Mutational Events in ESBL-Cefotaximases of the CTX-M-1 Cluster Involved in Ceftazidime Resistance

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

^* To whom correspondence should be addressed. Email: jgalanm.hrc{at}salud.madrid.org.

	Abstract

CTX-M -lactamases, which show a high cefotaxime hydrolytic activity, constitute the most prevalent 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, capable of hydrolyzing both cefotaxime and ceftazidime, especially among the CTX-M-1 cluster. A combined strategy of in-vitro step-wise 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 -lactam antibiotics in triggering the diversity found among enzymes belonging to this cluster. Two types of mutants, P167S and D240G, displaying high ceftazidime MIC values but reduced resistance to cefotaxime and/or cefepime respectively, were identified. Such antagonistic pleiotropic effect was particularly evident with P167S/T mutations. The incompatibility between P167S and D240G changes was demonstrated, as double mutants reduced susceptibility to both ceftazidime and cefotaxime-cefepime, which may explain the absence of strains containing both mutations in the hospital 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 third generation cephalosporins.