Characterization of mutations in the β-Lactamase AmpC from Pseudomonas aeruginosa CF isolates

COLQUE A; ALBARRACIN A; FELIZIANI S; MEINI R; VILA AJ; SMANIA A

Congreso; LII Reunión Anual de SAIB; 2016

Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of penicillin and cephalosporin resistance in Pseudomonas aeruginosa (PA). We previously sequenced the genomes of 14 mutator isolates of PA from a cystic fibrosis patient, spanning 20 years of infection history. Results revealed uncharacterized mutations in the ampC gene which led to 4 new allele variants. In silico analysis showed that most of the mutations were located in the omega loop, which is part of the AmpC active site and a hot spot for mutations able to extend the substrate specificity of β-lactamases. Whereas earliest isolates harbor unaltered ampC alleles, those obtained later accumulate 2 to 4 mutations and constitute the different allelic variants. Of note, in those late isolates carrying ampC alleles with 4 mutations, we found a high resistance level to the β-lactam cefepime. In order to explore the impact of these mutations on β-lactam MICs, we designed a strategy to express the 4 ampC variants in an ampC-deficient background of PA. The ΔampC mutant was obtained by allelic replacement using the pKNG101 suicide vector. The ampC alleles were amplified by PCR and cloned into the pMBLe vector which allowed us to tightly control the expression of the gene. MICs to different antibiotics, including carbapenems, are being performed to determine if they lead to extended-spectrum AmpCs.