A NEW HIGH-LEVEL GENTAMICIN RESISTANCE GENE, APH(2'')-ID, IN ENTEROCOCCUS SPP

Enterococcus casseliflavus UC73 is a clinical blood isolate with high- level resistance to gentamicin. DNA preparations from UC73 failed to h ybridize with intragenic probes for aac(6')-Ie-aph(2 '')-Ia and aph(2 '')-Ic. A 4-kb fragment from UC73 was cloned and found to confer resis tance to gentamicin in Escherichia coli DH5 alpha transformants. Nucle otide sequence analysis revealed the presence of a 906-bp open reading frame whose deduced amino acid sequence had a region with homology to the aminoglycoside-modifying enzyme APH(2 '')-Ic and to the C-termina l domain of the bifunctional enzyme AAC(6')-APH(2 ''). The gene is des ignated aph (2 '')-Id, and its observed phosphotransferase activity is designated APH(2 '')-Id, A PCR-generated intragenic probe hybridized to the genomic DNA from 17 of 118 enterococcal clinical isolates (108 with high-level gentamicin resistance) from five hospitals. All 17 wer e vancomycin-resistant Enterococcus faecium isolates, and pulsed-field typing revealed three distinct clones. The combination of ampicillin plus either amikacin or neomycin exhibited synergistic killing against E. casseliflavus UC73. Screening and interpretation of high-level ami noglycoside resistance in enterococci may need to be modified to inclu de detection of APH(2 '')-Id.