Mecanismes de resistència als ß-lactàmics en enterobacteris, 1994-1996

All clinically relevant Enterobacteriaceae strains isolated between 1994 and 1996 without inducible chromosomal b-lactamase, that showed decreased susceptibility to broad-spectrum cephalosporins and/or aztreonam, were selected. The mechanism implicated in the decreased susceptibility was determined by analytical isoelectric focusing, PCR and/or sequenciation. The results obtained showed that the most frequent mechanism implicated in the decreased susceptibility to broad-spectrum cephalosporins and/or aztreonam in E. coli and K. oxytoca was the hyperproduction of the chromosomal b-lactamase, followed by the SHV-1 hyperproduction in E. coli and K. pneumoniae. In our hospital, the incidence of plasmid-mediated extended-spectrum b-lactamases (ESBLs) between 1994 and 1996 was low (0.14%). The b-lactamase that couldn't be identified by the techniques previously described, was characterised by conjugation studies, clonation, and sequenciation experiments, allowing us to describe a new b-lactamase, CTX-M-9, that was the most frequent ESBL detected in our laboratory. The CTX-M-type b-lactamases has been described in various species of the family Enterobacteriaceae in geographically and temporally widely distant areas, most of them being plasmid-encoded. To know more about the dissemination of these enzymes around the world, we decided to study the environment of blaCTX-M-9. This study suggested us that blaCTX-M-9 is contained in a new complex integron, In60. This integron has the common 5'-CS and 3'-CS conserved sequences of class 1 integron and two gene cassettes encoding for a dihydrofolate reductase (dfrA16) and aminoglycoside-adenylytransferase (aadA2). Downstream of the first sul1 gene is present the orf513. Following this region there is the blaCTX-M-9 and an orf3-like region showing both about 80% identity with blaKLUA-1 and orf3 of K. ascorbata, respectively. Downstream of the orf3-like region, a new insertion sequence designated IS3000, is found. This IS is flanked by two imperfect inverted repeats and its deduced amino acid sequence presents the DD(35)-E motif highly conserved in the transposases. Close to the downstream IS3000 inverted repeat the second 3'-CS, is found. The presence of In60 was evaluated in a total of 37 enterobacteria isolated between 1994 and 1999 carrying a b-lactamase compatible with CTX-M-9. The results obtained showed that 33 strains had the environment compatible with In60 and four strains showed modifications or did not share this environment.