Susceptibility and resistance of multidrug-resistant gram-negative bacteria to novel beta-lactam/beta-lactamase inhibitor combinations

Project Summary/Abstract Antibiotics in the b-lactam/b-lactamase inhibitor (BLBLI) class are among the mainstays of antimicrobial treatment for gram-negative bacteria such as E. coli and Pseudomonas aeruginosa. Until recently, all b- lactamase inhibitors in these combinations were themselves b-lactam compounds that lacked direct antimicrobial activity. However, bacteria are increasingly developing resistance to all currently available BLBLIs. To address this problem, new combinations are being developed that incorporate novel diazabicyclooctane (DBO) b- lactamase inhibitors, which are non-b-lactam compounds that possess intrinsic direct antimicrobial activity mediated by binding to penicillin-binding protein 2 (PBP2). While the expanded spectrum of these new combinations is promising, the highly multidrug-resistant bacteria they will be used to treat are prone to the development of additional resistance mechanisms, and PBP2-mediated antibacterial activity in particular is known to be vulnerable to the emergence of resistance during treatment. The overall goal of this project is to characterize the development of resistance to novel DBO-containing BLBLIs in order to discover how best to make use of them while preventing the emergence of resistance. In Aim #1, rates of resistance to DBO- containing BLBLIs will be assessed among a large, diverse collection of gram-negative bacterial strains including E. coli, Klebsiella pneumoniae, Enterobacter cloacae complex, and P. aeruginosa. In Aim #2, mechanisms of resistance to these agents will be investigated using two different 'omics approaches. First, whole genome sequencing will be used to identify mutations in strains in which resistance has developed. Second, gene expression profiling using RNA-Seq will be employed to investigate the transcriptomic response of both susceptible and resistant bacteria to DBO-containing BLBLI treatment. The goal of Aim #3 is to understand how to prevent resistance to DBO-containing BLBLIs in models that better simulate in vivo treatment conditions. A time-kill assay, a hollow-fiber infection model, which allows for simulation of changing antibiotic concentrations over time, and a mouse thigh infection model will be employed to identify combinations that prevent resistance over longer periods of drug exposure, and whole genome sequencing of resistant isolates will be used to compare resistance-conferring mutations that occur in these models to those observed in standard in vitro assays. The proposed project, when completed, will provide a guide to the most effective ways to utilize novel DBO-containing BLBLIs in order to effectively treat patients with MDR infections while preventing the emergence of resistance.