Studies of environmental microbiota typically target only specific groups of microorganisms, with most focusing on bacteria through taxonomic classification of 16S rRNA gene sequences. For a more holistic understanding of a microbiome, a strategy to characterize the viral, bacterial, and eukaryotic components is necessary. We developed a method for metagenomic and amplicon-based analysis of freshwater samples involving the concentration and size-based separation of eukaryotic, bacterial, and viral fractions. Next-generation sequencing and culture-independent approaches were used to describe and quantify microbial communities in watersheds with different land use in British Columbia. Deep amplicon sequencing was used to investigate the distribution of certain viruses (g23 and RdRp), bacteria (16S rRNA and cpn60), and eukaryotes (18S rRNA and ITS). Metagenomic sequencing was used to further characterize the gene content of the bacterial and viral fractions at both taxonomic and functional levels. This study provides a systematic approach to separate and characterize eukaryotic-, bacterial-, and viral-sized particles. Methodologies described in this research have been applied in temporal and spatial studies to study the impact of land use on watershed microbiomes in British Columbia.

Background: Wastewater treatment plants are an essential part of maintaining the health and safety of the general public. However, they are also an anthropogenic source of antibiotic resistance genes. In this study, we characterized the resistome, the distribution of classes 1–3 integron-integrase genes (intI1, intI2, and intI3) as mobile genetic element biomarkers, and the bacterial and phage community compositions in the North End Sewage Treatment Plant in Winnipeg, Manitoba. Samples were collected from raw sewage, returned activated sludge, final effluent, and dewatered sludge. A total of 28 bacterial and viral metagenomes were sequenced over two seasons, fall and winter. Integron-integrase genes, the 16S rRNA gene, and the coliform beta-glucuronidase gene were also quantified during this time period.Results: Bacterial classes observed above 1% relative abundance in all treatments were Actinobacteria (39.24% ± 0.25%), Beta-proteobacteria (23.99% ± 0.16%), Gamma-proteobacteria (11.06% ± 0.09%), and Alpha-proteobacteria (9.18 ± 0.04%). Families within the Caudovirales order:Siphoviridae(48.69% ± 0.10%),Podoviridae(23.99% ± 0.07%), andMyoviridae(19.94% ± 0.09%) were the dominant phage observed throughout the NESTP. The most abundant bacterial genera (in terms of average percent relative abundance) in influent, returned activated sludge, final effluent, and sludge, respectively, includesMycobacterium(37.4%, 18.3%, 46.1%, and 7.7%),Acidovorax(8.9%, 10.8%, 5.4%, and 1.3%), andPolaromonas(2.5%, 3.3%, 1.4%, and 0.4%). The most abundant class of antibiotic resistance in bacterial samples was tetracycline resistance (17.86% ± 0.03%) followed by peptide antibiotics (14.24% ± 0.03%), and macrolides (10.63% ± 0.02%). Similarly, the phage samples contained a higher prevalence of macrolide (30.12% ± 0.30%), peptide antibiotic (10.78% ± 0.13%), and tetracycline (8.69% ± 0.11%) resistance. In addition,intI1was the most abundant integron-integrase gene throughout treatment (1.14 × 10⁴gene copies/mL) followed byintI3(4.97 × 10³gene copies/mL) whileintI2abundance remained low (6.4 × 10¹gene copies/mL).Conclusions: Wastewater treatment successfully reduced the abundance of bacteria, DNA phage and antibiotic resistance genes although many antibiotic resistance genes remained in effluent and biosolids. The presence of integron-integrase genes throughout treatment and in effluent suggests that antibiotic resistance genes could be actively disseminating resistance between both environmental and pathogenic bacteria.

The dissemination of antibiotic resistant bacteria from anthropogenic sources into the environment poses an emerging public health threat. Antibiotic resistance genes (ARGs) and gene-capturing systems such as integron-associated integrase genes (intI) play a key role in alterations of microbial communities and the spread of antibiotic resistant bacteria into the environment. In order to assess the effect of anthropogenic activities on watersheds in southwestern British Columbia, the presence of putative antibiotic resistance and integrase genes was analyzed in the microbiome of agricultural, urban influenced, and protected watersheds. A metagenomics approach and high-throughput quantitative PCR (HT qPCR) were used to screen for elements of resistance including ARGs and intI. Metagenomic sequencing of bacterial genomic DNA was used to characterize the resistome of microbial communities present in watersheds over a 1-year period. There was a low prevalence of ARGs relative to the microbial population (<1%). Analysis of the metagenomic sequences detected a total of 60 elements of resistance including 46 ARGs, intI1, and groEL/intI1 genes and 12 quaternary ammonium compounds (qac) resistance genes across all watershed locations. The relative abundance and richness of ARGs was found to be highest in agriculture impacted watersheds compared to urban and protected watersheds. A downstream transport pattern was observed in the impacted watersheds (urban and agricultural) during dry months. Similar to other reports, this study found a strong association between intI1 and ARGs (e.g., sul1), an association which may be used as a proxy for anthropogenic activities. Chemical analysis of water samples for three major groups of antibiotics was below the detection limit. However, the high richness and gene copy numbers (GCNs) of ARGs in impacted sites suggest that the effects of effluents on microbial communities are occurring even at low concentrations of antimicrobials in the water column. Antibiotic resistance and integrase genes in a year-long metagenomic study showed that ARGs were driven mainly by environmental factors from anthropogenized sites in agriculture and urban watersheds. Environmental factors such as land-use and water quality parameters accounted for 45% of the variability observed in watershed locations.

The composition, abundance and distribution of macroplastics (MAPs) and microplastics (MPs) in the Vinces and Los Tintos rivers were determined in three sites (Pueblo Nuevo, Santa Marianita, Los Tintos) from the low basin in the coastal province of Guayas, Ecuador. MAPS were recorded by visual census, covering a total distance of 140 m, and MPs were extracted in the intertidal sediments via density separation using a saturated NaCl solution, and these were counted using a stereomicroscope. A total of 940 plastic items were identified. The predominant debris was plastic with 85.2%, followed by manufactured materials and metals. The Vinces River contained the highest abundance of plastic in the locality of Pueblo Nuevo. The most abundant plastic was MPs. The most common MAPs were plastic bags (23%), food packaging (17%) and foamed plastic (8%). MP size classes quantified between 0.15 and 2.52 mm in intertidal, very fine sandy sediment and decreased in abundance with increasing grain size. The most common MPs were fibres (65.2%) (black (43.8%) and blue (25.8%)), and their distribution has a high correlation with population density and water flow direction: Santa Marianita 5.55 g⁻¹, Pueblo Nuevo 7.39 g⁻¹, Los Tintos 8.17⁻¹. A significant abundance of fibres was identified in Pueblo Nuevo. The plastic spatial distribution revealed major plastic pollution in areas where recreational and tourism activities have been developed. Therefore, we recommend implementing awareness campaigns by educating businesses, residents and tourists on managing solid waste (especially plastic) and wastewater. Our results can serve as a baseline for future plastic monitoring in the area.

COVID-19, caused by SARS-CoV-2, was declared a global pandemic on 11 March 2020 by the World Health Organization. The pandemic has triggered an unprecedented increase in the production, consumption and disposal of multiple types of plastic-based personal protective equipment (PPE) as a measure to reduce the infection. Recent research shows that plastic surfaces can serve as a fomite for coronavirus transmission as it can remain stable and be viable on polypropylene for up to 72 h or on other plastic surfaces for up to 9 days. While it is unknown whether or to what extent macroplastic debris and ubiquitous microplastics emitted into the environment can serve as physical vectors or fomites of pathogenic viruses, recent studies have reported that both macroplastic and microplastics can serve as vectors for harmful pathogens and invasive species (biological pollution). Here, hypothetical scenarios based on the weight of evidence are proposed to plausibly state the role of plastic debris (e.g., single-use-plastics), discarded PPE supplies, including facemasks, sanitizer bottles, gloves, and plastic bags, as well as microplastics as potential physical vectors of SARS-CoV-2, serving as a route of exposure to humans and wildlife in the terrestrial, freshwater and marine ecosystems.

We investigated the potential use and quantification of human enteric viruses in municipal wastewater samples of Winnipeg (Manitoba, Canada) as alternative indicators of contamination and evaluated the processing stages of the wastewater treatment plant. During the fall 2019 and winter 2020 seasons, samples of raw sewage, activated sludge, effluents, and biosolids (sludge cake) were collected from the North End Sewage Treatment Plant (NESTP), which is the largest wastewater treatment plant in the City of Winnipeg. DNA (Adenovirus and crAssphage) and RNA enteric viruses (Pepper mild mottle virus, Norovirus genogroups GI and GII, Rotavirus Astrovirus, and Sapovirus) as well as the uidA gene found in Escherichia coli were targeted in the samples collected from the NESTP. Total nucleic acids from each wastewater treatment sample were extracted using a commercial spin-column kit. Enteric viruses were quantified in the extracted samples via quantitative PCR using TaqMan assays. Overall, the average gene copies assessed in the raw sewage were not significantly different (p-values ranged between 0.1023 and 0.9921) than the average gene copies assessed in the effluents for DNA and RNA viruses and uidA in terms of both volume and biomass. A significant reduction (p-value ≤ 0.0438) of Adenovirus and Noroviruses genogroups GI and GII was observed in activated sludge samples compared with those for raw sewage per volume. Higher GCNs of enteric viruses were observed in dewatered sludge samples compared to liquid samples in terms of volume (g of sample) and biomass (ng of nucleic acids). Enteric viruses found in gene copy numbers were at least one order of magnitude higher than the E. coli marker uidA, indicating that enteric viruses may survive the wastewater treatment process and viral-like particles are being released into the aquatic environment. Viruses such as Noroviruses genogroups GI and GII, and Rotavirus were detected during colder months. Our results suggest that Adenovirus, crAssphage, and Pepper mild mottle virus can be used confidently as complementary viral indicators of human fecal pollution.