Meta-taxonomic analysis of prokaryotic and eukaryotic gut flora in stool samples from visceral leishmaniasis cases and endemic controls in Bihar State India

Abstract

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.

Inhibitory Effect of a Microecological Preparation on Azoxymethane/Dextran Sodium Sulfate-Induced Inflammatory Colorectal Cancer in Mice

“…Furthermore, the integrated analysis of 16s rRNA and metabolomics data showed that there were 19 functional relationship pairs, including eight altered microbiota (e.g., Ruminiclostridium and Prevotellaceae_UCG_001) and 16 disturbed metabolites (e.g., C10H11NO3 and C21H32O8) between the JK5G and model groups. Actually, Alloprevotella has been regarded as the cancer-related bacteria in colon cancer or visceral leishmaniasis (30,31). Moreover, a previous study showed that the abundance of the Ruminiclostridium genus was related to the enhancement of animal immune responses to spore adsorbent antigen and probiotics (32).…”

Genetics, Transcriptomics and Meta-Taxonomics in Visceral Leishmaniasis

“…However, this does not provide data on the influence these pathogens might have on the broader gut microflora. We therefore undertook a study to evaluate the use of amplicon-based sequencing of 16S ribosomal RNA (16S rRNA) gene regions to identify prokaryotic flora and 18S rRNA gene regions to identify eukaryotes using a meta-taxonomic approach (Lappan et al, 2019).…”

“…In this small scoping study we examined 16S rRNA and 18S rRNA profiles in 23 active cases of VL compared to 23 geographically matched healthy endemic controls from Bihar State India. Using QIIME software (Caporaso et al, 2010;Bolyen et al, 2018) to analyze the amplicon sequence libraries (Figure 4) the most abundant bacterial taxa identified across all fecal samples 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%) (Lappan et al, 2019). It was of interest to compare these results to previous reports of the composition of gut microflora across India.…”

“…(Novais et al, 2015;Kong et al, 2017;Ashwin et al, 2018)]. Here we review results from studies using three different omics-based approaches to study VL caused by L. donovani in India: (i) chip-based analysis of single nucleotide variants used to carry out the first genomewide association study (GWAS) of VL to identify host genetic risk factors (Fakiola et al, 2013); (ii) chip-based transcriptional profiling used to compare active with cured cases (Fakiola et al, 2019), including the influence of anti-IL-10 on whole blood responses to soluble leishmania antigen (SLA) (Singh et al, 2020); and (iii) a meta-taxonomic approach based on sequencing amplicons derived from regions of 16S ribosomal RNA (16S rRNA) and 18S rRNA genes that allowed us to determine composition of both prokaryotic and eukaryotic gut microflora in VL cases compared to endemic controls (Lappan et al, 2019).…”

Comparison of gut microbiota of healthy and diseased walking sticks, Phasmotaenia lanyuhensis

“…The amplicon products of the V3/V4 region of 16S rDNA gene were subjected to sequencing library construction as instructed by standard protocol (NEBNext Ultra II DNA Library Prep Kit, Illumina) and sequenced by Illumina MiSeq sequencer with paired-end (PE) technology of 300 bp × 2. QIIME TM 2 (Lappan et al, 2019) was applied for metagenomics analysis and the overall flowchart was briefly described ( Figure S1). In quality trimming step, the PCR primer sequences were trimmed and the reads were filtered by DADA2 module including length trimming, denoising, chimera removal.…”