Published in: Applied and Environmental Microbiology. Document Version: Peer reviewed version

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1 Fecal microbiota transplant from highly feed efficient donors shows little effects on age-related changes in feed efficiency-associated fecal microbiota in chickens Siegerstetter, S-C., Petri, R. M., Magowan, E., Lawlor, P. G., Zebeli, Q., O'Connell, N., & Metzler-Zebeli, B. U. (2017). Fecal microbiota transplant from highly feed efficient donors shows little effects on age-related changes in feed efficiency-associated fecal microbiota in chickens. Applied and Environmental Microbiology. Published in: Applied and Environmental Microbiology Document Version: Peer reviewed version Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Publisher rights Copyright 2017 American Society for Microbiology. This work is made available online in accordance with the publisher s policies. Please refer to any applicable terms of use of the publisher. General rights Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Download date:18. Mar. 2019

2 1 2 Fecal microbiota transplant from highly feed efficient donors shows little effects on age- related changes in feed efficiency-associated fecal microbiota in chickens 3 4 Running title: Fecal transplant and feed efficiency Sina-Catherine Siegerstetter a, Renée M. Petri a, Elizabeth Magowan b, Peadar G. Lawlor c, Qendrim Zebeli a, Niamh E. O Connell d, Barbara U. Metzler-Zebeli a a Institute of Animal Nutrition and Functional Plant Compounds, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Vienna, Austria. b Agri-Food and Biosciences Institute, Agriculture Branch, Hillsborough, Northern Ireland, UK. c Teagasc, Pig Development Department, Animal & Grassland Research & Innovation Centre, Moorepark, Ireland. d Institute for Global Food Security, Queen s University Belfast, Northern Ireland, UK Address correspondence to Barbara U. Metzler-Zebeli, barbara.metzler@vetmeduni.ac.

3 a Observed OTUs Sequences Per Sample b Observed OTUs dph 29 dph FMT chickens CT chickens Low RFI High RFI FMT inoculum Diets Sequences Per Sample FIG S1 Rarefaction curves showing microbiota diversity based on Observed operational taxonomic units (OTUs): (a) separate for each of the fecal samples (n = 112), fecal microbiota transplant (FMT) (n = 8) and diet sample (n = 6); (b) summarized to day post-hatch (dph), FMT and control (CT) group, residual feed intake (RFI), FMT inoculum and diet sample means. Water samples were not included in the graphs due to their low number of sequencing reads/sample (162 and 180 sequences/sample, respectively). 2

4 FIG S2 Principal coordinate analysis (PCoA) plot of weighted UniFrac analysis of fecal and diet samples. Fecal samples, n = 111, diet samples, n = 6. Rarefaction depth of 10,000 sequences per sample removed 1 sample from the dataset (male, low residual feed intake, control transplant, 16 day post-hatch). 33 3

5 34 35 TABLE S1 Descriptive statistics for the most abundant bacterial phyla in diet and water samples Item n Mean SE Minimum Maximum Starter Diet Cyanobacteria Proteobacteria Firmicutes Unclassified Actinobacteria Tenericutes Spirochaetes TM Grower Diet Cyanobacteria Proteobacteria Firmicutes Actinobacteria Unclassified Bacteroidetes Tenericutes Finisher Diet Cyanobacteria Proteobacteria Firmicutes Unclassified Actinobacteria Bacteroidetes Spirochaetes Water Firmicutes Proteobacteria Cyanobacteria Actinobacteria Tenericutes Unclassified Bacteroidetes Spirochaetes

6 37 38 TABLE S2 Descriptive statistics for the most abundant operational taxonomic units (OTUs) in diet and water samples Item Taxonomy (Genus) a n Mean SE Minimum Maximum Starter Diet OTU14 Unclassified Streptophyta OTU17 Unclassified Streptophyta OTU25 Unclassified Streptophyta OTU38 Unclassified mitochondria OTU57 Unclassified Streptophyta OTU61 Unclassified mitochondria OTU87 Unclassified Streptophyta OTU122 Unclassified mitochondria OTU142 Unclassified Streptophyta OTU125 Unclassified mitochondria OTU375 Unclassified Streptophyta OTU381 Erwinia OTU557 Unclassified Streptophyta OTU508 Unclassified Streptophyta OTU53 Unclassified Enterobacteriaceae OTU643 Stenotrophomonas OTU752 Unclassified mitochondria OTU621 Vibrio OTU1 Unclassified Enterobacteriaceae OTU2 Lactobacillus OTU3 Turicibacter OTU5 Lactobacillus OTU8 Lactobacillus OTU20 Lactobacillus OTU127 Sphingomonas OTU245 Unclassified Comamonadaceae OTU32 Unclassified Clostridiales OTU7 Lactobacillus OTU4 Unclassified Ruminococcaceae OTU107 Unclassified Ruminococcaceae OTU214 Unclassified Sinobacteraceae OTU72 Unclassified Ruminococcaceae OTU105 Unclassified family G OTU63 Unclassified Ruminococcaceae OTU10 Unclassified Clostridiales OTU31 Unclassified Lachnospiraceae

7 OTU15 Unclassified Ruminococcaceae OTU33 Unclassified Enterobacteriaceae Grower Diet OTU14 Unclassified Streptophyta OTU17 Unclassified Streptophyta OTU25 Unclassified Streptophyta OTU38 Unclassified mitochondria OTU61 Unclassified mitochondria OTU57 Unclassified Streptophyta OTU87 Unclassified Streptophyta OTU125 Unclassified mitochondria OTU122 Unclassified mitochondria OTU142 Unclassified Streptophyta OTU381 Erwinia OTU375 Unclassified Streptophyta OTU508 Unclassified Streptophyta OTU752 Unclassified mitochondria OTU557 Unclassified Streptophyta OTU621 Vibrio OTU53 Unclassified Enterobacteriaceae OTU1 Unclassified Enterobacteriaceae OTU7 Lactobacillus OTU643 Stenotrophomonas OTU3 Turicibacter OTU26 Unclassified Clostridiales OTU127 Sphingomonas OTU5 Lactobacillus OTU2 Lactobacillus OTU105 Unclassified family G OTU110 Phenylobacterium OTU8 Lactobacillus OTU172 Slackia OTU20 Lactobacillus OTU214 Unclassified Sinobacteraceae OTU19 Ruminococcus OTU245 Unclassified Comamonadaceae OTU29 Unclassified Clostridiales Finisher Diet OTU14 Unclassified Streptophyta

8 OTU17 Unclassified Streptophyta OTU25 Unclassified Streptophyta OTU38 Unclassified mitochondria OTU57 Unclassified Streptophyta OTU61 Unclassified mitochondria OTU87 Unclassified Streptophyta OTU142 Unclassified Streptophyta OTU122 Unclassified mitochondria OTU125 Unclassified mitochondria OTU53 Unclassified Enterobacteriaceae OTU375 Unclassified Streptophyta OTU621 Vibrio OTU381 Erwinia OTU557 Unclassified Streptophyta OTU643 Stenotrophomonas OTU508 Unclassified Streptophyta OTU752 Unclassified mitochondria OTU1 Unclassified Enterobacteriaceae OTU3 Turicibacter OTU9 Unclassified Clostridiales OTU2 Lactobacillus OTU5 Lactobacillus OTU32 Unclassified Clostridiales OTU105 Unclassified family G OTU8 Lactobacillus OTU31 Unclassified Lachnospiraceae OTU7 Lactobacillus OTU214 Unclassified Sinobacteraceae OTU19 Ruminococcus OTU127 Sphingomonas OTU26 Unclassified Clostridiales OTU20 Lactobacillus OTU232 Unclassified family G OTU4 Unclassified Ruminococcaceae OTU63 Unclassified Ruminococcaceae OTU15 Unclassified Ruminococcaceae OTU75 Unclassified Clostridiales OTU364 Unclassified Peptostreptococcaceae OTU29 Unclassified Clostridiales OTU22 Turicibacter OTU411 Unclassified Clostridiales

9 Water OTU9 Unclassified Clostridiales OTU1 Unclassified Enterobacteriaceae OTU32 Unclassified Clostridiales OTU11 Unclassified Clostridiales OTU5 Lactobacillus OTU4 Unclassified Ruminococcaceae OTU26 Unclassified Clostridiales OTU72 Unclassified Ruminococcaceae OTU3 Turicibacter OTU8 Lactobacillus OTU2 Lactobacillus OTU105 Unclassified family G OTU63 Unclassified Ruminococcaceae OTU39 Unclassified Lachnospiraceae OTU20 Lactobacillus OTU10 Unclassified Clostridiales OTU28 Unclassified Clostridiales OTU251 Unclassified Clostridiales OTU182 Unclassified Clostridiales OTU225 Unclassified Clostridiales OTU79 Ruminococcus OTU65 Ruminococcus OTU94 Unclassified Ruminococcaceae OTU214 Unclassified Sinobacteraceae OTU19 Ruminococcus OTU31 Unclassified Lachnospiraceae OTU245 Unclassified Comamonadaceae OTU15 Unclassified Ruminococcaceae OTU35 Unclassified Clostridiales OTU232 Unclassified family G OTU172 Slackia OTU75 Unclassified Clostridiales OTU48 Unclassified Ruminococcaceae OTU74 Unclassified Ruminococcaceae OTU342 Ruminococcus OTU360 Unclassified Lachnospiraceae OTU127 Sphingomonas OTU29 Unclassified Clostridiales OTU22 Turicibacter

10 39 OTU165 Unclassified Lachnospiraceae OTU363 Unclassified RF OTU14 Unclassified Streptophyta OTU17 Unclassified Streptophyta OTU25 Unclassified Streptophyta OTU57 Unclassified Streptophyta OTU7 Lactobacillus OTU110 Phenylobacterium OTU107 Unclassified Ruminococcaceae OTU364 Unclassified Peptostreptococcaceae OTU411 Unclassified Clostridiales OTU33 Unclassified Enterobacteriaceae a Taxonomic classification based on the 13_8 Greengenes default database in Qiime (version 1.9.1). 9

11 40 41 TABLE S3 Differences in relative abundance (%) of most abundant operational taxonomic units (OTUs) in feces at 16 and 29 days post-hatch (dph) in low and high residual feed intake (RFI) broiler chickens receiving either a fecal microbiota transplant (FMT) or a control transplant (CT) 16 dph 29 dph FMT CT FMT CT P value Item Taxonomy (Genus) a,b Low RFI High RFI Low RFI High RFI Low RFI High RFI Low RFI High RFI SEM T c FMT RFI FMT RFI T FMT RFI OTU1 Escherichia/Shigella OTU2 Lactobacillus OTU3 Turicibacter < OTU7 Lactobacillus < OTU4 Eubacterium OTU6 Escherichia/Shigella OTU5 Lactobacillus OTU8 Lactobacillus OTU16 Anaerobacterium OTU15 Eubacterium OTU22 Turicibacter < OTU26 Gracilibacter OTU28 Anaerobacterium OTU13 Acinetobacter OTU21 Lactobacillus OTU34 Turicibacter < OTU46 Lactobacillus < OTU33 Escherichia/Shigella < OTU27 Lactobacillus OTU30 Lactobacillus

12 OTU35 Eisenbergiella OTU58 Anaerobacterium OTU44 Turicibacter < OTU31 [Ruminococcus] OTU32 Saccharofermentans OTU56 Anaerobacterium OTU42 Lactobacillus OTU47 Lactobacillus OTU39 [Clostridium] OTU43 Lactobacillus OTU65 [Clostridium] OTU80 Acetivibrio OTU81 Anaerobacterium OTU89 Hespellia OTU53 Klebsiella OTU18 Klebsiella OTU68 Flintibacter OTU51 Lactobacillus OTU74 Eubacterium OTU71 Lactobacillus OTU95 Escherichia/Shigella < OTU119 Anaerobacterium OTU96 Pseudoflavonifractor OTU67 Lactobacillus OTU84 Flintibacter OTU129 Lactobacillus

13 OTU107 Flintibacter OTU103 Turicibacter < OTU113 Comamonas OTU132 Lactobacillus OTU93 Lactobacillus OTU117 [Clostridium] OTU130 Anaerobacterium OTU150 Oscillibacter OTU77 Oscillibacter OTU98 Lactobacillus Data are presented as least-square means and pooled SEM. Low RFI FMT females, n = 8/time point; low RFI FMT males, n = 7/time point; high RFI FMT females, n = 7/time point; high RFI FMT males, n = 6/time point; low RFI CT females, n = 7/time point; low RFI CT males, n = 7/time point; high RFI CT females, n = 7/time point; high RFI CT males, n = 7/time point. a Taxonomic classification based on the National Center for Biotechnology Information (NCBI) nucleotide database using Blastn for taxonomic classification and the database limited to the 16S rrna target ( b Sequences not distinguishable between Escherichia and Shigella. c T, time point. 12

14 49 TABLE S4 BLAST search results for selected operational taxonomic units (OTUs) a,b OTU Blast hit (16S NCBI database) Accession Number Percent idendity OTU1 Escherichia coli strain JCM 1649 NR_ Shigella flexneri strain ATCC NR_ OTU2 Lactobacillus salivarius strain HO 66 NR_ OTU3 Turicibacter sanguinis strain MOL361 NR_ OTU4 Eubacterium desmolans strain ATCC NR_ OTU5 Lactobacillus crispatus strain DSM NR_ OTU6 Escherichia coli strain JCM 1649 NR_ Shigella flexneri strain ATCC NR_ OTU7 Lactobacillus johnsonii strain CIP NR_ OTU8 Lactobacillus crispatus strain DSM NR_ OTU13 Acinetobacter bereziniae strain ATCC NR_ OTU15 Eubacterium desmolans strain ATCC NR_ OTU16 Anaerobacterium chartisolvens strain T-1-35 NR_ OTU18 Klebsiella variicola strain F2R9 NR_ OTU21 Lactobacillus crispatus strain DSM NR_ OTU22 Turicibacter sanguinis strain MOL361 NR_ OTU24 Romboutsia timonensis strain DR1 NR_ OTU26 Gracilibacter thermotolerans strain JW/YJL-S1 NR_ OTU27 Lactobacillus salivarius strain HO 66 NR_ OTU28 Anaerobacterium chartisolvens strain T-1-35 NR_ OTU29 Anaerobacterium chartisolvens strain T-1-35 NR_ OTU30 Lactobacillus crispatus strain DSM NR_ OTU31 [Ruminococcus] torques strain VPI B2-51 NR_ OTU32 Saccharofermentans acetigenes strain P6 NR_ OTU33 Escherichia coli strain JCM 1649 NR_ Shigella flexneri strain ATCC NR_ OTU34 Turicibacter sanguinis strain MOL361 NR_ OTU35 Eisenbergiella massiliensis strain AT11 NR_ OTU39 [Clostridium] saccharolyticum strain WM1 NR_ OTU42 Lactobacillus crispatus strain DSM NR_ OTU43 Lactobacillus salivarius strain HO 66 NR_ OTU44 Turicibacter sanguinis strain MOL361 NR_ OTU46 Lactobacillus johnsonii strain CIP NR_ OTU47 Lactobacillus salivarius strain HO 66 NR_ OTU48 Negativibacillus massiliensis strain Marseille-P3213 NR_ OTU50 Gracilibacter thermotolerans strain JW/YJL-S1 NR_ OTU51 Lactobacillus crispatus strain DSM NR_ OTU52 [Clostridium] leptum strain DSM 753 NR_

15 OTU53 Klebsiella oxytoca strain NBRC NR_ OTU55 Clostridium disporicum strain DS1 NR_ OTU56 Anaerobacterium chartisolvens strain T-1-35 NR_ OTU58 Anaerobacterium chartisolvens strain T-1-35 NR_ OTU60 [Clostridium] leptum strain DSM 753 NR_ OTU64 Hespellia stercorisuis strain PC18 NR_ OTU65 [Clostridium] leptum strain DSM 753 NR_ OTU67 Lactobacillus crispatus strain DSM NR_ OTU68 Flintibacter butyricus strain BLS21 NR_ OTU70 Negativibacillus massiliensis strain Marseille-P3213 NR_ OTU71 Lactobacillus salivarius strain HO 66 NR_ OTU74 Eubacterium desmolans strain ATCC NR_ OTU75 Eisenbergiella massiliensis strain AT11 NR_ OTU77 Oscillibacter ruminantium strain GH1 NR_ OTU79 [Clostridium] leptum strain DSM 753 NR_ OTU80 Acetivibrio cellulolyticus strain CD2 NR_ OTU81 Anaerobacterium chartisolvens strain T-1-35 NR_ OTU83 Anaerotruncus colihominis strain WAL NR_ OTU84 Flintibacter butyricus strain BLS21 NR_ OTU88 Gracilibacter thermotolerans strain JW/YJL-S1 NR_ OTU89 Hespellia porcina strain PC80 NR_ OTU93 Lactobacillus salivarius strain HO 66 NR_ OTU95 Escherichia coli strain JCM 1649 NR_ Shigella flexneri strain ATCC NR_ OTU96 Pseudoflavonifractor phocaeensis strain Marseille-P3064 NR_ OTU97 Oscillibacter ruminantium strain GH1 NR_ OTU98 Lactobacillus crispatus strain DSM NR_ OTU103 Turicibacter sanguinis strain MOL361 NR_ OTU107 Flintibacter butyricus strain BLS21 NR_ OTU113 Comamonas thiooxydans strain S23 NR_ OTU117 [Clostridium] viride strain T2-7 NR_ OTU119 Anaerobacterium chartisolvens strain T-1-35 NR_ OTU129 Lactobacillus johnsonii strain CIP NR_ OTU130 Anaerobacterium chartisolvens strain T-1-35 NR_ OTU132 Lactobacillus johnsonii strain CIP NR_ OTU150 Oscillibacter ruminantium strain GH1 NR_ a BLAST search was performed for the most abundant OTUs in the fecal microbiota transplant as well as for the OTUs in feces differently affected by time point, fecal microbiota transplant or residual feed intake. b National Center for Biotechnology Information (NCBI) nucleotide database ( last accessed 12/10/

16 TABLE S5 Dietary ingredients and chemical composition of diets (on as-fed basis) Item Ingredient (%) Starter 1-8 days post-hatch Grower 9-20 days post-hatch Finisher days post-hatch Corn Soybean meal Soybean oil Monocalcium phosphate Calcium carbonate DL-Methionine Natrium bicarbonate Lysine-HCL Salt L-Threonine Premix Analyzed chemical composition, g/kg dry matter (DM) DM Crude protein Crude fat Crude fiber Crude ash Starch Sugar Calcium Phosphorus ME (MJ/kg) Provided per kilogram of complete starter diet (Garant - Tiernahrung GmbH, Pöchlarn, Austria): 12,500 IU of vitamin A, 5,000 IU of vitamin D 3, 75.0 mg of vitamin E, 6.0 mg of vitamin K3, 2.50 mg of vitamin B 1, 7.0 mg of vitamin B 2, 4.50 mg of vitamin B 6, mg of vitamin B 12, 60.0 mg of nicotinic acid, 15.0 mg of pantothenic acid, 1.0 mg of folic acid, 0.25 mg of biotin, 1, mg of choline, mg of choline chloride, mg of betaine, mg of ethoxyquin, FTU of 6Phytase, g of ß-glucan, % of C 18:2, % of polyenic acid, mg of F-Xanto (total). Provided per kilogram of complete grower diet (Garant - Tiernahrung GmbH, Pöchlarn, Austria): 12,500 IU of vitamin A, 5,000 IU of vitamin D 3, 75.0 mg of vitamin E, 6.0 mg of vitamin K3, 2.50 mg of vitamin B 1, 7.0 mg of vitamin B 2, 4.50 mg of vitamin B 6, mg of vitamin B 12, 60.0 mg of nicotinic acid, 15.0 mg of pantothenic acid, 1.0 mg of folic acid, 0.25 mg of biotin, 1, mg of choline, mg of choline chloride, mg of betaine, mg of ethoxyquin, FTU of 6Phytase, g of ß-glucan, % of C 18:2, 3.103% of polyenic acid, mg of F-Xanto (total). 15

17 Provided per kilogram of complete finisher diet (Garant - Tiernahrung GmbH, Pöchlarn, Austria): 5,000 IU of vitamin D 3, 75.0 mg of vitamin E, 6.0 mg of vitamin K3, 2.50 mg of vitamin B 1, 7.0 mg of vitamin B 2, 4.5 mg of vitamin B 6, mg of vitamin B 12, 60.0 mg of nicotinic acid, 15.0 mg of pantothenic acid, 1.0 mg of folic acid, 0.25 mg of biotin, 1, mg of choline, mg of choline chloride, mg of betaine, mg of ethoxyquin, FTU of 6Phytase, g of ß-glucan, % of C 18:2, % of polyenic acid, mg of F-Xanto (total). 16