Quantification of Salmonella enterica serovar Typhimurium Population Dynamics in Murine Infection Using a Highly Diverse Barcoded Library

Abstract

Murine models are often used to study the pathogenicity and dissemination of the enteric pathogen Salmonella enterica serovar Typhimurium. Here, we quantified S. Typhimurium population dynamics in mice using the STAMPR analytic pipeline and a highly diverse S. Typhimurium barcoded library containing [~]55,000 unique strains distinguishable by genomic barcodes by enumerating S. Typhimurium founding populations and deciphering routes of spread in mice. We found that a severe bottleneck allowed only one in a million cells from an oral inoculum to establish a niche in the intestine. Furthermore, we observed compartmentalization of pathogen populations throughout the intestine, with few barcodes shared between intestinal segments and feces. This severe bottleneck widened and compartmentalization was reduced after streptomycin treatment, suggesting the microbiota plays a key role in restricting the pathogens colonization and movement within the intestine. Additionally, there was minimal sharing between the intestine and extraintestinal organ populations, indicating dissemination to extraintestinal sites occurs rapidly, before substantial pathogen expansion in the intestine. Bypassing the intestinal bottleneck by inoculating mice via intravenous or intraperitoneal injection revealed that Salmonella re-enters the intestine after establishing niches in extraintestinal sites by at least two distinct pathways. One pathway results in a diverse intestinal population. The other re-seeding pathway is through the bile, where the pathogen is often clonal, leading to clonal intestinal populations and correlates with gallbladder pathology. Together, these findings deepen our understanding of Salmonella population dynamics. Significance StatementSalmonella is a prevalent food-borne pathogen that infects hundreds of millions of people worldwide. Here, we created a highly complex barcoded Salmonella enterica serovar Typhimurium library containing [~]55,000 barcodes to further understand and quantify Salmonella population dynamics in experimental murine infection. Through comparisons of barcode abundance and frequency in different samples and following different routes of inoculation, we quantify key facets of Salmonella infection, including bottleneck sizes and dissemination patterns, and uncover hidden routes of spread that drive heterogeneity in infection outcome. These observations provide a detailed map of Salmonella infection and demonstrate the power of high-diversity barcoded libraries in deciphering microbial population dynamics.