This study investigated the enhancement of winter nitrification performance in a pilot-scale moving bed biofilm reactor (MBBR) post-lagoon treatment under extreme cold conditions using ammonia supplementation (5 and 50 mg N/L) and split-feed operations. The enhanced train demonstrated faster adaptation to low temperatures and superior overall nitrification performance. In Year 1, with a lower ammonium dose (5 mg N/L), the enhanced train recovered faster after an initial performance decline, achieving 81.8 +/- 9.3 % total ammonium nitrogen (TAN) removal (surface area removal rate (SARR): 0.60 +/- 0.07 g N/m(2)& centerdot;d), compared to 57.1 +/- 5.6 % (SARR: 0.40 +/- 0.05 g N/m(2)& centerdot;d) in the control train. In Year 2, with high-concentration and prolonged ammonia supplementation (50 mg N/L), the enhanced train consistently achieved over 90 % TAN removal (SARR: 0.91 +/- 0.08 g N/m(2)& centerdot;d) at 1 degrees C for three months, outperforming the control train (84.4 +/- 3.9 %, SARR: 0.77 +/- 0.08 g N/m(2)& centerdot;d). Microbial analysis revealed seasonal shifts in the bacterial community, with ammonia-oxidizing bacteria (AOB) dominated by Nitrosomonadaceae and nitrite-oxidizing bacteria (NOB) dominated by Nitrospira. Notably, NOB showed greater cold tolerance than AOB, functioning effectively with minimal populations (<1 %). While enhanced strategies enriched nitrifiers and improved system performance, operational challenges were observed: the split-feed operation compromised the performance of the first reactor in the enhanced train, leading to TAN accumulation and placing stress on downstream reactors. Overall, this study provides valuable insights into the design and operation of MBBR systems in cold climates, offering a framework to improve nitrification performance under extreme environmental conditions.

Acinetobacter radioresistens was identified in UV-treated wastewater effluent from a First Nation reserve in Manitoba, Canada, by whole-genome sequencing (3,195,655 bp; 41.7% GC) and digital DNA-DNA hybridization with A. radioresistens DSM 6976 (average contig nucleotide identity: 90.6%). Draft genome analysis revealed the presence of the clinically relevant carbapenem-resistance gene, blaOXA-23.

This pilot-scale study investigated nitrifying moving bed biofilm reactors (MBBRs) in a post-lagoon treatment setup over two years to evaluate the impact of seasonal ammonia fluctuations on winter nitrification. In Year 2, reactors without fall ammonia starvation achieved significantly higher winter ammonia removal (97.2 ± 1.5 %) and surface area ammonia removal rates (SARR) (0.69 ± 0.06 g N/m2·d) compared to Year 1 (63.7 ± 2.5 % ammonia removal, SARR of 0.35 ± 0.04 g N/m2·d), demonstrating the critical role of fall ammonia availability for winter nitrification. Biofilms in Year 2 were thinner and denser, with higher biomass concentrations, potentially supporting more active biomass and improved substrate uptake. Seasonal shifts and diversity loss were observed within the biofilm microbial community, and nitrifiers were identified as Nitrosomonadaceae and Nitrospiraceae. Moreover, linear relationships were explored between winter ammonia removals and two ratios: (1) days with influent ammonia levels ≤ 5 mg N/L to days with temperatures above 5 °C, and (2) average ammonia concentration during fall to peak winter ammonia concentration. The modeling results indicated that winter ammonia removal performance could be enhanced by minimizing low-ammonia periods in the fall and maximizing pre-winter ammonia concentration. Overall, this study not only provided a deeper understanding of the year-round nitrifying MBBR process but also highlighted the importance of maintaining adequate substrate levels during fall to ensure sufficient biomass accumulation and activity for robust winter nitrification performance. These findings are essential for enhancing wastewater treatment performance in cold climates and offer practical guidance for optimizing biofilm-based nitrification systems.

This study evaluated the performance of pilot-scale post-lagoon nitrifying moving bed biofilm reactors (MBBRs) across seasonal temperature and total ammonia nitrogen (TAN) variations from fall to spring over two years, focusing on the impact of split-feed operation on winter nitrification. The investigation revealed that Train 2, with split-feed operation, consistently exhibited better nitrification performance during winter compared to Train 1 (control train) over two years. In Year 1, Train 2 achieved over 90 % TAN removal with a total surface area ammonia removal rate (SARR) of 0.69 +/- 0.05 g/m(2)d in winter, while Train 1 only achieved 58.3 +/- 5.3 % TAN removal with a SARR of 0.41 +/- 0.04 g/m(2)d. In Year 2, although Train 2 initially maintained better winter performance, higher pre-winter TAN concentrations enhanced Train 1's performance, allowing it to achieve similar outcomes, with over 90 % TAN removal (> 0.7 g/m(2)d of SARR) by the end of winter. The enhanced performance of Train 2 was attributed to improved nitrification in its second reactor (R2-2), despite a slight compromise in the performance of the first reactor (R2-1). Biofilm microbial community analyses revealed distinct seasonal shifts and identified Nitrosomonadaceae and Nitrospiraceae as the dominant nitrifiers. The split-feed strategy increased nitrifier abundances in R2-2 during the fall and maintained high levels throughout the winter, supporting Train 2's superior winter nitrification performance. Overall, the split-feed strategy proved effective in enhancing winter nitrification and supporting the more efficient year-round operation of post-lagoon MBBR systems, particularly during years with pre-winter ammonia starvation.

Although wastewater treatment plants harbor many pathogens, traditional methods that monitor the microbial quality of surface water and wastewater have not changed since the early 1900s and often disregard the presence of other types of significant waterborne pathogens such as viruses. We used metagenomics and quantitative PCR to assess the taxonomy, functional profiling, and seasonal patterns of DNA and RNA viruses, including the virome distribution in aquatic environments receiving wastewater discharges. Environmental water samples were collected at 11 locations in Winnipeg, Manitoba, along the Red and Assiniboine rivers during the Spring, Summer, and Fall 2021. Samples were filtered and underwent skimmed milk flocculation for viral concentration. The taxonomy of classified DNA was primarily bacteriophages such as Autographiviridae, Kyanoviridae, and Peduoviridae, which were abundant to a lesser extent. Phage-related functionalities such as portal proteins and bacteriophage T4 major head proteins accounted for approximately 20%-40% of aquatic samples across seasons, which may possibly correspond to the DNA phages that were previously identified. RNA viruses went unclassified in the study; however, similar to DNA viruses, many were found to be non-enveloped or "naked viruses" such as Partiviridae, Picobirnaviridae, Tombusviridae, and Picornaviridae, which accounted for 3%-30% of RNA viruses in the study locations across season. Interestingly, aquatic samples were revealed to maintain an abundance of RNA phage-related functionalities such as long tail fiber protein and putative tail proteins, which accounted for approximately 2%-5% of aquatic samples during the Fall of 2021. IMPORTANCE:Municipal wastewater effluents discharged into the Red and Assiniboine rivers of Winnipeg, Manitoba, rely on traditional methods that monitor the microbial quality of effluents and receiving surface waters focus solely on the detection of coliforms, which are not necessarily good indicators of viruses or other pathogens. There is also a lack of current wastewater system effluent regulations at the federal and provincial level. Furthermore, previous literature has shown that when viral DNA and RNA sequences are blasted against current genomic databases, approximately 50% of the viral reads are classified as unknown. The significance of our research in characterizing the virome distribution in aquatic environments addresses a knowledge gap in the current effluent guidelines and a need for regulatory practices. In the long run, fecal indicator bacteria, combined with the detection of enteric viruses, may complement assessment of water quality in effluents discharged into rivers.

This study investigated the impact of varying total ammonia nitrogen (TAN) feed levels along with water temperature decreases on the performance of nitrifying moving bed biofilm reactor (MBBR) at 1 °C and its recovery at 3 °C. Five MBBR reactors were operated with different TAN concentrations as water temperature decreased from 20 to 3 °C: reactor R1 at 30 mg N/L, reactor R2 at 20 mg N/L, reactor R3 at 15 mg N/L, reactor R4 at 10 mg N/L and reactor R5 at 0 mg N/L. The corresponding biofilm characteristics were also analyzed to understand further nitrifying MBBR under different TAN feeding scenarios. The findings revealed that the higher TAN levels were before reaching 1 °C, the better nitrification performance and the more biomass grew. However, the highest TAN concentration (30 mg N/L) might negatively affect the nitrification performance, the activity of nitrifiers, and the growth of biofilms at 1 °C because of the toxic effects of un-ionized or free ammonia (FA). It was observed that the activities of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were affected by FA concentrations ranging from 0.2 to 0.7 mg N/L at 1 °C, but they could gradually be adapted to such inhibitory environment, with NOB recovering more quickly and robustly than AOB. The study identified 20 mg N/L (67 % of maximum influent TAN at 1 °C in R2 as the optimal TAN feeding concentration, achieving over 90 % TAN removal and a surface area removal rate (SARR) of 0.78 ± 0.02 g N/m2·d at 1 °C. Meanwhile, R2 also exhibited the highest biofilm mass, with total solids at 13.3 mg/carrier and volatile solids at 11.3 mg/carrier. As TAN was removed, nitrite accumulation was observed at 1 °C, and higher influent TAN concentrations prior to 1 °C appeared to delay the accumulation. When water temperature increased from 1 °C to 3 °C, nitrification performance improved significantly in all reactors without nitrite accumulation, and the higher TAN feeding in the previous stage led to faster recovery. Compared with 20 °C, biofilm became thinner and denser at 1 °C and 3 °C. Furthermore, this study revealed significant shifts in microbial community composition and nitrifier abundances in response to changes in water temperature and influent TAN levels. The dominant nitrifiers were identified as Nitrosomonadaceae (AOB) and Nitrospiraceae (NOB). At 1 °C, the nitrifier abundances were significantly correlated with SARRs, FA, and biofilm density. R2, which exhibited the best nitrification performance, maintained higher nitrifier abundances at 1 °C.

Wastewater surveillance represents an alternative approach to regulating contamination and the early detection of infectious agents and outbreaks of diseases of public health importance. This study evaluated domestic wastewater effects on recreational waters in estuarine and seawater bodies in Guayas and Santa Elena provinces in Ecuador, South America. Fecal indicator bacteria (thermotolerant coliforms) served as key indicators for evaluation. Physical, chemical, and microbiological quality markers following the Ecuadorian environmental quality standard and the discharge of effluents to the water resource were analyzed. Samples were collected from 44 coastal sites and 2 oxidation lagoons during the dry and rainy seasons of 2020 and 2021, respectively. SARS-CoV-2 RNA was detected in samples with higher E. coli concentrations using reverse transcription quantitative PCR to detect the genes N and ORF1ab. All samples analyzed for SARS-CoV-2 showed Ct ˂ 40 for at least one gene. Four samples showed at least 20 genome copies of gene N per reaction. These were at an artisanal fishing port, an estuarine area (Palmar), a recreational bay, and an oxidation lagoon. A moderate correlation was found between SARS-CoV-2 RNA, thermotolerant coliform and E. coli (p-value ≤ 0.0037), and a strong and positive correlation between thermotolerant coliform and E. coli. (p-value ≤ 0.00001), highlighting the utility of these established parameters as a proxy of the virus. Significant differences were found in the concentrations of thermotolerant coliforms between seasons (p-value = 0.016) and sites (p-value = 0.005). The highest levels of coliforms were found in the dry season (63000 MPN/100 mL) in Anconcito and during the rainy season (14000 MPN/100 mL) at Esterillo in Playas County. It is recommended that the decentralized autonomous governments of the surveyed provinces in Ecuador implement urgent corrective actions and establish medium-term mechanisms to minimize a potential contamination route. Additional parameters must be included in the monitoring, such as Enterococcus and intestinal parasites, due to their public health implications. In the oxidation lagoons, maintenance actions must be carried out, including the dissolution of sediments, an increase in water retention times, and in situ treatment of the sludge, to improve the system’s performance.

ABSTRACTThe microbiological content of water is an ongoing concern in First Nations communities in Canada. Many communities lack water treatment plants and continue to be under drinking water advisories. However, lack of access to treatment plants is only a part of the problem as poor water distribution systems also contribute to the failure to provide safe drinking water. Here, we studied the microbial diversity and antibiotic resistome from water stored in cisterns from two First Nations communities in Manitoba, Canada. We found that the cistern water contained a high number of bacteria and showed the presence of diverse antimicrobial resistance genes. Interestingly, the bacterial diversity and antimicrobial resistance genes varied considerably from that of the untreated source water, indicating that the origin of contamination in the cistern water came from within the treatment plant or along the delivery route to the homes. Our study highlights the importance of proper maintenance of the water distribution system in addition to access to water treatment facilities to ensure a supply of safe water to First Nations communities in Canada.IMPORTANCEThe work described addresses a critical issue in First Nations communities in Canada—the microbiological content of water. Many of these communities lack access to water treatment plants and frequently experience drinking water advisories. This study focused on the microbial diversity and antibiotic resistome in water stored in cisterns within two First Nations communities in Manitoba, Canada. These findings reveal that cistern water, a common source of drinking water in these communities, contains a high number of bacteria and a wide range of antimicrobial resistance genes. This highlights a serious health risk as exposure to such water can lead to the spread of drug-resistant infections, posing a threat to the well-being of the residents.

Changing climates are allowing the geographic expansion of ticks and their animal hosts, increasing the risk of Borrelia-caused zoonoses in Canada. However, little is known about the genomic diversity of Borrelia from the west of the Canadian Rockies and from the tick vectors Ixodes pacificus, Ixodes auritulus and Ixodes angustus. Here, we report the whole-genome shotgun sequences of 51 Borrelia isolates from multiple tick species collected on a range of animal hosts between 1993 and 2016, located primarily in coastal British Columbia. The bacterial isolates represented three different species from the Lyme disease-causing Borrelia burgdorferi sensu lato genospecies complex [Borrelia burgdorferi sensu stricto (n=47), Borrelia americana (n=3) and Borrelia bissettiae (n=1)]. The traditional eight-gene multi-locus sequence typing (MLST) strategy was applied to facilitate comparisons across studies. This identified 13 known Borrelia sequence types (STs), established 6 new STs, and assigned 5 novel types to the nearest sequence types. B. burgdorferi s. s. isolates were further differentiated into ten ospC types, plus one novel ospC with less than 92 % nucleotide identity to all previously defined ospC types. The MLST types resampled over extended time periods belonged to previously described STs that are distributed across North America. The most geographically widespread ST, ST.12, was isolated from all three tick species. Conversely, new B. burgdorferi s. s. STs from Vancouver Island and the Vancouver region were only detected for short periods, revealing a surprising transience in space, time and host tick species, possibly due to displacement by longer-lived genotypes that expanded across North America. This article contains data hosted by Microreact.

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities. The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 µm and 0.2 µm membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA. On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities. The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 µm and 0.2 µm membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA. On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

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.

For decades, multiple anthropogenic stressors have threatened the Galápagos Islands. Widespread marine pollution such as oil spills, persistent organic pollutants, metals, and ocean plastic pollution has been linked to concerning changes in the ecophysiology and health of Galápagos species. Simultaneously, illegal, unreported, and unregulated fishing are reshaping the composition and structure of endemic and native Galápagos pelagic communities. In this novel review, we discuss the impact of anthropogenic pollutants and their associated ecotoxicological implications for Galápagos species in the face of climate change stressors. We emphasize the importance of considering fishing pressure and marine pollution, in combination with climate‐change impacts, when assessing the evolutionary fitness of species inhabiting the Galápagos. For example, the survival of endemic marine iguanas has been negatively affected by organic hydrocarbons introduced via oil spills, and endangered Galápagos sea lions exhibit detectable concentrations of DDT, triggering potential feminization effects and compromising the species' survival. During periods of ocean warming (El Niño events) when endemic species undergo nutritional stress, climate change may increase the vulnerability of these species to the impacts of pollutants, resulting in the species reaching its population tipping point. Marine plastics are emerging as a deleterious and widespread threat to endemic species. The Galápagos is treasured for its historical significance and its unparalleled living laboratory and display of evolutionary processes; however, this unique and iconic paradise will remain in jeopardy until multidisciplinary and comprehensive preventative management plans are put in place to mitigate and eliminate the effects of anthropogenic stressors facing the islands today. We present a critical analysis and synthesis of anthropogenic stressors with some progress from local and international institutional efforts and call to action more precautionary measures along with new management philosophies focused on understanding the processes of change through research to champion the conservation of the Galápagos. Integr Environ Assess Manag 2023;19:870–895. © 2022 SETAC

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.

Abstract 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%), and Myoviridae (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, includes Mycobacterium (37.4%, 18.3%, 46.1%, and 7.7%), Acidovorax (8.9%, 10.8%, 5.4%, and 1.3%), and Polaromonas (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, intI1 was the most abundant integron-integrase gene throughout treatment (1.14 × 104 gene copies/mL) followed by intI3 (4.97 × 103 gene copies/mL) while intI2 abundance remained low (6.4 × 101 gene 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.

Although numerous studies have detected SARS-CoV-2 RNA in wastewater and attempted to find correlations between the concentration of SARS-CoV-2 RNA and the number of cases, no consensus has been reached on sample collection and processing, and data analysis. Moreover, the fate of SARS-CoV-2 in wastewater treatment plants is another issue, specifically regarding the discharge of the virus into environmental settings and the water cycle. The current study monitored SARS-CoV-2 RNA in influent and effluent wastewater samples with three different concentration methods and sludge samples over six months (July to December 2020) to compare different virus concentration methods, assess the fate of SARS-CoV-2 RNA in wastewater treatment plants, and describe the potential relationship between SARS-CoV-2 RNA concentrations in influent and infection dynamics. Skimmed milk flocculation (SMF) resulted in 15.27 ± 3.32% recovery of an internal positive control, Armored RNA, and a high positivity rate of SARS-CoV-2 RNA in stored wastewater samples compared to ultrafiltration methods employing a prefiltration step to eliminate solids in fresh wastewater samples. Our results suggested that SARS-CoV-2 RNA may predominate in solids, and therefore, concentration methods focusing on both supernatant and solid fractions may result in better recovery. SARS-CoV-2 RNA was detected in influent and primary sludge samples but not in secondary and final effluent samples, indicating a significant reduction during primary and secondary treatments. SARS-CoV-2 RNA was first detected in influent on September 30th, 2020. A decay-rate formula was applied to estimate initial concentrations of late-processed samples with SMF. A model based on shedding rate and new cases was applied to estimate SARS-CoV-2 RNA concentrations and the number of active shedders. Inferred sensitivity of observed and modeled concentrations to the fluctuations in new cases and test-positivity rates indicated a potential contribution of newly infected individuals to SARS-CoV-2 RNA loads in wastewater.

Climate change and marine pollution are intertwined in the Galapagos’ living laboratory, where unabated anthropogenic pollution is reshaping evolution in the Anthropocene.

Early virus detection and characterization is key to successful avian influenza virus (AIV) surveillance for the health of humans as well as domestic poultry. We explored a novel sampling approach and molecular strategy using sediment from wetlands and outdoor waterbodies on poultry farms as a population-level proxy of AIV activity in waterfowls. RNA was extracted using the MoBio RNA PowerSoil Total RNA isolation kit with additional chloroform extraction steps to reduce PCR inhibition. AIV matrix protein (MP) gene was detected in 42/345 (12.2%) samples by RT-qPCR; an additional 64 (18.6%) samples showed evidence of amplification below the threshold and were categorized as "suspect positive." Enrichment-based targeted resequencing (TR) identified AIV sequences in 79/345 (22.9%) samples. TR probes were designed for MP, hemagglutinin (HA), and neuraminidase (NA), however PB2 and PA were also identified. Although RT-qPCR and TR only had fair-moderate agreement, RT-qPCR positivity was predictive of TR-positivity both when using only strictly positive RT-qPCR samples (OR = 11.29) and when coding suspect positives as positive (OR = 7.56). This indicates that RT-qPCR could be used as a screening tool to select samples for virus characterization by TR and that future studies should consider RT-qPCR suspect positives to be positive samples for subsequent resequencing when avoiding false negatives is the priority, for instance in a diagnostic test, and to consider suspect positives to be negative samples when cost efficiency over a large number of samples is the priority, for instance in a surveillance program. A total of 13 HA (H1-7, H9-13, H16) and 9 NA (N1-9) subtypes were identified, with a maximum of 8 HA and 8 NA subtypes detected in a single sample. The optimized RNA extraction and targeted resequencing methods provided increased virus detection and subtyping characterization that could be implemented in an AIV surveillance system.

Myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) is a syndrome of unknown etiology characterized by profound fatigue exacerbated by physical activity, also known as post-exertional malaise (PEM). Previously, we did not detect evidence of immune dysregulation or virus reactivation outside of PEM periods. Here we sought to determine whether cardiopulmonary exercise stress testing of ME/CFS patients could trigger such changes. ME/CFS patients (n = 14) and matched sedentary controls (n = 11) were subjected to cardiopulmonary exercise on 2 consecutive days and followed up to 7 days post-exercise, and longitudinal whole blood samples analyzed by RNA-seq. Although ME/CFS patients showed significant worsening of symptoms following exercise versus controls, with 8 of 14 ME/CFS patients showing reduced oxygen consumption ([Formula: see text]) on day 2, transcriptome analysis yielded only 6 differentially expressed gene (DEG) candidates when comparing ME/CFS patients to controls across all time points. None of the DEGs were related to immune signaling, and no DEGs were found in ME/CFS patients before and after exercise. Virome composition (P = 0.746 by chi-square test) and number of viral reads (P = 0.098 by paired t-test) were not significantly associated with PEM. These observations do not support transcriptionally-mediated immune cell dysregulation or viral reactivation in ME/CFS patients during symptomatic PEM episodes.