Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children

Abstract

Undernourished children exhibit impaired development of their gut microbiota. Transplanting microbiota from 6- and 18-month-old healthy or undernourished Malawian donors into young germ-free mice that were fed a Malawian diet revealed that immature microbiota from undernourished infants and children transmit impaired growth phenotypes. The representation of several age-discriminatory taxa in recipient animals correlated with lean body mass gain; liver, muscle, and brain metabolism; and bone morphology. Mice were cohoused shortly after receiving microbiota from healthy or severely stunted and underweight infants; age- and growth-discriminatory taxa from the microbiota of the former were able to invade that of the latter, which prevented growth impairments in recipient animals. Adding two invasive species, Ruminococcus gnavus and Clostridium symbiosum, to the microbiota from undernourished donors also ameliorated growth and metabolic abnormalities in recipient animals. These results provide evidence that microbiota immaturity is causally related to undernutrition and reveal potential therapeutic targets and agents.

Fig. 1. Sparse Random Forests-derived model of gut microbiota maturation obtained from concordant healthy Malawian twins/triplets

(A) Random-Forests regression of fecal bacterial 97%ID OTUs from a training set of healthy Malawian infants/children (n=31) to chronological age yielded a rank order of age-discriminatory taxa. Random Forests assigns a mean squared error (MSE), or feature importance score, to each OTU that indicates the extent to which each OTU contributes to the accuracy of the model. The 25 most age-discriminatory taxa, ranked by MSE, yielded a sparse model that predicted microbiota age and accounted for ~80% of the observed variance in the healthy cohort (see Table S3A for a complete list of OTUs and MSE values). The top 25 most discriminatory OTUs with their taxonomic assignments are shown ranked by feature importance (mean±SD of the MSE). The insert shows the results of 10-fold cross-validation; as OTUs are added to the model in order of their feature importance rank, the model’s error decreases. Taxa highlighted in red indicate OTUs with >97% nucleotide sequence identity with an OTU present in a sparse Random Forests-based model of microbiota maturation in healthy Bangladeshi infants/children (11). (B) A heatmap of changes over time in relative abundances of the 25 OTUs in fecal microbiota collected from healthy Malawian infants/children comprising the test set (n=29). OTUs are hierarchically clustered according to pairwise distances by Pearson correlation. (C) Predictions of chronological age using the sparse 25 OTU model of microbiota age in healthy children comprising the test set cohort. r² calculated from Pearson correlation.

Fig. 2. Transplantation of microbiota from 6- and 18-month old donors to young germ-free mice provides evidence of a causal relationship between gut microbiota maturity and growth phenotypes

(A) Experimental design of microbiota screen. Mice (4.5-weeks old) were switched to the M8 diet three days prior to gavage with the selected microbiota donor’s fecal sample (n=5 mice per donor). Fecal samples, body weight and body composition were defined at the indicated time. (B,C) Gnotobiotic mice colonized with fecal samples from healthy children gain more total body weight (panel B) and lean mass (panel C) than mice colonized with microbiota from undernourished donors (mean±SEM shown; p-values shown for donor status effect based on 2-way ANOVA). All recipient mice harbor microbiota that represent >50% of OTU diversity present in the intact uncultured donor’s sample. (D) The 30 most weight-gain discriminatory OTUs and their taxonomic assignments, ranked by feature importance (mean MSE±SD values are plotted). The weight gain model explained ~66% of the observed phenotypic variation (p<0.0001, permutation test; 999 permutations). Taxa in red indicate OTUs that appear within the 30 most discriminatory OTUs for both the weight and lean mass gain Random Forests-based models. Taxa in purple indicate species that appear in the 25-member sparse Random Forests-derived model of Malawian gut microbiota maturation. Bars to the right of the OTU ID numbers represent Spearman’s rank correlation of the same OTU ID to chronological age within the healthy Malawian infant/child cohort (see Table S9).

Fig. 3. Co-housing results in transfer of species from the microbiota of cagemates colonized with the healthy donor’s community into the microbiota of cagemates containing the severely stunted/underweight donor’s community and prevention of growth faltering

(A) Experimental design for co-housing experiments. Dually housed 4.5-week old mice were switched to the M8 diet and colonized 3 days later with either the intact uncultured healthy or stunted/underweight donor microbiota. Four days after gavage, subsets of the mice were co-housed (H^CH and Un^CH, respectively), while healthy and stunted/underweight control mice remained in their original isolators and were paired with a new cagemate from that isolator (H-H and Un-Un controls). Fecal samples were collected throughout the experiment; growth was assayed by changes in total body weight and body composition (the latter by qMR). Mice were sacrificed three weeks after colonization. (B) H^CH and Un^CH mice have increased lean mass gain relative to the Un-Un controls 15 days post colonization (Un-Un vs. H^CH p = 0.0447, Un-Un vs. Un^CH p=0.0121, Mann-Whitney test; n=6 cages of co-housed mice, 3 cages of each dually housed control group/experiment; two independent experiments). To quantify invasion further, we used the mean and standard deviation of the null distribution of invasion scores (defined as the scores from recipients of the H or Un microbiota that had never been co-housed with each other) to calculate a z-value and a Benjamini-Hochberg adjusted p-value for the invasion score of each species in H^CH and Un^CH mice (see Materials and Methods). We defined a taxon as a successful invader if it (i) had a Benjamini-Hochberg adjusted p≤0.05, and (ii) had a relative abundance of ≤0.05% before cohousing and ≥0.5% in the fecal microbiota at the time of sacrifice. Fig. 3C and Table S12 provide information about the direction and success of invasion. (C) Heatmap showing results of the invasion assay. Each row represents a species-level taxon, while each column represents a mouse at a given day post colonization (dpc); rows of the heatmap were hierarchically clustered according to pair-wise distances using Pearson correlation. Bars at the right side of each experimental arm represent the fold-change (fc) in that species’ relative abundance before and after cohousing (fold-change defined by the log₂[(average relative abundance of species post cohousing (days 7 through 22))/(average relative abundance of species before cohousing (day 4))]. Species in red represent those identified as one of the top 30 growth-discriminatory taxa by the weight or lean mass gain Random Forests-based models shown in Fig. 2.

Fig. 4. A consortium of cultured growth-discriminatory OTUs augments the growth of mice colonized with the 6-month severely stunted/underweight donor’s microbiota

(A) Experimental design including the composition of the 5-member consortium of cultured bacterial strains. (B) Weight gain and lean body mass gain 21 days after gavage of the donor microbiota with or without the cultured consortium. (C) Comparison of the fecal microbiota of mice belonging to untreated control and treated experimental group showing the establishment of OTUs from the consortium at 21 days post colonization. (D) The effects of treatment with the consortium on host metabolism (p<0.05 for all metabolites shown, Student’s t-test). Each row represents a metabolite from a given tissue, while each column represents an individual mouse. Tissues were collected 21 days after colonization.