Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci

Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens, One hundred clinical isolates of enterococci (E, casseliflavus/E. flavescens [n = 10], E, faecalis [n = 34], E, faecium [n = 43], E. avium [n = 1], E, gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed directly with colonies from blood agar plates, Six previously characterized VRE strains which carry either vanA, vanB, vanC-1, or vanC-2 genes were used as controls, To discriminate among van genes, the PCR amplicons were digested with MspI and were electrophoresed on agarose gels, Because of significant sequence homology between vanC-2 and vanC-3 genes, this assay is unable to discriminate these genes from each other; therefore, these are referred to as vanC-2/3 genes, PCR products were detected in 63 of the 100 clinical isolates, The restriction fragment length patterns were consistent with vanA for 10 strains, vanB for 30 strains, vanC-1 for 12 strains, vanC-2 for 6 strains, and vanA and vanC-1 for 1 strain, The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanA and vanB were all greater than or equal to 64 mu g/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 mu g/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 mu g/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to >256 mu g/ml) with restriction fragment length patterns differing from those for the control vanA, vanB, vanC-1, and vanC-2 isolates, DNA sequencing of these amplicons revealed that two of the four isolates had nucleic acid sequences which were closely relater! to the published sequence for the vanC-2 gene and two had nucleic acid sequences which were closely related to the published sequence for the vanC-2 and vanC-3 genes, Multiplex PCR-restriction fragment length polymorphism appears to be a useful and convenient method for rapidly detecting and discriminating genotypes for vancomycin-resistant Enterococcus spp, in the clinical laboratory, In instances in which unusual restriction fragment patterns of PCR amplicons occur, DNA sequencing can be performed to discriminate van genotypes.