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Antimicrobial Agents and Chemotherapy, July 2008, p. 2377-2382, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.01658-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutational Events in Cefotaximase Extended-Spectrum β-Lactamases of the CTX-M-1 Cluster Involved in Ceftazidime Resistance

Hospital Universitario Ramón y Cajal, IMSALUD, Madrid, Spain,¹ Unidad de Resistencia a Antibióticos y Virulencia Bacteriana (RYC-CSIC), and CIBER-ESP, Madrid, Spain,² Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Valencia, Spain³

Received 21 December 2007/ Returned for modification 3 March 2008/ Accepted 18 April 2008

CTX-M β-lactamases, which show a high cefotaxime hydrolytic activity, constitute the most prevalent extended-spectrum β-lactamase (ESBL) type found among clinical isolates. The recent explosive diversification of CTX-M enzymes seems to have taken place due to the appearance of more efficient enzymes which are capable of hydrolyzing both cefotaxime and ceftazidime, especially among the CTX-M-1 cluster. A combined strategy of in vitro stepwise evolution experiments using bla_CTX-M-1, bla_CTX-M-3, and bla_CTX-M-10 genes and site-directed mutagenesis has been used to evaluate the role of ceftazidime and other β-lactam antibiotics in triggering the diversity found among enzymes belonging to this cluster. Two types of mutants, P167S and D240G, displaying high ceftazidime MICs but reduced resistance to cefotaxime and/or cefepime, respectively, were identified. Such an antagonistic pleiotropic effect was particularly evident with P167S/T mutations. The incompatibility between P167S and D240G changes was demonstrated, since double mutants reduced susceptibility to both ceftazidime and cefotaxime-cefepime; this may explain the absence of strains containing both mutations in the clinical environment. The role of A77V and N106S mutations, which are frequently associated with P167S/T and/or D240G, respectively, in natural strains, was investigated. The presence of A77V and N106S contributes to restore a high-level cefotaxime resistance phenotype, but only when associated with mutations P167S and D240G, respectively. However, A77V mutation increases resistance to both cefotaxime and ceftazidime when associated with CTX-M-10. This suggests that in this context this mutation might be considered a primary site involved in resistance to broad-spectrum cephalosporins.

Published ahead of print on 28 April 2008.