Evaluation of the Velogene Genomic Assay for Detection of vanA and vanB Genes in Vancomycin-Resistant Enterococcus Species

Abstract

The Velogene (VEL) genomic assay is a qualitative DNA probe for vanA and vanB in enterococci. 150 clinical isolates were tested with the VEL assay and characterized by pigment production, catalase levels, motility, growth in 6 g of vancomycin/ml, vancomycin and teicoplanin susceptibility, API 20S assay, and genotyping by multiplex PCR. The VEL assay identified all enterococcal strains with vanA and vanB genes.During the last three decades, the prevalence of enterococci as nosocomial pathogens has been increasing. Vancomycinresistant enterococci (VRE) were first recognized in 1986 (4) and are now a worldwide clinical problem, especially in acute care hospitals and intensive care units (3).There are three predominant genotypes of VRE (2). The VanA gene confers high-level vancomycin resistance (vancomycin MIC Ն 64 g/ml), with accompanying teicoplanin resistance (Ͼ16 g/ml). The vanB gene confers low-to high-level vancomycin resistance (vancomycin MICs between 16 and Ն1,000 g/ml) and no teicoplanin resistance. Both vanA and vanB are acquired and transferable and are most commonly seen in Enterococcus faecalis and E. faecium. VanC1 (associated with E. casseliflavus), vanC2 (associated with E. gallinarum), and vanC3 (associated with E. flavescens) confer intrinsically low-level vancomycin resistance (3, 6).Conventional testing with the vancomycin screen agar containing 6 g of vancomycin/ml and conventional vancomycin susceptibility tests do not reliably differentiate between vanC and low-level vanB resistance. Identification of the isolated species of VRE requires a minimum of 18 to 24 h. Hospitals that would implement infection control measures to control VRE with vanA and vanB genes, but not those with vanC genes, need the differentiation. Homebrew PCR assays may detect resistance genes, but they are available only to those laboratories with the expertise to develop their own tests. An efficient, simple, and reliable test to differentiate genotypes would help rapid treatment and initiation of appropriate infection control procedures (3).The Velogene (VEL) assay (ID Biomed, Bothell, Wash.) is based on a cycling probe technology that can be used to detect small amounts of DNA (1). Bacterial DNA is released from cells by suspending 1 loopful of growth from an 18-to 24-h culture in a lysing reagent that contains 20 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] (pH 6.8), 0.05% Triton X-100, 150 U of achromopeptidase/ml, and 50 U of mutanolysin/ml. The DNA is denatured at 95°C for 5 min. When the target DNA (vanA and vanB genes) is hybridized with a fluorescein-labeled, biotinylated DNA-RNA-DNA chimeric probe, the RNase H cleaves the RNA portion of the probe. The mixture is transferred into streptavidin-coated microwells, and the uncleaved probe (negative reaction) is detected with the addition of fluorescein antibody conjugated with horseradish peroxidase, which converts the substrate to a blue end product. A positive-reaction product is colorless. The results can be detected visually or by spectrophotom...

Epidemiology and mechanisms of glycopeptide resistance in enterococci

“…In an initial study, the rapid chemiluminescence assay (developed by Eiken Chemicals, Tokyo, Japan) showed 100% sensitivity in determining vanA, vanB, vanC-1, and vanC2/3 genotypes in a small number of isolates [46]. Similar results, using isolates, were reported using two recently developed detection kits for VRE which use DNA probe hybridization: the EVIGENE assay (Statens Serum Institute, Denmark) and the Velogene Genomic Assay (ID Biomed, Bothell, Washington, USA) [47,48].…”

Evaluation of ChromocultR enterococci agar for the isolation and selective enumeration of Enterococcus spp. in broilers

“…One hundred pure cultures of presumptive enterococcus colonies from each medium were characterized by colony and cell morphology, their haemolytic character, Gram stain, and oxidase and catalase activity. Following this preliminary identification, pure cultures were subcultured in Columbia agar with 5% sheep blood, harvested, and used to inoculate galleries of API 20 Strep (Biomerieux) (Appleman et al. 2004) for identification purposes.…”

Frequency Distribution of Fluoroquinolones-Resistant Enterococcus faecalis Isolates from Patients with Prostatitis in Golestan Province, Iran

“…Hospital-acquired infections prolong hospitalization period, which significantly increases health care costs (2). Enterococcus faecalis and Enterococcus faecium are the most common Enterococci that cause human infections, such as urinary tract infections (UTIs), ulcers and endocarditis (3,4). E. faecalis is an anaerobic, gram-positive and catalase-negative bacterium that can ferment glucose without gas production.…”