Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition

Abstract

Background: Diarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.

Results: We use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age.

Conclusions: Our findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.

Figure 1

Comparison of diarrheal and non-diarrheal stool. (A) Proportional abundance of genera in non-diarrheal controls and MSD cases in different age categories. Each color represents a different group. The order and color for each group is the same for controls (patients without MSD) and MSD cases. The eight groups most frequently found in controls (Prevotella, Bacteroides, Escherichia/Shigella, Veillonella, Streptococcus, Lactobacillus, Faecalibacterium, Megasphaera, plus unassigned and other) are depicted. (B) Shannon diversity index across ages and diarrheal status. Average Shannon diversity indices for the five different age strata as well as the corresponding 95% confidence intervals. Both cases and controls exhibited higher mean Shannon diversity index scores at higher age groups compared to lower age groups (P <0.001, one-way ANOVA). The diversity of healthy samples positively correlates with age in the first 2 years of life, as previously reported [12]. The diversity index for cases is significantly less than that for controls within each country (P <0.02, Tukey’s t-test corrected for multiple comparisons). Also see Additional file 2: Table S2 and Additional file 3: Figure S1.

Figure 2

Comparison of dysenteric and non-dysenteric stool. (A) Genus-level comparison of dysenteric and non-dysenteric diarrheal stool (top) stratified by age; (bottom) stratified by country. (B) Proportional abundance boxplots of Prevotella, Lactobacillus, and Streptococcus in dysenteric and non-dysenteric diarrheal stools by age category. The upper whisker extends from the 75th percentile to the highest value that is within 1.5 * IQR of the hinge, where IQR is the inter-quartile range, or distance between the first and third quartiles. The lower whisker extends from the hinge to the lowest value within 1.5 * IQR of the hinge. Data beyond the end of the whiskers are outliers and are not plotted. Asterisks above the whisker indicate a statistically significant difference (by t-test) between dysenteric and non-dysenteric stools placed in the panel with the more abundant mean. A single asterisk indicates P <0.05; double asterisks indicate P <0.01. Prevotella is significantly associated with non-dysenteric cases overall (P = 0.0003) and in age groups 0 to 6 months (P = 0.01), 12 to 17 months (P = 0.03), and 24 to 59 months (P = 0.001). Lactobacillus is significantly associated with non-dysenteric cases overall (P = 0.0002) and in children 6 to 11 months (0.02) and 12 to 17 months (P = 0.003), while the genus Streptococcus is associated with dysentery overall (P = 0.007), particularly in children aged 12 to 17 months (P = 0.01).