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

Abstract

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 >64 g/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 g/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 g/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to >256 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 related to the published sequence for the vanB 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.

vanA and vanB Incorporate into an Endemic Ampicillin-Resistant Vancomycin-Sensitive Enterococcus faecium Strain: Effect on Interpretation of Clonality

“…Multiplex PCR. Microbiologically identified VRE isolates were further characterized by multiplex PCR (6,15) with six primer pairs to identify vanA (5), vanB (20), vanC1 (10), vanC2-vanC3 (14), ddl E. faecalis, and ddl E. faecium encoding D-alanyl-D-alanyl ligase (6). Approximately 200 to 1,000 ng (1 l) of DNA was added to a PCR mixture (99 l) containing 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl 2 , 0.01% gelatin, 0.1% Triton X-100, 0.25 mM (each) the four deoxyribonucleotide triphosphates, 100 pmol of each primer, and 2 U of Taq DNA polymerase.…”

Detection of clinical vancomycin‐resistant enterococci in Denmark by multiplex PCR and sandwich hybridization

“…PCR assays to detect glycopeptide resistance genotypes in enterococci have often been described previously (9,(12)(13)(14)(15). In our PCR setup, 16s rDNA primers were included as a control of the DNA preparation, but also to ensure that template actually had been added to the test tube.…”

“…. In the USA, the rate of vancomycin resistance among clinical enterococcal isolates has increased dramatically from 5.6% in 1992 to 14.4% in 1996 (6, 7) and the continuous spread of enterococci resistant to the glycopeptides vancomycin and teicoplanin is now causing great concern in most parts of the world.…”

Genetic Methods for Assessing Antimicrobial Resistance

“…These methods are also provided in Table 1. For general bacteria, these combinations include the assessment of methicillin resistance in coaglase-negative Staphylococcus species (4,18,28,29,35,36,77,78), low-level vancomycin resistance in Enterococcus species (46), and extended-spectrum ␤-lactamase resistance in gram-negative facultatively anaerobic bacteria such as Escherichia coli and Klebsiella pneumonia (2,45). For Mycobacterium tuberculosis, genetic methods have been reported for detecting resistance to isoniazid (7,19,21,34,37,39,52,61,66,69,74,76), rifampin (16,39,48,51,66,67,68,79,81,82), streptomycin (39, 60) ethambutol (59), pyrazinamide (58), and the fluoroquinolones (65).…”

“…Recently, we have reported the use of RFLP analysis to differentiate among genes encoding vancomycin resistance (46). Because of conserved DNA sequences which exist among vancomycin resistance genes, more than one vancomycin resistance gene may be amplified with the same PCR oligonucleotide primers.…”