Molecular basis for the emergence of a new hospital endemic tigecycline-resistant Enterococcus faecalis ST103 lineage.

Enterococcus faecalis are a major cause of nosocomial infection worldwide, and the spread of vancomycin resistant strains (VRE) limits treatment options. Tigecycline-resistant VRE began to be isolated from inpatients at a Brazilian hospital within months following the addition of tigecycline to the hospital formulary. This was found to be the result of a spread of an ST103 E. faecalis clone. Our objective was to identify the basis for tigecycline resistance in this lineage. The genomes of two closely related tigecycline-susceptible (MIC = 0.06 mg/L), and three representative tigecycline-resistant (MIC = 1 mg/L) ST103 isolates were sequenced and compared. Further, efforts were undertaken to recapitulate the emergence of resistant strains in vitro. The specific mutations identified in clinical isolates in several cases were within the same genes identified in laboratory-evolved strains. The contribution of various polymorphisms to the resistance phenotype was assessed by trans-complementation of the wild type or mutant alleles, by testing for differences in mRNA abundance, and/or by examining the phenotype of transposon insertion mutants. Among tigecycline-resistant clinical isolates, five genes contained non-synonymous mutations, including two genes known to be related to enterococcal tigecycline resistance (tetM and rpsJ). Finally, within the in vitro-selected resistant variants, mutation in the gene for a MarR-family response regulator was associated with tigecycline resistance. This study shows that E. faecalis mutates to attain tigecycline resistance through the complex interplay of multiple mechanisms, along multiple evolutionary trajectories.

Daptomycin and vancomycin heteroresistance revealed among CC5-SCCmecII MRSA clone and in vitro evaluation of treatment alternatives.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is a threat to the success of clinical treatment. Besides high antimicrobial resistance rates, the presence of heterogeneous vancomycin-intermediate S. aureus (hVISA) and heterogeneous daptomycin-non-susceptible S. aureus (hDNSSA) in the hospital environment is underestimated and is associated with treatment failure. The aim of this study was to investigate MRSA dissemination in a Brazilian hospital and to evaluate the efficacy of various treatment options in vitro. METHODS: MRSA strains were typed by MLST, PFGE and SCCmec typing. Minimum inhibitory concentrations (MICs) to daptomycin, linezolid, quinupristin/dalfopristin, teicoplanin, tetracycline, tigecycline, vancomycin and tedizolid were determined by broth microdilution. The presence of a heterogeneous population was detected by population analysis profile (PAP). Regarding hVISA and hDNSSA strains, the sequences and expression levels of genes involved in resistance to daptomycin and vancomycin were determined as well as cell wall thickness and autolysis. RESULTS: ST5/ST105-SCCmecII lineage was prevalent amongst 27 clinical MRSA characterised in this study. Two hDNSSA strains (one also hVISA) were detected and were confirmed by PAP. Isolate SCMSC29 (hVISA and hDNSSA) showed increased expression of genes involved in cell wall metabolism, slight cell wall thickening, reduction of autolysis, and single nucleotide polymorphisms (SNPs) in the rpoB and mprF genes compared with the susceptible strain SCMSC31. SCMSC35 (hDNSSA) presented SNPs in the rpoB and mprF genes as well as a thickened cell wall. CONCLUSIONS: Despite this worrying and hard to detect phenotype, treatment alternatives such as teicoplanin, linezolid, tetracycline, tigecycline, quinupristin/dalfopristin and tedizolid were all active against these isolates.

Resistance in In Vitro Selected Tigecycline-Resistant Methicillin-Resistant Staphylococcus aureus Sequence Type 5 Is Driven by Mutations in mepR and mepA Genes.

A tigecycline-susceptible (TGC-S) Sequence Type (ST) 5 clinical methicillin-resistant Staphylococcus aureus (MRSA) strain was cultured in escalating levels of tigecycline, yielding mutants eightfold more resistant. Their genomes were sequenced to identify genetic alterations, resulting in resistance. Alterations in rpsJ, commonly related to tigecycline resistance, were also investigated. Tigecycline resistance was mediated by loss-of-function mutations in the transcriptional repressor mepR, resulting in derepression of the efflux pump mepA. Increased levels of resistance were obtained by successive mutations in mepA itself. No alterations in RpsJ were observed in selected strains, but we observed a K57M substitution, previously correlated with resistance, among TGC-S clinical strains. Thus, the pathway to tigecycline resistance in CC5 MRSA in vitro appears to be derepression of mep operon as the result of mepR loss-of-function mutation, followed by alterations in MepA efflux pump. This shows that other evolutionary pathways, besides mutation of rpsJ, are available for evolving tigecycline resistance in CC5 MRSA.