Arsenic exposure and intestinal microbiota in children from Sirajdikhan, Bangladesh

Background: Arsenic has antimicrobial properties at high doses yet few studies have examined its effect on gut microbiota. This warrants investigation since arsenic exposure increases the risk of many diseases in which gut microbiota have been shown to play a role. We examined the association between arsenic exposure from drinking water and the composition of intestinal microbiota in children exposed to low and high arsenic levels during prenatal development and early life. Results: 16S rRNA gene sequencing revealed that children with high arsenic exposure had a higher abundance of Proteobacteria in their stool compared to matched controls with low arsenic exposure. Furthermore, whole metagenome shotgun sequencing identified 332 bacterial SEED functions that were enriched in the high exposure group. A separate model showed that these genes, which included genes involved in virulence and multidrug resistance, were positively correlated with arsenic concentration within the group of children in the high arsenic group. We performed reference free genome assembly, and identified strains of E.coli as contributors to the arsenic enriched SEED functions. Further genome annotation of the E.coli genome revealed two strains containing two different arsenic resistance operons that are not present in the gut microbiome of a recently described European human cohort (Metagenomics of the Human Intestinal Tract, MetaHIT). We then performed quantification by qPCR of two arsenic resistant genes (ArsB, ArsC). We observed that the expression of these two operons was higher among the children with high arsenic exposure compared to matched controls. Conclusions: This preliminary study indicates that arsenic exposure early in life was associated with altered gut microbiota in Bangladeshi children. The enrichment of E.coli arsenic resistance genes in the high exposure group provides an insight into the possible mechanisms of how this toxic compound could affect gut microbiota

Selected characteristics of 50 children included in this nested case-control study.

<p>Selected characteristics of 50 children included in this nested case-control study.</p

Arsenic exposure and intestinal microbiota in children from Sirajdikhan, Bangladesh - Fig 4

<p>A) Comparison (displayed using Artemis Comparison Tool; doi <a href="https://doi.org/10.1093/bioinformatics/bti553" target="_blank">10.1093/bioinformatics/bti553</a>) of DNA sequences of <i>E</i>.<i>coli</i> strains in Bangladeshi children cohort. Gray bars represent the forward and reverse strands of DNA. The yellow lines between the sequences represent existence nucleotide similarity (blastn). Arsenic resistant operons are given green color. B) Comparison of DNA sequences of <i>E</i>.<i>coli</i> strains in Bangladeshi children and European cohort. Comparison is displayed for two <i>E</i>.<i>coli</i> strains in Bangladesh children (ST2747 and FHI98) and their most similar <i>E</i>.<i>coli</i> strain in European cohort (ATCC 25922). C) qPCR quantification of two arsenic resistance operon genes (ArsB, ArsC) found in Bangladeshi children <i>E</i>.<i>coli</i> strain ST2747. (* p value<0.05).</p

Microbial taxonomic composition in high arsenic exposure group and low arsenic exposure group based on 16S rRNA V4 gene region sequencing.

<p>(A) Relative abundance of phyla for high arsenic exposure samples (n = 25, orange color) and low exposure samples (n = 25, blue color). The boxplot represent interquartile range with the black bar indicating the median relative abundance and error bars represent minimum and maximum values. Outliers are represented by solid circles. *P<0.02, Mann–Whitney U test. (B) Relative abundance of three lower taxonomic ranks of phylum <i>Proteobacteria</i> for high arsenic exposure and unexposed samples. * P<0.03, # P<0.1(C) Correlation between relative abundance of phylum <i>Proteobacteria</i> and water arsenic level in high arsenic exposure group. p-value is calculated for two-tailedSpearman correlation coefficient.</p

Annotations of SEED functions associated with arsenic exposure.

<p>Frequency of SEED genes annotated under specific SEED level 2 function categories within 332 arsenic related SEED functions (black color) and all SEED functions detected in the samples. The SEED level function categories in the figure are significantly overrepresented or depleted in the 332 arsenic related functions (chi-square test P value <0.05).</p

Whole genome shotgun sequencing detected microbial SEED functions associated with arsenic level.

<p>(A) Heatmap showing fold change of 901 different abundant SEED functions between high exposure versus low exposure group and correlation coefficients (Spearman) with arsenic levels in high exposure group for 332 SEED functions. The 332 SEED functions either have higher abundance in high exposure group and positively correlated with arsenic level in high exposure group or have lower abundance in high exposure group and negatively correlated with arsenic level in high exposure group (Fisher combined FDR <0.1). Brackets mark 258 SEED functions that also had increased relative abundance in mice after treatment by four antibiotics (ampicillin, neomycin trisulfate, vancomycin, metronidasole) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188487#pone.0188487.ref027" target="_blank">27</a>]. (B). Fraction of the 332 overlapping SEED functions covered by reference microbial genomes. Those covering more than 70% of 332 gene set are listed. C) Distribution of Arsenic related and non-related SEED genes in assembled genome MGS0010 (<i>E</i>.<i>coli</i>) and other genomes. *Chi-squared test odds ratio 6.69, FDR 1.08e-48.</p