Korean J Clin Microbiol.  2011 Jun;14(2):60-66. 10.5145/KJCM.2011.14.2.60.

Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-Resistant Enterococcus faecium Isolated from the Chungcheong Area

• Cho HH
• Sung JY
• Kwon KC
• Lim JS
• Koo SH

• KMID: 2089816
• DOI: http://doi.org/10.5145/KJCM.2011.14.2.60

Abstract

BACKGROUND
Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal complex 17 (CC17). In the present study, characterization of the glycopeptide resistance mechanism, genetic relatedness, and pathogenicity in isolates of vancomycin-resistant E. faecium in the Chungcheong area were investigated.
METHODS
A total of 37 consecutive, non-duplicate, vancomycin-resistant E. faecium were isolated at three university hospitals in the Chungcheong area. The mechanism of glycopeptide resistance and pathogenicity factors were studied using PCR, and the genetic relatedness was determined via multilocus sequence type and esp repeat profile analysis. Additionally, the quinolone resistance-determining regions of parC and gyrA were sequenced to identify mutations involved in ciprofloxacin resistance.
RESULTS
Two genotypes of VRE were confirmed: VanA-phenotype vanA genotype VRE (25 isolates) and VanB-phenotype vanA genotype VRE (12 isolates). MLST analysis revealed five sequence types. A significant result was that ST414 and CNS4 (4-1-1-1-1-1-1) were considered as belonging to CC17. The esp and hyl genes were found in 100% and 86.4% of the isolates, respectively. A total of 37 isolates showed genetic mutations in parC and gyrA.
CONCLUSION
All isolated strains in the present study belonged to one of the CC17 genotypes including ST414 and CNS4 (4-1-1-1-1-1-1), which were not previously detected in Korea. The combination of MLST and the esp gene repeat profiles can be useful for genetic characterization of VREF isolates with regard to the evolutionary process and epidemiology of the clones.

Keyword

Vancomycin-resistant E. faecium; MLST; Clonal complex 17

MeSH Terms

Ciprofloxacin
Clone Cells
Enterococcus
Enterococcus faecium
Genotype
Hospitals, University
Korea
Multilocus Sequence Typing
Polymerase Chain Reaction
Virulence Factors
Ciprofloxacin
Virulence Factors

Reference

1. Song JY, Hwang IS, Eom JS, Cheong HJ, Bae WK, Park YH, et al. Prevalence and molecular epidemiology of vancomycin-resistant enterococci (VRE) strains isolated from animals and humans in Korea. Korean J Intern Med. 2005; 20:55–62.

2. Choi YH, Lee YS, Lee JK, Yoo JL, Kim CK, Kim BS. Molecular relationship of vanA glycopeptide resistance gene in enterococci from hospitalized patients and poultry in Korea. Korean J Infect Dis. 2001; 33:383–91.

3. Choi WS, Seo YB, Jo YM, Kim JY, Kee SY, Jeong HW, et al. Epidemiology and clinical significance of bacteriuria caused by vancomycin-resistant enterococci. Infect Chemother. 2006; 38:242–9.

4. Willems RJ, Top J, van Santen M, Robinson DA, Coque TM, Baquero F, et al. Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex. Emerg Infect Dis. 2005; 11:821–8.

5. Top J, Willems R, van der Velden S, Asbroek M, Bonten M. Emergence of clonal complex 17 Enterococcus faecium in the Netherlands. J Clin Microbiol. 2008; 46:214–9.

6. Ko KS, Baek JY, Lee JY, Oh WS, Peck KR, Lee N, et al. Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Korea. J Clin Microbiol. 2005; 43:2303–6.

7. Lee WG, Lee SM, Kim YS. Molecular characterization of Enterococcus faecium isolated from hospitalized patients in Korea. Lett Appl Microbiol. 2006; 43:274–9.

8. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; eighteenth informational supplement. M100-S18 (M2). Wayne, PA: Clinical and Laboratory Standards Institute;2008.

9. Ribeiro T, Abrantes M, Lopes Mde F, Crespo MT. Vancomycin-susceptible dairy and clinical enterococcal isolates carry vanA and vanB genes. Int J Food Microbiol. 2007; 113:289–95.

10. Homan WL, Tribe D, Poznanski S, Li M, Hogg G, Spalburg E, et al. Multilocus sequence typing scheme for Enterococcus faecium. J Clin Microbiol. 2002; 40:1963–71.

11. Khan MA, van der Wal M, Farrell DJ, Cossins L, van Belkum A, Alaidan A, et al. Analysis of VanA vancomycin-resistant Enterococcus faecium isolates from Saudi Arabian hospitals reveals the presence of clonal cluster 17 and two new Tn1546 lineage types. J Antimicrob Chemother. 2008; 62:279–83.

12. Leavis H, Top J, Shankar N, Borgen K, Bonten M, van Embden J, et al. A novel putative enterococcal pathogenicity island linked to the esp virulence gene of Enterococcus faecium and associated with epidemicity. J Bacteriol. 2004; 186:672–82.

13. Leavis HL, Willems RJ, Top J, Bonten MJ. High-level ciprofloxacin resistance from point mutations in gyrA and parC confined to global hospital-adapted clonal lineage CC17 of Enterococcus faecium. J Clin Microbiol. 2006; 44:1059–64.

14. Eom JS, Hwang IS, Hwang BY, Lee JG, Lee YJ, Cheong HJ, et al. Emergence of vanA genotype vancomycin-resistant enterococci with low or moderate levels of teicoplanin resistance in Korea. J Clin Microbiol. 2004; 42:1785–6.

15. Arthur M and Courvalin P. Genetics and mechanisms of glycopeptide resistance in enterococci. Antimicrob Agents Chemother. 1993; 37:1563–71.

16. Reynolds PE and Courvalin P. Vancomycin resistance in enterococci due to synthesis of precursors terminating in D-alanyl-D-serine. Antimicrob Agents Chemother. 2005; 49:21–5.

17. Lee WG, Huh JY, Cho SR, Lim YA. Reduction in glycopeptide resistance in vancomycin-resistant enterococci as a result of vanA cluster rearrangements. Antimicrob Agents Chemother. 2004; 48:1379–81.

18. Oh JY, Her SH, Seol SY, Lee YC, Lee JC, Kim JM, et al. Antimicrobial resistance and multilocus sequence typing of vancomycin-resistant Enterococcus faecium isolated from clinical specimens. J Bacteriol Virol. 2008; 38:19–27.

19. MLST. MLST web sites. Enterococcus faecium MLST database.http://efaecium.mlst.net/. [Online] (last visited on 10 May 2010).

20. Peta M, Carretto E, Barbarini D, Zamperoni A, Carnevale L, Perversi L, et al. Outbreak of vancomycin-resistant Enterococcus spp. in an Italian general intensive care unit. Clin Microbiol Infect. 2006; 12:163–9.

21. Shankar V, Baghdayan AS, Huycke MM, Lindahl G, Gilmore MS. Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infect Immun. 1999; 67:193–200.

22. Toledo-Arana A, Valle J, Solano C, Arrizubieta MJ, Cucarella C, Lamata M, et al. The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation. Appl Environ Microbiol. 2001; 67:4538–45.

23. Oancea C, Klare I, Witte W, Werner G. Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis. J Antimicrob Chemother. 2004; 54:232–5.

24. Freitas AR, Tedim AP, Novais C, Ruiz-Garbajosa P, Werner G, Laverde-Gomez JA, et al. Global spread of the hyl (Efm) colonization-virulence gene in megaplasmids of the Enterococcus faecium CC17 polyclonal subcluster. Antimicrob Agents Chemother. 2010; 54:2660–5.

25. Panesso D, Reyes J, Rincón S, Díaz L, Galloway-Peña J, Zurita J, et al. Molecular epidemiology of vancomycin-resistant Enterococcus faecium: a prospective, multicenter study in South American hospitals. J Clin Microbiol. 2010; 48:1562–9.

26. Rice LB, Carias L, Rudin S, Vael C, Goossens H, Konstabel C, et al. A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin. J Infect Dis. 2003; 187:508–12.

27. Camargo IL, Gilmore MS, Darini AL. Multilocus sequence typing and analysis of putative virulence factors in vancomycin-resistant and vancomycin-sensitive Enterococcus faecium isolates from Brazil. Clin Microbiol Infect. 2006; 12:1123–30.