A novel resistance reversion mechanism in a vancomycin-variableEnterococcus faeciumstrain

Abstract

Objectives: To investigate an outbreak of Enterococcus faecium in a hospital haematology ward and uncover the mechanism of a vancomycin resistance phenotype-genotype disparity in an isolate from this outbreak. Methods: Whole genome shotgun sequencing was used for the phylogenetic analysis of E. faecium isolates (n = 39) and to identify the carriage of antibiotic resistance genes. A long-read sequencing approach was adopted to identify structural variations in the vancomycin resistance region of a vancomycin-variable E. faecium (VVE) and to uncover the resistance reversion mechanism in this isolate. RT-qPCR and RT-PCR were used to determine differences in the expression of vanRS and vanHAX among strains. Results: The E. faecium strains isolated in the hospital haematology ward were extensively drug resistant and highly diverse. The notable expansion of ST262 among patients was the likely driver of a VRE outbreak. A VVE isolate was identified that could rapidly revert to a vancomycin-resistant state in the presence of vancomycin. Disruption of the vanR gene in this isolate by an IS6-family element impaired its response to vancomycin. However, when the isolate was evolved to vancomycin resistance, it could constitutively express the vanHAX genes at levels up to 36,000-fold greater than the parent isolate via co-transcription with a ribosomal RNA operon. Conclusion: In this study, we report a VVE isolate that was isolated during a VRE outbreak. This strain was capable of rapidly reverting to a resistant phenotype through a novel mechanism involving integration of vanHAX downstream of a ribosomal RNA operon. During VRE outbreaks, attention should be paid to contemporaneous vancomycin-susceptible strains as these may carry silent vancomycin resistance genes that can be activated through genomic rearrangements upon exposure to vancomycin.