Differentiation of Blastocystis and parasitic archamoebids encountered in untreated wastewater samples by amplicon-based next-generation sequencing

Christen Rune Stensvold; Marianne Lebbad; Anette Hansen; Jessica Beser; Salem Belkessa; Lee O'Brien Andersen; C Graham Clark

doi:10.1016/j.parepi.2019.e00131

Differentiation of Blastocystis and parasitic archamoebids encountered in untreated wastewater samples by amplicon-based next-generation sequencing

Parasite Epidemiol Control. 2019 Dec 21:9:e00131. doi: 10.1016/j.parepi.2019.e00131. eCollection 2020 May.

Authors

Christen Rune Stensvold¹, Marianne Lebbad², Anette Hansen², Jessica Beser², Salem Belkessa^{1

3

4}, Lee O'Brien Andersen¹, C Graham Clark⁵

Affiliations

¹ Laboratory of Parasitology, Department of Bacteria, Parasites and Fungi, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.
² Department of Microbiology, Public Health Agency of Sweden, SE-171 82 Solna, Sweden.
³ Department of Biochemistry and Microbiology, Faculty of Biological and Agronomic Sciences, Mouloud Mammeri University of Tizi Ouzou, 15000 Tizi Ouzou, Algeria.
⁴ Department of Natural and Life Sciences, Faculty of Exact Sciences and Natural and Life Sciences, Mohamed Khider University of Biskra, 07000 Biskra, Algeria.
⁵ Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.

Abstract

Background: Application of next-generation sequencing (NGS) to genomic DNA extracted from sewage offers a unique and cost-effective opportunity to study the genetic diversity of intestinal parasites. In this study, we used amplicon-based NGS to reveal and differentiate several common luminal intestinal parasitic protists, specifically Entamoeba, Endolimax, Iodamoeba, and Blastocystis, in sewage samples from Swedish treatment plants.

Materials and methods: Influent sewage samples were subject to gradient centrifugation, DNA extraction and PCR-based amplification using three primer pairs designed for amplification of eukaryotic nuclear 18S ribosomal DNA. PCR products were sequenced using ILLUMINA® technology, and resulting sequences were annotated to species and subtype level using the in-house BION software, sequence clustering, and phylogenetic analysis.

Results: A total of 26 samples from eight treatment plants in central/southern Sweden were analysed. Blastocystis sp. and Entamoeba moshkovskii were detected in all samples, and most samples (n = 20) were positive for Entamoeba coli. Moreover, we detected Entamoeba histolytica, Entamoeba dispar, Entamoeba hartmanni, Endolimax nana, and Iodamoeba bütschlii in 1, 11, 4, 10, and 7 samples, respectively. The level of genetic divergence observed within E. nana and E. moshkovskii was 20.2% and 7.7%, respectively, across the ~400-bp region studied, and two clades of E. moshkovskii were found. As expected, Blastocystis sp. subtypes 1-4 were present in almost all samples; however, ST8 was present in 10 samples and was the only subtype not commonly found in humans that was present in multiple samples.

Conclusions: Entamoeba and Blastocystis were identified as universal members of the "sewage microbiome". Blastocystis sp. ST8, which has been rarely reported in humans, was a very common finding, indicating that a hitherto unidentified but common host of ST8 contributed to the sewage influent. The study also provided substantial new insight into the intra-generic diversity of Entamoeba and Endolimax.

Keywords: Ameba, Archamoebae, Stramenopiles, NGS, Survey, Phylogeny, Ribosomal lineage; Amoebae; Scandinavia, Genetic diversity, Parasite; Sewage, Wastewater.