Gut microbiome of helminth-infected indigenous Malaysians is context dependent

Abstract

Background: While microbiomes in industrialized societies are well characterized, indigenous populations with traditional lifestyles have microbiomes that are more akin to those of ancient humans. However, metagenomic data in these populations remains scarce, and the association with soil-transmitted helminth infection status is unclear. Here, we sequenced 650 metagenomes of indigenous Malaysians from five villages with different prevalence of helminth infections.

Results: Individuals from villages with higher prevalences of helminth infections have more unmapped reads and greater microbial diversity. Microbial community diversity and composition were most strongly associated with different villages and the effects of helminth infection status on the microbiome varies by village. Longitudinal changes in the microbiome in response to albendazole anthelmintic treatment were observed in both helminth infected and uninfected individuals. Inference of bacterial population replication rates from origin of replication analysis identified specific replicating taxa associated with helminth infection.

Conclusions: Our results indicate that helminth effects on the microbiota were highly dependent on context, and effects of albendazole on the microbiota can be confounding for the interpretation of deworming studies. Furthermore, a substantial quantity of the microbiome remains unannotated, and this large dataset from an indigenous population associated with helminth infections is a valuable resource for future studies. Video Abstract.

Keywords: Albendazole; Helminth; Indigenous population; Metagenomic sequencing; Microbiome.

Fig. 1

Variation in the gut microbiome of 650 Malaysians from Orang Asli (OA) villages and Kuala Lumpur (KL). A Violin plots illustrating the percentage of mapped reads with RefSeq (i.e., bacteria, protozoa, fungi, viral, archaea), Unified Human Gastrointestinal Genome (UHGG), and human reference gut microbiome (HRGM) databases between OA (green) and KL (purple) samples. B Relative abundance of phyla from the 237 species of the core gut microbiota of OA and KL populations (left). The relative abundance of the main species from Firmicute A (right). C Bar plot shows the bacterial species that are differentially abundant between Orang Asli and Urban cohort from Kuala Lumpur based on the output of the microbiome multivariable association with linear models 2 (MaAsLin2). The length of the bar corresponds to the value of the significant association. Red color represents the bacterial species associated with the Orang Asli subjects, whereas blue color represents the bacterial species associated with the urban cohort. D The percentage of mapped reads to the HRGM database for samples from different OA villages and KL. E Comparison of pairwise beta diversity at species level within group to the KL cohort, assessed by Jaccard distance based on the distance of nucleotide k-mer sketches k = 21 (top) and genus-level classification (bottom). F Principal coordinates analysis (PCoA) of Jaccard distance based on the gut metagenomic profiles (species levels) in all samples, with individuals from different geographical locations denoted by specific color (ADONIS: p = 0.001, R² = 0.073; ANOSIM: p = 0.001, R = 0.215). The p-values for A, D, and E are computed using Wilcoxon rank-sum test

Fig. 2

Effects of intestinal helminth infection status on gut microbial diversity and composition for the 351 Orang Asli individuals. A The prevalence of intestinal helminth infection in the OA cohort based on overall infection status, as well as specific intestinal helminth infection (i.e., trichuriasis, ascariasis, and hookworm infection). B Principal coordinates analysis (PCoA) of Jaccard distances based on gut microbiota profiles (species levels) of the OA cohort. The individuals infected and uninfected with intestinal helminths are denoted by blue and red, respectively (ADONIS: p = 0.001, R² = 0.024; ANOSIM: p = 0.001, R = 0.145). C Alpha-diversity box plot of species richness based on different status of intestinal helminth infection, number of intestinal helminth infection, Trichuris infection, and intensity of Trichuris infection. Wilcoxon rank-sum test is used for two independent variables, while the Kruskal-Wallis test is used for more than two comparison groups. D The prevalence of intestinal helminth infection (top) and Trichuris infection (bottom) by different geographical locations. E Comparison of alpha diversity (species richness) between individuals from KL and specific OA villages. F Spearman correlation between the intensity of Trichuris infection and percentage of unmapped reads to the HRGM database (p = 3.200e⁻⁶, R = 0.250). The blue line represents the linear regression between intensity of Trichuris infection and percentage of unmapped reads. G Bar plot of the F statistic values from ADONIS analysis of variables that contribute to the gut microbiota composition. Colored bars indicate the variables that show significant effects on gut microbiota variation (p < 0.05). H Bar plot of effect size of the variables (village [p = 2.610e⁻⁰⁸, pseudo R² = 0.027], helminth infection [p = 0.029, pseudo R² = 0.004], and interactions between helminth village [p = 0.002, pseudo R² = 0.016]) that contribute significantly to the variance of the microbiota based on MDMR analysis. I Heatmap of bacterial species associated with village, helminth infections, and interactions from MaAsLin2. Blue for positive association and red for negative association

Fig. 3

Dynamic changes to the gut microbiota of 129 Orang Asli after albendazole treatment. A Line plots show changes of the infection intensity of Trichuris pre and post response to anthelmintic drugs stratified by full responders (n = 43), partial responders (n = 23), nonresponders (n = 5), and uninfected individuals (n = 58). B Principal coordinates analysis (PCoA) plot of Jaccard distances based on gut microbiota profiles (species levels) of responders (ADONIS: p = 0.001, R² = 0.014; ANOSIM: p = 0.001, R = 0.072), with pre-anthelmintic treatment (blue) and post-anthelmintic treatment (red). C Principal coordinates analysis (PCoA) plot of Jaccard distances based on gut microbiota profiles (species levels) of uninfected subjects (ADONIS: p = 0.006, R² = 0.012; ANOSIM: p = 0.001, R = 0.069) (Fig. 3C, Supplementary Table S2), with pre-anthelmintic treatment (blue) and post-anthelmintic treatment (red). D Venn diagram depicting the number of shared and exclusive bacteria species that are found to be differentially abundant (pre and post) between responders and uninfected individuals. The blue area includes 253 bacteria that are altered only in responders, while the yellow and mixed color area indicates the 873 bacteria that are altered in uninfected individuals. E Box plots show the bacterial taxa that are altered by deworming treatment in responders but not in nonresponders (host response). The differences in the abundance of (i) Sutterella HRGM Genome 4418 and (ii) Muricomes contorta_B in helminthic infections (left) and different groups of response (right), namely uninfected and responders in pre and post. F Box plots and line plots show the bacterial taxa that are associated with deworming treatment in both responders and nonresponders (drug response). The differences in the abundance of (i) Collinsella sp900540485 and (ii) Collinsella stercoris in helminthic infections (left) and different groups of response (right), namely uninfected and responders in pre and post. The p-values for E and F are computed using Wilcoxon signed-rank test (responders vs nonresponders) and Wilcoxon rank-sum test (helminthic infections)

Fig. 4

Gut bacterial replication in the context of intestinal helminth infection. A Heatmap of the growth rate index (GRiD) score, which infers an index of replication for top 20 gut bacteria in relation to helminth infection status of individuals based on Spearman correlation test followed by false discovery rate (FDR) correction. Samples are shown in rows, by village, whereas the GRiD score of each bacterium is shown in columns. The first vertical side bar color codes the intestinal helminth infection status, while the second side bar indicates the infection intensity of Trichuris. B GRiD score correlation between bacterial species with the infection intensity of Trichuris. The bar chart shows the Spearman’s rank correlation coefficient. Blue and gray colors represent the positive and negative correlations respectively. C Box plots of GRiD score for Prevotella stercorea (left), Bifidobacterium longum (middle), and Phocaeicola vulgatus (right) in Trichuris infected and uninfected individuals. The GRiD scores of these species between Trichuris infected and uninfected individuals were tested using Wilcoxon rank-sum test