Emergence of vancomycin-resistant enterococci in Australia: phenotypic and genotypic characteristics of isolates

Abstract

Enterococci with resistance to glycopeptides have recently emerged in Australia. We developed multiplex PCR assays for vanA, vanB, vanC1, and vanC2 or vanC3 in order to examine the genetic basis for vancomycin resistance in Australian isolates of vancomycin-resistant Enterococcus faecium and E. faecalis (VRE). The predominant genotype from human clinical E. faecium isolates was vanB. The PCR van genotype was consistent with the resistance phenotype in all but six cases. One vanA E. faecalis isolate had a VanB phenotype, one vanB E. faecium isolate had a VanA phenotype, and four E. faecalis isolates were consistently negative for vanA, vanB, vanC1, and vanC2 or vanC3, even though they exhibited a VanB phenotype. These four isolates were subsequently examined for the presence of vanD by published methods and were found to be negative. No vancomycin-susceptible strains produced a PCR product. On the basis of our findings the epidemiology of VRE in Australia appears to be different from that in either the United States or Europe. Our multiplex PCR assays gave a rapid and accurate method for determining the genotype and confirming the identification of glycopeptide-resistant enterococci. Rapid and accurate methods are essential, because laboratory-based surveillance is critical in programs for the detection, control, and prevention of the transmission of glycopeptide-resistant enterococci.

FIG. 1

PCR analysis of VRE. Enterococci were subjected to PCR analysis, as described in Materials and Methods, with primers VanABF, VanAR, and VanBR (A) or VanC1F, VanC1R, VanC23F, and VanC23R (B). The PCR mixtures were electrophoresed on 2% agarose gels and stained with ethidium bromide. Lanes: 1, E. faecalis ATCC 51299; 2, E. faecium ATCC 19434; 3, E. faecalis 91; 4, E. faecalis 3; 5, E. faecium 143; 6, E. faecium 135; 7, E. gallinarum 129; 8, E. casseliflavus 38; 9, E. faecalis 26; 10, E. faecalis 21; 11, E. faecium 30. Lane M₁, pUC19 DNA digested with HpaII (fragments of 501 and 489, 404, 331, 242, 190, and 147 bp are visible); lane M₂, bacteriophage SPP1 DNA digested with EcoRI (fragments of 1,950, 1,860, 1,510, 1,390, 1,160, 980, 720, 480, and 360 bp are visible.