The Impact of Anthelmintic Treatment on Human Gut Microbiota Based on Cross-Sectional and Pre- and Postdeworming Comparisons in Western Kenya

Abstract

Murine studies suggest that the presence of some species of intestinal helminths is associated with changes in host microbiota composition and diversity. However, studies in humans have produced varied conclusions, and the impact appears to vary widely depending on the helminth species present. To demonstrate how molecular approaches to the human gut microbiome can provide insights into the complex interplay among disparate organisms, DNA was extracted from cryopreserved stools collected from residents of 5 rural Kenyan villages prior to and 3 weeks and 3 months following albendazole (ALB) therapy. Samples were analyzed by quantitative PCR (qPCR) for the presence of 8 species of intestinal parasites and by MiSeq 16S rRNA gene sequencing. Based on pretreatment results, the presence of neither Ascaris lumbricoides nor Necator americanus infection significantly altered the overall diversity of the microbiota in comparison with age-matched controls. Following ALB therapy and clearance of soil-transmitted helminths (STH), there were significant increases in the proportion of the microbiota made up by Clostridiales (P = 0.0002; average fold change, 0.57) and reductions in the proportion made up by Enterobacteriales (P = 0.0004; average fold change, -0.58). There was a significant posttreatment decrease in Chao1 richness, even among individuals who were uninfected pretreatment, suggesting that antimicrobial effects must be considered in any posttreatment setting. Nevertheless, the helminth-associated changes in Clostridiales and Enterobacteriales suggest that clearance of STH, and of N. americanus in particular, alters the gut microbiota.IMPORTANCE The gut microbiome is an important factor in human health. It is affected by what we eat, what medicines we take, and what infections we acquire. In turn, it affects the way we absorb nutrients and whether we have excessive intestinal inflammation. Intestinal worms may have an important impact on the composition of the gut microbiome. Without a complete understanding of the impact of mass deworming programs on the microbiome, it is impossible to accurately calculate the cost-effectiveness of such public health interventions and to guard against any possible deleterious side effects. Our research examines this question in a "real-world" setting, using a longitudinal cohort, in which individuals with and without worm infections are treated with deworming medication and followed up at both three weeks and three months posttreatment. We quantify the impact of roundworms and hookworms on gut microbial composition, suggesting that the impact is small, but that treatment of hookworm infection results in significant changes. This work points to the need for follow-up studies to further examine the impact of hookworm on the gut microbiota and determine the health consequences of the observed changes.

Keywords: 16S RNA; epidemiology; helminths; hookworm; microbial communities; microbial ecology; microbiota.

FIG 1

Microbial diversity and richness in STH-infected individuals and age-matched uninfected individuals. (A) Shannon index comparisons for individuals infected with A. lumbricoides (red) and age-matched uninfected individuals (blue). For A-F in this figure, data from batch B are shown on the left of each graph (as circles), and data from batch A are shown on the right of each graph (as triangles). Each dot represents a single individual. (B) Shannon index comparisons for individuals infected with N. americanus (red) and age-matched individuals uninfected with any STH (blue). (C) Shannon index comparisons for individuals infected with either STH compared to STH-uninfected individuals. (D) Chao 1 comparisons for individuals infected with A. lumbricoides compared to STH-uninfected individuals. (E) Chao1 comparisons for individuals infected with N. americanus compared to STH-uninfected individuals. (F) Chao1 comparisons for individuals infected with either STH compared to STH-uninfected individuals.

FIG 2

Microbial diversity and richness pretreatment and 3 months posttreatment in longitudinal cohorts. Shannon diversity index measurements from individuals with results from both pretreatment and 3 months posttreatment are connected by lines in order to show changes in the diversity of their microbiota (A-D). Samples from individuals currently infected with any STH are shown in red, and samples from uninfected individuals are shown in blue. (A) Individuals infected with A. lumbricoides pretreatment, and STH-uninfected 3 months posttreatment. (B) Individuals infected with N. americanus pretreatment, and STH-uninfected 3 months posttreatment. (C) Individuals infected with either STH pretreatment, and STH-uninfected 3 months posttreatment. (D) STH-uninfected individuals pretreatment, who were treated with ALB and remained STH-uninfected 3 months posttreatment. Results are shown in panels E to H for the same individuals, using the Chao1 index to examined changes in microbial richness in these patients over time. The only significant change is a decrease in Chao1 among people uninfected with STH pretreatment (Wilcoxon matched-pairs signed rank test P value = 0.026).

FIG 3

Microbial diversity and richness pretreatment and 3 weeks posttreatment in longitudinal cohorts. Shannon diversity index measurements from individuals with results from both pretreatment and 3 months posttreatment are connected by lines in order to show changes in the diversity of their microbiota (A to C). Samples from individuals currently infected with any STH are shown in red, and samples from uninfected individuals are shown in blue. (A) STH-uninfected individuals pretreatment, who were treated with ALB and remained STH-uninfected 3 weeks posttreatment. (B) Individuals infected with either STH pretreatment, and STH-uninfected 3 weeks posttreatment. A. lumbricoides-infected and N. americanus-infected individuals are combined due to small samples sizes available from 3 weeks posttreatment. (C) All individuals with sequencing results at both pretreatment and 3 weeks posttreatment, regardless of initial infection status. Pretreatment samples are color coded based on infection status: those infected with any STH are shown in red, whereas all others are shown in blue. Results are shown in panels D to F for the same individuals, using the Chao1 index. Wilcoxon matched-pairs signed rank test P values were insignificant for all comparisons.

FIG 4

Principal-coordinate analysis (PCoA) of samples from individuals infected with N. americanus pretreatment, but uninfected 3 months posttreatment. PCoA of unweighted UniFrac distances based on 16s rRNA gene sequencing results from 17 individuals infected with N. americanus pretreatment, and from the same people 3 months posttreatment. This is a visualization of one of the comparisons summarized in Table 1. Pretreatment samples are shown in red, and posttreatment samples from the same 17 people are shown in green. Samples are labeled with the study ID.

FIG 5

Clostridiales and Enterobacteriales proportions at baseline versus 3 months posttreatment in individuals initially infected with N. americanus. The posttreatment changes shown here are a visualization of the longitudinal changes identified in Table 2. A) Barplots of the 17 individuals infected with N. americanus pretreatment and STH-uninfected 3 months posttreatment. Individuals are ordered by study ID both pre- and posttreatment. B) A volcano plot showing posttreatment changes in OTUs among these 17 individuals. Those OTUs shown in yellow, and labeled by the family to which they belong, have a log₂ fold change greater than 1 following treatment, and are significantly different (adjusted P < 0.05) posttreatment. (C) Pairwise comparison of the proportion of the microbiota assigned to the Clostridiales and Enterobacteriales orders pre- and 3 months posttreatment, among the 17 N. americanus-infected individuals pretreatment. (D) Pairwise comparison of the proportion of the microbiota assigned to the Clostridiales and Enterobacteriales orders pretreatment and 3 months posttreatment, among the 21 STH-uninfected individuals.