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Supplementary Figure 1 Multicenter Randomised Control Trial 2746 Randomised Supplementary Figure 1 Multicenter randomised control trial 2746 randomised 947 control 910 MNP without zinc 889 MNP with zinc 223 lost to follow up 219 lost to follow up 183 lost to follow up 34 refused 29 refused 37 refused 16 died 12 died 9 died 3 excluded 4 excluded 1 excluded 671 in follow-up 646 in follow-up 659 in follow-up at 24mo of age at 24mo of age at 24mo of age Selection for Microbiome sequencing 516 paired samples unavailable 469 paired samples unavailable 497 paired samples unavailable 69 antibiotic use 63 antibiotic use 67 antibiotic use 31 outside of WLZ criteria 37 outside of WLZ criteria 34 outside of WLZ criteria 6 diarrhea last 7 days 2 diarrhea last 7 days 7 diarrhea last 7 days 39 WLZ > -1 at 24 mo 10 WLZ < -2 at 24mo 58 WLZ > -1 at 24 mo 17 WLZ < -2 at 24mo 48 WLZ > -1 at 24 mo 8 WLZ < -2 at 24mo available for selection available for selection available for selection available for selection available for selection1 available for selection1 14 selected 10 selected 15 selected 14 selected 20 selected1 7 selected1 1 Two subjects (one in the reference WLZ group and one undernourished) had, at 12 months, no diarrhea within 1 day of stool collection but reported diarrhea within 7 days prior. Length, cm kg Weight, Supplementary Figure 2. Length (left) and weight (right) z-scores of children recruited into clinical trial NCT00705445 during the first 24 months of life. Median and quantile values are shown, with medians for participants profiled in current study indicated by red (undernourished) and black (reference WLZ) lines. Protozoa, rarefied to 1000 80 reference WLZ undernourished 60 hness c 40 Ri 20 0 CTL MNP MNP + Zn Supplementary Figure 3. Protozoan richness in stool samples, grouped by nutritional status and micronu- trient supplementation. Samples were rarefied to 1000 reads. a age (p<0.001) nutritional status b 12 month reference WLZ (rarefied to 25,000) 2.00 24 month undernourished 1.7 reference WLZ 1.75 undernourished 1.6 3 1.50 1.5 1.25 1.4 Shannon diversity (H’) Shannon diversity (H’) 0 10000 20000 30000 40000 50000 0 10000 20000 30000 40000 50000 2 Read depth Read depth supplementation site CTL rural MNP 1.7 urban MNP+Zn Shannon diversity (H') 1.8 1 1.6 1.6 1.5 1.4 1.4 Shannon diversity (H’) Shannon diversity (H’) 1.2 0 0 10000 20000 30000 40000 50000 0 10000 20000 30000 40000 50000 Urban Rural Read depth Read depth c (Bray-Curtis) ns ns ns ns ns 0.8 ns 0.7 0.6 0.5 Distance to centroid 0.4 CTL MNP CTL MNP MNP+Zn MNP+Zn 12 month 24 month Supplementary Figure 4. Bacterial evenness and beta diversity. a, Rarefaction curves comparing species evenness (H′) by age group, micronutrient supplementation arm, nuritional status and site. Shaded regions represent standard error. Significance was tested at 25,000 read depth using a generalized linear model with stewise AIC-based model selection to identify significant variables. b, Boxplots comparing bacterial evenness by nutritional status and site. c, Variance among Bray-Curtis dissimilarities, calculated as distance to the centroid, between samples (n=150) by treatment arm and age group. 12 month CTL MNP MNP + Zn Actinomucor Incertae Corynebacterium Cercozoa Lactobacillus Rhizopus Rothia Rhizomucor Lactobacillus ● ● Lactobacillus Rhizopus ● Candida−Lodderomyces clade ● ● ● ● ● Trichosporon Cercomonas ● ● Actinomyces Enterococcus Pichia ● ● ● Bifidobacterium ● ● ● Pediococcus Lactobacillus Wallemia Gemmiger Senegalimassilia ● Wallemia Blastobotrys Cryptosporida ● ● Toxoplasma ActinomucorTrichosporon ● ● Blautia ● ● Colpodella ● Blautia Clostridium_XlVb ● Terrisporobacter ● ● ● ● ● Dorea Pseudomonas ● ● ● Kodamaea Wallemia Eugregarinorida−distant−branch ● Rothia Romboutsia Eggerthella ● ● Conoidasida ● Enterococcus ● ● Romboutsia ● ● Flavonifractor● ● ●Clostridium_sensu_stricto Bifidobacterium● Blautia ● Kluyveromyces ● Trichomonascus ● Intestinibacter ● Candida Corynebacterium ● ● ● Senegalimassilia Saccharomyces Collinsella BlastobotrysAmoebozoa_unknown_group1 ● Pichia ● ● ● ● Lysinibacillus Acinetobacter ● ● ● ● Actinomyces ● ● ● ● Bacillus Collinsella ● ● StreptococcusLichtheimia Bifidobacterium CryptosporidiumSaccharomyces Lachnospiracea_incertae_sedis ● ● Clostridium_XlVb Bifidobacterium Streptococcus Bacillus StenotrophomonasLysinibacillus ● Colpodella ● ● ● ● ● ● ● ● ● ● ● Lactobacillus ● Lachnospiracea_incertae_sedis Ruminococcus2 ● Amylomyces Weissella Syndiniales● ● Trichomonascus ● Dorea Actinomyces Lactobacillus Dorea ● Streptococcus Candida ● ● ● Gregarina−distant−branch ● ● ● Paenibacillus Acinetobacter Eugregarinorida● Gregarinasina Clostridium_sensu_stricto Lachnospiracea_incertae_sedis ● Gregarinasina−distant−branch Gregarina ● Eggerthella ● ● Clostridium_XVIII Cercomonas ● Prevotella Dorea ● ● ● Olsenella Enterocytozoon ● ● Pseudomonas Rhizopus ● Actinomyces ● Bifidobacterium Streptococcus ● ● ● ● ● Anaerostipes ● Intestinibacter ● Bifidobacterium Bifidobacterium Clostridium_sensu_stricto ● ● Syndiniales ● Heteromita Actinomyces Anaerostipes ● ● ● ● ● FaecalibacteriumCandida−Lodderomyces clade ● ● Gemella ● Blastobotrys ● ● Neogregarinorida Actinomyces Collinsella ● ● Lachnospi● racea_incertae_sedis ● ● IncertaeKluyveromyces Lachnospiracea_incertae_sedis Actinomyces Bifidobacterium ● ● Conoidasida ● ● MegasphaeClostridium_XlVbra Faecalibacterium ● ● Lactobacillus Kodamaea ● Bacillus Penicillium Cryptomycota ● ● ● Streptococcus ● Clostridium_sensu_stricto ● Streptococcus ● ● Senegalimassilia Lichtheimiaceae Lichtheimia Eggerthella ● ● ● ● ● ● ● ● ● Pseudomonas ● Candida−Lodderomyces clade Lachnospiracea_incertae_sedisSlackia G●regarina−distant−branch Streptococcus Lachnospiracea_incertae_sedis Tritrichomonas● ● ● ● Urban Alveolata Colpodella Cryptosporidium ● Lachnospiracea_incertae_sedis● ● Lachnospiracea_incertae_sedis Gregarinasina Tritrichomonas ● ● Anaerococcus ● ● Streptococcus Gregarinasina Dorea ● Olsenella Holdemanella ● ● ● ● Lactobacillus ● ● Bromeliothrix ● ● ● ● ● Clostridium_XVIII Acinetobacter ● Bifidobacterium Pseudomonas Ruminococcus2 ● Lactobacillus Penicillium ● ● ● Streptococcus Clostridium_XI Collinsella ● Pediococcus ● Cryptosporidium Dorea Streptococcus ● ● ● ● ● ● Gregarinasina−distant−bCollinsellaranch Fusicatenibacter ● Streptococcus ● ● ● Enterococcus ● ● ● ● ● ● Streptococcus Varibaculum ● Faecalibacterium ● ● Lachnospiracea_incertae_sedis Alveolata ● Clostridium_XI uncultured ● Lactobacillus ● ● ● ● ● Prevotella● Bifidobacterium ● ● ● ● Lactobacillus Penicillium Lactobacillus ● Incertae Mucor Lactobacillus Lactobacillus ● ● ● Streptococcus Lachnospiracea_incertae_sedis Lichtheimia Streptococcus Anaerostipes Akkermansia Lactobacillus ● ● Rhizomucor ● Neogregarinorida● ● ● ● Collinsella Stenotrophomonas Bifidobacterium● Talaromyces ● Cryptosporida Cercozoa ● Lactobacillus Corynebacterium Pediococcus Gregarina Roseburia Lichtheimiaceae● ● ● ● Eubacterium ● ● Collinsella Pediococcus Alveolata Gemmiger ● ● ● Actinomyces ● Ruminococcus2 ● ● Clostridium_sensu_stricto Blautia Pediococcus ● ● Actinomucor ● Clostridium_XVIII ● ● ● Gregarina−distant−branch Blautia ● Pseudomonas ● ● ● ● Clostridium_sensu_stricto ● ● Roseburia ● Finegoldia Enterococcus uncultured Amylomyces ● ● ● ● ● Olsenella Flavonifractor Fusicatenibacter Prevotella ● ● ● Anaerococcus ● Streptococcus ● ● ● ● Paenibacillus ● ● ● ● ● Clostridium_sensu_stricto Saccharomyces Lichtheimiaceae ● Prevotella Flavonifractor Pichia Intestinibacter Finegoldia ● ● Mogibacterium Lactobacillus Blautia Lactobacillus Bifidobacterium ● Clostridium_XVIII Mucor ● ● ● ● ● ● AmylomycesAnaerostipes ● ● ● ● Hanseniaspora ● ● Lactobacillus ● ● ● ● Enterococcus ● ● Streptococcus Clostridium_XVIII Enterococcus ● Clostridium_XVIII Rhizomucor ● Lachnospiracea_incertae_sedis● ● ● ● ● (5) ● (10) ● (9) Saccharomyces AkkermansiaClostridium_XlVb BifidobacteriumRhizomucor Streptococcus Nakaseomyces−Candida clade Gemella Pseudomonas Lactobacillus ● ● Stenotrophomonas Pichia ● ● ● Dorea Bacillus ● ● Mucor Rhizopus ● ● Collinsella ● Penicillium ● CollinsellaAlveolata−distant−branch ● ● BifidobacteriumBifidobacterium ● Blautia Dorea Lactobacillus ● Clostridium_sensu_stricto● ● Pseudomonas Streptococcus ● ● ● ● Olsenella Cryptomycota ● Rothia ● ● Eugregarinorida−distant−branch ● PseudomonasEugregarinorida−distant−b● ranch ● ● ● Bifidobacterium CercomonasClostridium_XVIII ● Atopobium ● Geotrichum● Candida−Lodderomyces clade ● ● Blautia ● ● ● Neogregarinorida Syndiniales Stenotrophomonas Actinomyces Lactobacillus ● Cercozoa● PucciniaEugregarinorida−distant−branch Varibaculum ● ● ● Lactobacillus ● ● ● ● Lichtheimiaceae Lactobacillus Trichomonascus ● ● Lactobacillus Heteromita Geotrichum ● Lichtheimia ● ● Talaromyces Actinomyces● Lactobacillus● ● Streptococcus ● Actinomucor ● WallemiaSaccharomyces Cercomonas ● Lactobacillus● Euglyphida ● ● Atopobium ● ● Holdemanella Anaerococcus Lactobacillus Hanseniaspo● r●a ● ● ● Prevotella● ● Cercomonas ● ● ActinomycesPenicillium● ● ● ● LactobacillusCandida−Lodderomyces clade Gregarina Actinomyces ●LactobacillusPediococcus Lactobacillus ● Wicherhamiella−Candida clade Eugregarinorida ● ● ● ● Weissella● Streptococcus● Gregarinasina−distant−branch uncultured ● Incertae ● ● Amylomyces ● ● Lactobacillus Amoebozoa_unknown_group3 ● Romboutsia ● ● ● Prevotella Pediococcus ConoidasidaAnaerostipes ● Neogregarinorida ● Gemella Eugregarinorida Clostridium_XlVb Toxoplasma Gordonibacter Alveolata−distant−branch ● Mucor Collinsella● ● ● ● Enterococcus ● ● Eggerthella Gemmiger Lactobacillus ● ● ● ● Actinomyces Candida Weissella ● Hanseniaspora Alveolata−distant−branch
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