Identification of Escherichia coli CFT073 fitness factors during urinary tract infection using an ordered transposon library.

Urinary tract infections (UTI), the second most diagnosed infectious disease worldwide, are caused primarily by uropathogenic Escherichia coli (UPEC), placing a significant financial burden on the healthcare system. High-throughput transposon mutagenesis combined with genome-targeted sequencing is a powerful technique to interrogate genomes for fitness genes. Genome-wide analysis of E. coli requires random libraries of at least 50,000 mutants to achieve 99.99% saturation; however, the traditional murine model of ascending UTI does not permit testing of large mutant pools due to a bottleneck during infection. To address this, an E. coli CFT073 transposon mutant ordered library of 9216 mutants was created and insertion sites were identified. A single transposon mutant was selected for each gene to assemble a condensed library consisting of 2913 unique non-essential mutants. Using a modified UTI model in BALB/c mice, we identified 36 genes important for colonizing the bladder, including: purB, yihE, and carB Screening of the condensed library in vitro identified yigP and ubiG to be essential for growth in human urine. Additionally, we developed a novel qPCR technique to identify genes with fitness defects within defined subgroups of related genes (e.g, fimbria, toxins, etc) following UTI. The number of mutants within these subgroups circumvents bottleneck restriction and facilitates validation of multiple mutants to generate individual competitive indices. Collectively, this study investigates the bottleneck effects during UTI, provides two techniques for evading those effects that can be applied to other disease models, and contributes a genetic tool in prototype strain CFT073 to the field.Importance Uropathogenic Escherichia coli cause most uncomplicated urinary tract infections (UTI), one of the most common infectious diseases worldwide. Random transposon mutagenesis techniques have been utilized to identify essential bacterial genes during infection; however, this has been met with limitations when applied to the murine UTI model. Conventional high-throughput transposon mutagenesis screens are not feasible because of inoculum size restrictions due to a bottleneck during infection. Our study utilizes a condensed ordered transposon library, limiting the number of mutants while maintaining the largest possible genome coverage. Screening of this library in vivo, and in human urine in vitro, identified numerous candidate fitness factors. Additionally, we have developed a novel technique using qPCR to quantify bacterial outputs following infection with small subgroups of transposon mutants. Molecular approaches developed in this study will serve as useful tools to probe in vivo models that are restricted by anatomical, physiological, or genetic bottleneck limitations.