vanB2 Enterococcus faecium with Vancomycin Susceptible Phenotype - Improved Detection by Etest Using Oxgall Supplementation

Microbiology, and Infectious Diseases Departments, Austin Health; Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia; Department of Medicine, University of Melbourne, Melbourne, Australia

^* To whom correspondence should be addressed. Email: Elizabeth.Grabsch{at}austin.org.au.

	Abstract

We have isolated a number of vanB E. faecium on bile esculin screening agar containing 6 mg/L vancomycin (EVA), which on subsequent susceptibility testing using Etest have repeatedly demonstrated MICs to vancomycin of 4 mg/L. To investigate this genotype-phenotype incongruence of "Low-MIC vancomycin-resistant enterococci" (LM-VRE), we examined the molecular characteristics of these isolates including the presence of the vanB operon using PCR amplification and DNA sequencing. LM-VRE all contained vanB associated with the transposon Tn1549 and were polyclonal. Sequencing of the vanB ligase gene showed no differences from the prototype vanB2. In addition, we examined supplemented media to improve phenotypic detection of these isolates. Etest detection of LM-VRE improved when Mueller Hinton and Brain Heart Infusion were supplemented with 10g/L oxgall (MHA-Oxg, BHIA-Oxg). We assessed the sensitivity and specificity of these media to detect vancomycin resistance using vancomycin-resistant vanB E. faecium (VRE, n=11), vancomycin-susceptible (van-negative), E. faecium (VSEfm, n=11), vancomycin-susceptible (van-negative) E. faecalis (VSEfs, n=11) and our LM-VRE isolates (n=23). After 48 h incubation, both MHA-Oxg and BHIA-Oxg were 100% (34/34) sensitive and 100% (22/22) specific in the identification of vancomycin resistance. These findings suggest that supplementation of MHA or BHIA with oxgall 10 g/L should be considered in laboratories where VRE detection protocols rely primarily on strain phenotype rather than early vanB gene detection by PCR.