Investigating ploidy modulation as a strategy to improve antibiotic activity

Project Summary
Persisters are antibiotic-tolerant cells that are genetically identical to the overall population that succumbs to
treatment, but they occupy a favorable phenotypic niche that enables survival. Persisters are an important
health concern because they are thought to contribute to chronic and recurrent infections, and recently, studies
have demonstrated that persisters can foster the development of antibiotic resistance. Fluoroquinolone (FQ)
persisters are particularly worrisome because FQs are one of the few antibiotic classes that can kill growth-
limited bacteria, and it has been shown that FQ persisters from stationary-phase populations experience de
novo mutation following treatment that not only accelerates resistance development for FQs but for
independent antibiotics as well (e.g., rifampicin, carbenicillin, D-cycloserine, fosfomycin). Recent work has
found that the chromosome copy number in individual bacteria (ploidy) is an important determinant of FQ
persistence, due to the inability of monoploids to conduct highly efficient homologous recombination to repair
FQ-induced DNA damage. Interestingly, polyploidy has been reported to increase the tolerances of bacteria to
diverse stresses. Here, we hypothesize that ploidy modulation could be a strategy to improve antibiotic killing
of growth-limited bacteria and reduce relapse infections. To test this hypothesis, we will identify DNA repair
requirements of monoploid and diploid persisters to FQs (levofloxacin, ciprofloxacin, moxifloxacin: most
commonly prescribed FQs); examine whether ploidy impacts killing by other antibiotics (UTI treatments:
nitrofurantoin, trimethoprim-sulfamethoxazole; digestive tract treatment: rifaximin); and screen for compounds
(e.g., FDA-approved compound repurposing library) that enhance monoploidy (least tolerant ploidy state). To
accomplish these tasks, we will use Escherichia coli MG1655 (laboratory model), CFT073 (urosepsis isolate),
and UTI89 (uropathogenic), and measure ploidy with Hoechst 33342 (live cell nucleic acid stain), PicoGreen
(dsDNA stain for fixed cells), and a fluorescent protein-based origin reporter where the number of fluorescent
foci indicate the number of chromosomes. We will use fluorescence activated cell sorting (FACS) to segregate
populations based on ploidy and then conduct persistence and antibiotic tolerance assays on those
subpopulations for wild-type and DNA repair mutants. In addition, we will screen a focused (FDA-approved
compound repurposing library) and diverse small molecule library for the ability to increase monoploidy in
growth-inhibited bacteria using Hoechst 33342 and high-throughput flow cytometry. Collectively, these
experiments will investigate how a phenotypic variable (ploidy) that often goes ignored impacts persistence to
FQs and other antibiotics. Further, this work will begin to examine that feature as a druggable target, with an
eye toward ploidy modulators (foster monoploidy) as new antibiotic adjuvants with anti-persister activity.

Grant Number: 5R21AI182745-02
NIH Institute/Center: NIH
Principal Investigator: Mark Brynildsen