Chickens, pigs, and cattle are key reservoirs of Salmonella enterica, a foodborne pathogen of worldwide importance. Though a decade has elapsed since publication of the first Salmonella genome, thousands of genes remain of hypothetical or unknown function, and the basis of colonization of reservoir hosts is ill-defined. Moreover, previous surveys of the role of Salmonella genes in vivo have focused on systemic virulence in murine typhoid models, and the genetic basis of intestinal persistence and thus zoonotic transmission have received little study. We therefore screened pools of random insertion mutants of S. enterica serovar Typhimurium in chickens, pigs, and cattle by transposon-directed insertion-site sequencing (TraDIS). The identity and relative fitness in each host of 7,702 mutants was simultaneously assigned by massively parallel sequencing of transposon-flanking regions. Phenotypes were assigned to 2,715 different genes, providing a phenotype–genotype map of unprecedented resolution. The data are self-consistent in that multiple independent mutations in a given gene or pathway were observed to exert a similar fitness cost. Phenotypes were further validated by screening defined null mutants in chickens. Our data indicate that a core set of genes is required for infection of all three host species, and smaller sets of genes may mediate persistence in specific hosts. By assigning roles to thousands of Salmonella genes in key reservoir hosts, our data facilitate systems approaches to understand pathogenesis and the rational design of novel cross-protective vaccines and inhibitors. Moreover, by simultaneously assigning the genotype and phenotype of over 90% of mutants screened in complex pools, our data establish TraDIS as a powerful tool to apply rich functional annotation to microbial genomes with minimal animal use.
Salmonella Typhimurium is a major cause of human diarrhoeal infections, usually acquired from chickens, pigs, cattle, or their products. To understand the basis of persistence and pathogenesis in these reservoir hosts, and to inform the design of novel vaccines and treatments, we generated a library of 7,702 S. Typhimurium mutants, each bearing an insertion at a random position in the genome. Using DNA sequencing, we identified the disrupted gene in each mutant and determined its relative abundance in a laboratory culture and after experimental infection of mice, chickens, pigs, and cattle. The method allowed large numbers of mutants to be investigated simultaneously, drastically reducing the number of animals required to perform a comprehensive screen. We identified mutants that grow in culture but do not survive in one or more of the animals. The genes disrupted in these mutants are inferred to be important for the infection process. Most of these genes were required in all three food-producing animals, but smaller subsets of genes may mediate persistence in a specific host species. The data provide the most comprehensive map of virulence-associated genes for any bacterial pathogen in natural hosts and are highly relevant for the design of control strategies.