Neutral barcoding of genomes reveals the dynamics of Salmonella colonization in cattle and their peripheral lymph nodes.

Feedlot cattle often contain Salmonella. The number of bacteria that initiate colonization of different cattle organs and the bacterial migration within these large animals are poorly understood. To investigate these questions, we constructed wild-type isogenic tagged strains (WITS) of Salmonella by inserting 21-base barcodes flanked by Illumina sequencing primers into a neutral genome location. We then delivered several different pools of uniquely barcoded clones orally and into multiple intradermal sites, in individual Holstein steers, and subsequently performed Salmonella-directed sequence tag-based analysis of microbial populations (STAMP). Using high-throughput sequencing of the barcodes of Salmonella grown from steer lymph nodes, organs and feces, we monitored how individual barcoded clones travel from different entry sites within animals. Data showed that gastrointestinal colonization was established by up to hundreds of Salmonella founder cells, whereas peripheral lymph nodes were usually colonized by very low numbers of founding bacteria, often originating from the nearest draining intradermal delivery site. Transmission of Salmonella from the gastrointestinal tract to the lymphatic system was frequently observed, whereas entry of intradermally delivered bacteria into the gut was rare. Bacteria undergo limited extraintestinal proliferation within or prior to arrival at peripheral lymph nodes. Overall, the application of the STAMP technique facilitated characterization of the migration routes and founder population size of Salmonella within feedlot cattle and their organs and lymph nodes in unprecedented detail.