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.