AMINOGLYCOSIDE 6'-N-ACETYLTRANSFERASE VARIANTS OF THE IB TYPE WITH ALTERED SUBSTRATE PROFILE IN CLINICAL ISOLATES OF ENTEROBACTER-CLOACAE AND CITROBACTER-FREUNDII

Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Ci trobacter freundii CFr564, displayed an aminoglycoside resistance prof ile evocative of low-level 6'-N acetyltransferase type II [AAC(6')-II] production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin. Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6') which acet ylated amikacin practically as well as it acetylated gentamicin in vit ro. Both compounds, however, as well as isepamicin, retained good bact ericidal activity against the three strains. The aac genes were borne by conjugative plasmids (pLMM562 and pLMM564 of ca. 100 kb and pLMM563 of ca. 20 kb). By PCR mapping and nucleotide sequence analysis, an aa c(6')-Ib gene was found in each strain upstream of an ant(3'')-I gene in a sull-type integron. The size of the AAC(6')-Ib variant encoded by pLMM562 and pLMM564, AAC(6')-Ib(7), was deduced to be 184 (or 177) am ino acids long, whereas in pLMM563 a 21-bp duplication allowing the re cruitment of a start codon resulted in the translation of a variant, A AC(6')-Ib(8), of 196 amino acids, in agreement with size estimates obt ained by Western blot analysis. Both variants had at position 119 a se rine instead of the leucine typical for the AAC(6')-Ib variants confer ring resistance to amikacin. By using methods that predict the seconda ry structure, these two amino acids appear to condition an cy-helical structure within a putative aminoglycoside binding domain of AAC(6')-I b variants.