Genetic linkage of the vanB2 gene cluster to Tn5382 in vancomycin-resistant enterococci and characterization of two novel insertion sequences

VanB-type vancomycin resistance is encoded by the vanB gene cluster, which disseminates by horizontal gene transfer and clonal spread of vancomycin-re sistant enterococci (VRE). Genetic linkage of the vanB gene cluster to tran sposon Tn5382 and the insertion sequences IS16 and IS256-like has previousl y been shown. In this study linkage of defined vanB gene cluster subtypes t o these elements was examined. All the vanB2 subtype strains studied (n = 1 4) revealed co-hybridization of vanB and Tn5382, whereas the strains of van B1 (n = 8) and vanB3 (n = 1) subtypes were Tn5382 negative. Conjugative cot ransfer of the vanB2 gene cluster and Tn5382 was demonstrated for two strai ns. DNA sequencing of the vanX(B)-ORFC region in vanB2 strains confirmed th at the vanB2 gene cluster is an integral part of Tn5382. No general pattern of linkage was observed with regard to IS16 and IS256-like. Two novel inse rtion sequences were identified in specific vanB2 subtype strains. (i) A 16 11 bp element (ISEnfa110) was detected in the left flank of Tn5382, Its ins ertion site, lack of terminal inverted and direct repeats, and two conserve d motifs in its putative transposase all conform to the conventions of the IS110 family. (ii) A 787 bp element (ISEnfa200) was detected in the vanS(B) -vanY(B) intergenic region. Its ORF encoded a putative protein with 60-70 % identity to transposases of the IS200 family. No further copies of ISEnfa1 10 were found by colony hybridization of 181 enterococcal isolates, whereas ISEnfa200 was found in four additional vanB2 strains from the USA. The fiv e strains had identical ISEnfa200 element insertion sites, and Tn5382 was l ocated downstream from a pbp5 gene conferring high-level ampicillin resista nce. These isolates showed related PFGE patterns, suggesting possible clona l spread of a VRE strain harbouring a Tn5382-vanB2-ISEnfa200 element linked to a pbp5 gene conferring ampicillin resistance.