Aminoglycoside 6′-N-Acetyltransferase Variants of the Ib Type with Altered Substrate Profile in Clinical Isolates of Enterobacter cloacae and Citrobacter freundii
Abstract
Aminoglycoside 6′- N -Acetyltransferase Variants of the Ib Type with Altered Substrate Profile in Clinical Isolates of Enterobacter cloacae and Citrobacter freundii Isabelle Casin 1 , 2 , * , Florence Bordon 2 , † , Philippe Bertin 3 , Anne Coutrot 2 , Isabelle Podglajen 2 , Robert Brasseur 4 , and Ekkehard Collatz 2 Service de Microbiologie, Hôpital Saint-Louis, and Université Paris VII, 75010 Paris, 1 Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Paris VI, 75270 Paris Cedex 06, 2 and Unitéde Régulation de l’Expression Génétique, Institut Pasteur, 75724 Paris Cedex 15, 3 France, and Centre de Biophysique Moléculaire Numérique, Faculté des Sciences Agronomiques de Gembloux, B 5030 Gembloux, Belgium 4 ABSTRACT 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′)-Ib 7 , 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′)-Ib 8 , 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 α-helical structure within a putative aminoglycoside binding domain of AAC(6′)-Ib variants.