kjcm Korean Journal of Clinical Microbiology Korean J Clin Microbiol 1229-0025 Korean Journal of Clinical Microbiology 10.5145/KJCM.2011.14.2.60 kjcm-14-60 Original Article Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-Resistant Enterococcus faecium Isolated from the Chungcheong Area Cho Hye Hyun 1 Sung Ji Youn 2 Kwon Kye Chul 1 Lim Jin Sook 1 Koo Sun Hoe 1 Department of Laboratory Medicine, College of Medicine, Chungnam National University, Daejeon, Korea Department of Biomedical Laboratory Science, Far East University, Eumseong, Korea Correspondence: Sun Hoe Koo, Department of Laboratory Medicine, Chungnam National University Hospital, 640 Daesa-dong, Jung-gu, Daejeon 301-721, Korea. (Tel) 82-42-280-7798, (Fax) 82-42-257-5365, (E-mail) shkoo@cnu.ac.kr 62011 2162011 142 6066 06092010 29122010 29122010 Copyright © 2011 Korean Journal of Clinical Microbiology 2011 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 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.

 Vancomycin-resistant E. faecium MLST Clonal complex 17 REFERENCES Song JY Hwang IS Eom JS Cheong HJ Bae WK Park YH Prevalence and molecular epidemiology of vancomycin-resistant enterococci (VRE) strains isolated from animals and humans in Korea Korean J Intern Med 200520 5562 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 200133 38391 Choi WS Seo YB Jo YM Kim JY Kee SY Jeong HW Epidemiology and clinical significance of bacteriuria caused by vancomycin-resistant enterococci Infect Chemother 200638 2429 Willems RJ Top J van Santen M Robinson DA Coque TM Baquero F Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex Emerg Infect Dis 200511 8218 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 200846 2149 Ko KS Baek JY Lee JY Oh WS Peck KR Lee N Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Korea J Clin Microbiol 200543 23036 Lee WG Lee SM Kim YS Molecular characterization of Enterococcus faecium isolated from hospitalized patients in Korea Lett Appl Microbiol 200643 2749 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 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 2007113 28995 Homan WL Tribe D Poznanski S Li M Hogg G Spalburg E Multilocus sequence typing scheme for Enterococcus faecium J Clin Microbiol 200240 196371 Khan MA van der Wal M Farrell DJ Cossins L van Belkum A Alaidan A 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 200862 27983 Leavis H Top J Shankar N Borgen K Bonten M van Embden J A novel putative enterococcal pathogenicity island linked to the esp virulence gene of Enterococcus faecium and associated with epidemicity J Bacteriol 2004186 67282 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 200644 105964 Eom JS Hwang IS Hwang BY Lee JG Lee YJ Cheong HJ Emergence of vanA genotype vancomycin-resistant enterococci with low or moderate levels of teicoplanin resistance in Korea J Clin Microbiol 200442 17856 Arthur M and Courvalin P Genetics and mechanisms of glycopeptide resistance in enterococci Antimicrob Agents Chemother 199337 156371 Reynolds PE and Courvalin P Vancomycin resistance in enterococci due to synthesis of precursors terminating in D-alanyl-D-serine Antimicrob Agents Chemother 200549 215 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 200448 137981 Oh JY Her SH Seol SY Lee YC Lee JC Kim JM Antimicrobial resistance and multilocus sequence typing of vancomycin-resistant Enterococcus faecium isolated from clinical specimens J Bacteriol Virol 200838 1927 MLST MLST web sites Enterococcus faecium MLST database. http://efaecium.mlst.net/ [Online] (last visited on 10 May 2010) Peta M Carretto E Barbarini D Zamperoni A Carnevale L Perversi L Outbreak of vancomycin-resistant Enterococcus spp. in an Italian general intensive care unit Clin Microbiol Infect 200612 1639 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 199967 193200 Toledo-Arana A Valle J Solano C Arrizubieta MJ Cucarella C Lamata M The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation Appl Environ Microbiol 200167 453845 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 200454 2325 Freitas AR Tedim AP Novais C Ruiz-Garbajosa P Werner G Laverde-Gomez JA Global spread of the hyl (Efm) colonization-virulence gene in megaplasmids of the Enterococcus faecium CC17 polyclonal subcluster Antimicrob Agents Chemother 201054 26605 Panesso D Reyes J Rincón S Díaz L Galloway-Peña J Zurita J Molecular epidemiology of vancomycin-resistant Enterococcus faecium: a prospective, multicenter study in South American hospitals J Clin Microbiol 201048 15629 Rice LB Carias L Rudin S Vael C Goossens H Konstabel C A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin J Infect Dis 2003187 50812 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 200612 112330 Tables

Ant tibiotic susce eptibility profile s of VREFs

Isolates MIC (μg/mL)
Ampicillin Vancomycin Teicoplanin Ciprofloxacin
CNS3 ＞256 ＞256 16 64
CNS4 ＞256 ＞256 16 64
CNS5 ＞256 ＞256 128 256
CNS6 ＞256 ＞256 32 32
CNS8 256 ＞256 8 64
CNS9 ＞256 ＞256 128 256
CNS10 ＞256 ＞256 256 16
CNS11 ＞256 ＞256 8 32
CNS12 ＞256 ＞256 8 32
CNS13 256 ＞256 256 256
CNS14 256 ＞256 16 256
CNS15 256 ＞256 64 64
CNS16 ＞256 ＞256 64 32
CNS17 ＞256 ＞256 64 256
CNS18 ＞256 ＞256 64 64
CNS19 ＞256 ＞256 64 32
CNS20 ＞256 ＞256 256 64
CNS22 ＞256 ＞256 8 256
CNS23 ＞256 ＞256 128 64
CNS24 ＞256 ＞256 128 256
CNS25 ＞256 ＞256 128 2
CNS27 ＞256 ＞256 128 64
CBS2 ＞256 ＞256 128 128
CBS3 ＞256 ＞256 64 256
CBS6 ＞256 ＞256 128 256
CBS7 ＞256 ＞256 128 64
CBS8 ＞256 ＞256 128 256
CBS9 ＞256 ＞256 16 128
CBS11 ＞256 ＞256 128 256
CBS12 ＞256 ＞256 128 256
CBS14 ＞256 ＞256 64 128
EJS1 ＞256 ＞256 16 128
EJS2 ＞256 ＞256 256 128
EJS3 ＞256 ＞256 8 128
EJS4 ＞256 ＞256 64 128
EJS5 ＞256 ＞256 8 128
EJS7 256 ＞256 16 128

Abbreviations: CNS, isolates from CN hospital; CBS, isolates from CB hospital; EJS, isolates from EJ hospital.

Genotypic characteristics of vancomycin-resistant E. faecium isolates from three Korean hospitals based on multilocus sequence typing, esp repeat profiles and hyl gene

STRAIN ST Allelic profile esp gene repeat profile hyl
atpA ddl gdh purK gyd pstS adk esp-A esp-C
CNS3 78 15 1 1 1 1 1 1 4 8 ＋
CNS4 New 4 1 1 1 1 1 1 4 8 ＋
CNS5 192 15 1 1 1 1 7 1 4 8 ＋
CNS6 78 15 1 1 1 1 1 1 5 6 ＋
CNS8 192 15 1 1 1 1 7 1 5 6 ＋
CNS9 78 15 1 1 1 1 1 1 4 8 ＋
CNS10 78 15 1 1 1 1 1 1 5 6 ＋
CNS11 192 15 1 1 1 1 7 1 5 6 ＋
CNS12 192 15 1 1 1 1 7 1 4 8 ＋
CNS13 78 15 1 1 1 1 1 1 5 6 ＋
CNS14 192 15 1 1 1 1 7 1 4 8 ＋
CNS15 192 15 1 1 1 1 7 1 5 6 ＋
CNS16 192 15 1 1 1 1 7 1 4 8 −
CNS17 78 15 1 1 1 1 1 1 4 8 ＋
CNS18 192 15 1 1 1 1 7 1 5 6 ＋
CNS19 192 15 1 1 1 1 7 1 5 6 ＋
CNS20 78 15 1 1 1 1 1 1 5 6 ＋
CNS22 192 15 1 1 1 1 7 1 4 8 ＋
CNS23 192 15 1 1 1 1 7 1 5 6 ＋
CNS24 192 15 1 1 1 1 7 1 4 8 ＋
CNS25 192 15 1 1 1 1 7 1 4 8 ＋
CNS27 17 1 1 1 1 1 1 1 4 8 ＋
CBS2 17 1 1 1 1 1 1 1 4 8 ＋
CBS3 78 15 1 1 1 1 1 1 5 6 −
CBS6 78 15 1 1 1 1 1 1 4 8 −
CBS7 192 15 1 1 1 1 7 1 5 6 ＋
CBS8 192 15 1 1 1 1 7 1 4 8 ＋
CBS9 414 15 5 1 1 1 20 1 4 8 −
CBS11 192 15 1 1 1 1 7 1 4 8 ＋
CBS12 192 15 1 1 1 1 7 1 4 8 ＋
CBS14 78 15 1 1 1 1 1 1 5 6 ＋
EJS1 192 15 1 1 1 1 7 1 5 6 ＋
EJS2 192 15 1 1 1 1 7 1 5 6 ＋
EJS3 78 15 1 1 1 1 1 1 6 7 ＋
EJS4 78 15 1 1 1 1 1 1 4 8 −
EJS5 192 15 1 1 1 1 7 1 5 6 ＋
EJS7 17 1 1 1 1 1 1 1 4 8 ＋

Abbreviations: CNS, isolates from CN hospital; CBS, isolates from CB hospital; EJS, isolates from EJ hospital.

parC and gyrA mutation in 37 E. faecium isolates corresponding to ciprofloxacin MIC

Amino acid mutation in gene No. of isolates with ciprofloxacin MIC (μg/mL) Total no. of islates
parC (80) gyrA (83) 2 4 8 16 32 64 128 256 37
R R       1 3 6 4 3 17
R I         2 2 1 4 9
I R             1 1 2
I I 1         1 3 4 9

Abbreviations: R, arginine; I, isoleucine.