Frameshift Mutations\nin Genes Encoding PBP3 and PBP4\nTrigger an Unusual, Extreme β‑Lactam Resistance Phenotype\nin Burkholderia multivorans

In our curated panel of Burkholderia cepacia complex isolates, Burkholderia multivorans strain AU28442 was unusually highly β-lactam resistant. To\nexplore the molecular mechanisms leading to this phenotype, we performed\nwhole genome sequencing (WGS) and microbiological and biochemical\nassays. WGS analysis revealed that strain AU28442 produced two β-lactamases,\nAmpC22 and a novel PenA-like β-lactamase denominated PenA39.\nAdditionally, the strain presented frame-shift mutations in the genes\nencoding penicillin binding proteins 3 (PBP3) and 4 (PBP4). The antibiotic\nsusceptibilities of the parent AU28442 strain carrying <i>bla</i><sub>PenA39</sub> vs the isogenic E. colistrain producing <i>bla</i><sub>PenA39</sub> were discrepant\nwith ceftazidime MICs of >512 and 1 μg/mL, respectively.\nAccordingly,\nPenA39 was found to poorly hydrolyze β-lactams with <i>k</i><sub>cat</sub> values of ≤8.8 s<sup>–1</sup>. An overlay of the crystal structure of PenA39 with PenA1 revealed\na shift in the SDN loop in the variant, which may affect the catalytic\nefficiency of PenA39 toward substrates and inhibitors. Moreover, microscopic\nexamination of AU28442 revealed shortened rod-shaped cells compared\nto B. multivoransATCC 17616, which\ncarries a full complement of intact PBPs. Further complementation\nassays confirmed that the loss of PBP3 and PBP4 was the main factor\ncontributing to the high-level β-lactam resistance observed\nin B. multivoransAU28442. This information\nallowed us to revert susceptibility by pairing a potent β-lactamase\ninhibitor with a β-lactam with promiscuous PBP binding. This\ndetailed characterization of B. multivoransprovides an illustration of the myriad ways in which bacteria under\nantibiotic selection can develop resistance and demonstrates a mechanism\nto overcome it.