Frameshift Mutations in Genes Encoding PBP3 and PBP4 Trigger an Unusual, Extreme β-Lactam Resistance Phenotype in .

In our curated panel of complex isolates, 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 vs the isogenic strain producing were discrepant with ceftazidime MICs of >512 and 1 μg/mL, respectively. Accordingly, PenA39 was found to poorly hydrolyze β-lactams with values of ≤8.8 s. 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 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 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 provides an illustration of the myriad ways in which bacteria under antibiotic selection can develop resistance and demonstrates a mechanism to overcome it.