Nature of transposon-mediated high-level gentamicin resistance in Enterococcus faecalis isolated in the United...

Forty-two high-level gentamicin-resistant (MIC > 1000 mg/L) strains of Enterococcus faecalis, isolated from diverse geographical locations throughout the UK between 1993 and 1995, were studied to identify the nature of the high-level gentamicin-resistant determinants and the possibility of these determinants being associated with a transposon. High-level gentamicin resistance was attributed to the synthesis of the bifunctional (AAC6'-APH2") aminoglycoside-modifying enzyme. The aac6'-aph2" gene, which was present on a 70 kb plasmid in all 42 isolates, could be transferred by conjugation in association with the 70 kb plasmid in 39 of the isolates studied. In three E. faecalis isolates, however, the high-level gentamicin resistance was transferable independent of the 70 kb plasmid, suggesting the presence of a conjugative transposon. Long-PCR studies showed that all 42 clinical isolates harboured a transposon similar to Tn5281, originally identified in E. faecalis strain HH22 isolated in the USA. Restriction endonuclease and Southern hybridization analysis of the UK transposon showed that it is closely related to the high-level gentamicin resistance-conferring transposon Tn5281. However, the UK transposon lacks the HaeIII site identified in Tn5281. Pulsed-field gel electrophoresis analysis identified seven different patterns. Further studies with nine restriction endonucleases showed that the aac6'-aph2" gene was associated with nine different plasmid types in E. faecalis.