Campylobacter species, Salmonella serotypes and ribosomal RNA-based fecal source tracking in the Kokemäki River watershed.

Fecal contamination of surface water compromises the usability of surface water for drinking water production due to an increase in human health risks. In this study, we collected surface water samples for two years from the Kokemäki River (Finland). The downstream river stretch is used for feeding production of artificial ground water for a major drinking water treatment plant. The prevalence of Campylobacter species and Salmonella serotypes together with fecal source identifiers targeting general, human, gull, swine, and ruminant were evaluated at 16 sampling sites throughout the studied watershed. We detected Campylobacter spp. from all 16 sampling sites with Campylobacter jejuni and Campylobacter lari as the most detected species. Salmonella spp. was detected in 10 out of 16 sampling sites, with Salmonella Typhimurium being the most common serovar. Regarding spatial variation in the hygienic quality of surface water, the upstream area (urban proximity) and downstream area (agricultural proximity) had higher microbial loads than the middle section of the study area. Samples taken in fall and spring had higher microbial loads than summer and winter samples. The lower ratio of rRNA to rRNA-gene (rDNA) of studied microbes in the winter than in other seasons may indicate low metabolic activity of bacterial targets during winter. The number of gulls, swine, and cattle in the catchment area concorded with the number of fecal source identifiers in the surface water. Further, the prevalence of gull-specific source identifier agreed with the detection of C. coli, C. lari, and S. Typhimurim, whereas the prevalence of swine- and ruminant-specific source identifiers agreed with the detection of C. jejuni and C. coli. Thus, fecal source identifiers are shown to be important tools for monitoring zoonotic pathogens affecting microbial quality of surface water. Further, variation in fecal loads indicates such variation in health risks related to surface water use.

Characterization of methicillin resistant Staphylococcus Aureus in municipal wastewater in Finland.

Wastewater-based surveillance (WBS) of multidrug-resistant bacteria could complement clinical data, serving as a population-level early warning tool. This study evaluated WBS as a pandemic preparedness tool, by selectively isolating and culturing methicillin-resistant Staphylococcus aureus (MRSA) with CHROMagar MRSA. Some 24-h composite wastewater samples (n = 80) were collected from ten treatment plants across Finland between February 2021 and January 2022. MRSA prevalence in wastewater samples was 27.5% (n = 22/80), showing seasonal and temporal variations. Phenotypic antimicrobial susceptibility testing (AST) with microdilution showed that over 80% of isolates were drug-resistant to clindamycin, sulfamethoxazole/trimethoprim, tetracycline, fusidic acid, and erythromycin. Four isolates (18.2%) were vancomycin-resistant. WGS revealed that 31.8% (n = 7) of the isolates belonged to the ST8-t008 and ST6-t304 spa types, respectively. In addition, two spa types (t011 and t034) belong to the CC398 complex. The mecA gene was found in all isolates (n = 22) and three tetracycline resistance determinants (tet38, tetK, and tetM) were detected with tet38 being the most abundant (81.8%, n = 18/22). Three isolates harboured the plasmid-mediated sat4 gene that confers resistance to Streptothricin. In addition, resistance determinants to macrolide antibiotics (mph (C)/msr (A) and fosfomycin (fosB) were detected in the seven isolates that belonged to spa type t008. All isolates except one harboured the SCCmec_type_IVa(2B). Six ST8 isolates harboured the LukS/F-PV genes encoding the Panton-Valentine leukocidin (PVL) and were also positive for the Arginine Catabolic Mobile Element (ACME), suggesting they belong to the USA300 clone. The Inc18 plasmid was the most abundant as it was detected in 72.7% (n = 16/22) of the isolates. Other plasmid replicons detected were the rep_trans and repA_N which were detected in 45.4% (n = 10/22) and 40.9% (n = 9/22) of the isolates respectively. Ten isolates harboured at least three plasmid replicons and no plasmid replicons were detected in four isolates (ST6/t304). The cgMLST revealed that some isolates aggregated into two genomically indistinguishable clusters: ST6/t304 belonging to cluster type CT12405 (≤20 allelic differences) and ST8/t008 belonging to cluster type CT1925 (<8 allelic differences). Overall, we found a high genotypic concordance with the national clinical bacterial resistance data. Our study demonstrates the sensitivity of culture-based wastewater surveillance for MRSA using clinical media following pre-enrichment, reliably predicting pathogen occurrence at the population level.

Long-term longitudinal monitoring of SARS CoV-2 in urban rivers and sewers of Nepal.

In regions without adequate centralized wastewater treatment plants, sample collection from rivers and sewers can be an alternative sampling strategy for wastewater surveillance. This study aimed to assess the feasibility of alternative sampling strategies by testing samples collected from rivers (n = 246) and sewers (n = 244) in the Kathmandu Valley between March 2021 and February 2022. All samples were concentrated using the skimmed-milk flocculation method and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was quantified using the nucleocapsid (N) and envelope (E) genes qPCR assays. Of the total, 75 % (371/490) of the samples tested positive using at least one qPCR assay, with concentrations ranging from 3.0 to 8.3 log10 gene copies/L. No significant correlation between concentrations of SARS-CoV-2 from both sewers and river with the number of confirmed coronavirus disease 2019 (COVID-19) cases in the Kathmandu valley was observed (p > 0.05). Despite the high concentration of SARS-CoV-2 in rivers and sewers, we hypothesize this finding to be a result of inaccurate number of clinical cases possibly due to inadequate clinical testing. This longitudinal study further supports the statement to consider sampling strategies from sewers and rivers for WBS in Nepal and other low and middle-income countries.

Simultaneous biomass concentration and subsequent quantitation of multiple infectious disease agents and antimicrobial resistance genes from community wastewater.

Wastewater-based surveillance (WBS) of infectious disease agents is increasingly seen as a reliable source of population health data. To date, wastewater-based surveillance efforts have largely focused on individual pathogens. However, given that wastewater contains a broad range of pathogens circulating in the population, a more comprehensive approach could enhance its usability. We focused on the simultaneous detection of SARS-CoV-2, sapovirus, Campylobacter jejuni, Campylobacter coli, Salmonella spp., pathogenic Escherichia coli, Cryptosporidium spp., Giardia spp. and antimicrobial resistance genes (ARGs) of clinical relevance. To achieve this goal, biomass concentration and nucleic acid extraction methods were optimized, and samples were analyzed by using a set of (RT)-qPCR and (HT)-qPCR methods. We determined the prevalence and the spatial and temporal trends of the targeted pathogens and collected novel information on ARGs in Finnish wastewater. In addition, the use of different wastewater concentrates, namely the ultrafiltered concentrate of the supernatant and the centrifuged pellet, and the effect of freezing and thawing wastewater prior to sample processing were investigated with the indicator microbe crAssphage. Freeze-thawing of wastewater decreased the gene copy count of crAssphage in comparison to analyzing fresh samples (p < 0.001). Campylobacters were most abundant in two of the four studied summer months (30 % detection rate) and in wastewaters from regions with intensive animal farming. Salmonella, however, was detected in 40 % of the samples without any clear seasonal trends, and the highest gene copy numbers were recorded from the largest wastewater treatment plants. Beta-lactamase resistance genes that have commonly been detected in bacteria isolated from humans in Finland, namely blaCTX-M, blaOXA48, blaNDM, and blaKPC, were also frequently detected in wastewaters (100, 98, 98, and 70 % detection rates, respectively). These results confirm the reliability of using wastewater in public health surveillance and demonstrate the possibility to simultaneously perform WBS of multiple pathogens.

Key considerations for pathogen surveillance in wastewater.

Wastewater surveillance (WWS) has received significant attention as a rapid, sensitive, and cost-effective tool for monitoring various pathogens in a community. WWS is employed to assess the spatial and temporal trends of diseases and identify their early appearances and reappearances, as well as to detect novel and mutated variants. However, the shedding rates of pathogens vary significantly depending on factors such as disease severity, the physiology of affected individuals, and the characteristics of pathogen. Furthermore, pathogens may exhibit differential fate and decay kinetics in the sewerage system. Variable shedding rates and decay kinetics may affect the detection of pathogens in wastewater. This may influence the interpretation of results and the conclusions of WWS studies. When selecting a pathogen for WWS, it is essential to consider it's specific characteristics. If data are not readily available, factors such as fate, decay, and shedding rates should be assessed before conducting surveillance. Alternatively, these factors can be compared to those of similar pathogens for which such data are available.

Avian Influenza outbreaks: Human infection risks for beach users - One health concern and environmental surveillance implications.

Despite its popularity for water activities, such as swimming, surfing, fishing, and rafting, inland and coastal bathing areas occasionally experience outbreaks of highly pathogenic avian influenza virus (HPAI), including A(H5N1) clade 2.3.4.4b. Asymptomatic infections and symptomatic outbreaks often impact many aquatic birds, which increase chances of spill-over events to mammals and pose concerns for public health. This review examined the existing literature to assess avian influenza virus (AIV) transmission risks to beachgoers and the general population. A comprehensive understanding of factors governing such crossing of the AIV host range is currently lacking. There is limited knowledge on key factors affecting risk, such as species-specific interactions with host cells (including binding, entry, and replication via viral proteins hemagglutinin, neuraminidase, nucleoprotein, and polymerase basic protein 2), overcoming host restrictions, and innate immune response. AIV efficiently transmits between birds and to some extent between marine scavenger mammals in aquatic environments via consumption of infected birds. However, the current literature lacks evidence of zoonotic AIV transmission via contact with the aquatic environment or consumption of contaminated water. The zoonotic transmission risk of the circulating A(H5N1) clade 2.3.4.4b virus to the general population and beachgoers is currently low. Nevertheless, it is recommended to avoid direct contact with sick or dead birds and to refrain from bathing in locations where mass bird mortalities are reported. Increasing reports of AIVs spilling over to non-human mammals have raised valid concerns about possible virus mutations that lead to crossing the species barrier and subsequent risk of human infections and outbreaks.

Application of Skimmed-Milk Flocculation Method for Wastewater Surveillance of COVID-19 in Kathmandu, Nepal.

Wastewater surveillance (WS) has been used globally as a complementary tool to monitor the spread of coronavirus disease 2019 (COVID-19) throughout the pandemic. However, a concern about the appropriateness of WS in low- and middle-income countries (LMICs) exists due to low sewer coverage and expensive viral concentration methods. In this study, influent wastewater samples (n = 63) collected from two wastewater treatment plants (WWTPs) of the Kathmandu Valley between March 2021 and February 2022 were concentrated using the economical skimmed-milk flocculation method (SMFM). The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was tested by qPCR using assays that target the nucleocapsid (N) and envelope (E) genes. Overall, 84% (53/63) of the total samples were positive for SARS-CoV-2 according to at least one of the tested assays, with concentrations ranging from 3.5 to 8.3 log10 gene copies/L, indicating the effectiveness of the SMFM. No correlation was observed between the total number of COVID-19 cases and SARS-CoV-2 RNA concentrations in wastewater collected from the two WWTPs (p > 0.05). This finding cautions the prediction of future COVID-19 waves and the estimation of the number of COVID-19 cases based on wastewater concentration in settings with low sewer coverage by WWTPs. Future studies on WS in LMICs are recommended to be conducted by downscaling to sewer drainage, targeting a limited number of houses. Overall, this study supports the notion that SMFM can be an excellent economical virus-concentrating method for WS of COVID-19 in LMICs.

Advancement of membrane separation technology for organic pollutant removal.

In the face of growing global freshwater scarcity, the imperative to recycle and reuse water becomes increasingly apparent across industrial, agricultural, and domestic sectors. Eliminating a range of organic pollutants in wastewater, from pesticides to industrial byproducts, presents a formidable challenge. Among the potential solutions, membrane technologies emerge as promising contenders for treating diverse organic contaminants from industrial, agricultural, and household origins. This paper explores cutting-edge membrane-based approaches, including reverse osmosis, nanofiltration, ultrafiltration, microfiltration, gas separation membranes, and pervaporation. Each technology's efficacy in removing distinct organic pollutants while producing purified water is scrutinized. This review delves into membrane fouling, discussing its influencing factors and preventative strategies. It sheds light on the merits, limitations, and prospects of these various membrane techniques, contributing to the advancement of wastewater treatment. It advocates for future research in membrane technology with a focus on fouling control and the development of energy-efficient devices. Interdisciplinary collaboration among researchers, engineers, policymakers, and industry players is vital for shaping water purification innovation. Ongoing research and collaboration position us to fulfill the promise of accessible, clean water for all.

Developing wastewater-based surveillance schemes for multiple pathogens: The WastPan project in Finland.

Wastewater comprises multiple pathogens and offers a potential for wastewater-based surveillance (WBS) to track the prevalence of communicable diseases. The Finnish WastPan project aimed to establish wastewater-based pandemic preparedness for multiple pathogens (viruses, bacteria, parasites, fungi), including antimicrobial resistance (AMR). This article outlines WastPan's experiences in this project, including the criteria for target selection, sampling locations, frequency, analysis methods, and results communication. Target selection relied on epidemiological and microbiological evidence and practical feasibility. Within the WastPan framework, wastewater samples were collected between 2021 and 2023 from 10 wastewater treatment plants (WWTPs) covering 40 % of Finland's population. WWTP selection was validated for reported cases of Extended Spectrum Beta-lactamase-producing bacterial pathogens (Escherichia coli and Klebsiella pneumoniae) from the National Infectious Disease Register. The workflow included 24-h composite influent samples, with one fraction for culture-based analysis (bacteria and fungi) and the rest of the sample was reserved for molecular analysis (viruses, bacteria, antibiotic resistance genes, and parasites). The reproducibility of the monitoring workflow was assessed for SARS-CoV-2 through inter-laboratory comparisons using the N2 and N1 assays. Identical protocols were applied to same-day samples, yielding similar positivity trends in the two laboratories, but the N2 assay achieved a significantly higher detection rate (Laboratory 1: 91.5 %; Laboratory 2: 87.4 %) than the N1 assay (76.6 %) monitored only in Laboratory 2 (McNemar, p < 0.001 Lab 1, = 0.006 Lab 2). This result indicates that the selection of monitoring primers and assays may impact monitoring sensitivity in WBS. Overall, the current study recommends that the selection of sampling frequencies and population coverage of the monitoring should be based on pathogen-specific epidemiological characteristics. For example, pathogens that are stable over time may need less frequent annual sampling, while those that are occurring across regions may require reduced sample coverage. Here, WastPan successfully piloted WBS for monitoring multiple pathogens, highlighting the significance of one-litre community composite wastewater samples for assessing community health. The infrastructure established for COVID-19 WBS is valuable for monitoring various pathogens. The prioritization of the monitoring targets optimizes resource utilization. In the future legislative support in target selection, coverage determination, and sustained funding for WBS is recomended.

Application of wastewater-based epidemiology for monitoring COVID-19 in hospital and housing wastewaters.

An alternative and complementary diagnostic method of surveillance is provided by wastewater-based surveillance (WBS), particularly in low-income nations like Nepal with scant wastewater treatment facilities and clinical testing infrastructure. In this study, a total of 146 water samples collected from two hospitals (n = 63) and three housing wastewaters (n = 83) from the Kathmandu Valley over the period of March 2021-Febraury 2022 were investigated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using quantitative reverse transcription TaqMan PCR assays targeting the N and E genes. Of the total, 67 % (98/146) samples were positive for SARS-CoV-2 RNA either by using N- or E-gene assay, with concentrations ranging from 3.6 to 9.1 log10 copies/L. There was a significant difference found between positive ratio (Chi-square test, p < 0.05) and concentration (t-test, p = 0.009) of SARS-CoV-2 RNA detected from hospital wastewater and housing waters. Wastewater data are correlated with COVID-19 active cases, indicating significance in specific areas like the Hospital (APFH) (p < 0.05). According to the application of a bivariate linear regression model (p < 0.05), the concentrations of N gene may be used to predict the COVID-19 cases in the APFH. Remarkably, SARS-CoV-2 RNA was detected prior to, during, and following clinical case surges, implying that wastewater surveillance could serve as an early warning system for public health decisions. The significance of WBS in tracking and managing pandemics is emphasized by this study, especially in resource-constrained settings.