Trade-off between soil enzyme activities and hotspots area depends on long-term fertilization: In situ field zymography.

Mineral fertilizers and livestock manure have been found to impact soil enzyme activities and distributions, but their trade-off and subsequent effects on soil functioning related to nutrient cycling are rarely evaluated. Here, we investigated the long-term effects of manure and mineral fertilization on the spatial distribution of enzyme activities related to carbon, nitrogen, and phosphorus cycling under field-grown maize. We found that the legacy of mineral fertilizers increased the rhizosphere extension for ß-glucosidase and N-acetylglucosaminidase by 16-170 %, and the hotspots area by 37-151 %, compared to manure. The legacy of manure, especially combined with mineral fertilizers, increased enzyme activities and formed non-rhizosphere hotspots. Furthermore, we found a trade-off between hotspots area and enzyme activities under the legacy effect of long-term fertilization. This suggested that plants and microorganisms regulate nutrient investments by altering spatial distribution of enzyme activities. The positive correlation between hotspots area and nutrient contents highlights the importance of non-rhizosphere hotspots induced by manure in maintaining soil fertility. Compared to mineral fertilization, the legacy effect of manure expanded the soil functions for nutrient cycling in both rhizosphere and non-rhizosphere by >1.7 times. In conclusion, the legacy of manure expands non-rhizosphere hotspots and enhances soil functioning, while mineral fertilization expands rhizosphere extension and intensifies hotspots area for nutrient exploitation.

Microplastic contamination accelerates soil carbon loss through positive priming.

The priming effect, i.e., the changes in soil organic matter (SOM) decomposition following fresh organic carbon (C) inputs is known to influence C storage in terrestrial ecosystems. Microplastics (particle size <5 mm) are ubiquitous in soils due to the increasing use and often inadequate end-of-life management of plastics. Conventional polyethylene and bio-degradable (PHBV) plastics contain large amounts of C within their molecular structure, which can be assimilated by microorganisms. However, the extent and direction of the potential priming effect induced by microplastics is unclear. As such, we added 14C-labeled glucose to investigate how background polyethylene and PHBV microplastics (1 %, w/w) affect SOM decomposition and its potential microbial mechanisms in a short-term. The cumulative CO2 emission in soil contaminated with PHBV was 42-53 % higher than under Polyethylene contaminated soil after 60-day incubation. Addition of glucose increased SOM decomposition and induced a positive priming effect, as a consequence, caused a negative net soil C balance (-59 to -132 µg C g-1 soil) regardless of microplastic types. K-strategists dominated in the PHBV-contaminated soils and induced 72 % higher positive priming effects as compared to Polyethylene-contaminated soils (160 vs. 92 µg C g-1 soil). This was attributed to the enhanced decomposition of recalcitrant SOM to acquire nitrogen. The stronger priming effect associated in PHBVs can be attributed to cooperative decomposition among fungi and bacteria, which metabolize more recalcitrant C in PHBV. Moreover, comparatively higher calorespirometric ratios, lower substrate use efficiency, and larger enzyme activity but shorter turnover time of enzymes indicated that soil contaminated with PHBV release more energy, and have a more efficient microbial catabolism and are more efficient in SOM decomposition and nutrient resource uptake. Overall, microplastics, (especially bio-degradable microplastics) can alter biogeochemical cycles with significant negative consequences for C sequestration via increasing SOM decomposition in agricultural soils and for regional and global C budgets.

Microcosm experiment investigating climate-induced thermal effects on human virus viability in seawater: qPCR vs capsid integrity for enhanced risk management.

Climate change is intensifying extreme weather events in coastal areas, leading to more frequent discharge of untreated wastewater containing human viruses into coastal waters. This poses a health risk, especially during heatwaves when bathing activity increases. A study examined the survival and viability of seven common wastewater viruses in seawater at different temperatures. Viral genomes were quantified using direct qPCR, whilst viability was assessed using Capsid Integrity qPCR. Results showed that T90 values from direct qPCR were much higher than those from CI-qPCR, suggesting that risk mitigation should be based on viral integrity tests. All viruses remained potentially viable for at least 72 h in environmental seawater and longer in sterile artificial seawater, highlighting the importance of biotic processes in viral inactivation. Viral persistence decreased with increasing temperature. Whilst heatwaves may partially reduce risks from human viral pathogens in coastal waters, they do not eliminate them entirely.

Amino acid-sulphur decomposition in agricultural soil profile along a long-term recultivation chronosequence.

The significance of sulphur (S) availability for crop yield and quality is highlighted under the global S deficiency scenario. However, little is known about the temporal trend in belowground organic S mineralisation when restoring land to productive agricultural systems, particularly for the deeper soil parts. Therefore, we investigated the decomposition of 35S-labelled methionine in surface (0-30 cm) and subsurface soil (30-60 cm and 60-90 cm) over a 48-year recultivation chronosequence (sampled after1, 8, 14, 24 and 48 years). Soil total sulphur (TS) significantly (p < 0.05) increased in surface soil but not in subsurface soils after 48 years of recultivation. Overall, the immobilisation of 35S-methionine (35S-MB) in subsurface soils relative to year 1 significantly decreased over the chronosequence but did not change in the surface samples. The 35S-MB values in subsurface soils were positively corrected with soil carbon (C) stoichiometry (Pearson correlation, p < 0.05), suggesting the immobilisation of methionine was likely constrained by microbial C demand in deep soil. Compared to year 1, 35S-SO42- released from 35S-methionine significantly declined throughout the older (≥ 8 years) soil profiles. Significant (p < 0.05) changes in the organic 35S partition (35S immobilisation and 35S released as sulphate) were observed in year 8 after the soil was recultivated with N-fixing alfalfa or fertilisers. Whereas, after that (≥ 14 years), soil organic S partition remained affected when conventional tillage and agricultural crops dominated this site. Indicating that the effect of recultivation on organic S decomposition depends on the manner of recultivation management. Our study contributes to an improved understanding of amino acid S and organic S mineralisation under severe anthropogenic disturbance.

Implications of long-term sample storage on the recovery of viruses from wastewater and biobanking.

Wastewater-based monitoring has been widely implemented worldwide for the tracking of SARS-CoV-2 outbreaks and other viral diseases. In many surveillance programmes, unprocessed and processed wastewater samples are often frozen and stored for long periods of time in case the identification and tracing of an emerging health threat becomes necessary. However, extensive sample bioarchives may be difficult to maintain due to limitations in ultra-freezer capacity and associated cost. Furthermore, the stability of viruses in such samples has not been systematically investigated and hence the usefulness of bioarchives is unknown. In this study, we assessed the stability of SARS-CoV-2, influenza viruses, noroviruses and the faecal indicator virus, crAssphage, in raw wastewater and purified nucleic aacid extracts stored at -80 °C for 6-24 months. We found that the isolated viral RNA and DNA showed little signs of degradation in storage over 8-24 months, whereas extensive decay viral and loss of qPCR signal was observed during the storage of raw unprocessed wastewater. The most stable viruses were noroviruses and crAssphage, followed by SARS-CoV-2 and influenza A virus. Based on our findings, we conclude that bioarchives comprised of nucleic acid extracts derived from concentrated wastewater samples may be archived long-term, for at least two years, whereas raw wastewater samples may be discarded after one year.

Use of wastewater from passenger ships to assess the movement of COVID-19 and other pathogenic viruses across maritime international boundaries.

Objective: The worldwide spread of SARS-CoV-2 and the resulting COVID-19 pandemic has been driven by international travel. This has led to the desire to develop surveillance approaches which can estimate the rate of import of pathogenic organisms across international borders. The aim of this study was to investigate the use of wastewater-based approaches for the surveillance of viral pathogens on commercial short-haul (3.5 h transit time) roll-on/roll-off passenger/freight ferries operating between the UK and the Republic of Ireland. Methods: Samples of toilet-derived wastewater (blackwater) were collected from two commercial ships over a 4-week period and analysed for SARS-CoV-2, influenza, enterovirus, norovirus, the faecal-marker virus crAssphage and a range of physical and chemical indicators of wastewater quality. Results: A small proportion of the wastewater samples were positive for SARS-CoV-2 (8% of the total), consistent with theoretical predictions of detection frequency (4%-15% of the total) based on the national COVID-19 Infection Survey and defecation behaviour. In addition, norovirus was detected in wastewater at low frequency. No influenza A/B viruses, enterovirus or enterovirus D68 were detected throughout the study period. Conclusion: We conclude that testing of wastewater from ships that cross international maritime boundaries may provide a cost-effective and relatively unbiased method to estimate the flow of infected individuals between countries. The approach is also readily applicable for the surveillance of other disease-causing agents.

Earthworms alleviate microplastics stress on soil microbial and protist communities.

Microplastic (MP) pollution can exert significant pressure on soil ecosystems, however, the interactive effects of MPs on soil bacterial, fungal and protist communities remains poorly understood. Soil macrofauna, such as earthworms, can be directly affected by MPs, potentially leading to a range of feedbacks on the soil microbial community. To address this, we conducted a microcosm experiment to examine the effects of conventional (i.e., polyethylene, polystyrene) and biodegradable MPs (i.e. PBAT, polylactic acid) on the structure of the soil bacterial, fungal, and protist communities in the presence or absence of earthworms. We found that MP contamination negatively affected the diversity and composition of soil microbial and protist communities, with smaller-sized conventional MPs having the most pronounced effects. For example, compared with the unamended control, small-sized polyethylene MPs both significantly reduced the Shannon diversity of soil bacteria, fungi, and protist by 4.3 %, 37.0 %, and 9.1 %, respectively. Biodegradable MPs increased negative correlations among bacteria, fungi, and protists. However, earthworms mitigated these effects, enhancing the diversity and altering the composition of these communities. They also increased the niche width and stability of the soil microbial food web network. Our study indicated that earthworms help attenuate the response of soil microorganisms to MPs stress by influencing the diversity and composition of soil microorganisms and soil physicochemical properties and underscores the importance of considering macrofauna in MPs research.

Plastic pollution and human pathogens: Towards a conceptual shift in risk management at bathing water and beach environments.

Emerging evidence indicates that micro- and macro-plastics present in water can support a diverse microbial community, including potential human pathogens (e.g., bacteria, viruses). This interaction raises important concerns surrounding the role and suitability of current bathing water regulations and associated pathogen exposure risk within beach environments. In response to this, we critically evaluated the available evidence on plastic-pathogen interactions and identified major gaps in knowledge. This review highlighted the need for a conceptual shift in risk management at public beaches recognising: (i) interconnected environmental risks, e.g., associations between microbial compliance parameters, potential pathogens and both contemporary and legacy plastic pollution; and (ii) an appreciation of risk of exposure to plastic co-pollutants for both water and waterside users. We present a decision-making framework to identify options to manage plastic-associated pathogen risks alongside short- and longer-term research priorities. This advance will help deliver improvements in managing plastic-associated pathogen risk, acknowledging that human exposure potential is not limited to only those who engage in water-based activity. We argue that adopting these recommendations will help create an integrated approach to managing and reducing human exposure to pathogens at bathing, recreational water and beach environments.

National-scale antimicrobial resistance surveillance in wastewater: A comparative analysis of HT qPCR and metagenomic approaches.

Wastewater serves as an important reservoir of antimicrobial resistance (AMR), and its surveillance can provide insights into population-level trends in AMR to inform public health policy. This study compared two common high-throughput screening approaches, namely (i) high-throughput quantitative PCR (HT qPCR), targeting 73 antimicrobial resistance genes, and (ii) metagenomic sequencing. Weekly composite samples of wastewater influent were taken from 47 wastewater treatment plants (WWTPs) across Wales, as part of a national AMR surveillance programme, alongside 4 weeks of daily wastewater effluent samples from a large municipal hospital. Metagenomic analysis provided more comprehensive resistome coverage, detecting 545 genes compared to the targeted 73 genes by HT qPCR. It further provided contextual information critical to risk assessment (i.e. potential bacterial hosts). In contrast, HT qPCR exhibited higher sensitivity, quantifying all targeted genes including those of clinical relevance present at low abundance. When limited to the HT qPCR target genes, both methods were able to reflect the spatiotemporal dynamics of the complete metagenomic resistome, distinguishing that of the hospital and the WWTPs. Both approaches revealed correlations between resistome compositional shifts and environmental variables like ammonium wastewater concentration, though differed in their interpretation of some potential influencing factors. Overall, metagenomics provides more comprehensive resistome profiling, while qPCR permits sensitive quantification of genes significant to clinical resistance. We highlight the importance of selecting appropriate methodologies aligned to surveillance aims to guide the development of effective wastewater-based AMR monitoring programmes.

Fungal necromass contribution to carbon sequestration in global croplands: A meta-analysis of driving factors and conservation practices.

Fungal necromass carbon (FNC) contributes significantly to the build-up of soil organic carbon (SOC) by supplying abundant recalcitrant polymeric melanin present in the fungal cell wall. However, the influence of a wide range of conservation practices and associated factors on FNC accumulation and contribution to SOC in global croplands remains unexplored. Here, a meta-analysis was performed using 873 observations across three continents, together with structural equation modeling, to evaluate conservation practices and factors responsible for the enhancement of FNC and SOC. FNC content (8.39 g kg-1) of North American soils was highest compared to FNC content of Asian and European soils. The structural equation models showed a significant (p < 0.05) positive influence of microbial biomass carbon (MBC), soil pH, and clay contents on the accumulation of FNC. Soil C/N ratio and climate factors, however, had only minor influences on FNC accumulation. Notably, the main driver of FNC was MBC, which is mainly influenced by the soil total N and geographic factors in the study areas. Typical 5 cropland practices had significant effect size (p < 0.05) on FNC, leading to an increase of 12 % to 26 %, and the FNC content was greatest under straw amendment (26 %). Fungal necromass accumulation efficiency ranged from 23 % to 45 % depending on cropland practices: non- and reduced tillage was the most efficient (45 %), followed by crop coverage (32 %), straw amendment (30 %), and manure application (27 %), while N fertilization had the lowest efficiency (23 %). We conclude that FNC contributes to over a quarter of SOC, highlighting its major role in enhancing C sequestration worldwide. Conservation practices, particularly non-tillage or reduced tillage, are important to enhance C sequestration from FNC in croplands.

Examining the stability of viral RNA and DNA in wastewater: Effects of storage time, temperature, and freeze-thaw cycles.

Wastewater-based epidemiology (WBE) has been demonstrably successful as a relatively unbiased tool for monitoring levels of SARS-CoV-2 virus circulating in communities during the COVID-19 pandemic. Accumulated biobanks of wastewater samples allow retrospective exploration of spatial and temporal trends for public health indicators such as chemicals, viruses, antimicrobial resistance genes, and the possible emergence of novel human or zoonotic pathogens. We investigated virus resilience to time, temperature, and freeze-thaw cycles, plus the optimal storage conditions to maintain the stability of genetic material (RNA/DNA) of viral +ssRNA (Envelope - E, Nucleocapsid - N and Spike protein - S genes of SARS-CoV-2), dsRNA (Phi6 phage) and circular dsDNA (crAssphage) in wastewater. Samples consisted of (i) processed and extracted wastewater samples, (ii) processed and extracted distilled water samples, and (iii) raw, unprocessed wastewater samples. Samples were stored at -80 °C, -20 °C, 4 °C, or 20 °C for 10 days, going through up to 10 freeze-thaw cycles (once per day). Sample stability was measured using reverse transcription quantitative PCR, quantitative PCR, automated electrophoresis, and short-read whole genome sequencing. Exploring different areas of the SARS-CoV-2 genome demonstrated that the S gene in processed and extracted samples showed greater sensitivity to freeze-thaw cycles than the E or N genes. Investigating surrogate and normalisation viruses showed that Phi6 remains a stable comparison for SARS-CoV-2 in a laboratory setting and crAssphage was relatively resilient to temperature variation. Recovery of SARS-CoV-2 in raw unprocessed samples was significantly greater when stored at 4 °C, which was supported by the sequencing data for all viruses - both time and freeze-thaw cycles negatively impacted sequencing metrics. Historical extracts stored at -80 °C that were re-quantified 12, 14 and 16 months after original quantification showed no major changes. This study highlights the importance of the fast processing and extraction of wastewater samples, following which viruses are relatively robust to storage at a range of temperatures.

Selective microbial attachment to LDPE plastic beads during passage through the wastewater network.

Urban wastewater treatment plants (WWTP) represent key point-source discharges of microplastics (MP) into the environment, however, little is known about the microbial carrying capacity of plastics travelling through them. The purpose of this study was to quantify the number of cells that become associated with MP at different locations within a WWTP, and to assess differences in microbiome communities. We conducted a field experiment incubating low density polyethylene (LDPE) MP beads in WWTP influent and effluent, as well as tracking free floating beads during passage in wastewater from a large municipal hospital to an urban WWTP, where they were subsequently recovered. Using two cell counting methods - automated flow cytometric true absolute cell counts and indirect cell quantification via protein content based on a model E. coli cell - we quantified cell attachment to LDPE beads. LDPE associated counts ranged from 350 × 103 cells cm-2 after incubation in wastewater effluent, and 990 × 103 cells cm-2 after incubation in wastewater influent. 16S rRNA gene amplicon sequencing was used to determine the bacterial community structure of the plastic-associated microbiomes. Our results showed that distinct bacterial communities developed on the LDPE MP following exposure to each wastewater type. Influent (untreated) wastewater LDPE-associated microbiomes were dominated by Bacillota whereas the microbes that attached in wastewater effluent (tertiary treated) were dominated by Pseudomonadota. In conclusion, this study provides clear evidence that microplastics migrating through the sewer network and WWTP rapidly accumulate microbiomes with unique microbial community structures varying from sewage influent to effluent. These findings demonstrate the differential microbiological risk from MP associated with routine wastewater discharges to those released from intermittent combined sewer overflows (CSOs) during storm events.

Do Added Microplastics, Native Soil Properties, and Prevailing Climatic Conditions Have Consequences for Carbon and Nitrogen Contents in Soil? A Global Data Synthesis of Pot and Greenhouse Studies.

Microplastics threaten soil ecosystems, strongly influencing carbon (C) and nitrogen (N) contents. Interactions between microplastic properties and climatic and edaphic factors are poorly understood. We conducted a meta-analysis to assess the interactive effects of microplastic properties (type, shape, size, and content), native soil properties (texture, pH, and dissolved organic carbon (DOC)) and climatic factors (precipitation and temperature) on C and N contents in soil. We found that low-density polyethylene reduced total nitrogen (TN) content, whereas biodegradable polylactic acid led to a decrease in soil organic carbon (SOC). Microplastic fragments especially depleted TN, reducing aggregate stability, increasing N-mineralization and leaching, and consequently increasing the soil C/N ratio. Microplastic size affected outcomes; those <200 µm reduced both TN and SOC contents. Mineralization-induced nutrient losses were greatest at microplastic contents between 1 and 2.5% of soil weight. Sandy soils suffered the highest microplastic contamination-induced nutrient depletion. Alkaline soils showed the greatest SOC depletion, suggesting high SOC degradability. In low-DOC soils, microplastic contamination caused 2-fold greater TN depletion than in soils with high DOC. Sites with high precipitation and temperature had greatest decrease in TN and SOC contents. In conclusion, there are complex interactions determining microplastic impacts on soil health. Microplastic contamination always risks soil C and N depletion, but the severity depends on microplastic characteristics, native soil properties, and climatic conditions, with potential exacerbation by greenhouse emission-induced climate change.

Regulatory path for soil microbial communities depends on the type and dose of microplastics.

To reveal the feedbacks and regulating mechanisms of microplastic types and doses on microbial community, a microcosm experiment was carried out with two non-degradable microplastics [polyethylene (PE) and polyvinyl chloride (PVC)] and four biodegradable microplastics [poly(butylene succinate) (PBS), polyhydroxyalkanoates (PHA), poly(butyleneadipate-co-terephthalate) (PBAT), and polypropylene carbonate (PPC)] at different levels (1 %, 7 %, and 28 %). As a result, the content of total carbon (TC), soil organic carbon (SOC), and microbial biomass carbon (MBC) (expect MBC in PBS soil) increased with increasing doses of microplastics, and increased at the lowest PE dose rate. Biodegradable microplastics created a more active ecological niche while enriching more pathogens than non-degradable microplastics. Structural equation modeling indicated that microbial diversities were in a type-dependent assembly, whereas microbial compositions were more profoundly affected by the microplastic doses, ultimately. The standardized total effect coefficient of microplastic types on bacterial and fungal diversities was - 0.429 and - 0.282, and that of doses on bacterial and fungal compositions was 0.487 and 0.336, respectively. Both microplastic types and doses significantly impacted pH, electrical conductivity, total nitrogen, TC, SOC, and MBC, subsequently inhibiting microbial diversities and stimulating microbial compositions with particular pathways. The results provide a comprehensive understanding for evaluating the potential risk of microplastics.

Needles in haystacks: monitoring the potential escape of bioaerosolised antibacterial resistance genes from wastewater treatment plants with air and phyllosphere sampling.

Wastewater treatment plants are well-known point sources of emissions of antibacterial resistance genes (ARGs) into the environment. Although most work to date has focused on ARG dispersal via effluent, aerial dispersal in bioaerosols is a poorly understood, but likely important vector for ARG dispersal. Recent evidence suggests that ARG profiles of the conifer needle phyllosphere could be used to measure bioaerosol dispersal from anthropogenic sources. Here, we assessed airborne dispersal of ARGs from wastewater treatment plants in Wales, UK and Quebec, Canada, using conifer needles as passive bioaerosol monitors. ARG profiles of wastewater were compared to those of conifer phyllosphere using high-throughput qPCR. ARG richness was significantly lower in conifer phyllosphere samples than wastewater samples, though no differences were observed across the dispersal gradients. Mean copy number of ARGs followed a similar trend. ARG profiles showed limited, but consistent patterns with increasing distance from wastewater treatment plants, but these did not align with those of wastewater samples. For example, proportional abundance of aminoglycosides decreased over the dispersal gradient in Wales, whereas mobile genetic elements showed the inverse relationship. In summary, while distinct ARG profiles exist along dispersal gradients, links to those of wastewater were not apparent.

Simultaneous detection and characterization of common respiratory pathogens in wastewater through genomic sequencing.

Genomic surveillance of SARS-CoV-2 has given insight into the evolution and epidemiology of the virus and its variant lineages during the COVID-19 pandemic. Expanding this approach to include a range of respiratory pathogens can better inform public health preparedness for potential outbreaks and epidemics. Here, we simultaneously sequenced 38 pathogens including influenza viruses, coronaviruses and bocaviruses, to examine the abundance and seasonality of respiratory pathogens in urban wastewater. We deployed a targeted bait capture method and short-read sequencing (Illumina Respiratory Virus Oligos Panel; RVOP) on composite wastewater samples from 8 wastewater treatment plants (WWTPs) and one associated hospital site. By combining seasonal sampling with whole genome sequencing, we were able to concurrently detect and characterise a range of common respiratory pathogens, including SARS-CoV-2, adenovirus and parainfluenza virus. We demonstrated that 38 respiratory pathogens can be detected at low abundances year-round, that hospital pathogen diversity is higher in winter vs. summer sampling events, and that significantly more viruses are detected in raw influent compared to treated effluent samples. Finally, we compared detection sensitivity of RT-qPCR vs. next generation sequencing for SARS-CoV-2, enteroviruses, influenza A/B, and respiratory syncytial viruses. We conclude that both should be used in combination; RT-qPCR allowed accurate quantification, whilst genomic sequencing detected pathogens at lower abundance. We demonstrate the valuable role of wastewater genomic surveillance and its contribution to the field of wastewater-based epidemiology, gaining rapid understanding of the seasonal presence and persistence for common respiratory pathogens. By simultaneously monitoring seasonal trends and early warning signs of many viruses circulating in communities, public health agencies can implement targeted prevention and rapid response plans.

Microbial communities colonising plastics during transition from the wastewater treatment plant to marine waters.

BACKGROUND: Plastics pollution and antimicrobial resistance (AMR) are two major environmental threats, but potential connections between plastic associated biofilms, the 'plastisphere', and dissemination of AMR genes are not well explored. RESULTS: We conducted mesocosm experiments tracking microbial community changes on plastic surfaces transitioning from wastewater effluent to marine environments over 16 weeks. Commonly used plastics, polypropylene (PP), high density polyethylene (HDPE), low density polyethylene (LDPE) and polyethylene terephthalate (PET) incubated in wastewater effluent, river water, estuarine water, and in the seawater for 16 weeks, were analysed via 16S rRNA gene amplicon and shotgun metagenome sequencing. Within one week, plastic-colonizing communities shifted from wastewater effluent-associated microorganisms to marine taxa, some members of which (e.g. Oleibacter-Thalassolituus and Sphingomonas spp., on PET, Alcanivoracaceae on PET and PP, or Oleiphilaceae, on all polymers), were selectively enriched from levels undetectable in the starting communities. Remarkably, microbial biofilms were also susceptible to parasitism, with Saprospiraceae feeding on biofilms at late colonisation stages (from week 6 onwards), while Bdellovibrionaceae were prominently present on HDPE from week 2 and LDPE from day 1. Relative AMR gene abundance declined over time, and plastics did not become enriched for key AMR genes after wastewater exposure. CONCLUSION: Although some resistance genes occurred during the mesocosm transition on plastic substrata, those originated from the seawater organisms. Overall, plastic surfaces incubated in wastewater did not act as hotspots for AMR proliferation in simulated marine environments.

Comparative assessment of Nanotrap and polyethylene glycol-based virus concentration in wastewater samples.

Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4%-21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing.