Examining the influence of chromosome copy number on Pseudomonas aeruginosa persisters to fluoroquinolones

Project Summary
Persisters are antibiotic-tolerant cells that are genetically identical to the overall population that succumbs to
treatment but occupy a favorable phenotypic niche that allows survival. Persisters are an important health
concern because they are thought to contribute to chronic and recurrent infections, and recent studies have
shown that persisters can foster resistance development. Fluoroquinolone (FQ) persisters are particularly
worrisome, since FQs are one of the few antibiotic classes that can kill slow/non-growing bacteria, and it has
been shown that FQ persisters experience de novo mutation following treatment that accelerates resistance
development to various independent antibiotics. Recently, we found that the number of copies of the chromo-
some individual E. coli contain (#Chr) is a critical phenotypic variable for FQ persister survival, due to the lack
of a homologous template for homologous recombination (HR) in 1Chr cells compared to cells with 2Chr or
more. In this project, we will focus on P. aeruginosa, which is one of the ESKAPE pathogens responsible for
the majority of nosocomial infections, and FQs, which offer the only oral treatment options for P. aeruginosa.
We hypothesize that persistence to FQs will depend on #Chr in individual P. aeruginosa, and that the genes
that mediate FQ persister survival will depend on #Chr as well. For example, DNA double-strand breaks are
caused by FQs and they can be repaired by HR or non-homologous end joining (NHEJ). P. aeruginosa has HR
and NHEJ, but HR cannot function in 1Chr cells (E. coli only has HR). The role of #Chr in FQ persistence of P.
aeruginosa remains unexplored, and little is known of the genetic basis of FQ persistence in P. aeruginosa. To
test our hypotheses, we will count #Chr in individual P. aeruginosa with nucleic acid stains and a fluorescent
origin reporter, segregate populations based on #Chr with FACS, and measure persistence to FQ (Aim 1). We
will then investigate the role of DNA repair in FQ persisters with different #Chr using a genetic approach (Aim
2), because we recently found that 2Chr and 1Chr E. coli persisters use different DNA repair machinery to
survive FQs. Further, we will explore genetic mediators beyond DNA repair by performing Tn-seq on libraries
that will be segregated based on #Chr, subjected to FQ persistence assays, and sequenced (Aim 3). Through-
out this project, we will use stationary-phase cultures because they have well-resolved unit Chr (e.g., 1Chr,
2Chr) and reflect difficult-to-eradicate infections (slow/non-growing bacteria). Ciprofloxacin and P. aeruginosa
PA01 (lab model) will be the main experimental system, with the generality of findings assessed with levo-
floxacin, PA14 (distinct lab model), MRSN 1612 (recent human ear isolate), and MRSN 11976 (recent human
lung isolate). This work will fill knowledge gaps in understanding of persistence to an antibiotic class regarded
as essential medicine by the WHO in an opportunistic pathogen that causes chronic infections, and novel
strategies to reduce relapse infections and prevent resistance development will be identified.

Grant Number: 1R21AI185486-01A1
NIH Institute/Center: NIH
Principal Investigator: Mark Brynildsen