| Autor: |
Mojica MF; Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106, United States.; Research Service, Veterans Affairs Northeast Ohio Healthcare System, Cleveland, Ohio 44106, United States.; CASE-VA Center for Antimicrobial Resistance and Epidemiology, Cleveland, Ohio 44106, United States., Nukaga M; Pharmaceutical Sciences, Josai International University, Togane City, Chiba 283-8555, Japan., Becka SA; Research Service, Veterans Affairs Northeast Ohio Healthcare System, Cleveland, Ohio 44106, United States., Zeiser ET; Research Service, Veterans Affairs Northeast Ohio Healthcare System, Cleveland, Ohio 44106, United States., Hoshino T; Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba 263-8522, Japan., LiPuma JJ; Department of Pediatrics and Communicable Disease, University of Michigan Medical School, Ann Arbor, Michigan 48109, United States., Papp-Wallace KM; Research Service, Veterans Affairs Northeast Ohio Healthcare System, Cleveland, Ohio 44106, United States.; CASE-VA Center for Antimicrobial Resistance and Epidemiology, Cleveland, Ohio 44106, United States.; Departments of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, United States.; Departments of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States. |
| Abstrakt: |
In our curated panel of Burkholderia cepacia complex isolates, Burkholderia multivorans strain AU28442 was unusually highly β-lactam resistant. To explore the molecular mechanisms leading to this phenotype, we performed whole genome sequencing (WGS) and microbiological and biochemical assays. WGS analysis revealed that strain AU28442 produced two β-lactamases, AmpC22 and a novel PenA-like β-lactamase denominated PenA39. Additionally, the strain presented frame-shift mutations in the genes encoding penicillin binding proteins 3 (PBP3) and 4 (PBP4). The antibiotic susceptibilities of the parent AU28442 strain carrying bla PenA39 vs the isogenic E. coli strain producing bla PenA39 were discrepant with ceftazidime MICs of >512 and 1 μg/mL, respectively. Accordingly, PenA39 was found to poorly hydrolyze β-lactams with k cat values of ≤8.8 s -1 . An overlay of the crystal structure of PenA39 with PenA1 revealed a shift in the SDN loop in the variant, which may affect the catalytic efficiency of PenA39 toward substrates and inhibitors. Moreover, microscopic examination of AU28442 revealed shortened rod-shaped cells compared to B. multivorans ATCC 17616, which carries a full complement of intact PBPs. Further complementation assays confirmed that the loss of PBP3 and PBP4 was the main factor contributing to the high-level β-lactam resistance observed in B. multivorans AU28442. This information allowed us to revert susceptibility by pairing a potent β-lactamase inhibitor with a β-lactam with promiscuous PBP binding. This detailed characterization of B. multivorans provides an illustration of the myriad ways in which bacteria under antibiotic selection can develop resistance and demonstrates a mechanism to overcome it. |
