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

• DOI: 10.1128/aac.00822-16
• Publication Date: 2016-08-02T03:04:34Z
• Authors: Guo-Bao Tian; Yi-Qi Jiang; Ying-Min Huang; Yun Qin; Lian-Qiang Feng; Xue-Fei Zhang; Hong-Yu Li; Lan-Lan Zhong; Kun-Jiao Zeng; Sandip Patil; Yong Xing; Xi Huang
• Keywords: Threonine; 0301 basic medicine; Base Composition; Alanine; Hydrolysis; Gene Expression; Microbial Sensitivity Tests; Recombinant Proteins; beta-Lactamases; Anti-Bacterial Agents; Cephalosporins; 3. Good health; Isoenzymes; 03 medical and health sciences; Amino Acid Substitution; Genome Size; Drug Resistance, Bacterial; Mutation; Escherichia coli; Cloning, Molecular; Proteus mirabilis; Genome, Bacterial

ABSTRACT

CTX-M-140, a novel CTX-M-type extended-spectrum β-lactamase (ESBL), was identified in cephalosporin-resistant clinical isolates of Proteus 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 an Escherichia coli isogenic 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 the E. coli host 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 the E. coli clones. Compared with that of CTX-M-14, a lower hydrolytic activity against cephalosporins was observed for CTX-M-140. bla CTX-M-140 is located on the chromosome as determined by I-CeuI pulsed-field gel electrophoresis (I-CeuI-PFGE) and Southern hybridization. The genetic environment surrounding bla CTX-M-140 is identical to the sequence found in different plasmids with bla CTX-M-9-group genes among the Enterobacteriaceae . Genome sequencing and analysis showed that P. mirabilis strains with bla CTX-M-140 have 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.