Mutations in ampD cause hyperproduction of AmpC and CphA beta-lactamases and high resistance to beta-lactam antibiotics in Chromobacterium violaceum

Abstract

Bacterial resistance to beta-lactam antibiotics mediated by beta-lactamase enzymes is widespread worldwide. Chromobacterium violaceum, an environmental Gram-negative bacteria pathogen, is intrinsically resistant to some beta-lactam antibiotics. In this work, we found that mutations in an ampD gene, encoding a peptidoglycan-recycling amidase, cause hyperproduction of two chromosomal beta-lactamases (AmpC and CphA), conferring high beta-lactam resistance in C. violaceum. Susceptibility tests using {Delta}ampC, {Delta}cphA, and{Delta} ampC{Delta}cphA mutant strains revealed specific susceptibility profiles to penicillin, cephalosporin, and carbapenem beta-lactams, suggesting that AmpC is a broad-spectrum beta-lactamase (penicillinase and cephalosporinase), while CphA is a narrow-spectrum metallo-carbapenemase. Beta-galactosidase assays indicate that the expression of ampC and cphA increased in response to beta-lactams. We isolated C. violaceum spontaneous mutants resistant to the antibiotic ceftazidime and found that most mutants were also resistant to several other beta-lactams and overexpressed ampC and cphA. DNA sequencing of the three paralog genes encoding the C. violaceum AmpD amidases revealed mutations of different types in AmpD1 (CV_0566) in most of the spontaneous mutants, but no mutation was found in AmpD2 or AmpD3. Analysis of single and combined null amidase mutants revealed overexpression of both beta-lactamases and increased resistance to beta-lactams only in mutants with deleted ampD1. When introduced into ampD1 null or spontaneous mutants, the ampD1 gene rescued the antibiotic-related phenotypes. The AmpD1 amidase from C. violaceum has a unique architecture with an N-terminal acetyltransferase domain. Our work offers new insights into the mechanisms of beta-lactamase-mediated antibiotic resistance and open perspectives to improve the treatment of C. violaceum infections.