Alternative Vanhax Promoters And Increased Vana-Plasmid Copy Number Resurrect Silenced Glycopeptide Resistance In Enterococcus Faecium

Background: Vancomycin variable enterococci (VVE) are van-positive isolates with a susceptible phenotype that can convert to a resistant phenotype during vancomycin selection.Objectives: To describe a vancomycin-susceptible vanA-PCR positive ST203 VVE Enterococcus faeclum isolate (VVESwe-S) from a Liver transplantation patient in Sweden which reverted to resistant (VVESwe-R) during in vitro vancomycin exposure.Methods: WGS analysis revealed the genetic differences between the isolates. Expression of the van-operon was investigated by qPCR. Fitness and stability of the revertant were investigated by growth measurements, competition and serial transfer.Results: The VVESwe-R isolate gained high-Level vancomycin (MIC >256 mg/L) and teicoplanin resistance (MIC = 8 mg/L). VVESwe-S has a 5'-truncated vanR activator sequence and the VVESwe-R has in addition acquired a 44 bp deletion upstream of vanHAX in a region containing alternative putative constitutive promoters. In VVESwe-R the vanHAX-operon is constitutively expressed at a Level comparable to the non-induced prototype E. faecium BM4147 strain. The vanHAX operon of VVESwe is Located on an Inc18-Like plasmid, which has a 3-4-fold higher copy number in VVESwe-R compared with VVESwe-S. Resistance has a Low fitness cost and the vancomycin MIC of VVESwe-R decreased during in vitro serial culture without selection. The reduction in MIC was associated with a decreased vanA-plasmid copy number.Conclusions: Our data support a mechanism by which vancomycin-susceptible VVE strains may revert to a resistant phenotype through the use of an alternative, constitutive, vanR-activator-independent promoter and a vanA-plasmid copy number increase.