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. 1997 Mar;35(3):703–707. doi: 10.1128/jcm.35.3.703-707.1997

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

R Patel 1, J R Uhl 1, P Kohner 1, M K Hopkins 1, F R Cockerill 3rd 1
PMCID: PMC229654  PMID: 9041416

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

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Selected References

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  1. Clark N. C., Cooksey R. C., Hill B. C., Swenson J. M., Tenover F. C. Characterization of glycopeptide-resistant enterococci from U.S. hospitals. Antimicrob Agents Chemother. 1993 Nov;37(11):2311–2317. doi: 10.1128/aac.37.11.2311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dutka-Malen S., Blaimont B., Wauters G., Courvalin P. Emergence of high-level resistance to glycopeptides in Enterococcus gallinarum and Enterococcus casseliflavus. Antimicrob Agents Chemother. 1994 Jul;38(7):1675–1677. doi: 10.1128/aac.38.7.1675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dutka-Malen S., Evers S., Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol. 1995 Jan;33(1):24–27. doi: 10.1128/jcm.33.1.24-27.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dutka-Malen S., Molinas C., Arthur M., Courvalin P. Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a D-alanine:D-alanine ligase-related protein necessary for vancomycin resistance. Gene. 1992 Mar 1;112(1):53–58. doi: 10.1016/0378-1119(92)90302-6. [DOI] [PubMed] [Google Scholar]
  6. Dutka-Malen S., Molinas C., Arthur M., Courvalin P. The VANA glycopeptide resistance protein is related to D-alanyl-D-alanine ligase cell wall biosynthesis enzymes. Mol Gen Genet. 1990 Dec;224(3):364–372. doi: 10.1007/BF00262430. [DOI] [PubMed] [Google Scholar]
  7. Evers S., Reynolds P. E., Courvalin P. Sequence of the vanB and ddl genes encoding D-alanine:D-lactate and D-alanine:D-alanine ligases in vancomycin-resistant Enterococcus faecalis V583. Gene. 1994 Mar 11;140(1):97–102. doi: 10.1016/0378-1119(94)90737-4. [DOI] [PubMed] [Google Scholar]
  8. Evers S., Sahm D. F., Courvalin P. The vanB gene of vancomycin-resistant Enterococcus faecalis V583 is structurally related to genes encoding D-Ala:D-Ala ligases and glycopeptide-resistance proteins VanA and VanC. Gene. 1993 Feb 14;124(1):143–144. doi: 10.1016/0378-1119(93)90779-3. [DOI] [PubMed] [Google Scholar]
  9. Handwerger S., Raucher B., Altarac D., Monka J., Marchione S., Singh K. V., Murray B. E., Wolff J., Walters B. Nosocomial outbreak due to Enterococcus faecium highly resistant to vancomycin, penicillin, and gentamicin. Clin Infect Dis. 1993 Jun;16(6):750–755. doi: 10.1093/clind/16.6.750. [DOI] [PubMed] [Google Scholar]
  10. Hayden M. K., Trenholme G. M., Schultz J. E., Sahm D. F. In vivo development of teicoplanin resistance in a VanB Enterococcus faecium isolate. J Infect Dis. 1993 May;167(5):1224–1227. doi: 10.1093/infdis/167.5.1224. [DOI] [PubMed] [Google Scholar]
  11. Jordens J. Z., Bates J., Griffiths D. T. Faecal carriage and nosocomial spread of vancomycin-resistant Enterococcus faecium. J Antimicrob Chemother. 1994 Oct;34(4):515–528. doi: 10.1093/jac/34.4.515. [DOI] [PubMed] [Google Scholar]
  12. Karanfil L. V., Murphy M., Josephson A., Gaynes R., Mandel L., Hill B. C., Swenson J. M. A cluster of vancomycin-resistant Enterococcus faecium in an intensive care unit. Infect Control Hosp Epidemiol. 1992 Apr;13(4):195–200. doi: 10.1086/646509. [DOI] [PubMed] [Google Scholar]
  13. Kolbert C. P., Connolly J. E., Lee M. J., Persing D. H. Detection of the Staphylococcal mecA gene by chemiluminescent DNA hybridization. J Clin Microbiol. 1995 Aug;33(8):2179–2182. doi: 10.1128/jcm.33.8.2179-2182.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Leclercq R., Derlot E., Duval J., Courvalin P. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N Engl J Med. 1988 Jul 21;319(3):157–161. doi: 10.1056/NEJM198807213190307. [DOI] [PubMed] [Google Scholar]
  15. Montecalvo M. A., Horowitz H., Gedris C., Carbonaro C., Tenover F. C., Issah A., Cook P., Wormser G. P. Outbreak of vancomycin-, ampicillin-, and aminoglycoside-resistant Enterococcus faecium bacteremia in an adult oncology unit. Antimicrob Agents Chemother. 1994 Jun;38(6):1363–1367. doi: 10.1128/aac.38.6.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Navarro F., Courvalin P. Analysis of genes encoding D-alanine-D-alanine ligase-related enzymes in Enterococcus casseliflavus and Enterococcus flavescens. Antimicrob Agents Chemother. 1994 Aug;38(8):1788–1793. doi: 10.1128/aac.38.8.1788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Quintiliani R., Jr, Evers S., Courvalin P. The vanB gene confers various levels of self-transferable resistance to vancomycin in enterococci. J Infect Dis. 1993 May;167(5):1220–1223. doi: 10.1093/infdis/167.5.1220. [DOI] [PubMed] [Google Scholar]
  18. Sahm D. F., Free L., Handwerger S. Inducible and constitutive expression of vanC-1-encoded resistance to vancomycin in Enterococcus gallinarum. Antimicrob Agents Chemother. 1995 Jul;39(7):1480–1484. doi: 10.1128/aac.39.7.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Uttley A. H., George R. C., Naidoo J., Woodford N., Johnson A. P., Collins C. H., Morrison D., Gilfillan A. J., Fitch L. E., Heptonstall J. High-level vancomycin-resistant enterococci causing hospital infections. Epidemiol Infect. 1989 Aug;103(1):173–181. doi: 10.1017/s0950268800030478. [DOI] [PMC free article] [PubMed] [Google Scholar]

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