Characterization of CTX-M-140, a Variant of CTX-M-14 Extended-Spectrum β-Lactamase with Decreased Cephalosporin Hydrolytic Activity, from Cephalosporin-Resistant Proteus mirabilis

Abstract

ABSTRACTCTX-M-140, a novel CTX-M-type extended-spectrum β-lactamase (ESBL), was identified in cephalosporin-resistant clinical isolates ofProteus mirabilis. CTX-M-140 contained an alanine-to-threonine substitution at position 109 compared to its putative progenitor, CTX-M-14. When it was expressed in anEscherichia coliisogenic background, CTX-M-140 conferred 4- to 32-fold lower MICs of cephalosporins than those with CTX-M-14, indicating that the phenotype was attributable to this single substitution. For four mutants of CTX-M-14 that were constructed by site-directed mutagenesis (A109E, A109D, A109K, and A109R mutants), MICs of cephalosporins were similar to those for theE. colihost strain, which suggested that the alanine at position 109 was essential for cephalosporin hydrolysis. The kinetic properties of native CTX-M-14 and CTX-M-140 were consistent with the MICs for theE. coliclones. Compared with that of CTX-M-14, a lower hydrolytic activity against cephalosporins was observed for CTX-M-140.blaCTX-M-140is located on the chromosome as determined by I-CeuI pulsed-field gel electrophoresis (I-CeuI-PFGE) and Southern hybridization. The genetic environment surroundingblaCTX-M-140is identical to the sequence found in different plasmids withblaCTX-M-9-groupgenes among theEnterobacteriaceae. Genome sequencing and analysis showed thatP. mirabilisstrains withblaCTX-M-140have a genome size of ∼4 Mbp, with a GC content of 38.7% and 23 putative antibiotic resistance genes. Our results indicate that alanine at position 109 is critical for the hydrolytic activity of CTX-M-14 against oxyimino-cephalosporins.