Intestinal protozoan infections shape fecal bacterial microbiota in children from Guinea-Bissau

Abstract

Intestinal parasitic infections, caused by helminths and protozoa, are globally distributed and major causes of worldwide morbidity. The gut microbiota may modulate parasite virulence and host response upon infection. The complex interplay between parasites and the gut microbiota is poorly understood, partly due to sampling difficulties in remote areas with high parasite burden. In a large study of children in Guinea-Bissau, we found high prevalence of intestinal parasites. By sequencing of the 16S rRNA genes of fecal samples stored on filter paper from a total of 1,204 children, we demonstrate that the bacterial microbiota is not significantly altered by helminth infections, whereas it is shaped by the presence of both pathogenic and nonpathogenic protozoa, including Entamoeba (E.) spp. and Giardia (G.) lamblia. Within-sample diversity remains largely unaffected, whereas overall community composition is significantly affected by infection with both nonpathogenic E. coli (R2 = 0.0131, P = 0.0001) and Endolimax nana (R2 = 0.00902, P = 0.0001), and by pathogenic E. histolytica (R2 = 0.0164, P = 0.0001) and G. lamblia (R2 = 0.00676, P = 0.0001). Infections with multiple parasite species induces more pronounced shifts in microbiota community than mild ones. A total of 31 bacterial genera across all four major bacterial phyla were differentially abundant in protozoan infection as compared to noninfected individuals, including increased abundance of Prevotella, Campylobacter and two Clostridium clades, and decreased abundance of Collinsella, Lactobacillus, Ruminococcus, Veillonella and one Clostridium clade. In the present study, we demonstrate that the fecal bacterial microbiota is shaped by intestinal parasitic infection, with most pronounced associations for protozoan species. Our results provide insights into the interplay between the microbiota and intestinal parasites, which are valuable to understand infection biology and design further studies aimed at optimizing treatment strategies.

Fig 1. Stacked barplot of taxonomic profile of the gut microbiota across participants.

Illustration of the relative abundance (y-axis) of phyla across the 1,204 samples (x-axis) in the study, irrespective of infection status. The 10 most abundant phyla are colored and listed in the legend. For all samples, the two dominant phyla are Bacteroidetes and Firmicutes, followed by Proteobacteria and Actinobacteria, in line with that of a normal human gut microbiota.

Fig 2. Phylodiversity is altered by infection with Entamoeba spp.

Illustration of differences in phylodiversity levels between samples with different infection status. Phylodiversity, as a measure of alpha diversity, remains unaltered by infection with helminth species, G. lamblia and E. nana. Infection with E. spp. significantly increases phylodiversity. Each boxplot shows the non-infected (with any tested parasite (orange) versus samples infected with the parasite of interest as listed in plot title (grey). The adjusted significance level from the robust regression test is given in parentheses.

Fig 3. Gut microbiota is shaped by protozoan infections and infection load.

(A) Increasing infection load, determined by the number of different parasitic species identified by microscopy in each sample, induces increasing shifts in the microbiota, suggesting that multispecies infections have more pronounced effects on microbiota than single species infections. (B) Parasite infection (any positive finding, regardless of species) imposes a visual shift in microbiota structure. (C) Overall protozoan infection imposes a shift in gut microbiota composition very similar to the shift observed for overall parasite infection, indicating that that protozoa infection, and not helminth infection, has the strongest effect on microbiota composition, an observation supported by results from the adonis-based analysis. (D) Helminth infection has no apparent effect on microbiota structure. The figure is composed of four ordination plots build using genera relative abundance data and principal coordinates analysis (capscale function in R package vegan with Bray-Curtis dissimilarity and automatic data transformation (metaMDS = T)). Plots are made with R package ggplot2 and ellipses drawn using function stat_ellipse with default parameters. Each dot shows a sample, all of which are colored by infection status. (A) All 1,204 samples colored by infection load from no infection (0) to infected with four different parasite species (4). (B-D) Plots showing relationship between the microbiota community of samples either (B) non-infected (red) or infected with a parasite (blue), (C) non-infected (red) or infected with a protozoa (blue) and (D) non-infected (red) or infected with a helminth (blue).

Fig 4. Specific taxa are altered by different parasitic species.

A total of 32 genera are significantly associated with intestinal parasitic infection, either overall infections or specific parasite species. Most associations are seen with protozoan infections, whereas helminth infections have limited effects on taxa alterations. The figure illustrates significant associations between genera and different parasite infections (linear regression, P.adj<0.05). Summary statistics for the shown associations are given in Table 4. The color illustrates coefficient (blue for low values, green for high values), whereas size illustrates the significance (calculated by -log(p-value) (the larger bubble, the higher significance). The plot is made using ggplot2 package in R.