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Antimicrobial Agents and Chemotherapy, September 2005, p. 3937-3939, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3937-3939.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The vanB2 Gene Cluster of the Majority of Vancomycin-Resistant Enterococcus faecium Isolates from Taiwan Is Associated with the pbp5 Gene and Is Carried by Tn5382 Containing a Novel Insertion Sequence

Jang-Jih Lu,* Tein-Yao Chang, Cherng-Lih Perng, and Shih-Yi Lee

Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital, Taipei, Taiwan, Republic of China

Received 2 February 2005/ Returned for modification 17 April 2005/ Accepted 5 June 2005


   ABSTRACT
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Thirty-two vanB2 Enterococcus faecium isolates were found to harbor Tn5382. Twenty-four isolates had a 1,419-bp sequence inserted within the open reading frame (ORF) C of Tn5382. This 1,419-bp sequence contained a 638-bp ORF with a 72% amino acid sequence homology with the transposase gene of IS150. Thirty isolates had the pbp5 gene linked to Tn5382.


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Vancomycin-resistant enterococci (VRE) have emerged as a major nosocomial pathogen in many parts of the world (1, 11). Two main vancomycin resistance phenotypes, VanA and VanB, exist (1, 11, 12). The VanB-type resistance is encoded by the vanB gene cluster (6). A 64-kb transposable element, Tn1547, bound by IS16- and IS256-like elements, has been shown to transpose the vanB gene cluster from the chromosome to plasmids (15, 16). A 27-kb transposon, Tn5382, containing the vanB gene cluster has also been described (2, 7, 17). To determine whether the vanB gene cluster in vancomycin-resistant Enterococcus faecium (VREF) isolates from Taiwan is also carried by transposons, we examined VREF isolates for the presence of transposable elements that carry the vanB2 gene cluster.

Thirty-two clinical isolates of VREF collected from five different hospitals in Taiwan from May 1995 to October 1997 were studied. These isolates were previously determined to harbor the vanB2 gene cluster (13), and their vancomycin MICs were determined to be in the range of 3 to >256 µg/ml. Twenty-six (81.25%) isolates were highly resistant to vancomycin (MICs of ≥128 µg/ml); 31 isolates were also resistant to ampicillin. E. faecalis CDCV583 containing Tn1547, IS16, and IS256 and E. faecium TUH2-18 containing Tn5382 and the pbp5 gene were used as control strains for the study of mobile genetic elements (5).

Two regions, designated region I and region II, within Tn5382 were amplified by PCR (Fig. 1). Region I was a 311-bp area located 246 bp downstream from the left (nonintegrase) end of Tn5382 and was amplified with primers 362F and 650R. Region II was amplified with primers VB6564F and Tn5382R and encompassed the area between the last gene (vanXB) of the vanB2 gene cluster and the integrase gene of Tn5382, with an expected size of 2,718 bp (Table 1). All 32 VREF isolates were positive for both PCRs (Table 2). Four isolates had an expanded region I (1,922 bp) and a normal region II. The 1,922-bp region I PCR products from four VREF isolates were found to contain the 1,611-bp transposable element ISEnfa110 (Fig. 1) (4). Twenty-four isolates generated a normal region I (311 bp) and an expanded region II (4,137 bp) PCR product. The remaining four isolates produced expected region I and region II PCR products. Analyses of the nucleotide sequences of the 4,137-bp region II PCR products revealed the presence of an additional 1,419-bp sequence inserted into the open reading frame (ORF) C of Tn5382 (Fig. 1). A pair of 20-bp imperfect repeats flanking this 1,419-bp sequence was present. The sequence of the left imperfect repeat is 5'-TGAACTGAACCCCAAAAGTT-3', and that of the right imperfect repeat is 5'-AACTTTTGGGGTGCACATCA-3'. A direct 3-bp repeat of CCA is found flanking the two 20-bp imperfect repeats. Two ORFs, ORF A (638 bp) and ORF B (326 bp), are present in this 1,419-bp sequence. These two ORFs are transcribed in an opposite direction to the integrase gene of Tn5382. The deduced amino acid sequences of the 638-bp ORF A had 72% homology with that of the putative transposase of IS150; therefore, we tentatively designated this insertion sequence as ISEnfa150 (3).



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  		FIG. 1. Schematic representation of Tn5382 and genetic linkage between Tn5382 and pbp5. IRL is the left terminal repeat, and IRR is the right terminal repeat of Tn5382, which harbors the vanB2 gene cluster. The locations where ISEnfa110 and ISEnfa150 are inserted within Tn5382 are indicated.

 

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  		TABLE 1. PCR primers used in this study

 

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  		TABLE 2. Detection of mobile genetic elements by PCR, and antimicrobial MICs of the VREF isolates

 
Our previous data indicated that the vanB2-type VREF, which is also highly resistant to ampicillin, is the most prevalent type of VREF in Taiwan (13). Since the majority of the 32 VREF isolates were also resistant to ampicillin, experiments were conducted to determine whether they harbor the pbp5 gene that encodes a penicillin-binding protein. The results revealed that all isolates were positive for the pbp5 gene by PCR (Table 2). To determine whether the pbp5 gene in these isolates is linked to the vanB2 gene cluster, another PCR amplifying a 1,079-bp region spanning the 3' end of the pbp5 gene and the 5' end of Tn5382 was performed (2). All but two isolates (30 of 32, or 93.8%) were positive by this PCR. The result of this experiment indicated that Tn5382 is located 134 bp downstream from the pbp5 gene in the majority of our VanB VREF isolates. Our result is consistent with a previous report that the pbp5 gene and Tn5382 are linked (2) and provides an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance with E. faecium clinical isolates (2).

Tn5382, which carries the vanB2 gene cluster responsible for vancomycin resistance in E. faecium isolates, has been found in several countries. In a study conducted in Korea, 20% (5 of 25) of VREF isolates are found to harbor Tn5382 (9, 11). A higher frequency (65% to 87.5%) has been reported in VREF isolates from the United States and Europe (4, 8, 14). Surprisingly, all 32 (100%) VREF isolates examined in this study are found to harbor Tn5382.

In this study, we also found that Tn5382 in several of the 32 VREF isolates harbors insertion sequences. Insertion sequences that reside in the vanB gene cluster are not as common as those in the vanA gene cluster (10). The high prevalence of ISEnfa110 may reflect a high transposition frequency of this element in enterococci in Taiwan. Therefore, ISEnfa110 may serve as an epidemiological marker to monitor VRE transmission.

One novel insertion sequence, designated ISEnfa3, was found to be inserted in Tn5382 in all vanB2 E. faecium isolates in a study conducted in Korea (10). Surprisingly, ISEnfa3 was not found in any of the 32 VREF isolates examined in this study. Instead, a novel insertion sequence tentatively designated ISEnfa150, which is inserted within Tn5382, was found. This observation reflects an interesting geographical difference for VREF isolates and provides another marker for the epidemiological study of VRE.

We have previously described that pulsed-field gel electrophoresis (PFGE) type I vanB2 E. faecium isolates are prevalent in all hospitals in Taiwan (13). In this study, 17 of 24 E. faecium isolates containing the pbp5 gene linked to Tn5382 with an ISEnfa150 inserted in its ORF C were found to belong to PFGE type I (Table 2). This result suggests that intra- and interhospital transmissions of this type of E. faecium isolate have occurred. In addition to this clonal dissemination, horizontal transfer of vancomycin and ampicillin resistance genes may also have occurred, as four E. faecium isolates with the same linkage between pbp5 and Tn5382 with the ISEnfa150 element belong to different PFGE types (types II, VI, and VIII). Based on the results of this study, we recommend that both mobile genetic elements and PFGE types be determined in order to study the epidemiology of VRE infections.

Nucleotide sequence accession number. The ISEnfa150 sequence was submitted to GenBank under accession number AY093592.


   ACKNOWLEDGMENTS
 
This study was supported by grants TSGH-C91-59 and TSGH-C90-59 from the Tri-Service General Hospital and NSC90-2314-B-016-065 and NSC91-2320-B-016-025 from the National Science Council, Taiwan, Republic of China.

We thank Chao-Hung Lee for assistance with the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: No. 325, Section 2, Cheng-kung Rd. Neihu, Taipei, Taiwan 114, Republic of China. Phone: 886-2-8792-7227. Fax: 886-2-8792-7226. E-mail: jjl{at}mail.ndmctsgh.edu.tw. Back


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Antimicrobial Agents and Chemotherapy, September 2005, p. 3937-3939, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3937-3939.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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