JAC Advance Access originally published online on October 18, 2007
Journal of Antimicrobial Chemotherapy 2007 60(6):1403-1405; doi:10.1093/jac/dkm399
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Correspondence |
Molecular characterization of vancomycin-resistant enterococci in Hangzhou, China
1 State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79, Qing Chun Road, Hangzhou, Zhejiang 310003, China 2 Zhejiang Provincial People's Hospital, No. 158, Shang Tang Road, Hangzhou, Zhejiang 310014, China 3 Hangzhou First People's Hospital, No. 261, Wan Sha Road, Hangzhou, Zhejiang 310006, China 4 Zhejiang Hospital, No. 12, Ling Ying Road, Hangzhou, Zhejiang 310013, China 5 Hangzhou Third People's Hospital, No. 38, Western Lake Road, Hangzhou, Zhejiang 310009, China
* Corresponding author. Tel: +86-571-8723-6421; Fax: +86-571-8723-6423; E-mail: yvys119{at}163.com
accepted 27 September 2007
Keywords: Enterococcus faecium , resistance , transposons
Sir,Within the last 10 years, vancomycin-resistant enterococci (VRE) have become an important nosocomial pathogen in many countries; they remain rare in China, although glycopeptide antimicrobials have been used there for decades.1,2 Since the first clinical VRE isolate was detected in Hangzhou, China, in April 2006, the frequency of VRE isolation from patients has increased rapidly. Multi-locus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA) have been developed to recognize genetically related and potential epidemic isolates of Enterococcus faecium.3,4 In addition to strain spread, the horizontal transfer of the glycopeptide resistance gene cluster is also an important factor aiding the spread of VRE; the vanA gene cluster is carried as part of Tn1546-like elements, and horizontal transfer of these elements plays an important role in the spread of vanA-type VRE. In the present study, we investigated VRE isolates from patients among five different hospitals of Hangzhou in China by MLST, MLVA and molecular analysis of their Tn1546-like elements to elucidate the mechanism of spread of VRE.
Twenty-one non-repetitive vancomycin-resistant E. faecium (VREF) isolates were obtained from inpatients among five hospitals in Hangzhou, China, from April 2006 to April 2007. The MICs of 12 antimicrobial agents were determined by Etest (AB Biodisk, Solna, Sweden) (Table 1).
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The vanA gene was identified by PCR as the glycopeptide resistance determinant in all of the 21 isolates. A more thorough investigation of the Tn1546-like elements harbouring vanA in these isolates was performed by using overlapping PCR and nucleotide sequencing. The results indicated that 19 of 21 isolates possessed a novel Tn1546-like element, with IS1485 in the vanX-vanY intergenic region. The locations of the IS1485 insertion in this region were identical in all isolates, corresponding to nucleotide 8698 of Tn1546 with a 3 bp duplication of the target sequence (CTT) (GenBank M97297 [GenBank] ).
Filter matings were performed using E. faecium BM4105RF (resistant to fusidic acid and rifampicin) as the recipient. Transconjugants were selected on BHI agar plates containing vancomycin (32 mg/L) and rifampicin (256 mg/L). Vancomycin resistance was transferred at frequencies of 10–5–10–7/donor from 18 of the 21 donor isolates (exceptions were isolates ZY30041, HY512 and HY48). All transconjugants expressed resistance to vancomycin and teicoplanin, with MICs equal to or lower than those for the donor isolates. Co-transfer of high-level resistance to gentamicin, streptomycin and amikacin was variable, but neither ampicillin nor ciprofloxacin resistance was transferred. All of the transconjugants acquired a plasmid of 
54 kb (data not shown).
MLST and MLVA were performed as described previously,3,4 and the results are shown in Table 1. MLST identified seven different sequence types (STs) among the 21 VREF isolates, but 18 isolates (excluding three isolates of ST343) belonged to clonal complex CC17. A new allele of purK (purK39) and three new STs, 341 (15-5-1-1-1-1-1), 342 (7-5-1-1-1-1-1) and 343 (15-1-1-39-1-20-1), were detected and submitted to the MLST web site (http://efaecium.mlst.net/). The predominant ST was ST78 (15-1-1-1-1-1-1: seven isolates). The MLVA typing scheme for VREF is less discriminatory than MLST5 and divided the 21 VREF isolates into four types (MTs); MT285 (6-9-3-3-1-2), MT286 (6-9-3-3-2-2), MT288 (6-9-3-3-2-3) and MT289 (7-9-3-3-2-3). Importantly, ST343, which does not belong to CC17, could not be distinguished from CC17 isolates by MLVA.
The presence of genes presumptively involved in virulence or epidemicity, esp, hyl, cylA, gelE and asa1, coding for an enterococcal surface protein, hyaluronidase, cytolysin/haemolysin, gelatinase and aggregation substance, respectively, was tested by PCR. Different combinations of putative virulence/epidemicity (VIEP) markers were detected (Table 1), and isolates belonging to the same clonal type had diverse VIEP profiles.
In summary, we have characterized 21 VREF isolated from five hospitals in Hangzhou, China, by molecular methods. All of the isolates showed the VanA phenotype and had a vanA genotype. There were differences between the molecular characteristics of the prevalent VREF isolates in this study and those of isolates previously reported from mainland China.2 A Tn1546-like element with IS1485 inserted between vanXY was common in our isolates, but this variant has not been previously reported.6 Most of the 21 VREF isolates belonged to the pandemic virulent and hospital-adapted clonal complex CC17.7 Therefore, it may be predicted that this clonal group will become prevalent in China, as it is currently in Europe, the United States and Australia.7 CC17 has not been the main clonal complex of VREF isolates from mainland China in the past 5 years.2 Hence, it is important to detect and control this emerging lineage in order to prevent or reduce the spread of VREF in mainland China.
None to declare.
This work was supported by a research grant from the National Basic Research Program 973 of China (no. 2005CB523101) and by a grant from the Program for New Century Excellent Talents in University (no. NCET-04-0552).
References
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Zheng B, Tomita H, Xiao YH, et al. Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from mainland China. J Clin Microbiol (2007) 45:2813–8.
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Top J, Schouls LM, Bonten MJM, et al. Multiple-locus variable-number tandem repeat analysis, a novel typing scheme to study the genetic relatedness and epidemiology of Enterococcus faecium isolates. J Clin Microbiol (2004) 42:4503–11.
5 Werner G, Klare I, Witte W. The current MLVA typing scheme for Enterococcus faecium is less discriminatory than MLST and PFGE for epidemic-virulent, hospital-adapted clonal types. BMC Microbiol (2007) 7:28.[CrossRef][Medline]
6
Jensen LB, Ahrens P, Dons L, et al. Molecular analysis of Tn1546 in Enterococcus faecium isolated from animals and humans. J Clin Microbiol (1998) 36:437–42.
7 Willems RJ, Top J, van Santen M, et al. Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex. Emerg Infect Dis (2005) 11:821–8.[Web of Science][Medline]
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