Abstract
BackgroundDue to a lack of systematic diagnostics, our understanding of the diversity and role of eukaryotic microbiota in human health is limited. While studies have shown fungal communities to be significant modulators of human health, information on the prevalence of taxa such as protozoa and helminths has been limited to a small number of species for which targeted molecular diagnostics are available. To probe the diversity of eukaryotic microbes and their relationships with other members of the microbiota, we applied in silico and experimental approaches to design a novel two-amplicon surveillance tool, based on sequencing regions of ribosomal RNA genes and their internal transcribed spacers. We subsequently demonstrated the utility of our approach by characterizing the eukaryotic microbiota of 46 hospitalized Malawian children suffering from Severe Acute Malnutrition (SAM).ResultsThrough in silico analysis and validation on a diverse panel of eukaryotes, we identified 18S rRNA variable genetic regions 4 and 5 (18S V4 V5), together with a region encoding 28S rRNA variable genetic region 2 and the internal transcribed spacers (transITS), as optimal for the systematic classification of eukaryotes. Sequencing of these regions revealed protozoa in all stool samples from children with SAM and helminths in most, including several eukaryotes previously implicated in malnutrition and diarrheal disease. Clinical comparisons revealed no association between protozoan parasites and diarrhea or HIV reactivity. However, the presence of Blastocystis correlated with bacterial alpha diversity and increased abundance of specific taxa, including Sporobacter, Cellulosibacter, Oscillibacter, and Roseburia.ConclusionWe suggest this novel two-amplicon based strategy will prove an effective tool to deliver new insights into the role of eukaryotic microbiota in health and disease.
Highlights
Due to a lack of systematic diagnostics, our understanding of the diversity and role of eukaryotic microbiota in human health is limited
We subsequently demonstrate the effectiveness of our approach to survey eukaryotic microbiota in a study of stool samples from 46 Malawian children hospitalized for Severe Acute Malnutrition (SAM) [26, 27], a cohort with a high expected prevalence of intestinal parasites
Amplicon identification and primer design We explored the sequence variability in eukaryotic rRNA genes in the SILVA v128 database to identify taxonomically informative genetic regions
Summary
Due to a lack of systematic diagnostics, our understanding of the diversity and role of eukaryotic microbiota in human health is limited. While studies have shown fungal communities to be significant modulators of human health, information on the prevalence of taxa such as protozoa and helminths has been limited to a small number of species for which targeted molecular diagnostics are available. In attempts to better characterize the contribution of microbial eukaryotes to health, marker gene studies, analogous to bacterial 16S rRNA gene surveys, have been proposed. In these approaches, variable genetic regions are targeted for PCR amplification using universal DNA primers which bind to all organisms; sequencing of the resultant amplicons subsequently yields a readout of taxa present [21,22,23,24]. High sequence conservation in the region being targeted can limit resolution at the level of species or genus
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