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Journal of Clinical Microbiology, June 2003, p. 2641-2643, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2641-2643.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Diversity of Tn1546-Like Elements in Vancomycin-Resistant Enterococci Isolated from Humans and Poultry in Korea

Hak-Sun Yu, Sung-Yong Seol, and Dong-Taek Cho^*

Department of Microbiology, Kyungpook National University School of Medicine, Taegu, 700-422, Korea

Received 4 November 2002/ Returned for modification 10 January 2003/ Accepted 7 March 2003

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To investigate the possible spread of vancomycin resistance among enterococci through horizontal gene transfer between two sources of vancomycin-resistant enterococci (VRE) isolated from farm animals and humans in Korea, molecular characterization of VRE isolated from poultry (20 isolates) and humans (35 isolates) was performed by PCR-restriction fragment length polymorphism typing of the vanA gene cluster (Tn1546). PCR mapping of Tn1546 finally distinguished seven different transposon types (types A to G). Type A was observed only in the poultry isolates, while the other six types were present only in the human isolates. An insertion sequence and deleted sequence were detected in most of the human isolates in the orf2-vanR and vanX-vanY regions in the Tn1546-like element, but not in the poultry isolates. Tn1546-like elements were found in conjugal plasmids of most human VRE, whereas they were detected in the chromosomes of all poultry VRE. Accordingly, no evidence was found of any recent transmission of vancomycin resistance genes between poultry and humans in Korea.

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Vancomycin-resistant enterococci (VRE) were first reported in Europe in 1988 and have since emerged as important nosocomial pathogens (4). Dissemination of vancomycin resistance can occur through horizontal transmission of resistant genes (2). Horizontal transfer of vancomycin resistance can be explained by the fact that the genetic determinant for the VanA type of vancomycin resistance typically resides in a mobile DNA element, such as Tn1546, which can be transferred to enterococci by plasmids. Several recent studies have described the heterogeneity of the VanA resistance determinant (9, 10). In these studies, indistinguishable Tn1546-like elements were found in VRE isolated from farm animals and humans, suggesting the possible transfer of vancomycin-resistant elements from farm animals to humans. In Korea, there is only limited information on vancomycin resistance dissemination between farm animals and humans. Thus, the aim of this study was to investigate the dissemination of vancomycin resistance by horizontal transfer of resistant genes between farm animals and humans based on molecular characterization of VRE isolated from poultry and humans by PCR typing of the VanA gene cluster (Tn1546).

Fifty-five vancomycin-resistant Enterococcus faecium isolates were subjected to this study. Thirty-five human clinical isolates of vancomycin-resistant Enterococcus faecium were isolated from patients at four university hospitals in Korea from January 2001 to May 2001. During the same period, 20 vancomycin-resistant E. faecium strains were isolated from poultry feces. These isolates were then identified by using a Vitek system, and identifications were confirmed by using an APIstrep kit (both from bioMérieux Inc., Hazelwood, Mo.). The MICs of vancomycin (Eli Lilly, Indianapolis, Ind.) and teicoplanin (Aventis Pharmaceutical Co., Somerset, N.J.) for these isolates were evaluated by using the agar dilution method according to NCCLS standards (6). All the isolates were resistant to vancomycin (MICs at which 50% [MIC₅₀] and 90% [MIC₉₀] of the isolates were inhibited, 512 and 1,024 µg/ml, respectively). In contrast, with teicoplanin, there was some difference between the MICs for the human and poultry VRE isolates: the MIC₅₀ and MIC₉₀ for the human VRE were 32 and 128 µg/ml, respectively, while those for the poultry VRE isolates were 16 and 32 µg/ml, respectively.

Pulsed-field gel electrophoresis (PFGE) analysis and interpretation were performed as previously described (5, 7). The 55 isolates were very heterogeneous, with some discrete clusters of related isolates identified. No VRE isolates from the two groups belonged to the same cluster in the PFGE pattern.

The PCR amplicons of vanA and vanB were produced by methods described in previous reports (3). The PCR amplicons from the internal region of the Tn1546-like elements were produced by an initial cycle at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 3.5 min, and a final extension step at 72°C for 10 min. Primers are listed in Table 1. For a Long PCR of the Tn1546-like elements, an Expand Long Template PCR System (Roche, Mannheim, Germany) was used according to the manufacturer's instructions. The vanA gene was detected in all the isolates, while the vanB gene was not detected in any (data not shown). The polymorphic regions of Tn1546-like elements in the 55 VREs were analyzed by a Tn1546 internal-region PCR method. The PCR mapping finally distinguished seven different Tn1546-like element types (Fig. 1). The PCR product pattern for type A was identical to the predicted pattern for the published sequence of Tn1546 (1), and all the elements were conserved and indistinguishable from those of Tn1546. The PCR-restriction fragment length polymorphism (RFLP) patterns for isolates of this type were also identical to each other. All 20 VRE isolates from poultry were included in this type, yet none of the human VRE isolates belonged to it. Types B, C, and D exhibited larger PCR products for the orf2-vanR region, whereas types C and F exhibited larger PCR products for the vanX-vanY region than for those regions of type A, the Tn1546 element prototype. The PCR products that were larger than those of the prototype vanA elements were digested with AluI and DdeI (Roche), respectively, and separated by agarose gel electrophoresis (1.5% agarose gels). The isolates included in type F were further divided into four subtypes (subtypes F1 through F4) based on PCR-RFLP analysis. These subtypes differed in only one fragment, the left end region of the insertion (IS) elements. Types C and F2 produced identical PCR-RFLP results for the vanX-vanY region. The orf2-vanR, vanX-vanY, and vanY-vanZ region PCR products that were larger than expected were purified with a PCR purification kit (Qiagen Inc., Hilden, Germany) according to the manufacturer's instructions. The purified PCR products were sequenced using a T7 sequencing kit (Amersham Biosciences, Piscataway, N.J.). The results of the RFLP and sequencing analysis revealed that the IS elements in the orf2-vanR and vanX-vanY regions were IS1542 and IS1216V, respectively, in all the isolates bearing IS elements. IS1542 elements were detected in 6 (17%) of the 35 human isolates, while IS1216V elements were detected in 26 (74%). Type D included an IS element in the vanY-vanZ region of the Tn1546-like element. This IS element sequence did not match any previously reported sequence.

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TABLE 1. PCR primers for amplification of the Tn1546-like element internal region

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FIG. 1. Genetic maps for classification of VRE isolates by molecular type of Tn1546. IR, inverted repeat sequence. Human*, number of isolates isolated from humans. Poultry+, number of isolates isolated from poultry.

To determine the locations of Tn1546-like elements in plasmids or chromosomes, Southern hybridization analysis using a digoxigenin DNA labeling and detection kit (Roche) was performed according to the manufacturer's instructions. According to the results, most of the vanA gene clusters in the human VRE isolates (33 of 35) were detected on a conjugal plasmid, whereas all the vanA gene clusters in the poultry VRE isolates were detected on a chromosome. Previously, a difference in the rate of vanA gene transfer was also found between human and poultry VRE isolates in a conjugal transfer test using filter-mating with E. faecalis JH2-2 as the recipient (8). The vanA gene transfer rates of the human VRE isolates (about 10^-4 to 10^-5 per donor) were higher than those of the poultry VRE isolates (about 10^-8 to 10^-9 per donor).

In conclusion, we found that vanA gene clusters and DNA fingerprint patterns by PFGE were quite different for the two groups of VRE, those from humans and poultry, in Korea, suggesting that the VRE from the two sources were clonally unrelated. No evidence was found of any recent transmission of vancomycin-resistant genes between farm animals and humans in Korea.

 
* Corresponding author. Mailing address: Department of Microbiology, Kyungpook National University School of Medicine, 101 Dongin-dong, Chung-gu, Taegu, 700-422, Korea. Phone: 82-53-420-6951. Fax: 82-53-427-5664. E-mail: dtcho{at}knu.ac.kr.

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Journal of Clinical Microbiology, June 2003, p. 2641-2643, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2641-2643.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yu, H.-S. Articles by Cho, D.-T. Search for Related Content PubMed PubMed Citation Articles by Yu, H.-S. Articles by Cho, D.-T.