Nucleotide sequence of IS1678, an insertion sequence in the vanA cluster of enterococci.

Resistance to glycopeptide antibiotics in enterococci is mediated most frequently by the vanA gene cluster, which is found in both humans and animals (1, 8). This vanA gene cluster is carried on transposon Tn1546 or by closely related elements that contain vanR, vanS, vanH, vanA, vanX, and vanZ (1). Detailed molecular analyses of Tn1546-like elements in enterococcal isolates have revealed the presence of variations. These variations include the integration of insertion sequences (ISs) with or without a deletion around the insertion site, point mutations, and deletions (2, 6). Until now, six kinds of IS elements had been found in Tn1546-like elements: IS1542, IS19, IS1216V, IS1251, IS1216V-IS3-like, and IS1476 (3, 5, 9). For the first time, we report here an IS element located in Tn1546-like elements. This IS element has been detected in the vanX-vanY intergenic region of a poultry isolate of Enterococcus faecium, which is resistant to vancomycin. Six vanA enterococci, which were isolated from poultry feces between 1998 and 1999 in Korea, were investigated by PCR with primers specific for regions flanking the vanX-vanY region (7). E. faecium BM4147 with a prototype of Tn1546 was used as a control. Three isolates (SN119-1, SN128-1, and SN154-1) produced PCR amplicons larger than that produced by E. faecium BM4147, and the PCR amplicons from the three isolates were purified and sequenced. Two isolates, SN119-1 and SN128-1, had integration of IS1216V in the vanX-vanY intergenic region, while one isolate, SN154-1, had an unknown sequence in the vanX-vanY intergenic region which has the characteristics of an IS element. This previously unknown IS element has been now designated IS1678. IS1678 is characterized by (i) its size of 1,678 bp, which is larger than that of the other ISs found in the Tn1546-like element; (ii) its 1,320-bp open reading frame (ORF) for encoding a putative 440-amino-acid protein; (iii) its 24-bp terminal imperfect inverted repeating sequences; (iv) its 5-bp duplication of target DNA; and (v) its ORF showing high homology at amino acid levels with transposases of other IS elements in gram-positive bacteria, as reported in the GenBank database (Fig. 1). A BLAST search yielded the highest scores of identity with the transposase in Bacillus halodurans (52%) (GenBank accession no. NP_244854) and with the IS1380-SpnI transposase in Streptococcus pneunoniae (41%) (4). There were no similar IS elements at the DNA sequence level. Integration of IS1678 would not affect the function of the vanA gene cluster, because SN154-1 demonstrated a high level of resistance to vancomycin (MIC 1,024) and teicoplanin (MIC 128). In summary, we have found a new IS-like element, IS1678, in the vanX-vanY intergenic region of E. faecium that was isolated from poultry and that shows vancomycin resistance. Fifty-two vanA enterococci that were isolated from poultry meat and feces in 2003 were also investigated, but all enterococci had the same amplicons the same size as those in E. faecium BM4147, except one isolate, M44, which had no amplicons (W. K. Jung, J. Y. Lim, N. H. Kwon, J. M. Kim, S. K. Hong, H. C. Koo, S. H. Kim, Y. Ike, K. Tanimoto, and Y. K. Park, unpublished data). Nucleotide sequence accession number. The complete nucleotide sequence of IS1678 can be found in GenBank under accession no. AY859490.