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Abstract Reverse transcription-quantitative PCR (RT-qPCR) is widely used for monitoring viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in wastewater. Various materials, including plasmid DNA, synthetic nucleic acids, PCR amplicons, genomic DNA, and cDNA, are currently used for SARS-CoV-2 quantification by generating standard curves. We assessed three common standards on quantifying SARS-CoV-2 RNA across nine wastewater treatment plants in Finland, as part of the national wastewater surveillance effort. We pairwise compared RT-qPCR results from 148 wastewater samples, using both IDT (#10006625, IDT, USA) and CODEX standards (#SC2-RNAC-1100, CODEX DNA), and 179 samples using both IDT and EURM019 standards (#EURM-019, European Commission, Joint Research Centre) in our assessment. Amongst the tested standards, the CODEX standard consistently yielded more stable results than either the IDT or EURM019 standards. We found that SARS-CoV-2 levels were higher with the IDT standard (4.36 Log10 GC/100 mL) compared to the CODEX standard (4.05 Log10 GC/100 mL). Similarly, quantification using the IDT standard was higher (5.27 Log10 GC/100 mL) than values obtained with the EURM019 (4.81 Log10 GC/100 mL). SARS-CoV-2 RNA quantified with IDT and CODEX standards exhibited stronger concordance (Spearman’s correlation rho median of 0.79) compared to those quantified with IDT and EURM019 standards (rho median of 0.59). This study highlights the significant impact of standard material selection on SARS-CoV-2 RNA quantification, emphasizing the need for harmonization in standard material.

Antimicrobial resistance (AMR) poses a significant global One Health challenge that causes increased mortality and a high financial burden. Animal production contributes to AMR, as more than half of antimicrobials are used in food-producing animals globally. There is a growing body of literature on AMR in food-producing animals in African countries, but the surveillance practices across countries vary considerably. This pilot study aims to explore the potential of wastewater and environmental surveillance (WES) of AMR and its extension to the veterinary field. Floor drainage swab (n = 18, 3/abattoir) and wastewater (n = 16, 2-3/abattoir) samples were collected from six South African abattoirs that handle various animal species, including cattle, sheep, pig, and poultry. The samples were tested for Extended-Spectrum Beta-Lactamase (ESBL) and Carbapenemase-producing Enterobacterales, Methicillin-Resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococci (VRE), and Candida auris by using selective culturing and MALDI-TOF MS identification. The phenotype of all presumptive ESBL-producing Escherichia coli (n = 60) and Klebsiella pneumoniae (n = 24) isolates was confirmed with a disk diffusion test, and a subset (15 and 6 isolates, respectively), were further characterized by whole-genome sequencing. In total, 314 isolates (0–12 isolates/sample) withstood MALDI-TOF MS, from which 37 species were identified, E. coli and K. pneumoniae among the most abundant. Most E. coli (n = 48/60; 80%) and all K. pneumoniae isolates were recovered from the floor drainage samples, while 21 presumptive carbapenem-resistant Acinetobacter spp. isolates were isolated equally from floor drainage and wastewater samples. MRSA, VRE, or C. auris were not found. All characterized E. coli and K. pneumoniae isolates represented ESBL-phenotype. Genomic analyses revealed multiple sequence types (ST) of E. coli (n = 10) and K. pneumoniae (n = 5), including STs associated with food-producing animals globally, such as E. coli ST48 and ST10 and K. pneumoniae ST101. Common beta-lactamases linked to food-producing animals, such as blaCTX-M-55 and blaCTX-M-15, were detected. The presence of food-production-animal-associated ESBL-gene-carrying E. coli and K. pneumoniae in an abattoir environment and wastewater indicates the potential of WES in the surveillance of AMR in food-producing animals. Furthermore, the results of this pilot study encourage studying the topic further with refined methodologies.

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

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. HIGHLIGHTS Effective wastewater treatment helps to recycle and reuse water.; Membrane technologies are promising for treating wide organic contaminants.; This paper explores various advanced membrane techniques.; This review examines membrane fouling, including influencing factors and prevention strategies.; It illuminates the evolving membrane techniques, aiding the progress of wastewater treatment.;

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 (

ABSTRACT: Objectives: Analysing samples of municipal wastewater influent (before treatment) can help to map the status of antibiotic-resistant bacteria (ARB) at the population level in sewershed communities and may also help in predicting the public health risks of ARB in surface water because of the outfall of wastewater. In this study, we investigated the bacterial isolates carrying beta-lactamase genes in wastewater and compared their genotypic and phenotypic characteristics. Methods: A total of 399 bacterial isolates grown on CHROMagarESBL (n = 207) and CHROMagarKPC (n = 192) from composite wastewater influent samples (n = 7) from the Viikinmäki wastewater treatment plant (Helsinki) were subcultured, nucleic acid was extracted, and the prevalence of different beta-lactamase genes was screened with multiplex polymerase chain reaction (PCR). All PCR-positive isolates were identified with MALDI-TOF. Results: A total of 32.6% of isolates (130 of 399) were PCR positive for at least one resistance gene, and 13% of these positive isolates out of 130 had at least three resistance genes. Among the 22 detected genes, blaGES group was the most prevalent, at 25.8% (n = 198; many isolates carried multiple genes), followed by blaMOX (13.1%) and blaTEM (10.1%) as most frequently detected. Furthermore, out of 18 different bacterial species/genera detected as carrying beta-lactamase genes, A. hydrophila/caviae (28.5%), Enterobacter spp. (16.9%), and E. coli (14.6%) were the most prevalent. Enterobacter spp., Aeromonas spp., and K. cryocescens potentially carried AmpC genes, and E. coli carried ESBL genes. Conclusion: We recorded a huge variety of beta-lactamases (blaAmpC, blaESBL, and blaCARBA) genes in many potential pathogens that probably originated from both enteric and environmental sources.

The ongoing global pandemic caused by the SARS-CoV-2 virus, known as COVID-19, has disrupted public health, businesses, and economies worldwide due to its widespread transmission. While previous research has suggested a possible link between environmental factors and increased COVID-19 cases, the evidence regarding this connection remains inconclusive. The purpose of this research is to determine whether or not there is a connection between the presence of fine particulate matter (PM2.5) and meteorological conditions and COVID-19 infection rates in Bangkok, Thailand. The study employs a statistical method called the Generalized Additive Model (GAM) to find a positive and non-linear association between relative humidity (RH), absolute humidity (AH), and rainfall (R) and the number of verified COVID-19 cases. The impacts of the seasons (especially summer) and rainfall on the trajectory of COVID-19 cases were also highlighted, with an adjusted R-square of 0.852 and a deviance explained of 85.60%, both of which were statistically significant (p

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.

On July 23, 2022 the World Health Organization (WHO) has announced the Monkeypox disease (MPXD) as a worldwide public health issue. This study conducts a systematic review and meta-analysis to determine the overall case fatality rate (CFR) of MPXD worldwide during 1970–2022. The tenure-tracked MPXD outbreaks associated with CFR were calculated based on available published data from six different periods (i.e., 1970-79, 1980-89, 1990-99, 2000-09, 2010-19, and 2000-2022). A total of 229 peer-reviewed accessible articles were investigated, of which, 17 articles met the inclusion and exclusion criteria. Most of the studies on MPXD CFR were published in the Democratic Republic of the Congo (DRC) providing 47% of data for the current meta-analysis. The overall pooled CFR of MPXD was 4.14% (range: 0.62% – 9.51%) during 1970–2022. In this study, total of 379 death cases were found from published MPXV based research articles where the pooled estimate CFR was 1.87%. The pooled CFR was higher during the earlier outbreak of the MPXD such as 10.71% in 1970-1979. With the progress of time, the CFR from MPXD followed a decreasing trend and reached 5.38% in 1980-1999 and 4.45% in 2000-2022. Young male children aged< 10 years were found to experience the worst outcome with a CFR of >73.0%. This is the first meta-analysis using 52 years of data which indicates that the CFR of MPXV is decreasing from previous years. The findings of this meta-analysis might be paramount for the policymakers to tackle MPXD and minimize the overall CFR of MPXD through strategic actions.

Multi-drug resistance is emerging in Citrobacter freundii, which is the third most common carbapenemase-producing (CP) Enterobacteriaceae in humans in Finland due to recent outbreaks. The objective of this study was to determine if wastewater surveillance (WWS) could detect CP C. freundii strains causing infections in humans. Selective culturing was used to isolate CP C. freundii from the hospital environment, hospital wastewater, and untreated municipal wastewater in Helsinki, Finland, between 2019 and 2022. Species were identified using MALDI-TOF, and presumptive CP C. freundii isolates were subjected to antimicrobial susceptibility testing and further characterized by whole genome sequencing. A genomic comparison was conducted to compare isolates collected from the hospital environment, untreated municipal wastewater, and a selection of isolates from human specimens from two hospitals in the same city. We also examined the persistence of CP C. freundii in the hospital environment and the impact of our attempts to eradicate it. Overall, 27 blaKPC − 2-carrying C. freundii were detected in the hospital environment (ST18; n = 23 and ST8; n = 4), while 13 blaKPC − 2-carrying C. freundii (ST8) and five blaVIM − 1-carrying (ST421) C. freundii were identified in untreated municipal wastewater. CP C. freundii was not identified in hospital wastewater. We found three clusters (cluster distance threshold ≤ 10 allelic difference) after comparing the recovered isolates and a selection of isolates from human specimens. The first cluster consisted of ST18 isolates from the hospital environment (n = 23) and human specimens (n = 4), the second consisted of ST8 isolates from the hospital environment (n = 4), untreated municipal wastewater (n = 6), and human specimens (n = 2), and the third consisted of ST421 isolates from the untreated municipal wastewater (n = 5). Our results support previous studies suggesting that the hospital environment could act as a source of transmission of CP C. freundii in clinical settings. Furthermore, the eradication of CP Enterobacteriaceae from the hospital environment is challenging. Our findings also showed that CP C. freundii is persistent throughout the sewerage system and demonstrate the potential of WWS for detecting CP C. freundii.

Coronavirus disease 2019 (COVID-19) has spread across the globe producing hundreds of thousands of deaths, shutting down economies, closing borders and causing havoc on an unprecedented scale. Its potent effects have earned the attention of researchers in different fields worldwide. Among them, authors from different countries have published numerous research articles based on the environmental concepts of COVID-19. The environment is considered an essential receptor in the COVID-19 pandemic, and it is academically significant to look into publications to follow the pathway of hot topics of research and upcoming trends in studies. Reviewing the literature can therefore provide valuable information regarding the strengths and weaknesses in facing the COVID-19 pandemic, considering the environmental viewpoint. The present study categorizes the understanding caused by environmental and COVID-19-related published papers in the Scopus metadata from 2020 to 2021. VOSviewer is a promising bibliometric tool used to analyze the publications with keywords “COVID-19*” and “Environment.” Then, a narrative evaluation is utilized to delineate the most interesting research topics. Co-occurrence analysis is applied in this research, which further characterizes different thematic clusters. The published literature mainly focused on four central cluster environmental concepts: air pollution, epidemiology and virus transmission, water and wastewater, and environmental policy. It also reveals that environmental policy has gained worldwide interest, with the main keyword “management” and includes keywords like waste management, sustainability, governance, ecosystem, and climate change. Although these keywords could also appear in other environmental policy-related research studies, the importance of the COVID-19 pandemic requires such comprehensive research. The fourth cluster involves governance and management concerns encountered during the pandemic. Mapping the research topics in different clusters will pave the way for researchers to view future potential ideas and studies better. The scope for further research needs from the perspective of environmental concepts is reviewed and recommended, which can expand the vital role and value of environmental sciences in alerting, observing, and COVID-19 prediction for all four clusters. In other words, the research trend would shift from qualitative studies and perspectives to quantitative ones.

ObjectivesThe present study was conducted to detect the occurrence of β-lactamase and biofilm-producing Escherichia coli, Salmonella, and Klebsiella in broilers and native fowl reared in the Andaman and Nicobar Islands, India. The study also included molecular docking experiments to confirm the nature of the catalytic domains found in the β-lactamase variants obtained and to reveal the clonal relationship of the isolates with human clinical strains from the database.Materials and methodsA total of 199 cloacal swabs were collected from five poultry breeds/varieties (broiler, Vanraja, Desi, Nicobari, and layer) in three districts of the Andaman and Nicobar Islands. E. coli, Salmonella enterica, and Klebsiella pneumoniae were isolated by standard techniques and confirmed by PCR. Phenotypical β-lactamase producers were identified by a double-disc test. The genes (blaCTX, blaSHV, blaTEM, and blaAmpC) were screened, and selected sequences of β-lactamase variants were submitted to DDBJ. Homology modeling, model validation, and active site identification of different β-lactamase variants were done by the SWISS-MODEL. Molecular docking was performed to identify the catalytic domains of the β-lactamase variants. The selected β-lactamase sequences were compared with the Indian ESBL sequences from human clinical strains in NCBI-GenBank.ResultsIn total, 425 Enterobacteriaceae strains were isolated from the collected samples. Klebsiella pneumoniae (42.58%) was found to be the most prevalent, followed by Salmonella enterica (30.82%) and E. coli (26.58%). The phenotypical antibiogram of all 425 isolates showed the highest resistance against oxytetracycline (61–76%) and the lowest against gentamicin (15–20%). Phenotypical production of β-lactamase enzymes was observed in 141 (33.38%) isolates. The isolation rate of β-lactamase producing E. coli, Salmonella enterica, and Klebsiella pneumoniae was significantly higher (p < 0.05) in the birds reared in the South Andaman district (25.6, 17.5, and 18.7%, respectively) than in Nicobar (11.5, 7.6, 7.1%, respectively). Genotyping of the β-lactamase-producing isolates revealed the maximum possession of blaTEM, followed by blaSHV and blaCTX − M. The nucleotide sequences were found to be similar with blaCTX − M−15, blaSHV − 11, blaSHV − 27, blaSHV − 228, blaTEM − 1, and blaAmpC in BLAST search. Distribution of studied biofilm-associated genes in Enterobacteriaceae strains from different varieties of the birds revealed that the layer birds had the maximum possession, followed by Vanraja, Desi, broilers, and Nicobari fowls. The phylogenetic analysis of selected sequences revealed a partial clonal relationship with human clinical strains of the Indian subcontinent. Molecular docking depicted the Gibbs free energy release for 10 different macromolecules (proteins) and ligand (antibiotic) complexes, ranging from −8.1 (SHV-27 + cefotaxime) to −7 (TEM-1 + cefotaxime) kcal/mol.Conclusion and relevanceThe study revealed β-lactamase variants circulating in the fowl population of the Andaman and Nicobar Islands (India), even in remote places with low anthropogenic activity. Most of the strains possessed blaTEM − 1, followed by blaCTX − M−15. Possession of blaSHV − 11, blaSHV − 27, and blaSHV − 228 in poultry Enterobacteriaceae strains was not reported earlier from any part of the world. The phylogenetic analysis revealed a partial clonal relationship of β-lactamase sequences with the human clinical strains isolated from the Indian subcontinent.

Infectious diseases caused by antibiotic-resistant bacterial (ARB) pathogens are a serious threat to human and animal health. The active surveillance of ARB using an integrated one-health approach can help to reduce the emergence and spread of ARB, reduce the associated economic impact, and guide antimicrobial stewardship programs. Wastewater surveillance (WWS) of ARB provides composite samples for a total population, with easy access to the mixed community microbiome. This concept is emerging rapidly, but the clinical utility, sensitivity, and uniformity of WWS of ARB remain poorly understood especially in relation to clinical evidence in sewershed communities. Here, we systematically searched the literature to identify studies that have compared findings from WWS of ARB and antibiotic resistance genes (ARG) with clinical evidence in parallel, thereby evaluating how likely WWS of ARB and ARG can relate to the clinical cases in communities. Initially, 2,235 articles were obtained using the primary search keywords, and 1,219 articles remained after de-duplication. Among these, 35 articles fulfilled the search criteria, and an additional 13 relevant articles were included by searching references in the primary literature. Among the 48 included papers, 34 studies used a culture-based method, followed by 11 metagenomics, and three PCR-based methods. A total of 28 out of 48 included studies were conducted at the single sewershed level, eight studies involved several countries, seven studies were conducted at national or regional scales, and five at hospital levels. Our review revealed that the performance of WWS of ARB pathogens has been evaluated more frequently for Escherichia coli, Enterococcus spp., and other members of the family Enterobacteriaceae, but has not been uniformly tested for all ARB pathogens. Many wastewater-based ARB studies comparing the findings with clinical evidence were conducted to evaluate the public health risk but not to relate with clinical evidence and to evaluate the performance of WWS of ARB. Indeed, relating WWS of ARB with clinical evidence in a sewershed is not straightforward, as the source of ARB in wastewater cannot be only from symptomatic human individuals but can also be from asymptomatic carriers as well as from animal sources. Further, the varying fates of each bacterial species and ARG within the sewerage make the aim of connecting WWS of ARB with clinical evidence more complicated. Therefore, future studies evaluating the performance of many AMR pathogens and their genes for WWS one by one can make the process simpler and the interpretation of results easier.

Dengue virus (DENV) is an enveloped, single-stranded RNA virus that causes approximately 390 million infections, leading to 40,000 deaths annually. Due to the increasing trend of urbanization, water supply scarcity, and climate change, dengue is regarded as the “disease of the future,” requiring robust surveillance for the early detection of DENV infection. Since the virus is shed in urine and saliva and persists in wastewater at different temperatures, our study conducted wastewater surveillance as a novel approach to monitor dengue outbreaks in the Kathmandu Valley, Nepal. The viral concentrates (n = 34), which were previously collected and concentrated from municipal and hospital wastewater, and river water using the electronegative membrane-vortex method, were tested for DENV using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and digital PCR (RT-dPCR). Pepper mild mottle virus, a process control and endogenous biomarker, was detected in all the samples with concentrations ranging from 8.0 to 10.0 log10 copies/L, whereas DENV was not detected in any sample using RT-dPCR and RT-qPCR. The undetected DENV in this study could be attributed to the collection of grab wastewater samples during a low relative prevalence of dengue infection in the region, insufficient sample volume processed, probable viral nucleic acid degradation due to storage of viral concentrate at -25 °C for a long period of time, or inefficiency of the primary concentration method used. This study highlights critical gaps in knowledge and provides recommendations for future implementation of wastewater surveillance of dengue outbreaks, especially in regions where dengue was recently introduced, clinical surveillance is limited, and wastewater surveillance for polio has been adopted.

Wastewater surveillance has been widely used to track the prevalence of SARS-CoV-2 in communities. Although some studies have investigated the decay of SARS-CoV-2 RNA in wastewater, understanding about its fate during wastewater transport in real sewers is still limited. This study aims to assess the impact of sewer biofilms on the dynamics of SARS-CoV-2 RNA concentration in naturally contaminated real wastewater (raw influent wastewater without extra SARS-CoV-2 virus/gene seeding) using a simulated laboratory-scale sewer system. The results indicated that, with the sewer biofilms, a 90% concentration reduction of the SARS-CoV-2 RNA was observed within 2 h both in wastewater of gravity (GS, gravity-driven sewers) and rising main (RM, pressurized sewers) sewer reactors. In contrast, the 90% reduction time was 8–26 h in control reactors without biofilms. The concentration reduction of SARS-CoV-2 RNA in wastewater was significantly more in the presence of sewer biofilms. In addition, an accumulation of c.a. 260 and 110 genome copies/cm2 of the SARS-CoV-2 E gene was observed in the sewer biofilm samples from RM and GS reactors within 12 h, respectively. These results confirmed that the in-sewer concentration reduction of SARS-CoV-2 RNA in wastewater was likely caused by the partition to sewer biofilms. The need to investigate the in-sewer dynamic of SARS-CoV-2 RNA, such as the variation of RNA concentration in influent wastewater caused by biofilm attachment and detachment, was highlighted by the significantly enhanced reduction rate of SARS-CoV-2 RNA in wastewater of sewer biofilm reactors and the accumulation of SARS-CoV-2 RNA in sewer biofilms. Further research should be conducted to investigate the in-sewer transportation of SARS-CoV-2 and their RNA and evaluate the role of sewer biofilms in leading to underestimates of COVID-19 prevalence in communities.

Antimicrobial resistance profiling of pathogens helps to identify the emergence of rare or new resistance threats and prioritize possible actions to be taken against them. The analysis of wastewater (WW) can reveal the circulation of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARG) among the catchment communities. Here, we analyzed WW influent samples to determine the prevalence of carbapenemase genes-carrying Gram-negative bacteria (Carba-GNB) in Helsinki, Finland. This study set important historical reference points from the very early stage of the carbapenemase era, during the period 2011–2012. A total of 405 bacterial isolates grown on CHROMagarKPC (n = 195) and CHROMagarESBL (n = 210) from WW influent samples were collected between October 2011 and August 2012 and were analyzed. The bacterial DNA from the isolates was extracted, and the prevalence of carbapenemases genes blaKPC, blaNDM, blaGES, blaOXA-48, blaIMP, blaIMI, and blaVIM were screened with multiplexed PCR. All carbapenemase-positive isolates were identified taxonomically to species or genus level with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The nucleic acid extraction was successful for 399 isolates, of which 59 (14.8%) were found to carry carbapenemase genes. A total of 89.8% of the carbapenemase positive isolates (53 out of 59) were obtained from CHROMagarKPC plates and only 10.2% (six out of 59) were obtained from CHROMagar ESBL plates. Among the Carba-GNB isolates, 86.4% were blaGES (51 out of 59), 10.2% were blaKPC (six out of 59), and 3.4% were blaVIM (two out of 59). The most common carba-gene, blaGES, was carried by 10 different bacterial species, including Aeromonas spp., Enterobacter spp., and Kluyvera spp.; the blaKPC gene was carried by Escherichia coli, Klebsiella pneumoniae, and Kluyvera cryocescens; and the blaVIM gene was carried by Aeromonas hydrophila/caviae and Citrobacter amalonaticus. This study emphasizes that wastewater surveillance (WWS) can be an additional tool for monitoring antimicrobial resistance (AMR) at the population level.

Abstract Background Rivers and lakes are used for multiple purposes such as for drinking water (DW) production, recreation, and as recipients of wastewater from various sources. The deterioration of surface water quality with wastewater is well-known, but less is known about the bacterial community dynamics in the affected surface waters. Understanding the bacterial community characteristics —from the source of contamination, through the watershed to the DW production process—may help safeguard human health and the environment. Results The spatial and seasonal dynamics of bacterial communities, their predicted functions, and potential health-related bacterial (PHRB) reads within the Kokemäenjoki River watershed in southwest Finland were analyzed with the 16S rRNA-gene amplicon sequencing method. Water samples were collected from various sampling points of the watershed, from its major pollution sources (sewage influent and effluent, industrial effluent, mine runoff) and different stages of the DW treatment process (pre-treatment, groundwater observation well, DW production well) by using the river water as raw water with an artificial groundwater recharge (AGR). The beta-diversity analysis revealed that bacterial communities were highly varied among sample groups (R = 0.92, p

The emergence of new variants of SARS-CoV-2 associated with varying infectivity, pathogenicity, diagnosis, and effectiveness against treatments challenged the overall management of the COVID-19 pandemic. Wastewater surveillance (WWS), i.e., monitoring COVID-19 infections in communities through detecting viruses in wastewater, was applied to track the emergence and spread of SARS-CoV-2 variants globally. However, there is a lack of comprehensive understanding of the use and effectiveness of WWS for new SARS-CoV-2 variants. Here we systematically reviewed published articles reporting monitoring of different SARS-CoV-2 variants in wastewater by following the PRISMA guidelines and provided the current state of the art of this study area. A total of 80 WWS studies were found that reported different monitoring variants of SARS-CoV-2 until November 2022. Most of these studies (66 out of the total 80, 82.5%) were conducted in Europe and North America, i.e., resource-rich countries. There was a high variation in WWS sampling strategy around the world, with composite sampling (50/66 total studies, 76%) as the primary method in resource-rich countries. In contrast, grab sampling was more common (8/14 total studies, 57%) in resource-limited countries. Among detection methods, the reverse transcriptase polymerase chain reaction (RT-PCR)-based sequencing method and quantitative RT-PCR method were commonly used for monitoring SARS-CoV-2 variants in wastewater. Among different variants, the B1.1.7 (Alpha) variant that appeared earlier in the pandemic was the most reported (48/80 total studies), followed by B.1.617.2 (Delta), B.1.351 (Beta), P.1 (Gamma), and others in wastewater. All variants reported in WWS studies followed the same pattern as the clinical reporting within the same timeline, demonstrating that WWS tracked all variants in a timely way when the variants emerged. Thus, wastewater monitoring may be utilized to identify the presence or absence of SARS-CoV-2 and follow the development and transmission of existing and emerging variants. Routine wastewater monitoring is a powerful infectious disease surveillance tool when implemented globally.

Information on the co-occurrence of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) among bacterial communities in drinking water distribution systems (DWDSs) is scarce. This study characterized ARGs and MRGs in five well-maintained DWDSs in Finland. The studied DWDSs had different raw water sources and treatment methods. Two of the waterworks employed artificially recharged groundwater (ARGW) and used no disinfection in the treatment process. The other three waterworks (two surface and one groundwater source) used UV light and chlorine during the treatment process. Ten bulk water samples (two from each DWDS) were collected, and environmental DNA was extracted and then sequenced using the Illumina HiSeq platform for high-throughput shotgun metagenome sequencing. A total of 430 ARGs were characterized among all samples with the highest diversity of ARGs identified from samples collected from non-disinfected DWDSs. Furthermore, non-disinfected DWDSs contained the highest diversity of bacterial communities. However, samples from DWDSs using disinfectants contained over double the ratio of ARG reads to 16S rRNA gene reads and most of the MRG (namely mercury and arsenic resistance genes). The total reads and types of ARGs conferring genes associated with antibiotic groups namely multidrug resistance, and bacitracin, beta-lactam, and aminoglycoside and mercury resistance genes increased in waterworks treating surface water with disinfection. The findings of this study contribute toward a comprehensive understanding of ARGs and MRGs in DWDSs. The occurrence of bacteria carrying antibiotic or metal resistance genes in drinking water causes direct exposure to people, and thus, more systematic investigation is needed to decipher the potential effect of these resistomes on human health.

The knowledge about the members of active archaea communities in DWDS is limited. The current understanding is based on high-throughput 16S ribosomal RNA gene (DNA-based) amplicon sequencing that reveals the diversity of active, dormant, and dead members of the prokaryote (bacteria, archaea) communities. The sequencing primers optimized for bacteria community analysis may underestimate the share of the archaea community. This study characterized archaea communities at five full-scale drinking water distribution systems (DWDS), representing a variety of drinking water production units (A-E); A&B use artificially recharged non-disinfected groundwater (ARG), the other DWDS's supplied water disinfected by using ultraviolet (UV) light and chlorine compounds, C&D were surface waterworks and E was a ground waterworks. For the first time for archaea community analyses, this study employed the archaea-specific high-throughput sequencing primers for 16S ribosomal RNA (rRNA) as a target (reverse-transcribed cDNA; an RNA-based approach) in addition to the previously used 16S rRNA gene target (rDNA; a DNA-based approach) to reveal the active fraction of the archaea present in DWDS. The archaea community structure in varying environmental conditions in the water and biofilm of the five DWDSs were investigated by taking into consideration the system properties (cold or hot water system) and water age (distance from the treatment plants) in samples from each season of one year.The RNA-based archaea amplicon reads were obtained mostly from cold water samples from DWDSs (A–B) distributing water without disinfection where the DNA-based and RNA-based analysis created separate clusters in a weighted beta-diversity analysis. The season and location in DWDS A further affected the diversity of these archaea communities as was seen by different clusters in beta-diversity plots. The recovery of archaea reads was not adequate for analysis in any of the disinfected samples in DWDSs C–E or non-disinfected hot water in DWDSs A–B when utilizing RNA-based template. The metabolically active archaea community of DWDSs thus seemed to be effectively controlled by disinfection of water and in the hot water systems by the temperature. All biofilms regardless of DWDS showed lower species richness values (mainly Nitrososphaeria class) than non-disinfected water from DWDSs A–B where several archaea classes occurred (e.g. Woesearchaeia, Nitrososphaeria, Micrarchaeia, Methanomicrobia, Iairchaeia, Bathyarchaeia) indicating only part of the archaea members were able to survive in biofilms. Thus, Archaea has been shown as a significant part of normal DWDS biota, and their role especially in non-disinfected DWDS may be more important than previously considered.

For microbial source tracking (MST), the 16S ribosomal RNA genes (rDNA) of host-specific bacteria and mitochondrial DNA (mtDNA) of animal species, known to cause fecal contamination of water, have been commonly used as molecular targets. However, low levels of contamination might remain undetected by using these DNA-based qPCR assays. The high copy numbers of ribosomal RNA (rRNA) could offer a solution for such applications of MST. This study compared the performance of eight MST assays: GenBac3 (general Bacteroidales), HF183 (human), BacCan (dog), Rum-2-Bac (ruminant), Pig-2-Bac (swine), Gull4 (gull), GFD, and Av4143 (birds) between rRNA-based and rDNA-based approaches. Three mtDNA-based approaches were tested: DogND5, SheepCytB, and HorseCytB. A total of 151 animal fecal samples and eight municipal sewage samples from four regions of Finland were collected for the marker evaluation. The usability of these markers was tested by using a total of 95 surface water samples with an unknown pollution load. Overall, the performance (specificity, sensitivity, and accuracy) of mtDNA-based assays was excellent (95–100%), but these markers were very seldom detected from the tested surface water samples. The rRNA template increased the sensitivity of assays in comparison to the rDNA template. All rRNA-based assays (except Av4143) had more than 80% sensitivity. In contrast, only half (HF183, Rum-2-Bac, Pig-2-Bac, and Gull4) of rDNA-based assays reached this value. For markers targeted to bird feces, the use of the rRNA-based assay increased or at least did not change the performance. Regarding specificity, all the assays had >95% specificity with a DNA template, except the BacCan assay (71%). While using the RNA template for the assays, HF183 and BacCan exhibited only a low level of specificity (54 and 55%, respectively). Further, the HF183 assay amplified from multiple non-targeted animal fecal samples with the RNA template and the marker showed cross-amplification with the DNA template as well. This study recommends using the rRNA-based approach for MST assays targeting bird fecal contamination. In the case of mammal-specific MST assays, the use of the rRNA template increases the sensitivity but may reduce the specificity and accuracy of the assay. The finding of increased sensitivity calls for a further need to develop better rRNA-based approaches to reach the required assay performance.

Enterococci are fecal indicator bacteria (FIB) used for monitoring the microbial quality of bathing water. However, the reliability of health protection by monitoring FIB is questioned. This study evaluated the decay pattern of Enterococcus faecalis in beach environment and compared it with decay patterns of the pathogen Vibrio cholerae and the virus indicator MS2 coliphage. Tests were done in an experimental mesocosm simulating natural Nordic summer daylight conditions with and without the aquatic plant Myriophyllum sibiricum. The decay of the spiked test microbes (E. faecalis, V. cholera, and MS2) was enumerated up to 27 days from two coastal bathing water mesocosms with subtidal sediment. E. faecalis and V. cholerae exhibited non-linear biphasic decay patterns and were detected upmost toward the end of the experiment in the water, sediment, and vegetation. The gene copies of V. cholerae dropped to a minimum by days 6–8 but then the numbers increased back up to nearly the spiked level. The MS2 coliphage was detected only up to 8–10 days into the experiment solely in the water where a log-linear decay pattern was seen. The test microbe, sample origin (water, sediment or vegetation) and, as determined for E. faecalis, the enumeration method (culture-based or qPCR) affected the decay pattern. E. faecalis decayed faster in water than in sediment and vegetation. Positive correlations between culturable E. faecalis counts with V. cholerae gene copies and MS2 counts were recorded on the first few days of the experiment. This study demonstrated the important role of water, sediment and vegetation regarding the partitioning of pathogens and fecal indicators in coastal environment. The enumeration of the enterococci counts alone was not sufficient for predicting the numbers of pathogens such as Vibrio spp. in bathing water. The growth of Vibrio spp. in the Baltic Sea deserves more attention and might require water quality monitoring to be applied for these pathogens in the coming years due to the predicted rise in sea surface temperature favoring Vibrio spp. growth. Further, different decay patterns observed between MS2 and enterococci emphasize the need for and importance of a viral indicator in assessing water quality more comprehensively.