Improved Detection of vanB2 -Containing Enterococcus faecium with Vancomycin Susceptibility by Etest Using Oxgall Supplementation

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Abstract
We have isolated a number of vanB -containing Enterococcus faecium isolates on bile esculin screening agar containing 6 mg/liter vancomycin, which on subsequent susceptibility testing using Etest have repeatedly demonstrated vancomycin MICs of ≤4 mg/liter. 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. All LM-VRE isolates contained vanB associated with the transposon Tn 1549 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 agar (MHA) and brain heart infusion agar (BHIA) were supplemented with 10 g/liter oxgall (MHA-Oxg and BHIA-Oxg, respectively). We assessed the sensitivity and specificity of these media to detect vancomycin resistance using vancomycin-resistant vanB -containing E. faecium ( n = 11), vancomycin-susceptible ( van negative) E. faecium ( n = 11), vancomycin-susceptible ( van negative) E. faecalis ( n = 11), and our LM-VRE ( n = 23) isolates. After 48 h of 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 10 g/liter oxgall should be considered in laboratories where VRE detection protocols rely primarily on strain phenotype rather than early vanB gene detection by PCR.

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