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

Abstract

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 faecium 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.

A Simple and Rapid Low-Cost Procedure for Detection of Vancomycin-Resistance Genes in Enterococci Reveals an Outbreak of Vancomycin-Variable Enterococcus faecium

“…Anti-neoplastic chemotherapy and the consumption of specific groups of antibiotics, including third-generation cephalosporins, glycopeptides, and metronidazole, were also risk factors for infections by vancomycin-resistant enterococci [ 15 ]. Subclinical transmissions between hospitalized patients was well-described [ 17 ], and efficient surveillance was recommended to prevent hospital transmission of vancomycin-resistant enterococci [ 18 ].…”

“…E. faecium carrying vanA gene without showing in vitro resistance to vancomycin have been reported from various parts of the world [ 6 , 7 , 8 , 9 , 10 , 11 ] and have caused hospital outbreaks [ 12 , 13 , 14 ]. Resistance to vancomycin may be induced by exposure to vancomycin in some of these strains and they are commonly referred to as vancomycin variable enterococci (VVE) [ 15 , 16 , 17 , 18 , 19 ]. Due to the potential induction of resistance to vancomycin, VVE may be treated as VREfm with regards to hospital infection control precautions.…”

Unbiased Antimicrobial Resistance Detection from Clinical Bacterial Isolates Using Proteomics

“…It is known that some Van ligase PCR positive isolates are nonfunctional, because of changes in their regulatory system. It remains elusive if proteomics is able to distinguish functional from nonfunctional isolates, which is not possible by PCR. − This shows that the full potential of untargeted proteomics for AMR detection is still hidden and must be evaluated in large-scale studies of clinical isolates.…”

Transiently silent acquired antimicrobial resistance: an emerging challenge in susceptibility testing

“…Likewise, a 44 bp deletion in the promoter region leading to constitutive vanHAX expression was observed in a VVE strain with a 5′-truncated vanR gene. 15 Moreover, constitutive vanHBX expression and teicoplanin resistance in E. faecium were caused by an 18 bp deletion in the vancomycin sensor gene vanS B . In combination with an insertion in the housekeeping ddl gene inactivating the host D-alanine: D-alanine ligase, the constitutive synthesis of D-alanyl-D-lactate terminating precursors supported peptidoglycan synthesis.…”

“…Several of the referred deletions appeared between tandem repeats. 8 , 15 , 60 , 72 , 73 Therefore, it has been hypothesized that the repeats in the AMR gene loci may act as emergency points allowing bacteria to express resistance genes when challenged. Apart from the mechanisms described here, it is likely that unknown trans -acting elements can provide resistance on and off switches.…”