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.
