title: Comparison of Methods to Identify Pathogens and Associated Virulence Functional Genes in Biosolids from Two Different Wastewater Treatment Facilities in Canada.
abstract: The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.
authors.affiliation: National Research Council Canada, Energy Mining and Environment, Montreal, Qc, Canada.
authors.forename: Etienne
authors.identifier: http://orcid.org/0000-0002-7112-3425
authors.initials: E
authors.lastname: Yergeau
authors.affiliation: National Research Council Canada, Human Health Therapeutics, Montreal, Qc, Canada.
authors.forename: Luke
authors.initials: L
authors.lastname: Masson
authors.affiliation: National Research Council Canada, Energy Mining and Environment, Montreal, Qc, Canada.
authors.forename: Miria
authors.initials: M
authors.lastname: Elias
authors.affiliation: Environment Canada, Biological Assessment and Standardization Section, Ottawa, On, Canada.
authors.forename: Shurong
authors.initials: S
authors.lastname: Xiang
authors.affiliation: Canadian Food Inspection Agency, Fertilizer Safety Office, Plant Health & Biosecurity Directorate, Ottawa, On, Canada.
authors.forename: Ewa
authors.initials: E
authors.lastname: Madey
authors.affiliation: Canadian Food Inspection Agency, Ottawa Laboratory - Fallowfield, Ottawa, On, Canada.
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Huang
authors.affiliation: Canadian Food Inspection Agency, Ottawa Laboratory - Fallowfield, Ottawa, On, Canada.
authors.forename: Brian
authors.initials: B
authors.lastname: Brooks
authors.affiliation: Environment Canada, Biological Assessment and Standardization Section, Ottawa, On, Canada.
authors.forename: Lee A
authors.initials: LA
authors.lastname: Beaudette
mesh_terms: D002170:Canada; D004784:Environmental Monitoring; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology; D005798:Genes, Bacterial / Q000235:genetics*; D056186:Metagenomics; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D012722:Sewage / Q000382:microbiology; D014774:Virulence / Q000235:genetics*; D018505:Waste Management; D062065:Wastewater / Q000382:microbiology; D014873:Water Pollutants / Q000302:isolation & purification*
chemical_list: D012336:RNA, Ribosomal, 16S; D012722:Sewage; D062065:Wastewater; D014873:Water Pollutants