S. Typhimurium burden following orogastric inoculation of untreated or streptomycin- treated mice. A) Percentage of initial weight over time; means and standard deviations are shown. B) Bacterial fecal burden. Box and whisker plots represent max-to-min and interquartile ranges. C) Bacterial burden in organs and fluids. Open circles indicate when the whole gallbladder was used instead of bile. Box and whisker plots represent max-to-min and interquartile ranges. Untreated n = 16 (8 males & 8 females), streptomycin (SM) pretreated n = 5 (female) unless otherwise noted. Sex- disaggregated data in Figure S4 Mann-Whitney tests were used for statistical analyses. Values with p < 0.1 shown. Abbreviations: Adipose, left perigonadal adipose tissue; MLN, mesenteric lymph node; PP, Peyer’s patches; SI, small intestine; wash, peritoneal wash

The bottleneck to S. Typhimurium intestinal colonization in orogastrically inoculated mice is widened after streptomycin treatment. A) Schematic depicting the effect of a tight versus wide bottleneck on a diverse inoculum and the STAMPR analytical methods used to calculate the founding population (FP) and compare populations at separate sites of infection using genetic distance (GD) analysis. B) Founding populations (Ns) of intestinal samples. Truncated violin plot with all points shown. C) Average CFU per founder (CFU/Ns) in intestinal tissues. Bars are geometric means and geometric standard deviations. D) Heatmaps of average genetic distance comparisons throughout the GI tract in untreated (left) and streptomycin-treated (right) mice. E) Comparison of genetic distance of GI tract to fecal samples. Mann-Whitney tests were used for statistical analyses. Values with p < 0.1 shown. Abbreviations: PP, Peyer’s patches; SI, small intestine; SM, streptomycin

S. Typhimurium disseminates from the intestine before substantial replication. A)

Founding populations and C) genetic distance from the liver after orogastric inoculation of S. Typhimurium with (+SM) or without (-SM) streptomycin pretreatment. B) Scheme depicting proposed dissemination patterns during infection. D-F) Organ CFUs (D), founding populations (E), and genetic distance from the liver (F) after inoculation via drinking (+SM, Drinking) are not statistically different from orogastric gavage (+SM). Data for +SM mice repeated from Figure 1-3. G-H) Organ CFUs (G) and founding populations (H) in untreated mice at 5 or 120 hours after inoculation. 120 hour data repeated from Figure 1-3. +SM, Drinking n = 5, untreated 5 hours n = 5. Mann-Whitney tests were used for statistical analyses. Values with p < 0.1 shown. Abbreviations: GI, gastrointestinal; MLN, mesenteric lymph node; PP, Peyer’s patches; SI, small intestine

S. Typhimurium population dynamics following different routes of inoculation. A)

Infections were performed via 3 routes: orogastric gavage with (Oral +SM) or without (Oral -SM) streptomycin pretreatment, intraperitoneal injection (IP), and intravenous injection (IV). B-C) Bacterial burden (B) and founding populations (C) in immune-rich extraintestinal organs. D-E) Bacterial burden (D) and founding population (E) in extraintestinal samples. F) Heatmaps of average genetic distance between extraintestinal organs after IP (top) and IV (bottom) inoculation. Box and whisker plots represent max-to-min and interquartile ranges. Truncated violin plots with all points displayed. Oral -SM n = 5, Oral +SM n = 4, IP n = 16 (8 male, 8 female), IV n = 16 (8 male, 8 female). Sex-disaggregated data in Figure S6 Mann-Whitney tests were used for statistical analyses. Values with p < 0.1 shown. Abbreviations: MLN, mesenteric lymph node

S. Typhimurium population dynamics after IV and IP inoculation differ compared to those after oral gavage. A)

Fecal shedding after orogastric gavage with (Oral +SM) or without (Oral - SM) streptomycin pretreatment, intraperitoneal injection (IP), and intravenous injection (IV) with S. Typhimurium. B) Bacterial burdens in the intestine. Box and whisker plots represent max-to-min and interquartile ranges. C) Founding population of GI organs. D) Genetic distance comparison of colon samples of IV and IP animals to other sites disaggregated by number of colon founders. Means and standard deviations displayed. Mann-Whitney tests were used for statistical analyses. Values with p < 0.1 shown. Abbreviations: M N, mesenteric lymph node; PP, Peyer’s patches; SI, small intestine

Bile re-seeding the intestine is correlated with gallbladder pathology. A)

Barcode frequency of clonal and diverse colons and bile in mice after IV inoculation. Asterisks indicate visible pathology in the bile. B) Gross anatomy and C) H&E-stained sections of normal, dark, cloudy, and hardened bile after S. Typhimurium infection. Red arrows indicate the luminal side of the gallbladder. D) CFU in bile after all infection routes disaggregated by bile phenotype. Box and whisker plots represent max-to-min and interquartile ranges. E) FRD of bile barcodes also found in colon samples. Bars are means with standard deviations. F) Fecal shedding correlates with bile burden when hardened or cloudy bile is present. Line of best fit displayed. Mann-Whitney tests were used for statistical analyses. Values with p < 0.1 shown