Convergent in vivo and in vitro selection of ceftazidime resistance mutations at position 167 of CTX-M-3 β-lactamase in hypermutable Escherichia coli strains

We report on a novel CTX-M extended-spectrum P-lactamase (ESBL), designated CTX-M-42, with enhanced activity toward ceftazidime. CTX-M-42 was identified in a hypermutable Escherichia coli nosocomial isolate (isolate Irk2320) and is a Pro167Thr amino acid substitution variant of CTX-M-3. By molecular typing of ESBL-producing E. coli strains previously isolated in the same hospital ward, we were able to identify a putative progenitor (strain Irk1224) of Irk2320, which had a mutator phenotype and harbored the CTX-M-3 beta-lactamase. To reproduce the natural evolution of CTX-M-3, we selected for ceftazidime resistance mutations in bla(CTX-M-3) gene in vitro both in clinical isolate Irk1224 and in laboratory-derived hypermutable (mutD5) strain GM2995. These experiments yielded CTX-M-3(Pro167Ser) and CTX-M-3(Asn136Lys) mutants which conferred higher levels of resistance to ceftazidime than to cefotaxime. CTX-M-3(Asn136Lys) had a level of low activity toward ampicillin, which may explain its absence from clinical isolates. We conclude that the selection of CTX-M-42 could have occurred in vivo following treatment with ceftazidime and was likely facilitated by the hypermutable background.