Novel OXA-10-derived extended-spectrum beta-lactamases selected in vivo or in vitro

Abstract

A clinical isolate of Pseudomonas aeruginosa, PAe191, was found to be highly resistant to all anti-Pseudomonas beta-lactam antibiotics (except imipenem) and resistant also to aminoglycosides. It produced a beta-lactamase (with an apparent pI of 7.6) which was not inhibited by clavulanic acid. Cloning and characterization of the beta-lactamase gene showed that it coded for a novel extended-spectrum OXA-10 variant, called OXA-19, which differed from OXA-10 by nine amino acids and from OXA-13 by two, i.e., Asn in position 73 (Asn73) instead of Ser and Asp157 instead of Gly. Asparagine in position 157 is implicated in resistance to ceftazidime, while the amino acid in position 73, in this variant, seems to condition the level of resistance to penicillins. The oxa19 gene was found to be inserted, in a typical integron structure, immediately downstream from an aac(6')-Ib gene coding for an aminoglycoside acetyltransferase variant, which was called AAC(6')-Ib9.

FIG. 1

Amino acid differences between the β-lactamases of the OXA-10 group. Amino acid numbering is according to Huovinen et al. (13); the conserved motifs typical for class D enzymes are boxed. The amino acids shown in boldface contribute to the substrate profile, with asparagine in position 157 leading to extended-spectrum variants. The amino acids of OXA-10 that are not numbered are G20, S27, S50, D55, V89, T107, Y174, A197, E229, T230, S245, and E259; −, absence of an amino acid.

FIG. 2

Structure of the integron encoding the β-lactamases OXA-19 and OXA-13-1. Nucleotide sequences are identical in both clusters, except for three differences in the −35 sequences of the promoter and in the aac(6′)-Ib and the oxa genes. Amino acid numbering is from Tran Van Nhieu and Collatz (36) and Huovinen et al. (13), respectively. Arrows represent the following open reading frames: intI1, for integrase; aac(6′)-Ib, for the aminoglycoside acetyltransferases genes aac(6′)-Ib₁₀ on pAZ310 (20) and aac(6′)-Ib₉ on pAZ316; oxa, for the OXA variants indicated on the left; and qacEΔ1, for the typically truncated quaternary ammonium resistance-conferring gene associated with the 3′ conserved segment of integrons. The intI1 and qacEΔ1 genes have been only partially sequenced here (indicated in boldface). Data for pAZ309 are from reference , and data for pAZ310 and pAZ316 are from the present study. Boldface, differences with respect to the OXA-13-encoding plasmid pAZ309; dashed line, the oxa13-1 region that has been sequenced.

FIG. 3

Isoelectric focusing of extended-spectrum OXA-10-related β-lactamases. Enzymes SHV-1 (pI_app, 7.6), SHV-5 (pI_app, 8.2), OXA-13 (pI_app, 8 [18]), and OXA-10 (pI_app, 6.1) were used as pI standards. The two novel β-lactamases, OXA-13-1 and OXA-19, had estimated pI_apps of 7.8 and 7.6, respectively. β-Lactamase activity was revealed by overlaying the gel with nitrocefin.