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

Bacterial resistance to β-lactam antibiotics mediated by β-lactamase enzymes is widespread worldwide. Chromobacterium violaceum , an environmental Gram-negative bacterial pathogen, is intrinsically resistant to some β-lactam antibiotics. In this work, we found that mutations in an ampD gene, encoding a peptidoglycan-recycling amidase, cause hyperproduction of two chromosomal β-lactamases (AmpC and CmcB), conferring high β-lactam resistance in C. violaceum . Susceptibility tests using Δ ampC , Δ cmcB , and Δ cmcBΔampC mutant strains revealed specific susceptibility profiles to penicillin, cephalosporin, and carbapenem β-lactams, suggesting that AmpC is a broad-spectrum β-lactamase (penicillinase and cephalosporinase), while CmcB is a narrow-spectrum metallo-carbapenemase. β-galactosidase assays indicate that the expression of ampC and cmcB increased in response to β-lactams. We isolated C. violaceum spontaneous mutants resistant to the antibiotic ceftazidime and found that most mutants were also resistant to several other β-lactams and overexpressed ampC and cmcB . 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 β-lactamases and increased resistance to β-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 β-lactamase-mediated antibiotic resistance and opens perspectives to improve the treatment of C. violaceum infections.

IMPORTANCE

Resistance to β-lactam antibiotics reduces the options for treating bacterial infections, posing a threat to public health. In this work, we demonstrated that the intrinsic resistance to β-lactam antibiotics in the environmental pathogen Chromobacterium violaceum is mediated by two chromosomally encoded β-lactamases, AmpC and CmcB, and revealed the mechanism that contributes to their simultaneous expression. Our data indicate that mutations in the peptidoglycan recycling amidase ampD1 , but not in its paralogs ampD2 and ampD3 , lead to stable overexpression of both β-lactamases and increased resistance to β-lactam antibiotics. Remarkably, AmpD1 possesses a unique N-terminal acetyltransferase domain, suggesting a distinct functional mechanism for this enzyme. Our work offers an explanation for the limited effectiveness of many β-lactams in treating C. violaceum infections. Understanding the mechanism of antimicrobial resistance is crucial for developing effective treatments and mitigating the spread of β-lactam-resistant bacteria.