Differentiation of and Parasitic Archamoebids Encountered in Untreated Wastewater Samples by Amplicon-based Next-generation Sequencing

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 , , , and , 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. sp. and were detected in all samples, and most samples ( = 20) were positive for . Moreover, we detected , , , , and in 1, 11, 4, 10, and 7 samples, respectively. The level of genetic divergence observed within and was 20.2% and 7.7%, respectively, across the ~400-bp region studied, and two clades of were found. As expected, 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: and were identified as universal members of the "sewage microbiome". 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 and .