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 Citrobacter
freundii CFr564, displayed an aminoglycoside resistance profile 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 acetylated amikacin practically as well as it
acetylated gentamicin in vitro. Both compounds, however, as well as isepamicin,
retained good bactericidal 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 aac(6')-Ib gene
was found in each strain upstream of an ant(3")-I gene in a sulI-type integron.
The size of the AAC(6')-Ib variant encoded by pLMM562 and pLMM564, AAC(6')-Ib7,
was deduced to be 184 (or 177) amino acids long, whereas in pLMM563 a 21-bp
duplication allowing the recruitment of a start codon resulted in the translation
of a variant, AAC(6')-Ib8, of 196 amino acids, in agreement with size estimates
obtained by Western blot analysis. Both variants had at position 119 a serine
instead of the leucine typical for the AAC(6')-Ib variants conferring resistance
to amikacin. By using methods that predict the secondary structure, these two
amino acids appear to condition an alpha-helical structure within a putative
aminoglycoside binding domain of AAC(6')-Ib variants.