Identification of eukaryotic microorganisms with 18S rRNA next-generation sequencing in wastewater treatment plants, with a more targeted NGS approach required for Cryptosporidium detection. (1st July 2019)

• Record Type: Journal Article
• Authors: Zahedi, Alireza; Greay, Telleasha L.; Paparini, Andrea; Linge, Kathryn L.; Joll, Cynthia A.; Ryan, Una M.
• Is Part Of: Water research. Volume 158(2019)
• Page Start: 301
• Page End: 312
• Publication Date: 2019-07-01
• Subjects: Wastewater -- Cryptosporidium -- Blastocystis -- Entamoeba -- Next-generation sequencing; Water -- Pollution -- Research -- Periodicals; 363.7394
• DOI: 10.1016/j.watres.2019.04.041
• Languages: English
• ISSNs: 0043-1354

Abstract

Abstract: While some microbial eukaryotes can improve effluent quality in wastewater treatment plants (WWTPs), eukaryotic waterborne pathogens are a threat to public health. This study aimed to identify Eukarya, particularly faecal pathogens including Cryptosporidium, in different treatment stages (influent, intermediate and effluent) from four WWTPs in Western Australia (WA). Three WWTPs that utilise stabilisation ponds and one WWTP that uses activated sludge (oxidation ditch) treatment technologies were sampled. Eukaryotic 18S rRNA (18S) was targeted in the wastewater samples ( n = 26) for next-generation sequencing (NGS), and a mammalian-blocking primer was used to reduce the amplification of mammalian DNA. Overall, bioinformatics analyses revealed 49 eukaryotic phyla in WWTP samples, and three of these phyla contained human intestinal parasites, which were primarily detected in the influent. These human intestinal parasites either had a low percent sequence composition or were not detected in the intermediate and effluent stages and included the amoebozoans Endolimax sp., Entamoeba sp. and Iodamoeba sp., the human pinworm Enterobius vermicularis (Nematoda), and Blastocystis sp. subtypes (Sarcomastigophora). Six Blastocystis subtypes and four Entamoeba species were identified by eukaryotic 18S NGS, however, Cryptosporidium sp. and Giardia sp. were not detected. Real-time polymerase chain reaction (PCR) also failed to detect Giardia, but Cryptosporidium -specific NGS detected Cryptosporidium in all WWTPs, and a total of nine species were identified, including five zoonotic pathogens. Although eukaryotic 18S NGS was able to identify some faecal pathogens, this study has demonstrated that more specific NGS approaches for pathogen detection are more sensitive and should be applied to future wastewater pathogen assessments. Graphical abstract: Image 1 Highlights: Western Australian WWTPs were studied for faecal pathogens with eukaryotic 18S NGS. Stabilisation ponds and activated sludge treatment technologies were assessed. Influent had the highest percent compositions of intestinal parasites. Six Blastocystis subtypes and four Entamoeba species were identified. Nine Cryptosporidium species/genotypes were only detected by Cryptosporidium -specific NGS.