Stunted children display ectopic small intestinal colonization by oral bacteria, which cause lipid malabsorption in experimental models

Abstract

Environmental enteric dysfunction (EED) is an inflammatory syndrome postulated to contribute to stunted child growth and to be associated with intestinal dysbiosis and nutrient malabsorption. However, the small intestinal contributions to EED remain poorly understood. This study aimed to assess changes in the proximal and distal intestinal microbiota in the context of stunting and EED and to test for a causal role of these bacterial isolates in the underlying pathophysiology. We performed a cross-sectional study in two African countries recruiting roughly 1,000 children aged 2 to 5 years and assessed the microbiota in the stomach, duodenum, and feces. Upper gastrointestinal samples were obtained from stunted children and stratified according to stunting severity. Fecal samples were collected. We then investigated the role of clinical isolates in EED pathophysiology using tissue culture and animal models. We find that small intestinal bacterial overgrowth (SIBO) is extremely common (>80%) in stunted children. SIBO is frequently characterized by an overgrowth of oral bacteria, leading to increased permeability and inflammation and to replacement of classical small intestinal strains. These duodenal bacterial isolates decrease lipid absorption in both cultured enterocytes and mice, providing a mechanism by which they may exacerbate EED and stunting. Further, we find a specific fecal signature associated with the EED markers fecal calprotectin and alpha-antitrypsin. Our study shows a causal implication of ectopic colonization of oral bacterial isolated from the small intestine in nutrient malabsorption and gut leakiness in vitro. These findings have important therapeutic implications for modulating the microbiota through microbiota-targeted interventions.

Keywords: environmental enteric dysfunction; lipid malabsorption; low-grade inflammation; small intestine; stunted child growth.

Fig. 1.

Differences in the fecal microbiota induced by different clinical cofactors. (A) Relative phylum abundance of the different samples analyzed in this study. (B) PCoA (Principal Coordinate Analysis) on the Bray–Curtis dissimilarity index of the samples rarefied to 5,000 sequences. Gastric samples: n = 212; duodenal samples: n = 137; fecal samples: n = 634. (C) PCoA on the Bray–Curtis dissimilarity index of the fecal samples rarefied to 5,000 sequences. Bangui, Central African Republic: n = 254; Antananarivo, Madagascar: n = 380. Alpha-diversity (D) and beta-diversity (E) in the fecal microbiota and the independent association with given clinical cofactors. (F) Significantly different genera according to stunting status in a multivariate DeSeq2 analysis using a generalized linear model and a likelihood ratio test correcting for age, gender, country of origin, and sequencing run on the pooled dataset from Bangui and Antananarivo.

Fig. 2.

Fecal inflammatory biomarkers of EED and their association with the fecal microbiota. (A) Alpha-diversity measures in fecal samples according to AAT and calprotectin levels. Significantly different genera according to (B) calprotectin levels and (C) AAT levels in a multivariate DeSeq2 analysis using a generalized linear model and a likelihood ratio test correcting for age, gender, country of origin, stunting status, and sequencing run on the pooled dataset from Bangui and Antananarivo. Taxa similarly affected by calprotectin and AAT are indicated in green. ns, P > 0.05. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3.

Small intestinal microbiota in children suffering from stunted growth. (A) Cytokines with a significantly different relative abundance between duodenal samples from children suffering from SIBO compared with children not suffering from SIBO in a logistic regression correcting for analysis batch, country of origin, and the presence or absence of anemia and using the Benjamini–Hochberg correction. (B) Average phylum relative abundance in Bangui, CAR and Antananarivo, Madagascar. (C) Core species at 0.01% relative abundance and a minimal prevalence of 75% in Antananarivo, Madagascar (green) or Bangui, Central African Republic (blue) or conserved in between both study countries (black). (D) Heat map of the Spearman correlation of duodenal samples co-occurrence/co-exclusion including all species with a relative abundance of at least 0.1%. Co-occurrence is indicated in red, and co-exclusion is in blue. Significant associations are indicated with a plus sign. (E) Heat map of co-occurrence and co-exclusion of different classes in the small intestine as measured by Spearman correlation on the relative abundance of given bacterial classes. Only classes with at least 0.1% relative abundance were kept in the analysis. A plus indicates a significant interaction (P < 0.05). Positive associations are indicated in red, and negative associations are in blue. Co-occurrences in Malagasy samples are indicated in the Left pannel and co-occurrences in Central African samples are indicated in the Right pannel. ns, P > 0.05. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4.

Duodenal isolates of oropharyngeal bacteria lead to decreased lipid absorption in vitro. (A) Experimental setup of the in vitro cell assay. (B) Expression of proinflammatory genes in m-IcCl2 cells overexposed to either a clinical, small intestinal isolate of S. salivarius or L. paracasei (control bacterium). Values are normalized to the geometric mean of the three housekeeping genes tbp, b2m, and gapdh. (C) Oleic acid absorption in polarized murine small intestinal cells (m-IcCl2) cocultured overnight with different clinical isolates from the duodenum of stunted children. (D) The sulfonic acid apical to basal permeability assay in polarized m-IcCl2 cells cocultured overnight with different clinical isolates from the duodenum of stunted children. (E) Oleic acid absorption in the context of live S. salivarius strain II, whole or filtered culture supernatant, or heat-killed cells of S. salivarius II or in a medium acidified to pH 5. (F) BODIPY C₁₂ (lauric acid) secretion in the context of live S. salivarius strain II, whole or filtered culture supernatant, or heat-killed cells of S. salivarius II or in a medium acidified to pH 5. (G) NMR-quantified levels of metabolites in the supernatant of polarized m-IcCl2 cells cocultured for 16 h with control medium, S. salivarius, or L. paracasei. Experiments were performed in three independent replicates. Groups are compared using the Mann–Whitney U test. ns, P > 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Duodenal isolates of oropharyngeal bacteria lead to decreased lipid absorption in vivo. (A) Schema of the experimental setup of the mouse experiments performed. (B) CFU for different groups of bacteria in the feces of mice prior to and after treatment with the antibiotic mixture for 7 consecutive days. (C) Average relative abundance of bacterial genera in the three treatment groups. (D) Expression of proinflammatory genes in the small intestine of mice overexposed to either S. salivarius or L. paracasei (control bacterium). Values are normalized to the geometric mean of the three housekeeping genes tbp, b2m, and gapdh. (E) PCoA (Principal Coordinate Analysis) on the Bray–Curtis index of the 16S amplicon data of the three treatment groups on ASV level; 95% CIs of the beta-dispersion of a given sample group are indicated with two ellipses. (F) BODIPY C₁₂ absorption in the jejunum and liver of mice overexposed to S. salivarius or L. paracasei. Groups are compared using the Mann–Whitney U test. ATB, antibiotics. ns, P > 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.