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

Abstract

BackgroundApplication 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 methodsInfluent 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.ResultsA 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.ConclusionsEntamoeba 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.

A Study on the Prevalence and Subtype Diversity of the Intestinal Protist Blastocystis sp. in a Gut-Healthy Human Population in the Czech Republic

“…Nevertheless, our finding of the avian subtypes ST6 and ST7 in humans as well as ST5 (often found in pigs) and ST8, could reflect cases of zoonotic transmission (Stensvold and Clark, 2016a ). Interestingly, rats might play a role in spreading of Blastocystis ST1-ST4 and possible also of ST8 as suggested by a study focused on a survey of wastewater samples in Sweden (Stensvold et al, 2020b ).…”

First Report on the Prevalence and Subtype Distribution of Blastocystis sp. in Edible Marine Fish and Marine Mammals: A Large Scale-Study Conducted in Atlantic Northeast and on the Coasts of Northern France

“…Consequently, the presence of ST8 in fish samples might be related to local water contamination by animal or human wastes. In a recent study conducted in central/southern Sweden [35], ST8 was surprisingly identified in almost 40% of the untreated wastewater samples tested. However, as discussed by the authors, the host, whether human or animal, contributing to the sewage influent remains as yet unidentified.…”

Entamoeba gingivalis: epidemiology, genetic diversity and association with oral microbiota signatures in North Eastern Tanzania

“…Genomic DNAs extracted from the mouthwash samples were subject to microbiota profiling (metabarcoding), using the recently described 16S + 18S assay in place at Statens Serum Institut [16][17][18]. Briefly, PCR used one primer pair targeting the 16S (341F3/ 806R5 [5ʹ-ACT CCT AYG GGR BGC ASC AG-3ʹ/5ʹ-AGC GTG GAC TAC NNG GGT ATC TAA T-3ʹ]) and three primer pairs targeting the 18S (G3F1/ G3R1 [5ʹ-GCC AGC AGC CGC GGT AAT TC-3ʹ/5ʹ-ACA TTC TTG GCA AAT GCT TTC GCA G-3ʹ], G4F3/G4R3 [5ʹ-CAG CCG CGG TAA TTC CAG CTC-3ʹ/5ʹ-GGT GGT GCC CTT CCG TCA AT-3ʹ], and G6F1/G6R1 [3ʹ-TGG AGG GCA AGT CTG GTG CC-3ʹ/5ʹ-ACG GTA TCT GAT CGT CTT CGA TCC C-3ʹ]).…”