Antimicrobials and antimicrobial resistance genes in the shadow of COVID-19 pandemic: A wastewater-based epidemiology perspective.

Higher usage of antimicrobial agents in both healthcare facilities and the communities has resulted in an increased spread of resistant bacteria. However, the improved infection prevention and control practices may also contribute to decreasing antimicrobial resistance (AMR). In the present study, wastewater-based epidemiology (WBE) approach was applied to explore the link between COVID-19 and the community usage of antimicrobials, as well as the prevalence of resistance genes. Longitudinal study has been conducted to monitor the levels of 50 antimicrobial agents (AAs), 24 metabolites, 5 antibiotic resistance genes (ARGs) and class 1 integrons (intI 1) in wastewater influents in 4 towns/cities over two years (April 2020 - March 2022) in the South-West of England (a total of 1,180 samples collected with 87,320 individual AA measurements and 8,148 ARG measurements). Results suggested higher loads of AAs and ARGs in 2021-22 than 2020-21, with beta-lactams, quinolones, macrolides and most ARGs showing statistical differences. In particular, the intI 1 gene (a proxy of environmental ARG pollution) showed a significant increase after the ease of the third national lockdown in England. Positive correlations for all quantifiable parent AAs and metabolites were observed, and consumption vs direct disposal of unused AAs has been identified via WBE. This work can help establish baselines for AMR status in communities, providing community-wide surveillance and evidence for informing public health interventions. Overall, studies focused on AMR from the start of the pandemic to the present, especially in the context of environmental settings, are of great importance to further understand the long-term impact of the pandemic on AMR.

Assessment of restriction measures implemented during COVID pandemics on community lifestyle choices via wastewater-based epidemiology.

SARS-CoV-2 pandemic affected lifestyle habits, and the mental health and wellbeing of people around the world. In this manuscript, two towns (Paulton and Radstock) and two cities (Bath and Bristol) in Southwest England (> 1 million people) were monitored for two years using Wastewater-Based Epidemiology to assess impacts of COVID (including management measures such as lockdowns and movement restrictions) on community lifestyle choices: illicit drugs, legal stimulants, abused pharmaceuticals and pain pharma usage. Results were triangulated with key dates captured during the pandemic (national lockdowns, restrictions and social distancing measures, etc.). This highlighted a reduction in cocaine intake (as benzoylecgonine) (community average: -36 %) during the first lockdown and an increase in illicit drugs usage after the 3rd national lockdown (community averages for amphetamine: +8 %, cocaine/benzoylecgonine: +39 %, ketamine:+70 %) when restrictions were removed, and social interaction and recreational activities increased. There was a reduction in the intake of caffeine (as 1,7-dimethylxanthine) (community average:-39 %) after 3rd national lockdown coinciding with pubs reopening while nicotine intake (via cotinine) remained stable indicating lack of impact of COVID on smoking habits. Pain pharma often used in pain management resulting from injuries linked with sport activities (naproxen and diclofenac) showed decrease in usage due to lockdown restrictions in physical exercise and access to gyms/sport facilities.

Understanding treatment of pain during SARS-CoV-2 pandemic in a two-year intercity longitudinal study using wastewater-based epidemiology.

SARS-CoV-2 pandemic had a significant impact on the society, economy, and health of people around the world with consequences that need to be better understood for future pandemic preparedness. This manuscript provides insights into the usage of pharmaceuticals for pain treatment management throughout SARS-CoV-2 pandemic. Four towns and cities with a total population of > 1 million people covering an area of 2000 km2 in South West England were monitored for twenty-four months. Results showed different patterns in pain pharma usage, with small towns having higher population normalised daily loads (PNDLs) than big cities for majority of pain killers studied. This is likely due to demographics of these cities with smaller cities having older population. Per capita consumption of non-steroidal anti-inflammatory drugs (NSAIDs) increased compared to pre-pandemic usage in line with SARS-CoV-2 infections (ibuprofen and acetaminophen), while body pain drugs (diclofenac and naproxen) decreased in line with restrictions and closure of sports facilities. Changes in population normalised daily intake (PNDI) of pain killers were particularly apparent during the 1st and 3rd national lockdown. Comparison of PNDIs with prescriptions highlighted differences related to medication availability (OTC drugs) and patients' nonadherence (prescribed drugs). In addition, several instances of direct disposal events across the catchments were observed which raises an issue of lack of pharma compliance and general understanding of potential environmental impacts from pharma usage.

Understanding associations between antimicrobial agents usage and antimicrobial resistance genes prevalence at the community level using wastewater-based epidemiology: A Spanish pilot study.

Understanding the development and spread of antimicrobial resistance (AMR) is important for combating this global threat for public health. Wastewater-based epidemiology (WBE) is a complementary approach to current surveillance programs that minimizes some of the existing limitations. The aim of the present study is to explore WBE for monitoring antibiotics and antibiotic resistance genes (ARGs) in wastewater samples collected during 2021/2022 from the city of Castellon (Spain). Eighteen commonly prescribed antibiotics have been selected and measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), with triple quadrupole mass analysers. Moreover, qPCR for specific ARGs has been performed to obtain information of these genes in co-presence with antibiotics. All selected ARGs, along with a total of 11 antibiotics, were identified. The highest population-normalized daily loads were observed for the macrolide azithromycin, followed by the quinolones ciprofloxacin and levofloxacin. Subsequently, daily consumption estimates based on wastewater data were compared with prescription data of antibiotics. Statistical analyses were conducted to explore if there is correlation between antibiotics and ARGs. While no correlations were found between antibiotics and their corresponding ARGs, certain correlations (p < 0.05) were identified among non-corresponding ARGs. In addition, a strong positive correlation was found between the sum of all antibiotics and the intl1 gene. Moreover, population-normalized ARG loads significantly correlate with the 16S rRNA-normalized ARG loads, serving as an indicator for population size. Results provide a baseline for future work and a proof-of-concept emphasising the need for future work and long-term surveillance, and highlight the need of similar programs at a regional and global levels worldwide.

Influent wastewater analysis to investigate emerging trends of new psychoactive substances use in Europe.

Wastewater-based epidemiology (WBE) can provide objective and timely information on the use of new psychoactive substances (NPS), originally designed as legal alternatives of internationally controlled drugs. NPS have rapidly emerged on the global drug market, posing a challenge to drug policy and constituting a risk to public health. In this study, a WBE approach was applied to monitor the use of more than 300 NPS, together with fentanyl and its main metabolite norfentanyl, in influent wastewater collected from 12 European cities during March-June 2021. Quantitative and qualitative analysis of NPS in composite 24 h influent wastewater samples were based on solid phase extraction and liquid chromatography-mass spectrometry. In-sample stability tests demonstrated the suitability of most investigated biomarkers, except for a few synthetic opioids, synthetic cannabinoids and phenetylamines. Fentanyl, norfentanyl and eight NPS were quantified in influent wastewater and at least three substances were found in each city, demonstrating their use in Europe. N,N-dimethyltryptamine and 3-methylmethcathinone (3-MMC) were the most common NPS found, with the latter having the highest mass loads (up to 24.8 mg/day/1000 inhabitants). Seven additional substances, belonging to five categories of NPS, were identified in different cities. Spatial trends of NPS use were observed between cities and countries, and a changing weekly profile of use was observed for 3-MMC. WBE is a useful tool to rapidly evaluate emerging trends of NPS use, complementing common indicators (i.e. population surveys, seizures) and helping to establish measures for public health protection.

Microbial community and antimicrobial resistance niche differentiation in a multistage, surface flow constructed wetland.

Free-living (FL) and particulate-associated (PA) communities are distinct bacterioplankton lifestyles with different mobility and dissemination routes. Understanding spatio-temporal dynamics of PA and FL fractions will allow improvement to wastewater treatment processes including pathogen and AMR bacteria removal. In this study, PA, FL and sediment community composition and antimicrobial resistance gene (ARG; tetW, ermB, sul1, intI1) dynamics were investigated in a full-scale municipal wastewater free-water surface polishing constructed wetland. Taxonomic composition of PA and FL microbial communities shifted towards less diverse communities (Shannon, Chao1) at the CW effluent but retained a distinct fraction-specific composition. Wastewater treatment plant derived PA communities introduced the bulk of AMR load (70 %) into the CW. However, the FL fraction was responsible for exporting over 60 % of the effluent AMR load given its high mobility and the effective immobilization (1-3 log removal) of PA communities. Strong correlations (r2>0.8, p < 0.05) were observed between the FL fraction, tetW and emrB dynamics, and amplicon sequence variants (ASVs) of potentially pathogenic taxa, including Bacteroides, Enterobacteriaceae, Aeromonadaceae, and Lachnospiraceae. This study reveals niche differentiation of microbial communities and associated AMR in CWs and shows that free-living bacteria are a primary escape route of pathogenic and ARG load from CWs under low-flow hydraulic conditions.

A perceptual approach to address complex water management issues in lowland permeable catchments.

Water quality management is a pressing global concern, and an increasingly complex issue due to climate and land-use change, legacy pollution, and the persistent release of well-known and emerging contaminants from diffuse and point sources. The increasing availability of high-frequency monitoring data is leading existing, often heuristic approaches, to be judged inadequate. Water managers frequently rely on simple qualitative and/or quantitative approaches for decision-making, but a lack of tangible improvement in freshwater quality outcomes is demanding new and innovative approaches that rely more on physical process understanding, rather than precedent. In this study, we drew upon local geological, hydrogeological, and hydrological data to infer a high-level perceptual model of surface/groundwater interactions in a chalk stream in Dorset, UK. We used the perceptual model to interrogate spatial and temporal trends in historical water quality data and to construct reach-scale nutrient mass balances. Through novel representation with Sankey diagrams, the perceptual model highlighted the relative importance of different hydrological features. Surface/groundwater interactions were found to occur predominantly by spring flow. We demonstrate that river flow accretion was dominated by the Chalk aquifer despite only occupying ca. 15 % of the surface bedrock area, and that spring sources, whilst vital to dilute treated sewage inputs in baseflow conditions, were also major sources of legacy nitrate. Nutrient mass balances showed that sewage treatment works contributed ca. 13 % to soluble reactive phosphorus load, with groundwater accounting for ca. 48 %. Thus, a determinand often associated with point-source pollution was shown to be diffuse dominated in this river. The study demonstrated how a multi-disciplinary approach to water management, based on a comprehensive perceptual modelling approach, could identify hitherto unknown sources and relative contributors to freshwater pollution and allow flow and load apportionment studies to provide useful decision-support to manage nutrient pollution. The novel application of perceptual modelling tools, such as the Sankey diagram, allows different source attributions to be presented in an accessible manner, and can be readily transferred to other study areas.

LoCKAmp: lab-on-PCB technology for <3 minute virus genetic detection.

The recent COVID-19 outbreak highlighted the need for lab-on-chip diagnostic technology fit for real-life deployment in the field. Existing bottlenecks in multistep analytical microsystem integration and upscalable, standardized fabrication techniques delayed the large-scale deployment of lab-on-chip solutions during the outbreak, throughout a global diagnostic test shortage. This study presents a technology that has the potential to address these issues by redeploying and repurposing the ubiquitous printed circuit board (PCB) technology and manufacturing infrastructure. We demonstrate the first commercially manufactured, miniaturised lab-on-PCB device for loop-mediated isothermal amplification (LAMP) genetic detection of SARS-CoV-2. The system incorporates a mass-manufactured, continuous-flow PCB chip with ultra-low cost fluorescent detection circuitry, rendering it the only continuous-flow µLAMP platform with off-the-shelf optical detection components. Ultrafast, SARS-CoV-2 RNA amplification in wastewater samples was demonstrated within 2 min analysis, at concentrations as low as 17 gc µL-1. We further demonstrate our device operation by detecting SARS-CoV-2 in 20 human nasopharyngeal swab samples, without the need for any RNA extraction or purification. This renders the presented miniaturised nucleic-acid amplification-based diagnostic test the fastest reported SARS-CoV-2 genetic detection platform, in a practical implementation suitable for deployment in the field. This technology can be readily extended to the detection of alternative pathogens or genetic targets for a very broad range of applications and matrices. LoCKAmp lab-on-PCB chips are currently mass-manufactured in a commercial, ISO-compliant PCB factory, at a small-scale production cost of £2.50 per chip. Thus, with this work, we demonstrate a high technology-readiness-level lab-on-chip-based genetic detection system, successfully benchmarked against standard analytical techniques both for wastewater and nasopharyngeal swab SARS-CoV-2 detection.

Assessment of the stability of antimicrobials and resistance genes during short- and long-term storage condition: accounting for uncertainties in bioanalytical workflows.

Unravelling complexities in antimicrobial agent-microbe interactions in the context of antimicrobial resistance (AMR) requires robust analytical workflows accounting for all uncertainties. Temporal storage of wastewater samples under refrigerated or frozen conditions prior to chemical and biological analysis is widely used to facilitate laboratory routine but may affect stability of analytes over time. Yet, little knowledge exists regarding stability of biological and chemical determinants in environmental samples, which hampers validity of research outputs. This study examines, for the first time, the stability of 32 antimicrobials (AAs) including commonly used classes of antibiotics and their representative metabolites and variation of 5 antibiotic resistance genes (ARGs) (ermB, sul1, tetW, blaCTX-M, qnrS), as well as intI1 and 16S rRNA genes in a reference wastewater sample stored under freezing condition for up to 1 year. Ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) and quantitative PCR (qPCR) techniques were adopted to measure concentration of AAs and ARGs, respectively. Results suggested that parent compounds are less affected by freezing storage compared to the metabolites. ß-Lactams, clindamycin, and N-desmethyl clindamycin are the most-affected compounds which were poorly recovered (34-67%) from the starting concentration. By contrast, sulfonamides, macrolides, quinolones, and azoles are generally stable under freezing condition. No consistent differences were observed in gene copies between fresh and frozen samples, and ermB and tetW showed the highest variabilities at 30% under freezing condition. Overall, this study adds to the current knowledge on environmental AMR monitoring and emphasises the need for standardised protocols for AMR monitoring in the wastewater samples.

Wastewater profiling of illicit drugs, an estimation of community consumption: A case study of eThekwini Metropolitan Municipality, South Africa.

Estimation of community-wide consumption of illicit drugs through wastewater analysis is a new concept in Africa although widely applied in developed nations as wastewater-based epidemiology (WBE). It is an important tool that can be used in understanding supply and demand patterns of illicit drug use on a local, national, and international scale. Information on illicit drug use is currently limited in Africa, because of a lack of monitoring structures by governments and financial constraints. This study hopes to bridge that gap by contributing to Africa's baseline information on illicit drug use. This study provides the first application of wastewater analysis to quantitatively evaluate daily illicit drug use in the eThekwini Metropolitan Municipality of KwaZulu-Natal Province of South Africa by quantifying the major urinary excreted metabolites, called drug target residues (DTRs), in raw wastewater from four major wastewater treatment plants (WWTPs) sampled for a week. The results showed that cocaine was the dominant illicit drug consumed in the catchment followed by amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine, with a per capita use of 360-3000 mg day-1 1000 inh-1, 47-800 mg day-1 1000 inh-1, 19-120 mg day -1 1000 inh-1, not detected (ND) to 4.9 mg day-1 1000 inh-1, and ND to 410 g day-1 1000 inh-1 respectively. The weekly usage patterns between the four WWTPs differed probably due to the lifestyle of the populace serving the different WWTPs. These results provide useful data on illicit drug use in eThekwini that can be utilised by public health agencies to implement suitable response strategies.

An integrated One Health framework for holistic evaluation of risks from antifungal agents in a large-scale multi-city study.

A new framework for retrospective mass spectral data mining for antifungal agents (AFs) and Wastewater-Based Epidemiology (WBE) was developed as part of One Health framework to tackle risks from AFs. A large scale, multi-city study was undertaken in South-West England. Key drivers of AFs in the catchment were identified with communal wastewater discharges being the main driver for human AFs (fluconazole, ketoconazole) and agricultural runoff being the main driver for pesticide AFs (prochloraz, prothioconazole and tebuconazole). Average WBE-estimated human used fluconazole and ketoconazole PNDIs (population normalised daily intake) exceeded 300 mg day-1 1000 inh-1 and 2000 mg day-1 1000 inh-1. This is much higher than PNDPs (population normalised daily prescriptions <40 mg day-1 1000 inh-1 and <80 mg day-1 1000 inh-1 for fluconazole and ketoconazole respectively). This was expected due to both prescription and over-the-counter usage, and both oral and topical applications. Pesticide AF, prothioconazole had PNDIs <40,000 mg day-1 1000 inh -1, which gave intake: 0.43, 0.26, 0.07 mg kg-1 in City A, B, and C, likely due to accounting for external/non-human sources. This is higher than the acceptable daily intake (ADI) of 0.01 mg kg-1bw day-1, which warrants further study. Intake per kg of body weight estimated using tebuconazole was 0.86, 1.39, 0.12, 0.13, and 2.7 mg kg-1 in City A-E respectively and is likely due to external/non-human sources. Intake calculated using its metabolite was 0.02 and 0.01 mg kg-1 in City B and C respectively, which aligned with ADI (0.03 mg kg-1bw day-1). The environmental risk assessment of AFs indicated low/medium risk from fluconazole, prochloraz, and tebuconazole, medium risk from epoxiconazole, prothioconazole's metabolite, and tebuconazole, and high risk for prothioconazole in river water. High risk was estimated from fluconazole, epoxiconazole, prothioconazole and its metabolite, tebuconazole, ketoconazole in wastewater samples, which is important during raw sewage discharge events via sewer overflows.

Antimicrobials and antimicrobial resistance genes in a one-year city metabolism longitudinal study using wastewater-based epidemiology.

This longitudinal study tests correlations between antimicrobial agents (AA) and corresponding antimicrobial resistance genes (ARGs) generated by a community of >100 k people inhabiting one city (Bath) over a 13 month randomised monitoring programme of community wastewater. Several AAs experienced seasonal fluctuations, such as the macrolides erythromycin and clarithromycin that were found in higher loads in winter, whilst other AA levels, including sulfamethoxazole and sulfapyridine, stayed consistent over the study period. Interestingly, and as opposed to AAs, ARGs prevalence was found to be less variable, which indicates that fluctuations in AA usage might either not directly affect ARG levels or this process spans beyond the 13-month monitoring period. However, it is important to note that weekly positive correlations between individual associated AAs and ARGs were observed where seasonal variability in AA use was reported: ermB and macrolides CLR-clarithromycin and dmCLR-N-desmethyl clarithromycin, aSPY- N-acetyl sulfapyridine and sul1, and OFX-ofloxacin and qnrS. Furthermore, ARG loads normalised to 16S rRNA (gene load per microorganism) were positively correlated to the ARG loads normalised to the human population (gene load per capita), which indicates that the abundance of microorganisms is proportional to the size of human population and that the community size, and not AA levels, is a major driver of ARG levels in wastewater. Comparison of hospital and community wastewater showed higher number of AAs and their metabolites, their frequency of occurrence and concentrations in hospital wastewater. Examples include: LZD-linezolid (used only in severe bacterial infections) and AMX-amoxicillin (widely used, also in community but with very low wastewater stability) that were found only in hospital wastewater. CIP-ciprofloxacin, SMX-sulfamethoxazole, TMP-trimethoprim, MTZ-metronidazole and macrolides were found at much higher concentrations in hospital wastewater while TET-tetracycline and OTC-oxytetracycline, as well as antiretrovirals, had an opposite trend. In contrast, comparable concentrations of resistant genes were observed in both community and hospital wastewater. This supports the hypothesis that AMR levels are more of an endemic nature, developing over time in individual communities. Both hospital and community wastewater had AAs that exceeded PNEC values (e.g. CLR-clarithromycin, CIP-ciprofloxacin). In general, though, hospital effluents had a greater number of quantifiable AAs exceeding PNECs (e.g. SMX-sulfamethoxazole, ERY-erythromycin, TMP-trimethoprim). Hospitals are therefore an important consideration in AMR surveillance as could be high risk areas for AMR.

Wastewater monitoring for detection of public health markers during the COVID-19 pandemic: Near-source monitoring of schools in England over an academic year.

BACKGROUND: Schools are high-risk settings for infectious disease transmission. Wastewater monitoring for infectious diseases has been used to identify and mitigate outbreaks in many near-source settings during the COVID-19 pandemic, including universities and hospitals but less is known about the technology when applied for school health protection. This study aimed to implement a wastewater surveillance system to detect SARS-CoV-2 and other public health markers from wastewater in schools in England. METHODS: A total of 855 wastewater samples were collected from 16 schools (10 primary, 5 secondary and 1 post-16 and further education) over 10 months of school term time. Wastewater was analysed for SARS-CoV-2 genomic copies of N1 and E genes by RT-qPCR. A subset of wastewater samples was sent for genomic sequencing, enabling determination of the presence of SARS-CoV-2 and emergence of variant(s) contributing to COVID-19 infections within schools. In total, >280 microbial pathogens and >1200 AMR genes were screened using RT-qPCR and metagenomics to consider the utility of these additional targets to further inform on health threats within the schools. RESULTS: We report on wastewater-based surveillance for COVID-19 within English primary, secondary and further education schools over a full academic year (October 2020 to July 2021). The highest positivity rate (80.4%) was observed in the week commencing 30th November 2020 during the emergence of the Alpha variant, indicating most schools contained people who were shedding the virus. There was high SARS-CoV-2 amplicon concentration (up to 9.2x106 GC/L) detected over the summer term (8th June - 6th July 2021) during Delta variant prevalence. The summer increase of SARS-CoV-2 in school wastewater was reflected in age-specific clinical COVID-19 cases. Alpha variant and Delta variant were identified in the wastewater by sequencing of samples collected from December to March and June to July, respectively. Lead/lag analysis between SARS-CoV-2 concentrations in school and WWTP data sets show a maximum correlation between the two-time series when school data are lagged by two weeks. Furthermore, wastewater sample enrichment coupled with metagenomic sequencing and rapid informatics enabled the detection of other clinically relevant viral and bacterial pathogens and AMR. CONCLUSIONS: Passive wastewater monitoring surveillance in schools can identify cases of COVID-19. Samples can be sequenced to monitor for emerging and current variants of concern at the resolution of school catchments. Wastewater based monitoring for SARS-CoV-2 is a useful tool for SARS-CoV-2 passive surveillance and could be applied for case identification and containment, and mitigation in schools and other congregate settings with high risks of transmission. Wastewater monitoring enables public health authorities to develop targeted prevention and education programmes for hygiene measures within undertested communities across a broad range of use cases.

Quantifying community-wide antibiotic usage via urban water fingerprinting: Focus on contrasting resource settings in South Africa.

There has been a significant increase in antimicrobial agents (AAs) usage, globally - however the relative consumption is unevenly distributed between nations. Inappropriate use of antibiotics can harbour inherent antimicrobial resistance (AMR); therefore, it is important to understand and monitor community-wide prescribing and consumption behaviours throughout different communities around the world. Wastewater-Based Epidemiology (WBE) is a novel tool enabling low cost and large scale studies focussed on AA usage patterns. The back-calculation of community antimicrobial intake was performed from quantities measured in municipal wastewater and informal settlement discharge in the city of Stellenbosch, utilising WBE. Seventeen antimicrobials, and their human metabolites, were evaluated, in concordance with prescription records corresponding to the catchment region. The proportional excretion, biological/chemical stability, and method recovery of each analyte were all crucial factors in the efficacy of the calculation. Mass per day measurements were normalised to the catchment area via population estimates. Municipal wastewater treatment plant population estimates were used to normalise the wastewater samples and prescription data (mg/day/1000 inhabitants). Population estimates for the informal settlements were less accurate due to a lack of reliable sources that were relevant to the sampling time period. Both mass loads and normalised loads suggested higher than average usage throughout the settlements, relative to municipal wastewater. This was seen most prominently in emtricitabine and lamivudine; but also, sulfamethoxazole, trimethoprim, sulfadiazine, clindamycin, ciprofloxacin, ofloxacin, and doxycycline. Urban water fingerprinting (UWF) data triangulation with prescription datasets showed good correlations for several antimicrobial agents (AAs) (e.g., clindamycin, clarithromycin, ofloxacin, and doxycycline). It also revealed discrepancies in usage for some compounds (e.g., tetracycline and sulfapyridine). This might be linked with a lack of pharma compliance in prescription datasets; erroneous association of prescription boundaries with the sewerage catchment; and/or uncertainties within the sewerage catchment (e.g., population estimations). The UWF tool provided a comprehensive overview of multiclass AAs usage, both prescription and over-the counter. For example, tetracycline was not reported in available prescription statistics, but was detected at an average of 18.4 mg/day/1000inh; and no antiviral prescriptions were obtained, but emtricitabine and lamivudine were quantified at 2415.4 and 144.4 mg/day/1000inh, respectively. A lack of clarity regarding prescriptions and a lack of inclusion of several critical (often over-the-counter) medications in public health databases makes WBE a useful and comprehensive epidemiology tool for tracking pharma usage within a catchment.

Microplastic biofilm, associated pathogen and antimicrobial resistance dynamics through a wastewater treatment process incorporating a constructed wetland.

Microplastics in wastewater are colonized by biofilms containing pathogens and antimicrobial resistance (AMR) genes that can be exported into receiving water bodies. This study investigated establishment and changes in microplastic-associated biofilm and AMR during a conventional full-scale 2100 population equivalent wastewater treatment process combined with a free water surface polishing constructed wetland. Sequential microplastic colonization experiments were conducted at different stages of the wastewater treatment process, including in raw sewage, treated effluent and the constructed wetland. Two scenarios were tested in which the constructed wetland served as either (i) a polishing step or (ii) as primary recipient of sewage inoculated microplastics. Bacterial 16S rRNA gene sequencing was carried out for qualitative bacterial community analysis. qPCR was applied for quantitative analysis of AMR genes (sul1, ermB, tetW, intiI1), bacterial biomass (16S rRNA) and a human fecal marker (HF183). Microbial diversity on microplastics increased with incubation time. The initial sewage-derived biofilm composition changed more significantly in the wastewater effluent compared to the constructed wetland. Pathogen and AMR load decreased by up to two orders of magnitude after coupled conventional and constructed wetland treatment, while less impact was observed when sewage-inoculated microplastic material was directly transferred into the constructed wetland. Aeromonas, Klebsiella, and Streptococcus were key pathogenic genera correlated with AMR in microplastic-associated biofilms. Despite decreasing trends on human pathogens and AMR load along the treatment process, microplastic-associated biofilms were a considerable potential hotspot for AMR (intI1 gene) and accommodated Cyanobacteria and fish pathogens.

Community infectious disease treatment with antimicrobial agents - A longitudinal one year study of antimicrobials in two cities via wastewater-based epidemiology.

Antimicrobial resistance (AMR) is one of the most significant global health threats. Inappropriate and over-usage of antimicrobial agents (AAs) is a major driver for AMR. Wastewater-based epidemiology (WBE) is a promising tool for monitoring AA usage in communities which is, for the first time, explored in this large scale, longitudinal study. Two contrasting urban catchment areas have been investigated: one city and one small town in the Southwest of the UK over a 13-month period in 2018-2019. Per capita daily intake of 17 AAs and metabolites has been estimated and obtained estimates were triangulated with catchment specific AA prescription data to understand AA usage patterns (both seasons driven prescription and AA prescription compliance). Results have demonstrated positive correlations for all quantifiable parent AAs and metabolites in wastewater, and spatial variability in AA usage was observed even in neighbouring urban areas. WBE and catchment specific prescription data showed similar seasonal trends but with low correlation in intake. The reasons might be variable prescribing patterns, prescription/intake outside the studied catchment, and/or lack of patient compliance. WBE proved useful in differentiating between consumption vs topical usage and/or direct disposal of unused AA. WBE is considered superior to prescription data as it provides information on AAs prescribed outside of the monitoring catchment, e.g. HIV antivirals and TB drugs. However, data triangulation, of both prescription data and wastewater data, provides the most comprehensive approach to understanding AA usage in communities.

Wastewater-based epidemiology for the assessment of population exposure to chemicals: The need for integration with human biomonitoring for global One Health actions.

WBE has now become a complimentary tool in SARS-CoV-2 surveillance. This was preceded by the established application of WBE to assess the consumption of illicit drugs in communities. It is now timely to build on this and take the opportunity to expand WBE to enable comprehensive assessment of community exposure to chemical stressors and their mixtures. The goal of WBE is to quantify community exposure, discover exposure-outcome associations, and trigger policy, technological or societal intervention strategies with the overarching aim of exposure prevention and public health promotion. To achieve WBE's full potential, the following key aspects require further action: (1) Integration of WBE-HBM (human biomonitoring) initiatives that provide comprehensive community-individual multichemical exposure assessment. (2) Global WBE monitoring campaigns to provide much needed data on exposure in low- and middle-income countries (LMICs) and fill in the gaps in knowledge especially in the underrepresented highly urbanised as well as rural settings in LMICs. (3) Combining WBE with One Health actions to enable effective interventions. (4) Advancements in new analytical tools and methodologies for WBE progression to enable biomarker selection for exposure studies, and to provide sensitive and selective multiresidue analysis for trace multi-biomarker quantification in a complex wastewater matrix. Most of all, further developments of WBE needs to be undertaken by co-design with key stakeholder groups: government organisations, health authorities and private sector.

Wastewater-based epidemiology for comprehensive community health diagnostics in a national surveillance study: Mining biochemical markers in wastewater.

This manuscript showcases results from a large scale and comprehensive wastewater-based epidemiology (WBE) study focussed on multi-biomarker suite analysis of both chemical and biological determinants in 10 cities and towns across England equating to a population of ∼7 million people. Multi-biomarker suite analysis, describing city metabolism, can provide a holistic understanding to encompass all of human, and human-derived, activities of a city in a single model: from lifestyle choices (e.g. caffeine intake, nicotine) through to health status (e.g. prevalence of pathogenic organisms, usage of pharmaceuticals as proxy for non-communicable disease, NCD, conditions or infectious disease status), and exposure to harmful chemicals due to environmental and industrial sources (e.g. pesticide intake via contaminated food and industrial exposure). Population normalised daily loads (PNDLs) of many chemical markers were found, to a large extent, driven by the size of population contributing to wastewater (especially NCDs). However, there are several exceptions providing insights into chemical intake that can inform either disease status in various communities or unintentional exposure to hazardous chemicals: e.g. very high PNDLs of ibuprofen in Hull resulting from its direct disposal (confirmed by ibuprofen/2-hydroxyibuprofen ratios) and bisphenol A (BPA) in Hull, Lancaster and Portsmouth likely related to industrial discharge. An importance for tracking endogenous health markers such as 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA, an oxidative stress marker) as a generic marker of health status in communities was observed due to increased levels of HNE-MA seen at Barnoldswick wastewater treatment plant that coincided with higher-than-average paracetamol usage and SARS-CoV-2 prevalence in this community. PNDLs of virus markers were found to be highly variable. Being very prevalent in communities nationwide during sampling, SARS-CoV-2 presence in wastewater was to a large extent community driven. The same applies to the fecal marker virus, crAssphage, which is very prevalent in urban communities. In contrast, norovirus and enterovirus showed much higher variability in prevalence across all sites investigated, with clear cases of localized outbreaks in some cities while maintaining low prevalence in other locations. In conclusion, this study clearly demonstrates the potential for WBE to provide an integrated assessment of community health which can help target and validate policy interventions aimed at improving public health and wellbeing.

PERK: An R/Shiny application to predict and visualise concentrations of pharmaceuticals in the aqueous environment.

Predicting the concentration of active pharmaceuticals ingredients (API) in the environment using modelling approaches is an important aspect in the assessment of their environmental risk, especially for the API with no or limited analytical detection methods. However, handling, validating, and incorporating diverse datasets, including API prescription/consumption data, metabolism, flow data, removal efficiency during wastewater treatment, and dilution factor for the modelling is often laborious and time-consuming. The aim of this manuscript is to evaluate R/Shiny based tool, PERK, to facilitate automated modelling and reporting predicted environmental concentration (PEC) of a comprehensive set of API in different environmental matrices. PERK helped to calculate PEC in wastewater influent, effluent, and river, and compare with measured environmental concentrations (MEC) for five catchments located in England. Prediction accuracy (PA), the ratio between PEC and MEC, can be also generated with the tool. PERK provides consistent interactive user-interface, enabling user to visualise the results with limited programming knowledge.

Microplastic dynamics in a free water surface constructed wetland.

This study investigates microplastic (MPs) dynamics of a recently established surface flow 2100 population equivalent polishing constructed wetland (CW) receiving 1.4 ML per day of secondary treated wastewater. MPs type, size ranges and concentrations were measured along the CW at a 2-months sampling campaign. The CW received an average of 5·106 MPs per day (6 MPs per liter), mostly 100-1000 µm-sized synthetic fibers followed by fragments in the same size range. 95 % of MPs were retained, resulting in 0.30 ± 0.09 MPs per liter in CW effluent. Most MPs (97 %) were trapped within the first 20 % of the CW which consisted of a settling pond and shallow vegetated treatment cells and provided an areal removal rate > 4000 MP m-2 d-1. Data and microscopic analysis indicate MPs erosion and fragmentation in the CW. Turbidity and suspended solids were no indicator for MP removal due to water fowl activity, algal growth, and preferential flow conditions. This is the first study on MP dynamics in an independently operating full scale free water surface CW incorporated into a municipal wastewater treatment scheme. Surface flow CWs can retain MPs effectively but accumulation in CW sediments and substrate needs to be considered when further utilized or recycled.