Enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome

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1 Authors: Robert P. Dickson MD 1, Benjamin H. Singer MD, PhD 1, Michael W. Newstead 1, Nicole R. Falkowski 1, John R. Erb-Downward PhD 1, Theodore J. Standiford MD 1,3, Gary B. Huffnagle PhD 1,2,3 SUPPLEMENTARY INFORMATION ARTICLE NUMBER: DOI: /NMICROBIOL Enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome Affiliations: 1 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan; 2 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan. 3 Co-senior authors Contents: 1) Supplementary Table 2) Supplemental Figure 1: Altered bacteria in the lung microbiome 24 hours after sepsis and sham surgery. 3) Supplemental Figure 2: Bacterial communities in the lungs of mice at extended timepoints. 4) Supplemental Figure 3: Enriched lung bacteria in experimental sepsis and human ARDS. 5) Supplemental Figure 4: Phylogenetic tree of prominent OTUs and closely-aligned sequences from a reference database. 6) Supplemental Figure 5: Time course of gut-lung community similarity following abdominal sepsis. 7) Supplemental Figure 6: Effect of experimental sepsis on the relative abundance of OTU008 Bacteroidales sp. in the murine gut. NATURE MICROBIOLOGY 1

2 8) Supplemental Figure 7: Gut-associated bacteria in the lungs of mice following LPS-induced shock. 9) Supplemental Figure 8: Stability of OTU009 (Bacteroides sp.) in serial BAL specimens from patients with ARDS. 2

3 Supplemental Table 1. Human subject characteristics. ARDS subjects (n=68) Age (47.4 ± 14.5) Patient demographics Male 46 (67%) Race: Caucasian 45 (66%) Race: African-American 19 (28.0%) Race: Other 4 (5.8%) Pneumonia 20 (29.4%) ARDS risk factor Aspiration 14 (20.6%) Sepsis 18 (26.4%) Other 16 (11.8%) Illness severity at study enrollment Outcomes Oxygenation index (16.0 ± 11.5) PaO 2 :FiO 2 Ratio (117.6 ± 55.8) APACHE III index (57.2 ± 17.1) 28-day mortality 14 (20.5%) Ventilator-free days 0-27 (8.7 ± 9.3) Healthy subjects (n=7) Age 47.4 ± 14.5 Subject demographics Male 5 (71%) Smoking history (Active/Former/Never) 0/0/7

4 Phyla Bacteroides Firmicutes Proteobacteria Actinobacteria Sham surgery Abdominal sepsis (CLP) Enterobacteriaceae : unclassified Porphyromonadaceae : unclassified Streptococcaceae : Streptococcus Porphyromonadaceae : unclassified Staphylococcaceae : Staphylococcus Lactobacillaceae : Lactobacillus Lachnospiraceae : unclassified Flavobacteriaceae : Flavobacterium Dermacoccaceae : Kytococcus Lachnospiraceae : unclassified Lachnospiraceae : unclassified Caulobacteraceae : Phenylobacterium Sphingomonadaceae : unclassified Lachnospiraceae : unclassified Ruminococcaceae : unclassified Sphingomonadaceae : Sphingobium Erysipelotrichaceae : Turicibacter Alcaligenaceae : Castellaniella unclassified : unclassified Ruminococcaceae : unclassified Supplemental Figure 1 Altered bacteria in the lung microbiome 24 hours after sepsis and sham surgery. The 20 most abundant operational taxonomic units detected in the lungs of untreated mice are shown across experimental arms. OTUs are ranked in descending order of mean abundance in the lungs of untreated mice. Values presented as means ± standard error of the mean.

5 a Lung communities: 14 days after intervention b Lung communities: 8 weeks after intervention PC2 (11.3% Explained) Imipenem only Imipenem + sham surgery Imipenem + sepsis (CLP) Imipenem: 14 days Sham: 14 days Sepsis: 14 days PC2 (11.3% Explained) Antibiotics only Antibiotics + sham surgery Antibiotics + sepsis (CLP) Sepsis: 8 weeks Imipenem: 8 weeks Sham: 8 weeks PC1 (16.9% Explained) PC1 (16.9% Explained) Supplemental Figure 2 Bacterial communities in the lungs of mice at extended timepoints. Mice were exposed to abdominal sepsis (cecal ligation and puncture and imipenem) and compared at multiple timepoints to three experimental control groups: untreated, imipenem only, and imipenem with sham surgery. Lung communities are shown at 14 days following exposure (a) and 8 weeks following exposure (b). Post-sepsis lungs were not collectively distinct from the lungs of control mice at either timepoint (P > 0.05). Post-sepsis lung communities within the red circle were enriched with four OTUs classified at the family level as Lachnospiraceae, comprising 37.2% of sequences. These same OTUs comprised only 5.7% of sequences in the remaining 14-day CLP lungs, and 6.9% of sequences in all other 14-day lungs.

6 a b OTU001: Bacillus sp. OTU002: Pseudomonas sp. OTU004: Streptococcus sp. OTU006: Pseudomonas sp. OTU009: Bacteroides sp. OTU005: Lactobacillus sp. OTU003: Streptococcus sp. OTU010: Burkholderiaceae sp. OTU012: Gammaproteobacteria sp. OTU021: Veillonella sp. OTU011: Gammaproteobacteria sp. OTU015: Neisseria sp. BAL: ARDS BAL: Healthy Reagent controls Abundances log y + 1 scale min Supplemental Figure 3 Enriched lung bacteria in experimental sepsis and human ARDS. (a) Biplot analysis of lungs of bacterial communities detected in lungs of untreated mice and at various timepoints following sepsis. A single taxonomic group (OTU008, Bacteroidales sp.) drove the community difference between post-sepsis lungs (5 days) and all other intervention arms. (b) Model-based analysis of multivariate abundance data (mvabund package in R15) of taxonomic groups relatively enriched in the BAL fluid of patients with ARDS compared to that of healthy subjects. Identified taxa corresponded to bacterial genera found in the lower intestinal tract (e.g. Bacillus, Bacteroidetes, Lactobacillus), the upper respiratory tract (Streptococcus, Burkholderiaceae, Neisseria) and both (Pseudomonas, Gammaproteobacteria spp.). OTU0009 (Bacteroides sp., highlighted in green) was selected for further study given its frequency and abundance in ARDS BAL specimens, its absence from healthy human BAL specimens and the previously reported microbiota of the upper respiratory tract, and its absence from reagent control specimens.

7 Sources of Prominent OTUs Human ARDS BAL Fluid Mouse Cecal Ligation and Puncture: 24 Hours Mouse Cecal Ligation and Puncture: Extended Timepoints 25_BAKK _168_1671_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_Bacteroides_faecichinchillae 43_HQ744283_1_1443_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_uncultured_organism 24_HQ814524_1_1435_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_uncultured_organism 0.12 Previously cultured bacterial species 23_HQ762161_1_1435_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_uncultured_organism 44_ASSM _161439_162955_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_Bacteroides_thetaiotaomicron 15_AB510710_1_1461_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_Bacteroides_thetaiotaomicron 33_HQ804178_1_1444_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_uncultured_organism 35_AB510701_1_1490_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_Bacteroides_fragilis 34_CS600341_10_1518_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_Bacteroides_fragilis 13_CR626927_ _ _Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidaceae_Bacteroides_Bacteroides_fragilis 4_Human_ARDS_OTU009 42_EU506153_1_1399_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 22_EU506168_1_1412_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 20_EU474236_1_1405_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 30_EU474413_1_1398_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 36_EF099195_1_1401_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium _EF099788_1_1401_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 10_EU474328_1_1399_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 1_CLP_24_Hours_OTU _EF099541_1_1401_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 19_EU454677_1_1401_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 29_EU453830_1_1403_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium _CLP_Extended_OTU008 7_EF100036_1_1412_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 26_EF097933_1_1412_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 18_EU451889_1_1411_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 17_EF099854_1_1383_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 38_EF406607_1_1506_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 40_EU455514_1_1230_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 27_EF099783_1_1409_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 8_EU452381_1_1411_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium _EF099203_1_1405_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 9_EU455451_1_1239_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 28_EF406381_1_1494_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 39_EU454660_1_1399_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 41_EU503946_1_1406_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 12_EU508238_1_1401_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 11_EU504412_1_1402_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium _EU504204_1_1357_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 14_JN012808_1_1459_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 31_EU504427_1_1402_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 32_EU791080_1_1490_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium 2_CLP_24_hours_OTU008 5_AB626933_1_1493_Bacteria_Bacteroidetes_Bacteroidia_Bacteroidales_Bacteroidales_S24-7_group_uncultured_bacterium Supplemental Figure 4 Phylogenetic tree of prominent OTUs and closely-aligned sequences from a reference database. Prominent OTUs detected the BAL fluid of humans with ARDS (green) and the lungs of mice following cecal ligation and puncture at 24 hours (blue) and extended timepoints (purple) were compared with 40 closely-aligned sequences obtained from the SILVA database ( Sequences obtained from cultivated organisms are highlighted in yellow. All related sequences were classified as belonging to the Bacteroidales order within the Bacteroidetes phylum. Tree generated with MAFFT v.7 (

8 a b Colon-Lung Similarity (Jaccard similarity coefficient) Days 14 Days 8 Weeks Feces-Lung Similarity (Jaccard similarity coefficient) Days 14 Days 8 Weeks Duration following abdominal sepsis (CLP) Duration following abdominal sepsis (CLP) Supplemental Figure 5 Time course of gut-lung community similarity following abdominal sepsis. For each mouse, community similarity was calculated for paired lung and colon communities (a) and paired lung and feces communities (b). Group means and standard errors of the mean are depicted. 30% Colon communities 30% Feces communities Relative abundance of OTU008 (Bacteroidales sp.) Imipenem only Imipenem + sham surgery Imipenem + sepsis 20% 10% * 20% 10% ** * 0% Day 5 Week 2 Week 8 Post-sepsis 0% Day 5 Week 2 Week 8 Post-sepsis Supplemental Figure 6 Effect of experimental sepsis on the relative abundance of OTU008 Bacteroidales sp. in the murine gut. Mice were exposed to abdominal sepsis (cecal ligation and puncture and imipenem) and compared at multiple timepoints to three experimental control groups: untreated, imipenem only, and imipenem with sham surgery. The taxonomic group enriched in post-sepsis lungs (OTU008, Bacteroidales sp.) decreased in relative abundance in colon and fecal communities at day 5 and week 2, normalizing by week 8. Statistical significance was determined by Kruskal Wallis one-way analysis of variance with Dunn s multiple comparisons test. ** P 0.01, * P 0.05.

9 a b LNG_LPS_6 LNG_LPS_5 PC2 (14.9% Explained) Systemic LPS: 1 day P = Systemic LPS: 4 days PC2 (14.9% Explained) LNG_LPS_4 Otu00026 LNG_UNT_1 Otu00021 Otu00138 Otu00047 Otu00018 Otu00027 Otu00051 Otu00046 Otu00053 Otu00114 Otu00150 Otu00061 Otu00062 Otu00088 Otu00013 Otu00055 Otu00028 Otu00069 Otu00009 Otu00039 Otu00017 Otu00086 Otu00087 Otu00029 Otu00030 Otu00032 Otu00033 Otu00034 Otu00035 Otu00036 Otu00037 Otu00038 Otu00040 Otu00041 Otu00042 Otu00043 Otu00044 Otu00045 Otu00049 Otu00052 Otu00054 Otu00056 Otu00057 Otu00058 Otu00059 Otu00060 Otu00082 Otu00083 Otu00001 Otu00002 Otu00003 Otu00006 Otu00007 Otu00008 Otu00011 Otu00012 Otu00014 Otu00015 Otu00016 Otu00019 Otu00020 Otu00022 Otu00023 Otu00024 Otu00025 Otu00091 Otu00092 Otu00093 Otu00094 Otu00096 Otu00097 Otu00098 Otu00099 Otu00063 Otu00064 Otu00065 Otu00066 Otu00067 Otu00068 Otu00070 Otu00071 Otu00072 Otu00073 Otu00074 Otu00076 Otu00077 Otu00078 Otu00079 Otu00100 Otu00115 Otu00116 Otu00118 Otu00119 Otu00121 Otu00125 Otu00126 Otu00128 Otu00130 Otu00131 Otu00106 Otu00108 Otu00109 Otu00110 Otu00112 Otu00102 Otu00117 Otu00132 Otu00178 Otu00157 Otu00181 Otu00154 Otu00330 Otu00141 Otu00195 Otu00217 Otu00539 LNG_UNT_3 Otu00146 Otu00134 Otu00202 Otu00313 Otu00182 Otu00183 Otu00184 Otu00185 Otu00186 Otu00188 Otu00189 Otu00190 Otu00193 Otu00194 Otu00196 Otu00198 Otu00200 Otu00204 Otu00135 Otu00136 Otu00137 Otu00139 Otu00140 Otu00142 Otu00143 Otu00147 Otu00149 Otu00152 Otu00153 Otu00155 Otu00156 Otu00159 Otu00161 Otu00163 Otu00164 Otu00165 Otu00166 Otu00167 Otu00168 Otu00170 Otu00171 Otu00172 Otu00173 Otu00175 Otu00176 Otu00179 Otu00205 Otu00224 Otu00223 Otu00261 Otu00258 Otu00226 Otu00227 Otu00229 Otu00230 Otu00232 Otu00234 Otu00236 Otu00240 Otu00243 Otu00245 Otu00246 Otu00251 Otu00252 Otu00253 Otu00255 Otu00256 Otu00257 Otu00263 Otu00267 Otu00270 Otu00272 Otu00277 Otu00279 Otu00285 Otu00288 Otu00291 Otu00292 Otu00293 Otu00296 Otu00299 Otu00300 Otu00301 Otu00302 Otu00305 Otu00306 Otu00307 Otu00309 Otu00310 Otu00315 Otu00318 Otu00319 Otu00326 Otu00327 Otu00336 Otu00337 Otu00208 Otu00209 Otu00210 Otu00212 Otu00214 Otu00215 Otu00216 Otu00218 Otu00219 Otu00220 Otu00221 Otu00311 Otu00101 Otu00278 Otu00298 Otu00287 Otu00081 Otu00338 Otu00342 Otu00343 Otu00344 Otu00346 Otu00351 Otu00345 Otu00352 Otu00355 Otu00464 Otu00384 Otu00358 Otu00373 Otu00376 Otu00359 Otu00360 Otu00361 Otu00362 Otu00367 Otu00368 Otu00377 Otu00459 Otu00408 Otu00475 Otu00391 Otu00476 Otu00468 Otu00494 Otu00578 Otu00807 Otu00772 Otu00718 Otu00603 Otu00508 Otu00478 Otu00574 Otu00615 Otu00559 Otu00511 Otu00432 Otu00379 Otu00638 Otu00655 Otu00872 Otu01027 Otu00507 Otu00856 Otu00910 Otu00913 Otu00765 Otu00866 Otu00995 Otu00971 Otu00697 Otu00715 Otu00719 Otu01048 Otu01084 Otu01023 Otu01105 Otu01312 Otu01070 Otu00622 Otu00632 Otu00640 Otu00644 Otu00652 Otu00669 Otu00727 Otu00730 Otu00763 Otu00770 Otu00774 Otu00784 Otu00832 Otu00833 Otu00921 Otu01009 Otu00899 Otu00378 Otu00380 Otu00392 Otu00396 Otu00513 Otu00520 Otu00558 Otu00410 Otu00411 Otu00412 Otu00418 Otu00428 Otu00431 Otu00433 Otu00436 Otu00445 Otu00449 Otu00451 Otu00453 Otu00458 Otu00462 Otu00465 Otu00470 Otu00498 Otu00502 Otu00506 Otu01146 Otu01305 Otu01309 Otu01279 Otu01268 Otu01213 Otu01198 Otu00895 Otu00454 Otu00123 Otu00397 Otu00393 Otu00160 Otu00323 Otu00371 Otu00197 Otu00050 Otu00174 Otu00237 Otu00005 Otu00129 Otu00225 Otu00105 Otu00084 Otu00151 Otu00127 Otu00080 Otu00004 Enterobacteriaceae sp. Otu00089 Otu00104 Otu00274 LNG_LPS_D Otu00120 Otu00370 Otu00048 Otu00158 Otu00111 Otu00122 Otu00095 Otu00180 LNG_LPS_C Otu00103 Otu00222 Otu00075 Otu00090 Otu00010 Otu00031 LNG_LPS_A LNG_LPS_B Otu00085 LNG_UNT_2 LNG_LPS_ PC1 (32.3% Explained) PC1 (32.3% Explained) c d OTU00004 Enterobacteriaceae sp. 1_OTU0004_enriched_in_post-LPS_lungs 3_KP064197_1_1596_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_uncultured_Enterobacter_sp 12_EF088367_1_1458_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_sp 05_01_120 26_JTBO _ _ _Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_cloacae 100% ** *** 27_JTBO _ _ _Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_cloacae 23_JTBO _241910_243463_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_cloacae 24_JTBO _ _ _Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_cloacae 28_JTBO _ _ _Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_cloacae 80% 60% 40% 20% 30_JTBO _ _ _Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_cloacae 29_JTBO _ _ _Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_cloacae 22_JTBO _7086_8637_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_cloacae 25_JTBO _995114_996665_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_cloacae 21_HQ787649_1_1448_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_uncultured_organism 14_JX317636_1_1424_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Pantoea_Enterobacter_cloacae 2_KJ577634_1_1407_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_asburiae 10_KF434761_1_1339_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_sp T1 4_KJ781381_1_1458_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_ludwigii 6_KF906826_1_1526_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_sp BR1 9_JX827717_1_1451_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Klebsiella_bacterium_RB5-FF-20 13_AB425050_1_1310_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_sp SCPB-1 17_HM462446_1_1447_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Klebsiella_Klebsiella_sp VITPGP5AB 20_HM462447_1_1249_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Klebsiella_Klebsiella_sp VITPGP23A 16_FJ445213_1_2097_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Pantoea_sp NIIST-167 OTU enriched in mouse lungs 1 day after systemic LPS Previously cultured bacterial species 7_KF906834_1_1528_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Cronobacter_Klebsiella_sp OR7 0% Reagent controls 1 day 4 days Time following systemic LPS 15_AY660027_1_1471_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Providencia_Xenorhabdus_bovienii 8_KF843010_1_1381_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Cronobacter_uncultured_bacterium 18_FJ626262_1_1425_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_endosymbiont_of_Sphenophorus_levis 11_KC463799_1_1243_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Klebsiella_uncultured_bacterium 5_LN558633_1_1352_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Klebsiella_Klebsiella_pneumoniae 19_HQ419280_1_1500_Bacteria_Proteobacteria_Gammaproteobacteria_Enterobacteriales_Enterobacteriaceae_Enterobacter_Enterobacter_sp NISOC_03 Supplemental Figure 7 Gut-associated bacteria in the lungs of mice following LPS-induced shock. Shock was induced via intraperitoneal injection of 5 mg/kg LPS, and lung bacterial communities were sequenced and analyzed after 1 day and 4 days. (a) One day after LPS-induced shock, lung communities were significantly distinct from those of untreated animals. This difference had resolved by 4 days. (b) Biplot analysis identified an Enterobacteriaceae-classified OTU (OTU00004) as the community member responsible for this difference. (c) This Enterobacteriaceae-classified OTU (OTU00004) had significantly increased relative abundance in the lung communities of LPS treated mice after 1 day; this difference had resolved by 4 days. (d) Phylogenetic tree of this OTU (OTU00004) compared with 30 neighboring sequences in the SILVA database ( Sequences obtained from cultivated organisms are highlighted in yellow. All sequences were genetically identical (distance = 0) to OTU00004, and belonged to the Enterobacteriaceae family within the Proteobacteria phylum. Tree generated with MAFFT v.7 ( ** P 0.01, *** P

10 Bacteroides sp. (OTU009) 100% 80% 60% 40% 20% 0% #1 #2 #3 Number of serial bronchoalveolar lavage specimen Supplemental Figure 8 Stability of OTU009 (Bacteroides sp.) in serial BAL specimens from patients with ARDS. The relative abundance of OTU009 (Bacteroides sp.) is shown for BAL specimens collected from the 25 ARDS patients who underwent serial bronchoscopy.