Identification of a novel carbapenem-hydrolysing class D β-lactamase RAD-1 inRiemerella anatipestifer

Abstract

Objectives To elucidate the role of a novel carbapenem-hydrolysing class D β-lactamase (RAD-1) from Riemerella anatipestifer. Methods We applied WGS and bioinformatic analysis to screen putative β-lactamase genes in R. anatipestifer SCVM0004. A putative class D β-lactamase gene was cloned into pET24a and transferred into Escherichia coli BL21 (DE3) for antibiotic susceptibility determination and protein purification. Meanwhile, the purified native protein was used to determine the enzymatic activities. Results A class D β-lactamase, RAD-1, was identified in the genome of R. anatipestifer SCVM0004. It was distinct from all characterized class D β-lactamases (≤42% amino acid sequence identity). Searching in GenBank showed that blaRAD-1 was widely disseminated among R. anatipestifer. Genomic environment analysis indicated that the chromosomal structures of blaRAD-1-located regions were relatively conserved. Expression of RAD-1 in E. coli results in elevated MICs for various β-lactam antibiotics, including penicillins, extended-spectrum cephalosporins, a monobactam and carbapenems. Moreover, kinetic analysis of purified RAD-1 revealed: (i) high-level activity against penicillins; (ii) highest affinity for carbapenems; (iii) moderate hydrolysis of extended-spectrum cephalosporins and a monobactam; and (iv) no activity for oxacillin and cefoxitin. Conclusions This study identified a novel chromosomally located class D carbapenemase RAD-1 (Bush–Jacoby functional group 2def) in R. anatipestifer SCVM0004. Moreover, bioinformatic analysis confirmed that the RAD-1 was widely prevalent and conserved in R. anatipestifer.

Epidemiological investigation and β-lactam antibiotic resistance of Riemerella anatipestifer isolates with waterfowl origination in Anhui Province, China

“…Through gene cloning, it was found that the mutant mediated resistance to penicillin, ampicillin, piperacillin, amoxicillin, cefotaxime, and cefazolin in β-lactam antibiotics in Escherichia coli . However, this gene did not exhibit high resistance in wild isolates, aligning with a previous study on tetX by Li et al (2022 ) , and the reported bla RAD-1 in R. anatipestifer ( Li et al, 2023 ). These observations suggest that while R. anatipestifer hosts to these resistance genes, they may manifest higher antibiotic resistance potential in other bacteria.…”