Genetic characterization of an extended-spectrum AmpC cephalosporinase with hydrolysing activity against fourth-generation cephalosporins in a clinical isolate of Enterobacter aerogenes selected in vivo.

Background: Extended-spectrum AmpC cephalosporinases (ESACs) have been reported in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we characterize a new AmpC variant presenting a broadened substrate activity towards fourth-generation cephalosporins, selected in vivo following cefepime treatment for Enterobacter aerogenes. Methods: Two consecutive clonally related isolates of E. aerogenes were evaluated. Screening for ESAC production was performed using plates containing 200 mg/L cloxacillin. MICs were determined by microdilution (CLSI guidelines). bla(AmpC) genes were cloned into a pCR-Blunt II-TOPO vector and expressed in Escherichia coli. The ampC genes were cloned into vector pGEX-6P-1 for protein purification. Results: Isolate Ea595 was resistant to two fourth-generation cephalosporins, cefepime and cefpirome; using plates containing cloxacillin, susceptibility to ceftazidime and cefepime was restored, suggesting overproduction of the ESAC beta-lactamase. Sequencing identified a new AmpC beta-lactamase variant presenting one amino acid substitution, Val291Gly, inside the H-10 helix. Recombinant plasmids harbouring this ESAC beta-lactamase conferred a broadened resistance profile to cefepime and cefpirome, with resistance levels increasing from 16-to 32-fold in E. coli. AmpC-Ea595 hydrolysed ceftazidime, cefepime and cefpirome at high levels, presenting a lower K-m and enabling us to classify the enzyme as an ESAC. Homology modelling suggested that the size of the active site could have increased. Conclusions: We characterized an ESAC beta-lactamase selected in vivo and conferring a high level of resistance to fourth-generation cephalosporins in E. aerogenes. The broadened spectrum was caused by a new modification to the H-10 helix, which modified the active site.