Reversible vancomycin susceptibility within emerging ST1421 Enterococcus faecium strains is associated with rearranged vanA-gene clusters and increased vanA plasmid copy number.

• ST1421 VVE E. faecium are associated with vanHAX and lack vanRS and vanZ. • Resistance reversion was due to increased vanA plasmid copy number and 44-bp deletion in vanHAX -promoter region. • Reversion occurred at a frequency below the detection limit of standard AST. • The reversion is initially associated with an increased fitness cost. Vancomycin variable enterococci (VVE) are van -positive enterococci with a vancomycin-susceptible phenotype (VVE-S) that can convert to a resistant phenotype (VVE-R) and be selected for during vancomycin exposure. VVE-R outbreaks have been reported in Canada and Scandinavian countries. The aim of this study was to examine the presence of VVE in whole genome sequenced (WGS) Australian bacteremia Enterococcus faecium (Efm) isolates collected through the Australian Group on Antimicrobial resistance (AGAR) network. Eight potential VVE Aus isolates, all identified as Efm ST1421, were selected based on the presence of vanA and a vancomycin-susceptible phenotype. During vancomycin selection, two potential VVE-S harboring intact vanHAX genes, but lacking the prototypic vanRS and vanZ genes, reverted to a resistant phenotype (VVE Aus -R). Spontaneous VVE Aus -R reversion occurred at a frequency of 4-6 × 10−8 resistant colonies per parent cell in vitro after 48 h and led to high-level vancomycin and teicoplanin resistance. The S to R reversion was associated with a 44-bp deletion in the vanHAX promoter region and an increased vanA plasmid copy number. The deletion in the vanHAX promoter region enables an alternative constitutive promoter for the expression of vanHAX. Acquisition of vancomycin resistance was associated with a low fitness cost compared with the corresponding VVE Aus -S isolate. The relative proportion of VVE Aus -R vs. VVE Aus -S decreased over time in serial passages without vancomycin selection. Efm ST1421 is one of the predominant VanA- Efm multilocus sequence types found across most regions of Australia, and has also been associated with a major prolonged VVE outbreak in Danish hospitals. [ABSTRACT FROM AUTHOR]