MMeettaaggeennoommiicc aannaallyysseess rreevveeaall aannttiibbiioottiicc--iinndduucceedd tteemmppoorraall aanndd ssppaattiiaall cchhaannggeess iinn iinntteessttiinnaall mmiiccrroobbiioottaa wwiitthh aassssoocciiaatteedd aalltteerraattiioonnss iinn iimmmmuunnee hhoommeeoossttaassiiss David A. Hilla, Christian Hoffmannb, Michael C. Abta, Yurong Dua, Thomas J. Kirna, Frederic D. Bushmanb, David Artisa aDept. of Pathobiology, bDept. of Microbiology, University of Pennsylvania, Philadelphia, PA 19104 Results Figure 4. Antibiotics alter stool bacterial communities over 1 9 y y a a D D time. c c d i i t t Abstract e e n o o u u i i l l A e B b b a a i i g Figure 1. Antibiotic administration mimics molecular, t t v v e n - n - L H2O ABX Phyl—um————————————Cl—as—s-—Or—de—r———————Family-Genus Antibiotic Day 0 A P A P Despite widespread use of antibiotics, few studies have measured their effects 100% Verrucomicrobi—a ——————Verrucomicrobiae-V—er—ruc—o—mi—cro—b—ial—es————————————Verrucomicrobiaceae-Akkermansia 0.0 6.1 0.015 anatomic, histologic, and immunologic characteristics of T—M7———————TM7 genera incertae sedis-Un—kn—ow—n———————————————————Unknown-Unknown 0.6 0.0 0.007 0.0 0.015 ————————Gammaproteobacteria-E—nte—ro—ba—c—ter—ial—es—————————————Enterobacteriaceae-Enterobacter on the burden or diversity of bacterial communities in the mammalian intestine. 90% Proteobact—eri—a-———————Betaproteobacteria-Burkholderial—es———————————————————Unknown-Unknown ———————————Alphaproteobacteria-Unknow—n———————————————————Unknown-Unknown reduced microbial stimulation. —————————E—ry—sip—e—lot—ric—hi-—Er—ys—ip—elo—tr—ich—al—es—————Erysipelotrichaceae-Erysipelotrichaceae Incertae S 0.0 0.6 0.042 We developed an oral antibiotic treatment protocol and characterized the effects 80% ————————————————————————Unknown-Unknown —————————————————————Ruminococcaceae-Unknown 2.2 0.0 0.007 0.3 0.037 ——————————————————Ruminococcaceae-Ruminococcaceae Incertae Sedis 0.4 0.0 0.025 0.0 0.044 —————————————————————Ruminococcaceae-Faecalibacterium of treatment on intestinal immune homeostasis and the burden and composition A B C D E 70% Mouse Weights 16s rDNA Copies Villus Length RELMβ in Stool —————————————————————Ruminococcaceae-Anaerotruncus 0.2 0.0 0.025 0.0 0.044 120 —————————————————Clostridia-Clostri—dia—le—s-———————————————Lachnospiraceae-Unknown 10.6 0.0 0.007 0.0 0.015 11 Firmicutes- of bacterial communities. Antibiotic administration resulted in cecal enlargement, 110 H2O 1.0×10 H2O *** ——————————————————Lachnospiraceae-Lachnospiraceae Incertae Sedis 1.0 0.0 0.007 0.0 0.015 110 60% ABX ABX H2O *** ——————————————————Lachnospiraceae-Coprococcus ** ——————————————————Lachnospiraceae-Butyrivibrio m 100 1.4 0.0 0.025 0.0 0.044 ——————————————————Lachnospiraceae-Bryantella lamina propria expansion, enterocyte hyperplasia, reduced RELMβ production, m 60 ** 10 50% 1.0×10 90 ——————————————————Lachnospiraceae-Acetitomaculum 105 ——————————————————Leuconostocaceae-Weissella 0.1 0.0 0.044 e 80 50 ————————————————————Bacilli-Lactobacillales- ——————————————————Lactobacillaceae-Lactobacillus and reduced production of the cytokines TNFα, IFNγ, IL-17A, IL-22, and IL-10 in g 70 40% Deferribacte—re—s ————————Deferribacteres-Deferr—ib—ac—ter—al—es—————————————Deferribacteraceae-Mucispirillum Copies/g n 09 40 ——————————————————U—nk—no—w—n-—Un—kn—ow—n———————————————————Unknown-Unknown 13.8 1.0 0.008 2.1 0.016 a 1.0×10 ABX H2O ABX GF ———————————————————————————Unknown-Unknown 41.9 1.8 0.008 4.3 0.016 the intestine, all consistent with reduced bacterial stimulation. Associated with h 100 0 1 5 9 30 30% ——————————————————Rikenellaceae-Unknown 1.9 0.1 0.010 C Day Villus Width ——————————————————Rikenellaceae-Marinilabilia 2.9 0.0 0.007 0.1 0.018 80 Pixel intensity 10 *** ——————————————————Rikenellaceae-Alistipes 7.3 1.9 0.016 these dramatic physiologic and anatomic changes, there was a ten-fold % 20 70 Bacteroidetes- ——————————————————Prevotellaceae-Xylanibacter 0.8 0.1 0.018 3.0 0.016 *** 20% ——————————Bacteroidetes-Bacteroidales- 60 ——————————————————Prevotellaceae-Unknown 1.2 0.1 0.031 5.2 0.016 m 10 ——————————————————Prevotellaceae-Prevotella 1.1 0.1 0.010 3.7 0.016 95 50 reduction in the amount of intestinal bacteria present. Sequencing of 16S rDNA GF m 1 ——————————————————Porphyromonadaceae-Unknown 0.9 0.0 0.010 0.0 0.015 Day 0 Day 1 Day 5 Day 9 10% 40 ——————————————————Porphyromonadaceae-Parabacteroides 0.4 47.0 0.008 21.3 0.016 sequences revealed a sharp reduction in the proportion of bacteria belonging to 30 ——————————————————Bacteroidaceae-Bacteroides 3.3 44.1 0.008 40.8 0.016 U—nk—no—w—n ——————————————Unknown-Un—kn—ow—n———————————————————Unknown-Unknown 0.7 0.0 0.010 0.1 0.034 20 0% 90 Fold change 0.1 H2O ABX GF 0 2 4 6 8 the Firmicutes phyla, while bacteria belonging to the Bacteroidetes and 1 3 5 7 9 0 1 5 9 Day Day Day Figure 4. A) Genus level phylogenetic classication of 16S rDNA frequencies in stool pellets collected from control-treated (H2O) or antibiotic-treated (ABX) Proteobacteria phyla persisted over time. In addition, we found significant animals from day 0 to day 9. B) Average frequency of bacterial groups before (Antibiotic Day 0) or during (Antibiotic Day 1, 9) antibiotic treatment. Mann- Figure 1. A) Weights of animals gavaged with unsupplemented (H2O; N=4) or antibiotic- Whitney P-values of changes in group frequency with antibiotic treatment. Frequency reductions upon antibiotic treatment in red, increases in green, non- temporal and spatial effects of antibiotics on luminal and mucosal-associated supplemented (ABX; N=5) water (± s.e.m). B) 16S rDNA gene copies as quantified from stool signicant changes blank. communities along the length of the colon including a reduction in the frequency pellets collected before (day 0) or over the course of antibiotic treatment (days 1, 5, 9) (N=5; ± Figure 5. Antibiotics modify luminal bacterial communities of mucosal-associated Lactobacillus. This comprehensive temporal and spatial s.e.m). C) Cecal images from control-treated (H2O), day 10 antibiotic-treated (ABX), or germ-free Cecum Prox. Colon Dist. Colon animals (GF) (bar 1 cm). D) Quantification of lamina propria expansion and enterocyte hyperpla- along the length of the colon. c c c d i i i t t t metagenomic analyses will provide a resource and framework to test the n e e e o o o u u u i i i e A B l l l b b b a a a g sia in the ceca from day 10 antibiotic-treated (ABX, N=5), as compared to control-treated (H2O; i i i O O O t t t v v v e 2 2 2 n - n - n - L influence of microbial communities in murine models of human metabolic and H2O ABX Phy—lum————————————Cl—as—s-—Or—de—r———————Family-Genus H A P H A P H A P N=4) or germ-free animals (GF; N=3) (***, p<0.001; ± s.e.m). E) Quantification of RELMβ protein 100% Verrucomicrob—ia ——————Verrucomicrobiae-V—er—ruc—o—mi—cro—b—ial—es————————————Verrucomicrobiaceae-Akkermansia 0.0 1.2 0.021 0.0 1.6 0.044 —T—M—7 ——————TM7 genera incertae sedis-Un—kn—ow—n———————————————————Unknown-Unknown inflammatory diseases. in stool pellets by western blot over the course of antibiotic treatment (N=5;**, P≤0.01; ± s.e.m). ————————Gammaproteobacteria-E—nte—ro—ba—c—ter—ial—es—————————————Enterobacteriaceae-Enterobacter 0.0 0.7 0.015 90% Proteobact—eri—a-———————Betaproteobacteria-Burkholderial—es———————————————————Unknown-Unknown 0.4 4.2 0.019 0.8 5.0 0.016 ———————————Alphaproteobacteria-Unknow—n———————————————————Unknown-Unknown —————————E—ry—sip—e—lot—ric—hi-—Er—ys—ip—elo—tr—ich—al—es—————Erysipelotrichaceae-Erysipelotrichaceae Incertae S 80% ————————————————————————Unknown-Unknown 1.2 0.3 0.029 Figure 2. Reduced cytokine production in the intestine —————————————————————Ruminococcaceae-Unknown 4.6 0.0 0.015 3.4 0.1 0.027 2.5 0.1 0.018 Introduction ——————————————————Ruminococcaceae-Ruminococcaceae Incertae Sedis 0.8 0.0 0.021 0.9 0.0 0.011 70% —————————————————————Ruminococcaceae-Faecalibacterium 0.9 0.0 0.042 of antibiotic-treated animals. —————————————————————Ruminococcaceae-Anaerotruncus 0.3 0.0 0.042 —————————————————Clostridia-Clostri—dia—le—s-———————————————Lachnospiraceae-Unknown 31.7 0.0 0.011 26.3 0.0 0.021 29.7 0.0 0.011 Firmicutes- ——————————————————Lachnospiraceae-Lachnospiraceae Incertae Sedis 1.7 0.0 0.011 1.8 0.0 0.021 1.9 0.0 0.011 60% • The human intestine is colonized by 100 trillion microorganisms ——————————————————Lachnospiraceae-Coprococcus 0.5 0.0 0.042 0.8 0.0 0.021 1.2 0.0 0.042 A tnfa ifng il17a il22 il10 ——————————————————Lachnospiraceae-Butyrivibrio 2.8 0.0 0.011 2.7 0.0 0.021 2.0 0.0 0.011 2 2 2 2 2 ——————————————————Lachnospiraceae-Bryantella 50% ——————————————————Lachnospiraceae-Acetitomaculum of which bacteria are the most abundant. ——————————————————Leuconostocaceae-Weissella ————————————————————Bacilli-Lactobacillales- ——————————————————Lactobacillaceae-Lactobacillus 40% Deferribacte—re—s————————Deferribacteres-Deferr—ib—ac—ter—al—es—————————————Deferribacteraceae-Mucispirillum 1.9 0.0 0.011 ——————————————————U—nk—no—w—n-—Un—kn—ow—n———————————————————Unknown-Unknown 8.4 1.6 0.016 10.6 1.6 0.029 10.4 2.1 0.016 • Intestinal bacteria promote proper digestion, metabolism, 1 1 1 1 1 ———————————————————————————Unknown-Unknown 26.0 2.7 0.016 31.5 3.8 0.029 28.8 3.8 0.016 30% ——————————————————Rikenellaceae-Unknown 1.3 0.3 0.029 *** * ——————————————————Rikenellaceae-Marinilabilia 1.4 0.2 0.018 ——————————————————Rikenellaceae-Alistipes 3.9 2.5 0.029 Relative Exp. *** epithelial cell function, angiogenesis, enteric nerve function, and Bacteroidetes- ——————————————————Prevotellaceae-Xylanibacter 1.3 2.5 0.029 *** ** 20% ——————————Bacteroidetes-Bacteroidales- 0 0 0 0 0 ——————————————————Prevotellaceae-Unknown ——————————————————Prevotellaceae-Prevotella 1.8 5.3 0.016 1.5 4.4 0.029 H2O ABX H2O ABX H2O ABX H2O ABX H2O ABX ——————————————————Porphyromonadaceae-Unknown 1.0 0.1 0.029 0.6 0.0 0.042 immune system development.