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

time. D a D a c c d t i t i

Abstract e e n o o u u i i l l A e

B b b a a g Figure 1. Antibiotic administration mimics molecular, t i t i 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 -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 - 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 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—————————————- 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 - ——————————————————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 10 ——————————————————Prevotellaceae-Prevotella 1.1 0.1 0.010 3.7 0.016 95 50 reduction in the amount of intestinal 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 classi cation of 16S rDNA frequencies in stool pellets collected from control-treated (H2O) or antibiotic-treated (ABX) phyla persisted over time. In addition, we found significant 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 signi cant 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 t i t i t i

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 sia in the ceca from day 10 antibiotic-treated (ABX, N=5), as compared to control-treated (H2O; g O O O t i t i t i v v v e 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 2 A P H 2 A P H 2 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. 10% ——————————————————Porphyromonadaceae-Parabacteroides 0.4 31.8 0.016 0.8 30.1 0.029 0.9 23.9 0.016 B TNFα IFNγ IL-17a IL-22 IL-10 ——————————————————Bacteroidaceae-Bacteroides 1.8 42.6 0.016 2.8 43.3 0.029 2.2 46.9 0.016 Unknown ——————————————Unknown-Un—kn—ow—n———————————————————Unknown-Unknown 0.50 4 17 2 10 0% • Patients with inflammatory bowel disease and atopic allergies Ce Prox Dist Ce Prox Dist Figure 5. A) Genus level phylogenetic classi cation of 16S rDNA frequencies in luminal samples collected from control-treated (H2O) or day 10 antibiotic- have altered intestinal communities, implicating microbial signals treated (Antibiotic) animals from the cecum (Ce), proximal colon (Prox), or distal colon (Dist). B) Average frequency of bacterial groups in samples from control- 0.25 2 8.5 1 5 treated (H2O) or antibiotic-treated (Antibiotic) animals. Mann-Whitney P-values of changes in group frequency with antibiotic treatment. Frequency reductions upon antibiotic treatment in red, increases in green, non-signi cant changes blank. in disease pathogenesis. *** ** ng/mL/g Tissue 0.00 0 0 0 0 Figure 6. Antibiotic treatment disrupts normal mucosal-associ- • Alterations to intestinal bacteria is an adverse effect of antibiotic H2O ABX H2O ABX H2O ABX H2O ABX H2O ABX ated bacterial communities along the length of the colon. treatment and antibiotic exposure is linked to increased risk of Figure 2. A) Cytokine mRNA expression on day 10 as assessed by real time RT-PCR of small Cecum Dist. Colon intestine samples from control-treated (H2O; N=4) or antibiotic-treated (ABX; N=5) animals. Re- c c d t i t i e e n o o i i u u e l l b Clostridium difficile colitis and asthma in humans. A b a B a g t i sults represent fold induction over control-treated animals ± s.e.m (*, p<0.05; **, p<0.01; ***, t i v v n n e - - A A L H2O H2O P H2O ABX Phyl—um————————————Cl—as—s-—Or—de—r———————Family-Genus P p<0.001). B) Cytokine production on Day 10 as assessed by ELISA of culture supernatants from 100% Verrucomicrobi—a ——————Verrucomicrobiae-V—er—ruc—o—mi—cro—b—ial—es————————————Verrucomicrobiaceae-Akkermansia • Antibiotic treatment of models has identified pro- T—M7———————TM7 genera incertae sedis-Un—kn—ow—n———————————————————Unknown-Unknown cecal explant tissue samples isolated from control-treated (H2O) or antibiotic-treated (ABX) ani- ————————Gammaproteobacteria-E—nte—ro—ba—c—ter—ial—es—————————————Enterobacteriaceae-Enterobacter 90% Proteobact—eri—a-———————Betaproteobacteria-Burkholderial—es———————————————————Unknown-Unknown 0.7 3.9 0.036 0.9 5.0 0.036 inflammatory and immunoregulatory roles for intestinal bacteria mals. Results represent ng cytokine/mL culture media/g tissue ± s.e.m (**, p<0.01; ***, p<0.001). ———————————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 2.7 0.4 0.036 —————————————————————Ruminococcaceae-Unknown 2.8 0.1 0.032 in modulating cytokine responses, altering resistance to enteric Figure 3. Global restructuring of intestinal bacterial ——————————————————Ruminococcaceae-Ruminococcaceae Incertae Sedis —————————————————————Ruminococcaceae-Faecalibacterium 70% —————————————————————Ruminococcaceae-Anaerotruncus 0.6 0.0 0.017 0.5 0.0 0.017 —————————————————Clostridia-Clostri—dia—le—s-———————————————Lachnospiraceae-Unknown 29.5 0.0 0.017 25.3 0.1 0.026 Firmicutes- pathogens, maintaining mucosal homeostasis, and controlling communities over time. ——————————————————Lachnospiraceae-Lachnospiraceae Incertae Sedis 3.4 0.0 0.017 3.3 0.0 0.026 60% ——————————————————Lachnospiraceae-Coprococcus 0.3 0.0 0.017 A Unweighted UniFrac B Weighted UniFrac ——————————————————Lachnospiraceae-Butyrivibrio 2.8 0.0 0.017 2.2 0.1 0.026 0.3 0.4 ——————————————————Lachnospiraceae-Bryantella 0.7 0.0 0.017 allergic inflammation. H2O ABX 50% ——————————————————Lachnospiraceae-Acetitomaculum 0.3 0.0 0.017 Pellet ——————————————————Leuconostocaceae-Weissella 0.2 0.3 ————————————————————Bacilli-Lactobacillales- Luminal ——————————————————Lactobacillaceae-Lactobacillus 13.5 0.4 0.036 5.2 0.8 0.036 0.1 0.2 Mucosal 40% Deferribacte—re—s ————————Deferribacteres-Deferr—ib—ac—ter—al—es—————————————Deferribacteraceae-Mucispirillum 8.5 0.0 0.017 0.8 0.0 0.017 % % ——————————————————U—nk—no—w—n-—Un—kn—ow—n———————————————————Unknown-Unknown 5.1 2.3 0.036 1 6 . . ———————————————————————————Unknown-Unknown While the use of oral broad-spectrum antibiotics in humans 4 19.7 4.1 0.036 22.7 4.0 0.036 0.0 0.1 5 1

——————————————————Rikenellaceae-Unknown 0.8 0.2 0.036

30% 2

2 ——————————————————Rikenellaceae-Marinilabilia 0.9 0.1 0.032 A -0.1 0.0 A

C ——————————————————Rikenellaceae-Alistipes C P Bacteroidetes- ——————————————————Prevotellaceae-Xylanibacter and animal models is common, knowledge of how antibiot- P 0.2 3.1 0.036 -0.2 -0.1 20% ——————————Bacteroidetes-Bacteroidales- H2O ABX ——————————————————Prevotellaceae-Unknown 0.7 4.0 0.036 Pellet -0.3 -0.2 ——————————————————Prevotellaceae-Prevotella 0.9 6.4 0.036 3.6 7.4 0.036 Luminal ——————————————————Porphyromonadaceae-Unknown Mucosal 10% ics modify intestinal bacterial communities is limited. ——————————————————Porphyromonadaceae-Parabacteroides 0.2 27.9 0.036 3.3 27.6 0.036 -0.4 -0.3 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 ——————————————————Bacteroidaceae-Bacteroides 1.4 39.7 0.036 4.2 37.6 0.036 U—nk—no—w—n ——————————————Unknown-Un—kn—ow—n———————————————————Unknown-Unknown PCA1 - 26.8% PCA1 - 75.4% 0% Ce Prox Dist Ce Prox Dist C Pellets Unweighted D Pellets Weighted Figure 6. A) Genus level phylogenetic classi cation of 16S rDNA frequencies in mucosal-associated samples collected from control-treated (H2O) or day 10 Methods treatment: H2O treatment: ABX treatment: H2O treatment: ABX 0.4 0.10 antibiotic-treated (Antibiotic) animals from the cecum (Ce), proximal colon (Prox), or distal colon (Dist). B) Average frequency of bacterial groups in mucosal- 2 2 2 2 Gavage daily for 10 days with 200μl: Sacrifice: r = 0.1454 r = 0.6428 0.08 r = 0.0106 r = 0.7347 associated samples from control-treated (H2O) or antibiotic-treated (Antibiotic) animals. Mann-Whitney P-values of changes in group frequency with antibiotic 0.3 p = 0.0661 p < 0.0001 p = 0.6322 p < 0.0001 0.06 treatment. Frequency reductions upon antibiotic treatment in red, increases in green, non-signi cant changes blank. Small Int. Large Int. 0.2 0.04 Sterile H O %

2 % 2 3 . 0.02 . 3 7

0.1

0.00 3 Or 2 A A 0.0 -0.02 C C P

P Conclusions -0.04 Sterile H2O + 0.5 g/L Vanc, -0.1 -0.06 -0.2 -0.08 Antibiotic administration results in molecular, anatomic, histologic, and 1 g/L Amp, 1 g/L Neo, Cecum Prox Colon Dist Colon -0.10 -0.3 -0.12 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 immunologic characteristics of reduced microbial stimulation including 1 g/L Met, 1 g/L Gent Day Day 1 2 3 4 5 6 7 8 9 Day H2O reduced production of intestinal TNFα, IFNγ, IL-17A, IL-22, and IL-10, and is ABX Day 10 Luminal Content Day 10 Immunology Figure 3. A) Unweighted or B) weighted UniFrac analysis of stool pellet, luminal content, and mucosal- associated with dramatic restructuring of stool pellet, luminal, and mucosal associated samples from control-treated or antibiotic-treated animals. C) Unweighted or D) weighted UniFrac Day 0 Stool Pellets Day 1-9 Stool Pellets associated bacterial communities in the intestine. analysis of stool pellet samples from control-treated (H2O) or antibiotic-treated (ABX) animals from day 1 to Day 10 Mucosal Associated day 9 post treatment initiation. Colored symbols represent treatment day, white squares represent means, Acknowledgements red line represents linear regression analysis, ± s.e.m. Research is supported by the National Institutes of Health (NIH), AI61570, AI74878 (DA), T32 AI060516 Training in Bacteriology (DH).