Strains of Extraintestinal Pathogenic Escherichia c oli (ExPEC) exhibit an array 
of virulence strategies and are a major cause of urinary tract infections, 
sepsis and meningitis. Efforts to understand ExPEC pathogenesis are challenged 
by the high degree of genetic and phenotypic variation that exists among 
isolates. Determining which virulence traits are widespread and which are 
strain-specific will greatly benefit the design of more effective therapies. 
Towards this goal, we utilized a quantitative genetic footprinting technique 
known as transposon insertion sequencing (Tn-seq) in conjunction with 
comparative pathogenomics to functionally dissect the genetic repertoire of a 
reference ExPEC isolate. Using Tn-seq and high-throughput zebrafish infection 
models, we tracked changes in the abundance of ExPEC variants within saturated 
transposon mutant libraries following selection within distinct host niches. 
Nine hundred and seventy bacterial genes (18% of the genome) were found to 
promote pathogen fitness in either a niche-dependent or independent manner. To 
identify genes with the highest therapeutic and diagnostic potential, a novel 
Trait Enrichment Analysis (TEA) algorithm was developed to ascertain the 
phylogenetic distribution of candidate genes. TEA revealed that a significant 
portion of the 970 genes identified by Tn-seq have homologues more often 
contained within the genomes of ExPEC and other known pathogens, which, as 
suggested by the first axiom of molecular Koch's postulates, is considered to be 
a key feature of true virulence determinants. Three of these Tn-seq-derived 
pathogen-associated genes--a transcriptional repressor, a putative 
metalloendopeptidase toxin and a hypothetical DNA binding protein--were deleted 
and shown to independently affect ExPEC fitness in zebrafish and mouse models of 
infection. Together, the approaches and observations reported herein provide a 
resource for future pathogenomics-based research and highlight the diversity of 
factors required by a single ExPEC isolate to survive within varying host 
environments.