Alternative vanHAX promoters and increased vanA-plasmid copy number resurrect silenced glycopeptide resistance in Enterococcus faecium

Abstract

<i>Background</i> - Vancomycin variable enterococci (VVE) are <i>van</i>-positive isolates with a susceptible phenotype that can convert to a resistant phenotype during vancomycin selection.<p>

<p><i>Objectives</i> - To describe a vancomycin-susceptible <i>vanA</i>-PCR positive ST203 VVE <i>Enterococcus faecium</i> isolate (VVE<i>Swe</i>-S) from a liver transplantation patient in Sweden which reverted to resistant (VVE<i>Swe</i>-R) during <i>in vitro</i> vancomycin exposure.<p>

<p><i>Methods</i> - WGS analysis revealed the genetic differences between the isolates. Expression of the <i>van</i>-operon was investigated by qPCR. Fitness and stability of the revertant were investigated by growth measurements, competition and serial transfer.<p>

<i>Results</i> - The VVE<i>Swe</i>-R isolate gained high-level vancomycin (MIC >256 mg/L) and teicoplanin resistance (MIC = 8 mg/L). VVE<i>Swe</i>-S has a 5′-truncated <i>vanR</i> activator sequence and the VVE<i>Swe</i>-R has in addition acquired a 44 bp deletion upstream of <i>vanHAX</i> in a region containing alternative putative constitutive promoters. In VVE<i>Swe</i>-R the <i>vanHAX</i>-operon is constitutively expressed at a level comparable to the non-induced prototype <i>E. faecium</i> BM4147 strain. The <i>vanHAX</i> operon of VVESwe is located on an Inc18-like plasmid, which has a 3–4-fold higher copy number in VVE<i>Swe</i>-R compared with VVE<i>Swe</i>-S. Resistance has a low fitness cost and the vancomycin MIC of VVE<i>Swe</i>-R decreased during <i>in vitro</i> serial culture without selection. The reduction in MIC was associated with a decreased vanA-plasmid copy number.<p>

<p><i>Conclusions</i> - Our data support a mechanism by which vancomycin-susceptible VVE strains may revert to a resistant phenotype through the use of an alternative, constitutive, <i>vanR</i>-activator-independent promoter and a <i>vanA</i>-plasmid copy number increase.