Volume 146, Issue 2, Pages 484-496.e4 (February 2014) Rifaximin Alters Intestinal Bacteria and Prevents Stress-Induced Gut Inflammation and Visceral Hyperalgesia in Rats  Dabo Xu, Jun Gao, Merritt Gillilland, Xiaoyin Wu, Il Song, John Y. Kao, Chung Owyang  Gastroenterology  Volume 146, Issue 2, Pages 484-496.e4 (February 2014) DOI: 10.1053/j.gastro.2013.10.026 Copyright © 2014 AGA Institute Terms and Conditions

Figure 1 Effect of chronic stress and antibiotics on VMR to CRD. (A) EMG amplitude expressed as mean change from baseline in rats subjected to chronic WAS or sham WAS. (B) EMG amplitude expressed as mean change from baseline after chronic treatment with vehicle (water) or rifaximin (150 mg/kg, twice daily, oral gavage) in rats previously subjected to chronic WAS or sham WAS. (C) EMG amplitude expressed as mean change from baseline after chronic treatment with vehicle or neomycin (150 mg/kg, twice daily, oral gavage) in rats previously subjected to chronic WAS or sham WAS. n = 8−9. ∗P < .05, compared with the sham WAS, or sham WAS + water gavage controls; #P < .05, compared with WAS + water gavage rats. (D) EMG amplitude expressed as mean change from baseline after chronic treatment with vehicle (water) or rifaximin (150 mg/kg, twice daily, oral gavage) in rats previously subjected to repeat RS or sham RS. n = 6. ∗P < .05, compared with sham RS + water gavage controls; #P <.05, compared with RS + water gavage rats. Gastroenterology 2014 146, 484-496.e4DOI: (10.1053/j.gastro.2013.10.026) Copyright © 2014 AGA Institute Terms and Conditions

Figure 2 Comparison of bacterial community composition and diversity, and total bacterial load in the ileal contents of rats previously subjected to WAS or sham WAS. (A) Phylotypes classified to the level of family in order of rank abundance as a percentage of the total community. (B) Diversity (Shannon diversity index and Shannon evenness) of bacterial communities. (C) Real-time qPCR analysis of total bacterial 16s rRNA gene copies (total bacterial load). n = 9. For all figures, ∗Significantly different than sham WAS (P < .05). Gastroenterology 2014 146, 484-496.e4DOI: (10.1053/j.gastro.2013.10.026) Copyright © 2014 AGA Institute Terms and Conditions

Figure 3 Effect of chronic stress and antibiotics on inflammatory cytokine expression in ileal tissue and gut permeability. (A, B, C, and D) Cytokine mRNA levels measured with real-time quantitative reverse transcription PCR. Data represent fold change in target mRNA levels relative to expression in control samples after normalization to GAPDH. (A) IL-17, IL-6, IL-10, TNF-α, IFN-gamma, and IL-1β mRNA levels in rats previously subjected to chronic WAS or sham WAS. (B) IL-17, IL-6, and TNF-α mRNA levels after chronic treatment with vehicle (water) or rifaximin (150 mg/kg, twice daily, oral gavage) in rats previously subjected to chronic WAS or sham WAS. (C) IL-17, IL-6, TNF-α, IL-10, IFN-gamma, and IL-1β mRNA levels in rats previously subjected to repeat RS or sham RS. NOTE. Rifaximin (150 mg/kg twice daily) significantly reduced the increase in IL-17, IL-6, and TNF-α mRNA evoked by chronic stress. Rifaximin also normalized the reduced IL-10 mRNA levels observed in repeat RS rats. (D) IL-17, IL-6, and TNF-α mRNA levels after chronic treatment with vehicle or neomycin (150 mg/kg, twice daily, oral gavage) in rats previously subjected to chronic WAS or sham WAS. n = 8−9. ∗P < .05, compared with sham WAS or sham RS. #P < .05, compared with WAS or RS + water gavage). Gastroenterology 2014 146, 484-496.e4DOI: (10.1053/j.gastro.2013.10.026) Copyright © 2014 AGA Institute Terms and Conditions

Figure 4 Effect of chronic treatment with rifaximin (150 mg/kg, twice daily, oral gavage) on total bacterial load and bacterial community composition in ileal contents. (A) Total bacterial 16s rRNA gene copies. n = 8. ∗P < .05, compared with sham WAS + water gavage controls; #P < .05, compared with WAS + water gavage rats. (B) Variation in bacterial community composition in the terminal ileum at phylum and family levels. (C) Relative abundance of selected phylotypes, identified at family level. n = 9 in sham WAS and WAS groups, n = 3 in WAS + rifaximin group. ∗Significantly different than sham WAS; #Significantly different than WAS (Kruskal-Wallis with Conover–Inman post-hoc test for multiple comparisons, P < .05). (D) Bacterial community composition analyzed with nonmetric multidimensional scaling (NMDS) plots using a θYC distance matrix of operational taxonomic unit−based data. Circles represent distinct bacterial communities identified in luminal contents of terminal ileum of individual rats. Circles that appear larger are closer in the axis not represented and circles that are smaller are farther away in the axis not represented; some circles have been made transparent. For this NMDS plot, stress = 0.09, r2 = 0.97. Gastroenterology 2014 146, 484-496.e4DOI: (10.1053/j.gastro.2013.10.026) Copyright © 2014 AGA Institute Terms and Conditions

Figure 5 Effect of chronic treatment with rifaximin (150 mg/kg, twice daily, oral gavage) on gut permeability and ileal tight junction protein expression. (A, B) Gut permeability measured by appearance of fluorescein isothiocyanate−labeled dextran in plasma. n = 8. ∗P < .05, compared with sham WAS + water gavage controls or sham RS + water gavage controls; #P < .05, compared with WAS + water gavage rats or RS + water gavage rats. (C) Densitometric quantification of occludin mRNA normalized to GAPDH band intensity. n = 8. *P < .05, compared with sham WAS + water gavage controls; #P < .05, compared with WAS + water gavage rats. (D) Densitometric quantification of occludin protein normalized to GAPDH band intensity. n = 6. *P < .05, compared with sham WAS + water gavage controls; #P < .05, compared with WAS + water gavage rats. (E) Densitometric quantification of occludin mRNA normalized to GAPDH band intensity, n = 6. *P < .05, compared with sham RS + water gavage controls; #P < .05, compared with RS + water gavage rats. (F) Densitometric quantification of occludin protein normalized to glyceraldehyde-3-phosphate dehydrogenase band intensity. n = 6. ∗P < .05, compared with sham RS + water gavage controls; #P <.05, compared with RS + water gavage. Gastroenterology 2014 146, 484-496.e4DOI: (10.1053/j.gastro.2013.10.026) Copyright © 2014 AGA Institute Terms and Conditions

Figure 6 Effect of chronic neomycin treatment (150 mg/kg, twice daily, oral gavage) on total bacterial load and bacterial community composition in ileal contents. (A) Total bacterial 16S rRNA gene copies. ∗P < .05, compared with sham WAS + water gavage controls; #P < .05, compared with WAS + water gavage rats. (B) Variation in bacterial community composition in the terminal ileum at phylum and family levels. (C) Relative abundance of selected phylotypes, identified at the family level. ∗Significantly different than sham WAS; #significantly different than WAS (Kruskal-Wallis with Conover-Inman post-hoc test for multiple comparisons, ∗P < .05). (D) Bacterial community composition analyzed with nonmetric multidimensional scaling (NMDS) plots using a θYC distance matrix of operational taxonomic unit−based data. For this NMDS plot, stress = 0.06, r2 = 0.98. n = 4. ND, not detected. Gastroenterology 2014 146, 484-496.e4DOI: (10.1053/j.gastro.2013.10.026) Copyright © 2014 AGA Institute Terms and Conditions

Figure 7 Effect of chronic treatment with neomycin (150 mg/kg, twice daily, oral gavage) on gut permeability and ileal tight junction protein expression. (A) Gut permeability measured by appearance of fluorescein isothiocyanate (FITC)−labeled dextran in plasma. n = 8. (B) Representative gel images of semi-quantitative reverse transcription PCR of tight junction protein occludin mRNA in ileal tissue. (C) Densitometric quantification of occludin mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band intensity. n = 6. (D) Representative immunoblots show occludin expression in ileal tissue. (E) Densitometric quantification of occludin protein normalized to GAPDH band intensity. n = 6. For all groups, ∗P < .05, compared with sham WAS + water gavage controls. Gastroenterology 2014 146, 484-496.e4DOI: (10.1053/j.gastro.2013.10.026) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 1 Effect of chronic treatment with rifaximin (150 mg/kg, twice daily, oral gavage) on bacterial community composition in the ileal contents (replicate experiment). (A) Relative abundance of selected phylotypes, identified at the level of family. (B) Nonmetric multidimensional scaling plots using a θYC distance matrix of the OTU-based data. n = 3 in each group (replicate experiment). For all studies, ND, not detected. ∗Significantly different than sham WA; #significantly different than WA (Kruskal-Wallis with Conover-Inman post-hoc test for multiple comparisons, P < .05). Gastroenterology 2014 146, 484-496.e4DOI: (10.1053/j.gastro.2013.10.026) Copyright © 2014 AGA Institute Terms and Conditions