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

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 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 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), vancomycinsusceptible (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.Vancomycin-resistant enterococci (VRE) are significant nosocomial pathogens worldwide, and both vanA and vanB strains of VRE may result in serious infections, including bacteremia (15, 23). In Australia, unlike the United States and Europe, vanB-containing Enterococcus faecium predominates (7,18). vanB-containing isolates show variable phenotypic resistance to vancomycin (MIC, 4 to 1,024 mg/liter) and can therefore be more difficult to detect than vanA-containing VRE that readily demonstrate high-level resistance to vancomycin (9,10,14,20).Both the vanA and vanB operons are associated with transposons, and transfer of resistance among enterococci appears to be mediated via acquisition of these elements (22). The vanA operon is located on the transposon Tn1546, while three transposons, Tn1547, Tn5382, and Tn1549, have been described which contain the vanB operon; sequencing data suggest Tn1549 is essentially identical to Tn5382 (22).Using Enterococcosel agar (BBL, Sparks, MD) containing 6 mg/liter vancomycin (EVA) for VRE screening, we have identified a number of vanB-containing E. faecium isolates that on subsequent susceptibility testing using the standard Etest (AB Biodisk, Solna, Sweden) method (inoculum of 0. We present here a description of the resistance genotypephenotype incongruence of these variants of VRE, including molecular characteristics. These LM-VRE strains can be ...

Current Trends of Emergence and Spread of Vancomycin-Resistant Enterococci

“…Preliminary findings direct to a similar trend in other European countries like Sweden (Soderblom et al, 2010;Fang et al, 2010). If this increased VanB-type prevalence is linked to a supposed reservoir of vanB among enterococcal or non-enterococcal intestinal colonizers (Stamper et al, 2007;Young et al, 2007;Graham et al, 2008;Usacheva et al, 2010;Bourdon et al, 2010;Werner et al, 2011c) or simply linked to an improved and better identification of low-level expressed VanB-type resistance (Pendle et al, 2008;Grabsch et al, 2008a;Grabsch et al, 2008b;Stamper et al, 2010) in relation to a reduced breakpoint as defined by EUCAST (EUCAST Clinical Breakpoint Table v. 1.1 2010-04-27) 6 remains to be elucidated in further studies.…”

Performance of the EUCAST Disk Diffusion Method, the CLSI Agar Screen Method, and the Vitek 2 Automated Antimicrobial Susceptibility Testing System for Detection of Clinical Isolates of Enterococci with Low- and Medium-Level VanB-Type Vancomycin Resistance: a Multicenter Study

“…Moreover, the CLSI agar screen method re- quires experienced personnel who can prepare and store the agar plates correctly, so that the vancomycin concentration in the plates is accurate. It is well known that VanB-type VRE can be difficult to detect, due to the inducible mechanism of resistance and the variable levels of vancomycin resistance expressed (12,37). The strain collection was designed to be genetically and epidemiologically diverse and to include troublesome isolates with low vancomycin MICs in addition to ATCC reference strains ( Table 1).…”

Comparative Study of Selective Chromogenic (chromID VRE) and Bile Esculin Agars for Isolation and Identification of vanB -Containing Vancomycin-Resistant Enterococci from Feces and Rectal Swabs

“…vanA-containing VRE typically have vancomycin MICs of Ն64 mg/liter (4,15) and would be expected to grow well in the presence of 8 mg/liter vancomycin. However, vanBcontaining VRE strains, which may exhibit much lower vancomycin MICs, including MICs of Յ4 mg/liter (4,8), may not be as readily isolated on cIDVRE as they are on bile esculin agar (Enterococcosel; BD, Sparks, MD) containing 6 mg/liter vancomycin (EVA). At Austin Health, where vanB-containing VRE predominate, we compared cIDVRE for the detection of vancomycin-resistant strains of E. faecalis and E. faecium with EVA for the isolation of VRE from fecal and rectal swab specimens.…”