MSSA) and -resistant (MRSA) clinical isolates of S. aureus (n=71) within 1–2h with 100% accuracy. Similarly, BCP correctly distinguished daptomycin susceptible (DS) from daptomycin non-susceptible (DNS) S. aureus strains (n=20) within 30min. Among MRSA isolates, BCP further identified two classes of strains that differ in their susceptibility to specific combinations of beta-lactam antibiotics. BCP provides a rapid and flexible alternative to gene-based susceptibility testing methods for S. aureus, and should be readily adaptable to different antibiotics and bacterial species as new mechanisms of resistance or multidrug-resistant pathogens evolve and appear in mainstream clinical practice.

Evaluation of tedizolid against Staphylococcus aureus and enterococci with reduced susceptibility to vancomycin, daptomycin or linezolid.

Tedizolid displays potent activity against Gram-positive pathogens. In vitro studies against clinical isolates of Staphylococcus aureus and enterococci demonstrated improved tedizolid activity over linezolid. However, this is not well-characterized against a large collection of resistant isolates, including vancomycin-intermediate S. aureus (VISA), heterogeneous VISA (hVISA), daptomycin-non-susceptible (DNS) S. aureus, linezolid-resistant (LR) S. aureus and VRE. Therefore, our objective was to determine tedizolid activity versus other agents, against MRSA and VRE with various resistance phenotypes. In total, 302 MRSA (75 DNS, 100 VISA, 120 hVISA and 7 LR) and 220 VRE [100 Enterococcus faecalis (all susceptible to daptomycin and linezolid) and 120 E. faecium (25 DNS and 10 LR)] were evaluated. LR isolates were analysed for the cfr gene. MICs of tedizolid, linezolid, ampicillin, clindamycin, daptomycin, gentamicin, levofloxacin, oxacillin, tigecycline, trimethoprim/sulfamethoxazole and vancomycin were determined in accordance with CLSI guidelines. Tedizolid MIC90 values for hVISA, VISA and DNS were 0.5 mg/L (versus 4, 4 and 2 mg/L, respectively, for linezolid). The tedizolid MIC range for LR MRSA was 0.063-1 mg/L. Two LR MRSA possessed the cfr gene with tedizolid MICs of 0.125 and 0.25 mg/L (linezolid MICs of 16 and 8 mg/L). The tedizolid MIC90 for vancomycin-resistant E. faecalis and E. faecium was 0.25 and 1 mg/L, respectively; three dilutions lower for E. faecalis and two dilutions lower for E. faecium compared with linezolid. Tedizolid MICs demonstrate activity against isolates with decreased susceptibility to alternative agents, including linezolid. Tedizolid may be a viable treatment option in clinical situations with MDR Gram-positive pathogens.

Emergence of daptomycin non-susceptibility in colonizing vancomycin-resistant Enterococcus faecium isolates during daptomycin therapy

Infections due to vancomycin-resistant enterococci (VRE) are of significant importance in high-risk populations, and daptomycin is a bactericidal antibiotic to treat multidrug-resistant VRE in these patients. The emergence of daptomycin non-susceptibility invasive VRE during daptomycin therapy is a major clinical issue. Here the hypothesis was tested that systemic daptomycin therapy also induces the emergence of daptomycin non-susceptible (DNS-) isolates in colonizing VRE populations. 11 vancomycin-resistant Enterococcus faecium strain pairs recovered from rectal swabs were available for analysis. All initial isolates exhibited daptomycin MICs within the wild type MIC distribution of E. faecium (MIC≤4mg/L). In follow-up isolates from five patients a 4–16-fold daptomycin MIC increase was detected. All patients carrying DNS-VRE received daptomycin (14–28 days) at 4mg/kg body weight, while two patients in whom no DNS-VRE emerged were only treated with daptomycin for 1 and 4 days, respectively. Comparative whole genome sequencing identified DNS-VRE-specific single nucleotide polymorphisms (SNP), including mutations in cardiolipin synthase (Cls), and additional SNPs in independent genes potentially relevant for the DNS phenotype. Mutations within cls were also identified in three additional, colonizing DNS-VRE. Of these, at least one strain was transmitted within the hospital. In none of the VRE isolates tested, pre-existing or de novo mutations in the liaFSR operon were detected. This is the first report documenting the emergence of DNS-VRE in colonizing strains during daptomycin treatment, putting the patient at risk for subsequent DNS-VRE infections and priming the spread of DNS-VRE within the hospital environment.

Stepwise decrease in daptomycin susceptibility in clinical Staphylococcus aureus isolates associated with an initial mutation in rpoB and a compensatory inactivation of the clpX gene.

Daptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of daptomycin-nonsusceptible S. aureus isolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) in rpoB (RpoB A477D) that decreased susceptibility not only to daptomycin but also to vancomycin, β-lactams, and rifampin. Furthermore, the rpoB mutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP in mprF (MprF S295L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin nonsusceptibility, growth fitness, and virulence traits.

Telavancin demonstrates activity against methicillin-resistant Staphylococcus aureus isolates with reduced susceptibility to vancomycin, daptomycin, and linezolid in broth microdilution MIC and one-compartment pharmacokinetic/pharmacodynamic models.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates have arisen with reduced susceptibility to several anti-MRSA agents. Telavancin (TLV), a novel anti-MRSA agent, retains low MICs against these organisms. Our objective was to determine the MICs for TLV, daptomycin (DAP), vancomycin (VAN), and linezolid (LZD) against daptomycin-nonsusceptible (DNS) S. aureus, vancomycin-intermediate S. aureus (VISA), heteroresistant VISA (hVISA), and linezolid-resistant (LZD(r)) S. aureus. We also evaluated these agents against each phenotype in pharmacokinetic/pharmacodynamic (PK/PD) models. Seventy DNS, 100 VISA, 180 hVISA, and 25 LZD(r) MRSA isolates were randomly selected from our library and tested to determine their MICs against TLV, DAP, VAN, and LZD via broth microdilution and a Trek panel. Four isolates were randomly selected for 168-h in vitro models to evaluate treatment with TLV at 10 mg/kg of body weight/day, DAP at 10 mg/kg/day, VAN at 1 g every 12 h (q12h), and LZD at 600 mg q12h. The MIC50/90 for TLV, DAP, VAN, and LZD against 70 DNS S. aureus isolates were 0.06/0.125 μg/ml, 2/4 μg/ml, 1/2 μg/ml, and 2/2 μg/ml, respectively. Against 100 VISA isolates, the MIC50/90 were 0.06/0.125 μg/ml, 1/1 μg/ml, 4/8 μg/ml, and 1/2 μg/ml, respectively. Against 170 hVISA isolates, the MIC50/90 were 0.06/0.125 μg/ml, 0.5/1 μg/ml, 1/2 μg/ml, and 1/2 μg/ml, respectively. Against 25 LZD(r) isolates, the MIC50/90 were 0.03/0.06 μg/ml, 1/1 μg/ml, 2/2 μg/ml, and 8/8 μg/ml, respectively. The TLV MIC was >0.125 μg/ml for 10/365 (2.7%) isolates. In PK/PD models, TLV was universally bactericidal at 168 h and statistically superior to all antibiotics against DNS S. aureus strain R2334. These data further establish the potency of TLV against resistant MRSA. The model data demonstrate in vitro bactericidal activity of TLV against hVISA, VISA, DNS S. aureus, and LZD(r) S. aureus strains. Further clinical research is warranted.

Frequency and Distribution of Single-Nucleotide Polymorphisms within mprF in Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides.

MprF is responsible for the lysinylation of phosphatidylglycerol (PG) to synthesize the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) are associated with a gain-in-function phenotype in terms of daptomycin resistance in Staphylococcus aureus. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation.) Using 22 daptomycin-susceptible (DAP(s))/daptomycin-resistant (DAP(r)) clinical methicillin-resistant S. aureus (MRSA) strain pairs, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs, (ii) the relationships of the SNPs to both daptomycin resistance and cross-resistance to the prototypical endovascular host defense peptide (HDP) thrombin-induced platelet microbicidal protein (tPMP), and (iii) the impact of mprF SNPs on positive surface charge phenotype and modifications of membrane PL profiles. Most of the mprF SNPs identified in our DAP(r) strains were clustered within the two MprF loci, (i) the central bifunctional domain and (ii) the C-terminal synthase domain. Moreover, we were able to correlate the presence and location of mprF SNPs in DAP(r) strains with HDP cross-resistance, positive surface charge, and L-PG profiles. Although DAP(r) strains with mprF SNPs in the bifunctional domain showed higher resistance to tPMPs than DAP(r) strains with SNPs in the synthase domain, this relationship was not observed in positive surface charge assays. These results demonstrated that both charge-mediated and -unrelated mechanisms are involved in DAP resistance and HDP cross-resistance in S. aureus.

Evaluation of Ceftaroline Alone and in Combination against Biofilm-Producing Methicillin-Resistant Staphylococcus aureus with Reduced Susceptibility to Daptomycin and Vancomycin in an In Vitro Pharmacokinetic/Pharmacodynamic Model.

Annually, medical device infections are associated with >250,000 catheter-associated bloodstream infections (CLABSI), with up to 25% mortality. Staphylococcus aureus, a primary pathogen in these infections, is capable of biofilm production, allowing organism persistence in harsh environments, offering antimicrobial protection. With increases in S. aureus isolates with reduced susceptibility to current agents, ceftaroline (CPT) offers a therapeutic alternative. Therefore, we evaluated whether CPT would have a role against biofilm-producing methicillin-resistant S. aureus (MRSA), including those with decreased susceptibilities to alternative agents. In this study, we investigated CPT activity alone or combined with daptomycin (DAP) or rifampin (RIF) against 3 clinical biofilm-producing MRSA strains in an in vitro biofilm pharmacokinetic/pharmacodynamic (PK/PD) model. Simulated antimicrobial regimens were as follows: 600 mg of CPT every 8 h (q8h) (free maximum concentration of drug [fCmax], 17.04 mg/liter; elimination half-life [t1/2], 2.66 h), 12 mg/kg of body weight/day of DAP (fCmax, 14.7 mg/liter; t1/2, 8 h), and 450 mg of RIF q12h (fCmax, 3.5 mg/liter; t1/2, 3.4 h), CPT plus DAP, and CPT plus RIF. Samples were obtained and plated to determine colony counts. Differences in log10 CFU/cm(2) were evaluated by analysis of variance with Tukey's post hoc test. The strains were CPT and vancomycin susceptible and DAP nonsusceptible (DNS). CPT displayed activity throughout the experiment. DAP demonstrated initial activity with regrowth at 24 h in all strains. RIF was comparable to the drug-free control, and little benefit was observed when combined with CPT. CPT plus DAP displayed potent activity, with an average log10 CFU/cm(2) reduction of 3.33 ± 1.01 from baseline. CPT demonstrated activity against biofilm-producing DNS MRSA. CPT plus DAP displayed therapeutic enhancement over monotherapy, providing a potential option for difficult-to-treat medical device infections.

Staphylococcus aureus Metabolic Adaptations during the Transition from a Daptomycin Susceptibility Phenotype to a Daptomycin Nonsusceptibility Phenotype

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The success of S. aureus as a pathogen is due in part to its many virulence determinants and resistance to antimicrobials. In particular, methicillin-resistant S. aureus has emerged as a major cause of infections and led to increased use of the antibiotics vancomycin and daptomycin, which has increased the isolation of vancomycin-intermediate S. aureus and daptomycin-nonsusceptible S. aureus strains. The most common mechanism by which S. aureus acquires intermediate resistance to antibiotics is by adapting its physiology and metabolism to permit growth in the presence of these antibiotics, a process known as adaptive resistance. To better understand the physiological and metabolic changes associated with adaptive resistance, six daptomycin-susceptible and -nonsusceptible isogenic strain pairs were examined for changes in growth, competitive fitness, and metabolic alterations. Interestingly, daptomycin nonsusceptibility coincides with a slightly delayed transition to the postexponential growth phase and alterations in metabolism. Specifically, daptomycin-nonsusceptible strains have decreased tricarboxylic acid cycle activity, which correlates with increased synthesis of pyrimidines and purines and increased carbon flow to pathways associated with wall teichoic acid and peptidoglycan biosynthesis. Importantly, these data provided an opportunity to alter the daptomycin nonsusceptibility phenotype by manipulating bacterial metabolism, a first step in developing compounds that target metabolic pathways that can be used in combination with daptomycin to reduce treatment failures.

Heterogeneity of genetic pathways toward daptomycin nonsusceptibility in Staphylococcus aureus determined by adjunctive antibiotics.

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

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.

Identification of a Novel Clone, ST736, among Enterococcus faecium Clinical Isolates and Its Association with Daptomycin Nonsusceptibility

Resistance to daptomycin in enterococcal clinical isolates remains rare but is being increasingly reported in the United States and worldwide. There are limited data on the genetic relatedness and microbiological and clinical characteristics of daptomycin-nonsusceptible enterococcal clinical isolates. In this study, we assessed the population genetics of daptomycin-nonsusceptible Enterococcus faecium (DNSE) clinical isolates by multilocus sequence typing (MLST) and whole-genome sequencing analysis. Forty-two nonduplicate DNSE isolates and 43 randomly selected daptomycin-susceptible E. faecium isolates were included in the analysis. All E. faecium isolates were recovered from patients at a tertiary care medical center in suburban New York City from May 2009 through December 2013. The daptomycin MICs of the DNSE isolates ranged from 6 to >256 μg/ml. Three major clones of E. faecium (ST18, ST412, and ST736) were identified among these clinical isolates by MLST and whole-genome sequence-based analysis. A newly recognized clone, ST736, was seen in 32 of 42 (76.2%) DNSE isolates and in only 14 of 43 (32.6%) daptomycin-susceptible E. faecium isolates (P < 0.0001). This report provides evidence of the association between E. faecium clone ST736 and daptomycin nonsusceptibility. The identification and potential spread of this novel E. faecium clone and its association with daptomycin nonsusceptibility constitute a challenge for patient management and infection control at our medical center.

Evaluation of Ceftaroline, Vancomycin, Daptomycin, or Ceftaroline plus Daptomycin against Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus in an In Vitro Pharmacokinetic/Pharmacodynamic Model of Simulated Endocardial Vegetations

Infective endocarditis (IE) caused by methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin and daptomycin has few adequate therapeutic options. Ceftaroline (CPT) is bactericidal against daptomycin (DAP)-nonsusceptible (DNS) and vancomycin-intermediate MRSA, but supporting data are limited for IE. This study evaluated the activities of ceftaroline, vancomycin, daptomycin, and the combination of ceftaroline plus daptomycin against DNS MRSA in a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). Simulations of ceftaroline-fosamil (600 mg) every 8 h (q8h) (maximum concentration of drug in serum [Cmax], 21.3 mg/liter; half-life [t1/2], 2.66 h), daptomycin (10 mg/kg of body weight/day) (Cmax, 129.7 mg/liter; t1/2, 8 h), vancomycin (1 g) q8h (minimum concentration of drug in serum [Cmin], 20 mg/liter; t1/2, 5 h), and ceftaroline plus daptomycin were evaluated against 3 clinical DNS, vancomycin-intermediate MRSA in a two-compartment, in vitro, PK/PD SEV model over 96 h with a starting inoculum of ∼8 log10 CFU/g. Bactericidal activity was defined as a ≥ 3-log10 CFU/g reduction from the starting inoculum. Therapeutic enhancement of combinations was defined as ≥ 2-log10 CFU/g reduction over the most active agent alone. MIC values for daptomycin, vancomycin, and ceftaroline were 4 mg/liter, 4 to 8 mg/liter, and 0.5 to 1 mg/liter, respectively, for all strains. At simulated exposures, vancomycin was bacteriostatic, but daptomycin and ceftaroline were bactericidal. By 96 h, ceftaroline monotherapy offered significantly improved killing compared to other agents against one strain. The combination of DAP plus CPT demonstrated therapeutic enhancement, resulting in significantly improved killing versus either agent alone against 2/3 (67%) strains. CPT demonstrated bactericidal activity against DNS, vancomycin-intermediate MRSA at high bacterial densities. Ceftaroline plus daptomycin may offer more rapid and sustained activity against some MRSA in the setting of high-inoculum infections like IE and should also be considered.