Prevalence of the vanB2 gene cluster in vanB glycopeptide-resistant enterococci in the United Kingdom and the Republic of Ireland and its association with a Tn5382-like element.

The vanB gene cluster of enterococci confers resistance to vancomycin but not teicoplanin (4). In the United Kingdom, this cluster is found in approximately 15% of glycopeptideresistant enterococci from hospitalized patients (8), although proportions as high as 64 and 90% have been reported in some hospitals (9, 11). Three subtypes, based on nucleotide variability, have been designated vanB1, vanB2, and vanB3 (2, 10), respectively, and have been found within different transposons (3). We previously analyzed vanB-mediated resistance in isolates from Scotland, showing that 28 (88%) of 32 belonged to subtype vanB2 (7). In this study we examined the prevalence of vanB subtypes among enterococci isolated in the United Kingdom and the Republic of Ireland and investigated whether vanB was located on a transferable element. A total of 204 vanB enterococcal isolates, isolated between 1989 and 1999 from patients in 59 different hospitals in England, Wales, and Scotland and from a single hospital in the Republic of Ireland, were examined. Nucleotide sequencing and HhaI digestion of a fragment of the vanB gene was used to distinguish between the vanB1, vanB2, and vanB3 gene clusters (7). Based on their vanB-HhaI restriction fragment length polymorphism (RFLP) profiles, 202 (99%) isolates contained vanB2. The presence of vanB1 in two isolates was confirmed by sequence analysis. vanB2 has been associated with the ca. 27-kb conjugative transposon, Tn5382 (1, 3), and the closely related Tn1549 transposon (5). Therefore, we selected 28 vanB2 isolates from the above collection (19 Enterococcus faecium isolates and 8 E. faecalis isolates from England and Wales, as well as 1 E. casseliflavus isolate from Dublin, Republic of Ireland), together with the 28 vanB2 E. faecium isolates from Scotland (7), to examine whether the vanB2 gene cluster was associated with a similar element. E. faecium strain C68, previously shown to carry the Tn5382 element (1), was kindly provided by L. B. Rice for use as a positive control. Using primers specific to sequences in the left end of Tn5382 (59-ACG CCA TGC TAT TTA CTT CCG GC-39 and 59-GTT CTT ATT CCG CAG GTG GTG ATT-39 [1]), a 311-bp PCR fragment was generated from strain C68, and a similarly sized fragment was generated from each of the 56 selected isolates. A second set of primers (59-TTG CAT GGT GTT CGT TGG-39 and 59-CGG CAT CAA CGC CTT TAG-39) was used to amplify a 1,581-bp fragment containing vanXB2 and part of the right end of Tn5382 from strain C68. A similarly sized fragment from each of the 56 isolates in this study was also amplified, suggesting that, in all cases, the vanB2 gene cluster was associated with sequences similar to those previously seen in Tn5382. In several VanB strains of E. faecium isolated in the United States, Tn5382 contains insertion sequences and is located directly downstream of pbp5, which encodes a low-affinity penicillin-binding protein responsible for high-level ampicillin resistance in E. faecium isolates (1, 3, 6). RFLP analysis of long PCR fragments spanning vanSB2-vanXB2 (7) from the 56 selected isolates showed that none contained ISEnfa200 (3) or any other additional DNA. Further PCR studies showed that Tn5382 was not located downstream of pbp5 in any of the 47 E. faecium isolates. Pulsed-field gel electrophoresis analysis of SmaI-digested DNA from the 56 isolates revealed 35 different types (12). Plate matings and subsequent PCR analysis of transconjugants confirmed that vanB2 and Tn5382-like sequences were transferred from each of 3 E. faecium and 1 E. faecalis donor isolates to both E. faecium GE-1 and E. faecalis JH2-2 recipients (13). Cotransfer of vancomycin and ampicillin resistance from the E. faecium donors, which has been reported for isolates from the United States (1, 6), was not detected for any of the transconjugants, further confirming the lack of linkage of the vanB2 cluster to pbp5. VanB resistance among enterococci in the United Kingdom and the Republic of Ireland is dominated by the vanB2 gene cluster and appears to have arisen by horizontal dissemination of the vanB2 gene cluster in association with a Tn5382-type element.