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

COLQUE CA; ALBARRACÍN ORIO A; FELIZIANI S; VILA A; SMANIA AM

Córdoba

Congreso; LII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2016

Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of antipseudomonal penicillin and cephalosporin resistance in Pseudomonas aeruginosa. A Whole-genome sequencing analysis of 14 MRS-deficient mutator strains obtained from a cystic fibrosis (CF) patient was performed. These results revealed five so far uncharacterized mutations in the blaampC gene, A89V, Q120K, H189Y, G216S and N321S, which combined with 1 to 2 already characterized substitutions lead to 4 new blaampC allele variants named as PDC-94 (2 mutations), PDC-95 (3 mutations), and PDC-96 and 97 (4 mutations). ,. AmpC variant PDC-97 was expressed by 8 isolates, PDC-96 by 2, PDC-94 and PDC-95 variants were found in single isolates, whereas earliest isolates harbor unaltered blaampC allelic variants (PDC-1). In silico analysis showed that most of the mutations were located in the o mega loop, which is part of the active site of the AmpC enzyme and a hot spot for mutations able to extend the substrate specificity of class C β-lactamases. Of note, MICs to different β-lactam antibiotics displayed high resistance to cefepime in those isolates carrying the four mutations-containing blaampC alleles. In order to explore the impact of PDCs on β-lactam MICs, the ampC genes of the 4 PDC-94/97 enzymes were amplified by PCR, cloned into vector pMBLe, and overexpressed in an AmpC-deficient P. aeruginosa PAO1 mutant constructed by allelic replacement using the suicide vector pKNG101. The same approach was used to clone the blaAmpC gene from the standard strain PAO1. MICs to different antibiotics, including carbapenems, are being performed to determine if they lead to extended-spectrum AmpCs (ESACs).