(a) Levels of AVNM-resistant bacteria in the intestinal-tract of AVNM-treated and water-treated wild-type male and female littermates 7 d post treatment-initiation. *P < 0.05 by Student's t-test and (n = 4) for both conditions
(b) Levels of AVNM-resistant bacteria in the lung of AVNM- plus DSS-treated and DSS-only treated male and female littermates 7 d post DSS treatment initiation. *P < 0.05 by Student's t-test and (n = 4) for both conditions. Red dashed line represents the limit of detection and black circles represent individual mice below the limit of detection.
(c) Levels of AVNM-resistant bacteria in the liver of AVNM- plus DSS-treated and DSS-only treated male and female littermates 7 d post DSS treatment initiation. *P < 0.05 by Student's t-test and (n = 4) for both conditions
(d) Representative images of disc diffusion assays and quantitation of the zone of inhibition to determine the susceptibility of an E. coli-O21:H+ isolate to AVNM or streptomycin. E. coli-K12 was used as a control. Error bars indicate standard deviation.
(e) Intestinal levels of total AVNM-resistant bacteria and AVNM-resistant E. coli-O21:H+ in AVNM-treated wild-type male and female mice compared to littermates that received a water control. DSS-treated mice (n = 4) and AVNM+DSS-treated mice (n = 4). Red dashed line represents the limit of detection and black circles represent individual mice below the limit of detection.
(f) Lung and liver levels of total AVNM-resistant bacteria and AVNM-resistant E. coli-O21:H+ in AVNM+DSS-treated wild-type male and female mice compared to littermates that received a DSS-only control. DSS-treated mice (n = 4) and AVNM+DSS-treated mice (n = 4).
(g) Intestinal levels of AVNM-resistant E. coli and streptomycin-resistant E. coli in AVNM and streptomycin-treated wild-type male and female mice were determined and compared to wild-type littermates that received a water control. Unmolested mice (n = 4); AVNM treated mice (n = 4) and streptomycin treated mice (n = 4).
(h) Intestinal levels of AVNM-resistant E. coli in single-housed female Jax mice and Jax mice co-housed with female colony-born (CB) mice after 7 d of AVNM treatment. Jax single-housed (n = 3); Jax co-housed (n = 3) and CB co-housed (n = 3).
(i) Temperature and survival of AVNM+DSS-treated female single-housed Jax mice and Jax mice co-housed with female colony-born mice. For survival, P = 0.0236 by Log rank analysis. Data represent two combined experiments; Jax single-housed (n = 11), Jax co-housed (n = 11); CB co-housed (n = 11) and DSS only (n = 5). Error bars indicate standard deviation.

(a) Survival of wild-type and Nlrc4^−/−Naip5^−/− male and female fostermates treated with AVNM+5% DSS. Kaplan-Meier plot is representative of two experiments and P = 0.0003 by log-rank analysis. Wild-type (n = 13) and Nlrc4^−/−Naip5^−/− (n = 14).
(b) Representative images of cecums and colons from wild-type and mutant female fostermates treated with AVNM+5% DSS at 3 d post DSS treatment initiation.
(c) Representative images of small intestines from AVNM+5% DSS treated wild-type and mutant female fostermates at 3 d post DSS treatment initiation.
(d) Rectal temperatures of wild-type and Nlrc4^−/−Naip5^−/− male and female fostermates treated with AVNM+5% DSS. Data are representative of three experiments. P < 0.05 by Student t-test. Wild-type (n = 10) and Nlrc4^−/−Naip5^−/− (n = 7).
(e) Serum BUN, CPK, AST and ALT levels at 2 d post DSS treatment initiation of wild-type and Nlrc4^−/−Naip5^−/− male and female fostermates treated with AVNM+5% DSS. Data are from three experiments and analyzed by Mann Whitney t-test. * P < 0.05. Wild-type (n = 3) and Nlrc4^−/− Naip5^−/− (n = 3).
(f) Levels of AVNM-resistant E. coli in the intestinal tracts of wild-type and Nlrc4^−/−Naip5^−/− male and female fostermates after 7 d of AVNM treatment. Wild-type (n = 6) and Nlrc4^−/−Naip5^−/− (n = 4).
(g) Lung and liver levels of total AVNM-resistant E. coli-O21:H+ in AVNM+DSS-treated wild-type mice and AVNM+DSS-treated Nlrc4^−/− Naip5^−/− male and female fostermates 3 d post DSS treatment initiation. Black dots represent tissues isolated from mice that had no bacteria above the limit detection.

(a) Survival of female wild-type and Naip5^−/−Nlrc4^−/− mutant mice infected with 5×10⁸ live E. coli. P = 0.0001 by Log rank analysis. Wild-type (n = 9) and Naip5^−/−Nlrc4^−/− (n = 11).
(b) Rectal temperature of female wild-type and Naip5^−/−Nlrc4^−/− mutant mice infected with 5×10⁸ live E. coli. Wild-type (n = 9) and Naip5^−/−Nlrc4^−/− (n = 11). *** P = 0.0003 by Mann-Whitney test.
(c) Intestinal pathology at 48 h post-infection of wild-type and Naip5^−/−Nlrc4^−/− mutant mice infected with 5×10⁸ live E. coli.
(d) Serum levels of BUN, CPK, ALT and AST female wild-type and Naip5^−/−Nlrc4^−/− mutant mice infected with 5×10⁸ live E. coli. at 24 h post-infection. *P < 0.05 by Mann-Whitney test. Wild-type (n = 7) and Naip5^−/−Nlrc4^−/− (n = 6).
(e) in vivo infection of Caspase-1^−/− and Il-1β^−/− female mutant mice. Survival was monitored and compared to infected wild-type female mice. Infectious dose was 5×10⁸ live E. coli O21:H+. For Caspase-1^−/− P = 0.0025 and for Il-1β^−/− P < 0.0001 by Log rank analysis. Wild-type (n = 9), Caspase-1^−/− (n = 6) and Il-1β^−/− (n = 12).
(f) Wild-type male and female mice were treated with AVNM for 7 d and then given AVNM+5% DSS. At 1, 24 and 48 h post DSS treatment initiation, mice were injected i.p. with 100 μg of α-IL-1R (n = 13) or control IgG antibody (n = 8) and survival was monitored. P = 0.0016 by Log rank analysis
(g) Wild-type and Naip5^−/−Nlrc4^−/− mutant female mice were infected with 5×10⁸ live E. coli. At 24 and 48 h post infection, tissues were harvested, homogenized and analyzed for levels of E. coli colonization. Data were analyzed by Student t-test.

(a) Survival of wild-type male and female mice that received AVNM+5% DSS (n = 5) and 5% DSS-only (n = 8). P = 0.0005 by Log rank analysis.
(b) Survival of wild-type male and female mice that received streptomycin+5% DSS (n = 6) and 5% DSS-only (n = 7). P = 0.2818 by Log rank analysis.
(c) Weight loss of wild-type male and female mice treated with 5% DSS compared to littermates that received drinking water supplemented with AVNM+5% DSS. Error bars indicate standard deviation; DSS-only (n = 9); AVNM+DSS (n = 9).
(d) Length of colons from wild-type male and female mice treated with 5% DSS compared to littermates that received drinking water supplemented with AVNM+5% DSS. Error bars indicate standard deviation. ***P = 0.0003 by Student's t-test. DSS-only (n = 4); AVNM+DSS (n = 4).
(e) Representative cecums and colons at 4 d post DSS treatment initiation of wild-type male mice treated with 5% DSS, AVNM+5% DSS or water-treated (unmolested) control littermates.
(f) Representative images of small intestine at 4 d post DSS treatment initiation of wild-type male mice treated with 5% DSS, AVNM+5% DSS or water-treated (unmolested) control littermates.
(g) Rectal temperature of wild-type male and female mice treated with AVNM+DSS compared to littermates that received 5% DSS only. Error bars represent standard deviation. *P < 0.05 by Student's t-test. DSS-only (n = 8); AVNM/DSS (n = 5).
(h) Serum levels of blood urea nitrogen (BUN), creatine phosphokinase (CPK), alanine transaminase and aspartate transaminase (AST) at 3 d post DSS treatment initiation of wild-type male and female mice. Data are representative of two combined experiments. **P < 0.01 and *P < 0.05 by Mann Whitney test. DSS-only (n = 7); AVNM/DSS (n = 7).
(i) Levels of culturable bacteria in the lung and liver of wild-type male and female mice 3 d post DSS treatment initiation. Data are representative of three independent experiments. *P < 0.05 by Student's t-test and (n = 4) for both conditions. Red dashed lines indicates limit of detection and black circles indicate individual mice below the limit of detection.

(a) Survival of wild-type female mice injected with live or dead 5×10⁸, 7.5×10⁸ or 1×10⁹ live E. coli-O21:H+. P < 0.0001 for live vs. dead comparisons by Log rank analysis, (n = 5) females for all conditions.
(b) Rectal temperature of wild-type female mice injected with 5×10⁸, 7.5×10⁸ or 1×10⁹ live or dead E. coli-O21:H+. At 6 h PI 1×10⁹ live vs. 1×10⁹ dead P = 0.0079, 7.5×10⁸ live vs. 7.5×10⁸ dead P = 0.0079, at 24 h PI 5×10⁸ live vs. 5×10⁸ dead P = 0.0195. (n = 5) for all conditions.
(c) Wild-type female mice were inoculated with 5×10⁸ live or dead E. coli-O21:H+ and serum levels of BUN, CPK, ALT and AST were analyzed 24 h post infection. (n = 4) for live infection and (n = 12) females for dead infection. *P < 0.05, **P < 0.01 and *** P < 0.0005 by Student t-test and error bars indicate standard deviation.
(d) E. coli-O21:H+ CFUs in liver, lung, spleen and kidneys of wild-type female mice infected with 5×10⁸ live bacteria at 24 and 48 h post infection. (n = 3) at both 24 h and 48 h PI and error bars indicate standard deviation.
(e) Survival of wild-type female mice infected with 5×10⁸ live E. coli-O21:H+ or E. coli-K12. P < 0.0001 by Log rank analysis. (n = 8) for both E. coli-O21:H+ and E. coli-K12 injections.
(f) Rectal temperature of wild-type female mice infected with 5×10⁸ live E. coli-O21:H+ or E. coli-K12. (n = 8) for both E. coli-O21:H+ and E. coli-K12 injections.

(a) Motility agar was inoculated with wild-type, motile S. Typhimurium LT2, flagellin-deficient S. Typhimurium ΔfliCΔfljB or E. coli-O21:H+ and incubated at 37 °C overnight. Representative images of the diffusion of motile bacteria are shown.
(b) Retroviral constructs expressing fliC from E. coli-O21:H+ or flaA from L. pneumophila followed by an IRES-GFP element or GFP alone were transduced into wild-type, Naip5^−/−, or Nlrc4^−/− macrophages, and the percentage of GFP-positive cells was enumerated by flow cytometry 4 d post-transduction.
(c) The percentage of cells expressing GFP from the retroviral lethality assay in (b) is indicated and charted.
Data are representative of three independent experiments.