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

Abstract

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.

Keywords: Tigecycline resistance; Vancomycin-resistant enterococci; rpsJ; tetM.

Fig. 1.

Dendrogram of vancomycin-resistant Enterococcus faecalis isolated from Risoleta Tolentino Neves Hospital in 2009 and 2011. Legend: PFGE – Pulsotype; ST – Sequence Type; TGC – Tigecycline; VAN – Vancomycin; LZD – Linezolid; DAP – Daptomycin. Breakpoints: TGC – S ≤ 0. 25 mg/L/R > 0.5 mg/L; VAN – S ≤ 4 mg/L/ I = 8–16 mg/L/R ≥ 32 mg/L; LZD – S ≤ 2 mg/L/I = 4 mg/L/R ≥ 8 mg/L; DAP – S ≤ 4 mg/L.

Fig. 2.

Relative mRNA abundance levels in tigecycline-susceptible and resistant E. faecalis clinical isolates. Mean levels of rpsJ, tetM, Multidrug ABC transporter ATP-binding protein QP83_01175 and Hypothetical protein QP83_08660 mRNA in tigecycline-susceptible VRE57 and VRE109, and tigecycline-resistant VRE65, VRE69, and VRE80 strains normalized to reference genes gdh, pyrC and gyrA (p > .05, as determined by ANOVA). Error bars represent standard deviations of at least three experimental replicates, and statistically significant differences are marked with ***.

Fig. 3.

Relative mRNA levels for MarR-family transcriptional regulator QP83_14295 (A), rpsJ (B) and tetM (C) for E. faecalis VRE109 and tigecycline-resistant variants selected in vitro. Mean expression is plotted relative to the reference genes gdh, pyrC and gyrA. Relative QP83_14295, rpsJ and tetM expression were normalized to VRE109 (p > .05, as determined by ANOVA). Error bars represent the standard deviations, and statistically significant differences are marked with *, ** or *** indicating the intensity of the difference, being *** the more intense.