Profiling pathogenic protozoan and their functional pathways in wastewater using 18S rRNA and shotgun metagenomics

Nonsikelelo P Mthethwa-Hlongwa; Isaac D Amoah; Andres Gomez; Sam Davison; Poovendhree Reddy; Faizal Bux; Sheena Kumari

doi:10.1016/j.scitotenv.2023.169602

Profiling pathogenic protozoan and their functional pathways in wastewater using 18S rRNA and shotgun metagenomics

Sci Total Environ. 2024 Feb 20:912:169602. doi: 10.1016/j.scitotenv.2023.169602. Epub 2023 Dec 26.

Authors

Nonsikelelo P Mthethwa-Hlongwa¹, Isaac D Amoah², Andres Gomez³, Sam Davison³, Poovendhree Reddy⁴, Faizal Bux⁵, Sheena Kumari⁶

Affiliations

¹ Institute for Water and Wastewater Technology, Durban University of Technology, Durban 4000, South Africa; Department Community Health Studies, Faculty of Health Sciences, Durban University of Technology, Durban 4000, South Africa.
² Institute for Water and Wastewater Technology, Durban University of Technology, Durban 4000, South Africa; Department of Environmental Science, The University of Arizona, Shantz Building Rm 4291177 E 4th St., Tucson, AZ 85721, USA.
³ Department of Animal Science, University of Minnesota, St. Paul, MN, USA.
⁴ Department Community Health Studies, Faculty of Health Sciences, Durban University of Technology, Durban 4000, South Africa.
⁵ Institute for Water and Wastewater Technology, Durban University of Technology, Durban 4000, South Africa.
⁶ Institute for Water and Wastewater Technology, Durban University of Technology, Durban 4000, South Africa. Electronic address: sheenak1@dut.ac.za.

PMID: 38154626
DOI: 10.1016/j.scitotenv.2023.169602

Abstract

Despite extensive research, little is known about the composition of eukaryotic protists in environmental samples. This is due to low parasite concentrations, the complexity of parasite diversity, and a lack of suitable reference databases and standardized protocols. To bridge this knowledge gap, this study used 18S rRNA short amplicon and shotgun metagenomic sequencing approaches to profile protozoan microbial communities as well as their functional pathways in treated and untreated wastewater samples collected from different regions of South Africa. Results demonstrated that protozoan diversity (Shannon index P-value = 0.03) and taxonomic composition (PERMANOVA, P-value = 0.02) was mainly driven by the type of wastewater samples (treated & untreated) and geographic location. However, these WWTPs were also found to contain a core community of protozoan parasites. The untreated wastewater samples revealed a predominant presence of free-living, parasitic, and potentially pathogenic protists typically found in humans and animals, ranging from Alveolata (27 %) phylum (Apicomplexa and Ciliophora) to Excavata (3.88 %) (Discoba and Parasalia) and Amoebozoa (2.84 %) (Entamoeba and Acanthamoeba). Shotgun metagenomics analyses in a subset of the untreated wastewater samples confirmed the presence of public health-importance protozoa, including Cryptosporidium species (3.48 %), Entamoeba hystolitica (6.58 %), Blastocystis hominis (2.91 %), Naegleria gruberi (2.37 %), Toxoplasma gondii (1.98 %), Cyclospora cayetanensis (1.30 %), and Giardia intestinalis (0.31 %). Virulent gene families linked to pathogenic protozoa, such as serine/threonine protein phosphatase and mucin-desulfating sulfatase were identified. Additionally, enriched pathways included thiamine diphosphate biosynthesis III, heme biosynthesis, Methylerythritol 4-Phosphate Pathway, methyl erythritol phosphate (MEP), and pentose phosphate pathways. These findings suggest that protozoan pathogens may possess metabolic and growth potential within WWTPs, posing a severe risk of transmission to humans and animals if inadequately disinfected before release. This study provides a baseline for the future investigation of diverse protozoal communities in wastewater, which are of public health importance.

Keywords: 18S rRNA amplicon sequencing; Metagenomics; Public health; Wastewater treatment plant; Waterborne protozoan pathogens.

MeSH terms

Animals
Cryptosporidiosis*
Cryptosporidium* / genetics
Entamoeba*
Eukaryota
Humans
Metagenomics / methods
Parasites*
Phosphates / analysis
RNA, Ribosomal, 18S
Wastewater

Substances

Wastewater
RNA, Ribosomal, 18S
Phosphates