Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1995 Jan;33(1):24–27. doi: 10.1128/jcm.33.1.24-27.1995

Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR.

S Dutka-Malen 1, S Evers 1, P Courvalin 1
PMCID: PMC227872  PMID: 7699051

Abstract

A PCR assay that allows simultaneous detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci (Enterococcus faecium, E. faecalis, E. gallinarum, and E. casseliflavus) was developed. This assay was based on specific amplification of internal fragments of genes encoding D-alanine:D-alanine ligases and related glycopeptide resistance proteins. The specificity of the assay was tested on 5 well-characterized glycopeptide-resistant strains and on 15 susceptible enterococcal type strains. Clinical isolates of enterococci that could not be identified to the species level by conventional methods were identified by the PCR test. This assay offers a specific and rapid alternative to antibiotic susceptibility tests, in particular for detection of low-level vancomycin resistance.

Full Text

The Full Text of this article is available as a PDF (198.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Billot-Klein D., Gutmann L., Sablé S., Guittet E., van Heijenoort J. Modification of peptidoglycan precursors is a common feature of the low-level vancomycin-resistant VANB-type Enterococcus D366 and of the naturally glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum. J Bacteriol. 1994 Apr;176(8):2398–2405. doi: 10.1128/jb.176.8.2398-2405.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bugg T. D., Wright G. D., Dutka-Malen S., Arthur M., Courvalin P., Walsh C. T. Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. Biochemistry. 1991 Oct 29;30(43):10408–10415. doi: 10.1021/bi00107a007. [DOI] [PubMed] [Google Scholar]
  3. Courvalin P. Genotypic approach to the study of bacterial resistance to antibiotics. Antimicrob Agents Chemother. 1991 Jun;35(6):1019–1023. doi: 10.1128/aac.35.6.1019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Devriese L. A., Pot B., Collins M. D. Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J Appl Bacteriol. 1993 Nov;75(5):399–408. doi: 10.1111/j.1365-2672.1993.tb02794.x. [DOI] [PubMed] [Google Scholar]
  6. Dutka-Malen S., Leclercq R., Coutant V., Duval J., Courvalin P. Phenotypic and genotypic heterogeneity of glycopeptide resistance determinants in gram-positive bacteria. Antimicrob Agents Chemother. 1990 Oct;34(10):1875–1879. doi: 10.1128/aac.34.10.1875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Eyers M., Chapelle S., Van Camp G., Goossens H., De Wachter R. Discrimination among thermophilic Campylobacter species by polymerase chain reaction amplification of 23S rRNA gene fragments. J Clin Microbiol. 1993 Dec;31(12):3340–3343. doi: 10.1128/jcm.31.12.3340-3343.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Facklam R. R., Collins M. D. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J Clin Microbiol. 1989 Apr;27(4):731–734. doi: 10.1128/jcm.27.4.731-734.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fantin B., Leclercq R., Arthur M., Duval J., Carbon C. Influence of low-level resistance to vancomycin on efficacy of teicoplanin and vancomycin for treatment of experimental endocarditis due to Enterococcus faecium. Antimicrob Agents Chemother. 1991 Aug;35(8):1570–1575. doi: 10.1128/aac.35.8.1570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feng D. F., Doolittle R. F. Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J Mol Evol. 1987;25(4):351–360. doi: 10.1007/BF02603120. [DOI] [PubMed] [Google Scholar]
  14. Frankel G., Giron J. A., Valmassoi J., Schoolnik G. K. Multi-gene amplification: simultaneous detection of three virulence genes in diarrhoeal stool. Mol Microbiol. 1989 Dec;3(12):1729–1734. doi: 10.1111/j.1365-2958.1989.tb00158.x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Leclercq R., Dutka-Malen S., Duval J., Courvalin P. Vancomycin resistance gene vanC is specific to Enterococcus gallinarum. Antimicrob Agents Chemother. 1992 Sep;36(9):2005–2008. doi: 10.1128/aac.36.9.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Murray B. E. The life and times of the Enterococcus. Clin Microbiol Rev. 1990 Jan;3(1):46–65. doi: 10.1128/cmr.3.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Oyofo B. A., Thornton S. A., Burr D. H., Trust T. J., Pavlovskis O. R., Guerry P. Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction. J Clin Microbiol. 1992 Oct;30(10):2613–2619. doi: 10.1128/jcm.30.10.2613-2619.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pompei R., Berlutti F., Thaller M. C., Ingianni A., Cortis G., Dainelli B. Enterococcus flavescens sp. nov., a new species of enterococci of clinical origin. Int J Syst Bacteriol. 1992 Jul;42(3):365–369. doi: 10.1099/00207713-42-3-365. [DOI] [PubMed] [Google Scholar]
  20. Predari S. C., Ligozzi M., Fontana R. Genotypic identification of methicillin-resistant coagulase-negative staphylococci by polymerase chain reaction. Antimicrob Agents Chemother. 1991 Dec;35(12):2568–2573. doi: 10.1128/aac.35.12.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Reynolds P. E., Snaith H. A., Maguire A. J., Dutka-Malen S., Courvalin P. Analysis of peptidoglycan precursors in vancomycin-resistant Enterococcus gallinarum BM4174. Biochem J. 1994 Jul 1;301(Pt 1):5–8. doi: 10.1042/bj3010005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sahm D. F., Kissinger J., Gilmore M. S., Murray P. R., Mulder R., Solliday J., Clarke B. In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis. Antimicrob Agents Chemother. 1989 Sep;33(9):1588–1591. doi: 10.1128/aac.33.9.1588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sahm D. F., Olsen L. In vitro detection of enterococcal vancomycin resistance. Antimicrob Agents Chemother. 1990 Sep;34(9):1846–1848. doi: 10.1128/aac.34.9.1846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schaberg D. R., Culver D. H., Gaynes R. P. Major trends in the microbial etiology of nosocomial infection. Am J Med. 1991 Sep 16;91(3B):72S–75S. doi: 10.1016/0002-9343(91)90346-y. [DOI] [PubMed] [Google Scholar]
  26. Swenson J. M., Hill B. C., Thornsberry C. Problems with the disk diffusion test for detection of vancomycin resistance in enterococci. J Clin Microbiol. 1989 Sep;27(9):2140–2142. doi: 10.1128/jcm.27.9.2140-2142.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tenover F. C., Tokars J., Swenson J., Paul S., Spitalny K., Jarvis W. Ability of clinical laboratories to detect antimicrobial agent-resistant enterococci. J Clin Microbiol. 1993 Jul;31(7):1695–1699. doi: 10.1128/jcm.31.7.1695-1699.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tomasz A. Multiple-antibiotic-resistant pathogenic bacteria. A report on the Rockefeller University Workshop. N Engl J Med. 1994 Apr 28;330(17):1247–1251. doi: 10.1056/NEJM199404283301725. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Vincent S., Knight R. G., Green M., Sahm D. F., Shlaes D. M. Vancomycin susceptibility and identification of motile enterococci. J Clin Microbiol. 1991 Oct;29(10):2335–2337. doi: 10.1128/jcm.29.10.2335-2337.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Willey B. M., Kreiswirth B. N., Simor A. E., Willaims G., Scriver S. R., Phillips A., Low D. E. Detection of vancomycin resistance in Enterococcus species. J Clin Microbiol. 1992 Jul;30(7):1621–1624. doi: 10.1128/jcm.30.7.1621-1624.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. al-Obeid S., Gutmann L., Shlaes D. M., Williamson R., Collatz E. Comparison of vancomycin-inducible proteins from four strains of Enterococci. FEMS Microbiol Lett. 1990 Jun 15;58(1):101–105. doi: 10.1016/0378-1097(90)90110-c. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES