A novel structure of Tn4001-truncated element, type V, in clinical enterococcal isolates and multiplex PCR for detecting aminoglycoside resistance genes

A multiplex polymerase chain reaction (PCR) was established for detecting aacA-aphD, aph(2')-Ib, aph(2')-Ic and aph(2')-Id, encoding high-level gentamicin resistance (HLGR), and aadA and aadE, encoding high-level streptomycin resistance (HLSR), in enterococci. The assay was implemented for 419 enterococcal blood and urine isolates recovered from patients at a university hospital in Thailand. Among the isolates tested, 56.1% (235 isolates) and 58.9% (247 isolates) contained aacA-aphD and aadE, respectively. The aph(2')-Ib, aph(2')-Ic, aph(2')-Id and aadA genes were not found in any isolate. Among the isolates carrying the aacA-aphD gene, 99.1% exhibited a HLGR phenotype. All 235 enterococcal isolates containing aacA-aphD were further studied by PCR to characterise the structure of the resistance determinants carrying the aacA-aphD gene. The result revealed that only 22.6% carried Tn4001-related element, whereas the remaining isolates contained Tn4001-truncated element. No Tn4001-IS257 hybrid structure was detected. The majority of isolates carrying Tn4001-related element were Enterococcus faecalis (77.4%). Among Tn4001-truncated elements detected, all previously reported types (types I-IV) were found. Furthermore, a novel Tn4001-truncated type, designated type V, was also identified.