Insertion sequence ISEcp1-like element connected with a novel aph(2") allele [aph(2"-le] conferring high-level gentamicin resistance and a novel streptomycin ad enylyltransferase gene in Enterococcus

Enterococcus casseliflavus HZ95 is an enterococcus with high-level resistance to aminoglycosides. Nine genes responsible for high-level aminoglycoside resistance, including aac(6')-leaph(2")-Ia, aph(2")-Ib, aph(2")-Ic, aph(2")-Id, aph(3')-IIIa, aac(6')-Ii, ant(3')-Ia, ant(4')-Ia and ant(6')-Ia, were not detected in HZ95. An 8 kb fragment from unconjugative plasmids of HZ95 was cloned, and expressed gentamicin resistance in Escherichia coli DH5 alpha. The genetic structures (similar to 8 kb DNA fragment) containing these aminoglycoside-modifying enzyme genes in Ent. casseliflavus HZ95 were determined. The deduced amino acid sequence of the novel aph(2") allele, aph(2")-Ie, had 93(.)7% amino acid identity with APH(2")-Id. The aph(2")-Ie gene was bracketed upstream by an insertion sequence (IS)Ecp1-like element and downstream by a streptomycin adenylyltransferase gene (str). The streptomycin adenylyltransferase encoded by the str gene had 80(.)3% amino acid identity with the protein encoded by aadE. The plasmid of similar to 16 kb could hybridize with a PCR-generated aph(2")-Ie intragenic probe. The ISEcp1-like element had 91 identity with ISEcp1. ISEcp1, which commonly acts as a key factor in the dissemination of CTX-M-type beta-lactamase genes in Gram-negative bacteria, has not been reported in Enterococcus.