Meta-taxonomic Analysis of Prokaryotic and Eukaryotic Gut Flora in Stool Samples from Visceral Leishmaniasis Cases and Endemic Controls in Bihar State India

Visceral leishmaniasis (VL) caused by Leishmania donovani remains of public health concern in rural India. Those at risk of VL are also at risk of other neglected tropical diseases (NTDs) including soil transmitted helminths. Intestinal helminths are potent regulators of host immune responses sometimes mediated through cross-talk with gut microbiota. We evaluate a meta-taxonomic approach to determine the composition of prokaryotic and eukaryotic gut microflora using amplicon-based sequencing of 16S ribosomal RNA (16S rRNA) and 18S rRNA gene regions. The most abundant bacterial taxa identified in faecal samples from Bihar State India were Prevotella (37.1%), Faecalibacterium (11.3%), Escherichia-Shigella (9.1%), Alloprevotella (4.5%), Bacteroides (4.1%), Ruminococcaceae UCG-002 (1.6%), and Bifidobacterium (1.5%). Eukaryotic taxa identified (excluding plant genera) included Blastocystis (57.9%; Order: Stramenopiles), Dientamoeba (12.1%; Family: Tritrichomonadea), Pentatrichomonas (10.1%; Family: Trichomonodea), Entamoeba (3.5%; Family: Entamoebida), Ascaridida (0.8%; Family: Chromodorea; concordant with Ascaris by microscopy), Rhabditida (0.8%; Family: Chromodorea; concordant with Strongyloides), and Cyclophyllidea (0.2%; Order: Eucestoda; concordant with Hymenolepis). Overall alpha (Shannon’s, Faith’s and Pielou’s indices) and beta (Bray-Curtis dissimilarity statistic; weighted UniFrac distances) diversity of taxa did not differ significantly by age, sex, geographic subdistrict, or VL case (N=23) versus endemic control (EC; N=23) status. However, taxon-specific associations occurred: (i) Ruminococcaceae UCG-014 and Gastranaerophilales_uncultured bacterium were enriched in EC compared to VL cases; (ii) Pentatrichomonas was more abundant in VL cases than in EC, whereas the reverse occurred for Entamoeba. Across the cohort, high Escherichia-Shigella was associated with reduced bacterial diversity, while high Blastocystis was associated with high bacterial diversity and low Escherichia-Shigella. Individuals with high Blastocystis had low Bacteroidaceae and high Clostridiales vadin BB60 whereas the reverse held true for low Blastocystis. This scoping study provides useful baseline data upon which to develop a broader analysis of pathogenic enteric microflora and their influence on gut microbial health and NTDs generally.Author SummaryVisceral leishmaniasis (VL), also known as kala-azar, is a potentially fatal disease caused by intracellular parasites of the Leishmania donovani complex. VL is a serious public health problem in rural India, causing high morbidity and mortality, as well as major costs to local and national health budgets. People at risk of VL are also at risk of other neglected tropical diseases (NTDs) including soil transmitted helminths (worms). Intestinal worms are potent regulators of host immune responses often mediated through cross-talk with gut bacteria. Here we have used a modern DNA sequencing approach to determine the composition of microbiota in stool samples from VL cases and endemic controls. This allows us to determine all bacteria, as well as all single-celled and multicellular organisms, that comprise the microorganisms in the gut in a single sequencing experiment from a single stool sample. In addition to providing valuable information concerning commensal and pathogenic gut micro-organisms prevalent in this region of India, we find some specific associations between single-celled gut pathogens and VL case status.