Protozoa contribute to water purification through predation in wastewater treatment systems. Full-scale aerobic granular sludge (AGS) reactors treating municipal wastewater contain AGS of varying sizes, with those larger than 2 mm dominating. These size fractions exhibit different sludge morphologies and microbial communities. To date, little is known about protozoan communities and their role in the removal of human-associated bacteria (like pathogens) in AGS plants, particularly across different size fractions. This study conducted uptake experiments with fluorescent Escherichia coli, as a model for human-associated bacteria, followed by microscopic observation to investigate protozoan communities and their predatory behavior in six AGS size fractions and activated sludge collected from full-scale municipal wastewater treatment plants. Sessile ciliates, particularly Epistylis and Vorticella, dominated protozoan populations across six AGS size fractions, with Epistylis being more abundant in larger AGS fractions (>1 mm) and Vorticella in smaller fractions (<1 mm). Additionally, microcosm experiments under aerobic (including predation) and anoxic conditions (excluding predation) revealed that predation was likely to be the main E. coli removal pathway, contributing an additional 0.5 to 2.5 log10CFU mL^–1reduction over a combination of non-predatory biological and abiotic processes. Larger AGS fractions showed greater predation capacity, linked to higher Epistylis abundance, while activated sludge, dominated by Vorticella, resembled smaller AGS fractions with lower predation capacity. These findings advance the understanding of the distribution of protozoan communities and their contribution to E. coli removal by predation in AGS wastewater treatment.

Water quality is under threat due to the presence of pathogenic and antibiotic-resistant bacteria. Escherichia coli (E. coli) serves as an indicator of faecal contamination and the potential presence of other harmful pathogens. Understanding E. coli concentrations helps in assessing the overall health risks associated with waterborne diseases and developing effective water management strategies. Therefore, we developed the first large-scale model, GloWPa-Ecoli C1 to simulate E. coli loads and concentrations in rivers and apply this model to China. The model provides the first comprehensive overview of microbial water quality across China's rivers. The model simulates E. coli concentrations in 2020 to range from 10−1.2 to 106.3 CFU/L, with 45.6 %–78.1 % of rivers exhibiting poor microbial water quality. Major hotspots of E. coli pollution are Haihe, Huaihe and Pearl River Basins. Direct discharge of human faecal waste contributes 80.2 % of the total E. coli load, while directly discharged livestock waste accounts for 13.1 %. To mitigate E. coli pollution in rivers in China, we recommend increasing human faecal waste collection rates, expanding wastewater treatment plant (WWTP) coverage, phasing out primary treatment WWTPs and eliminating direct livestock faecal waste discharge, particularly from smallholder farms. The study underscores the urgent need to improve microbial water quality in China's rivers. The findings provide actionable insights to inform policy development aimed at safeguarding water quality and public health. Furthermore, the modelling approach is applicable to other regions and microorganisms, offering a foundation for developing models to address antibiotic-resistant bacteria and other emerging water quality challenges.

Aerobic granular sludge (AGS) process is an effective wastewater treatment technology for nutrient and organic matter removal and is being widely applied worldwide. To date, its performance in removing organic micropollutants (OMPs), particularly under wet weather conditions when operation differs, remains poorly understood. This study evaluated the occurrence and removal of OMPs, including 19 pharmaceuticals and 2 industrial compounds, in a full-scale AGS plant during one year under both dry and wet weather conditions. Under dry weather conditions, influent concentrations of 5 pharmaceuticals and 1 industrial compound exceeded 1 μg L−1. Rainfall resulted in diluted OMP influent concentrations, but also caused a significant increase in the influent load of 6 OMPs with positively charged functional groups, likely due to mobilization of sewage sediments that had adsorbed these OMPs. Under dry weather conditions, average removal efficiencies of 14 compounds were greater than 20 %, with 6 of these compounds detected in the sludge phase, and thus likely removed through sorption. Under wet weather conditions, OMP removal efficiencies decreased by 8 % to 38 %. Shortened aeration reaction time significantly reduced (p-value<0.05; R2>0.5) the removal of 8 potentially biodegradable compounds, while the impact on sorption-driven removal was limited for 6 compounds. Effluent OMP load increased under wet weather conditions, mainly due to reduced removal efficiency, rather than the discharge of OMPs adsorbed onto suspended solids. Under dry weather conditions, the AGS plant exhibited comparable or slightly higher OMP removal efficiencyies than activated sludge plants; however, differences in performance under wet weather conditions remain unclear due to limited data on activated sludge systems. Overall, this study is the first to assess OMP removal in a full-scale AGS plant under wet weather, showing the impact of increased flow on the sorption and biotransformation of OMPs.

Pseudomonas aeruginosa may colonize humans, however, epidemiological data are scarce. Here, we determined overall and body site-specific carriage rates and associated risk factors among healthy individuals and newly admitted patients in three major cities. This cross-sectional study was conducted in Rotterdam (The Netherlands), Rome (Italy), and Jakarta (Indonesia) between 2022–2024. Adult healthy individuals and newly admitted patients were asked to provide throat, navel, and rectal/perianal swabs, and to complete a questionnaire. Univariable and multivariable analyses were performed to determine factors associated with P. aeruginosa carriage. Carriage rates differed significantly between cities (p < 0.001), and were lowest in Rome (healthy individuals 4.8%; patients 6.5%), followed by Rotterdam (healthy individuals 12.0%; patients 12.7%), and Jakarta (healthy individuals 28.6%; patients 24.0%). In carriers from Rotterdam, P. aeruginosa was most often detected in perianal swabs, while mostly in throat swabs among carriers from Rome and Jakarta. P. aeruginosa carriage had a seasonal association in patients from Rotterdam (p = 0.014) and Jakarta (p = 0.020). Among patients from Jakarta, female sex (aOR 1.98, 95% CI 1.02–3.84; p = 0.045) was associated with P. aeruginosa carriage. Overall, P. aeruginosa carriage rates and colonized body sites differ between cities and are likely associated with climate differences. Our findings warrant setting-specific adaptations of screening strategies and surveillance programs.

Aerobic granular sludge (AGS) is an effective wastewater treatment widely applied worldwide for the removal of nutrients and organic matter. However, limited information is available on the effectiveness of full-scale AGS plants for removing organic micropollutants (OMP), particularly during wet weather. This study investigated the occurrence and removal of OMPs, including 19 pharmaceuticals and 2 industrial compounds, in a full-scale AGS plant during both dry and wet weather over one year. We selected a full-scale AGS plant located in Utrecht, the Netherlands as the targeted AGS plant, and collected monthly 24-hour composite water samples and grab sludge samples f rom May 2023 to April 2024. 21 OMPs were extracted from water and sludge samples and measured by LC/MS. Concentrations of ammonia and organic matter (both particulate and soluble) were measured with HACH Lange GMBH kits.

The results show that Influent concentrations of 5 pharmaceuticals and 1 industrial compound exceeded 1 ?g L-1, which were diluted by rainfall. Influent loadings of 7 OMPs (absolute OMP amount in ?g day -1) were significantly increased during wet weather, likely from sewage sediment resuspen sion and urban runoff. Average removal efficiencies of 11 compounds achieved greater than 20%, with 5 of them exceeding 50%, in the AGS plants. Simple linear regression results between OMP removal efficiencies and flow rates showed that biodegradable OMPs were more strongly affected by increased influent volumes (as indicated by steeper slopes) than OMPs primarily removed through sorption. Additionally, correlation analysis results showed that the removal of soluble organic matter was significantly correlated (p-value < 0.05) with the removal of 14 OMPs, suggesting that organic matter removal may be an indicator for OMP removal in the AGS plant. After AGS treatment, 8 compounds in the effluent remained above their predicted no-effect concentration levels, indicating potential ecological and human health risks in the receiving water. During wet weather, effluent OMP loadings increased mainly driven by reduced removal efficiency. Additionally, compared to activated sludge plants, the AGS plant exhibited comparable or slightly higher OMP removal efficiency during dry weather. Overall, this study is the first to investigate OMP removal during wet weather in a full-scale AGS plant and propose the potential impact of increased flow rates on biodegradable OMPs or OMPs mainly removed through sorption.

One Health seeks to integrate and balance the health of humans, animals, and environmental systems, which are intricately linked through microbiomes. These microbial communities exchange microbes and genes, influencing not only human and animal health but also key environmental, agricultural, and biotechnological processes. Preventing the emergence of pathogens as well as monitoring and controlling the composition of microbiomes through microbial effectors including virulence factors, toxins, antibiotics, non-ribosomal peptides, and viruses holds transformative potential. However, the mechanisms by which these microbial effectors shape microbiomes and their broader functional consequences for host and ecosystem health remain poorly understood. Metaproteomics offers a novel methodological framework as it provides insights into microbial dynamics by quantifying microbial biomass composition, metabolic functions, and detecting effectors like viruses, antimicrobial resistance proteins, and non-ribosomal peptides. Here, we highlight the potential of metaproteomics in elucidating microbial effectors and their impact on microbiomes and discuss their potential for modulating microbiomes to foster desired functions.

Antimicrobial resistance (AMR) is among the top 10 public health threats, with nearly 5 million deaths in 2019 linked to AMR-related bacterial infections. (1) A One Health approach is needed to combat AMR.

Healthcare-based surveillance (HBS) of AMR provides incomplete information about the scope of the AMR threat. HBS screens only patients seeking medical attention, lacking community-level representativeness, and suffers from underreporting. (2) Consequently, researchers are turning to wastewater-based surveillance (WBS) to complement HBS. (3) WBS can provide information about AMR circulating within communities and hospitals, offering a comprehensive understanding of AMR prevalence. However, the surveillance targets and data obtained from WBS are distinct from those derived from HBS, creating uncertainty regarding their utility to the public health sector and ability to yield policy relevant information. In May 2024, participants in a workshop during the 7th Environmental Dimension of Antimicrobial Resistance (EDAR7) conference (Montréal, Canada) sought to answer four questions aimed at advancing the policy relevance of AMR data generated by WBS.

Background/Objectives: The global spread of carbapenem-resistant Pseudomonas aeruginosa (CRPA) warrants collaborative action. Guidance should come from integrated One Health surveillance; however, a surveillance strategy is currently unavailable due to insufficient knowledge on the sources and transmission routes of CRPA. The aim of the SAMPAN study (“A Smart Surveillance Strategy for Carbapenem-resistant Pseudomonas aeruginosa”) is to develop a globally applicable surveillance strategy. Methods: First, an international cross-sectional study will be conducted to investigate CRPA in clinical and environmental settings in Rotterdam (The Netherlands), Rome (Italy), and Jakarta (Indonesia). Screening cultures and risk factor questionnaires will be taken from healthy individuals and patients upon hospital admission. Clinical CRPA isolates will also be included. Additionally, samples will be taken twice from wet hospital environments and monthly from the hospitals’ (drinking) water system, hospital and municipal wastewater treatment plants, and receiving rivers. Whole-genome sequencing will be performed to characterize CRPA isolates and determine the genetic relatedness among the isolates from different reservoirs. Findings from the cross-sectional study, combined with expert elicitation using a Delphi method, will serve as the input for the surveillance strategy. Conclusions: The SAMPAN study will provide a broader understanding of the sources and transmission routes of CRPA. Therewith, the development of a globally applicable smart surveillance strategy will be made possible, delivering information that is needed to guide actions against the spread of CRPA.

Water systems can act as an important reservoir for Pseudomonas aeruginosa, which can pose public health risks during human contact. Carbapenem resistance is one of the most concerning resistances in P. aeruginosa making it a high-priority pathogen according to the World Health Organization (WHO), due to its ability to cause difficult-to-treat infections. Culture methods for detecting carbapenem-resistant P. aeruginosa (CRPA) have generally been developed for clinical samples but rarely for water. Therefore, the aim of this study was to develop a standardized and widely applicable method for detecting CRPA in water samples, both in surface water and wastewater. Growth of well-characterized strains of CRPA was determined in four different selective agar media which were each supplemented with imipenem, meropenem, or ceftazidime, including after an enrichment. Wastewater samples presumptively containing CRPA (naturally contaminated samples) and surface water samples spiked with well-characterized CRPA strains were filtered and cultured using selective agar plates supplemented with imipenem and meropenem, with and without a prior enrichment step. M-PA-C agar plates supplemented with imipenem performed best compared to other selective agar plates with respect to growth of well-characterized CRPA strains and suppression of background growth of other bacteria and carbapenem-susceptible P. aeruginosa (CSPA) with positive predictive value of 80–100 %. A prior step using asparagine proline broth with vancomycin is recommended for higher specificity considering direct plating was unsuccessful due to extensive background growth. A combination of nonselective enrichment with selective plating (i.e., addition of imipenem to the plate) is recommended to achieve low limits of detection. The addition of imipenem in the broth instead of in solid agar plates eliminated some of the CRPA strains, producing an increase in the lower limit of detection and a lower PPV (32 % vs 73 %).

Aerobic Granular Sludge (AGS) is a relatively new approach to wastewater treatment. As opposed to activated sludge with mainly flocculant biomass of relatively even composition, the gra nules in AGS differ in size and composition. Biomass ranges from flocs similar to activated sludge to granules of >6 mm. Granules also have distinct biomass due to the stratification of redox conditions across the granules. Typically organic micropollutants (OMPs) are removed via sorption and biodegradation during wastewater treatment, however this has yet to be thoroughly invested for AGS systems. We hypothesized that different sized granules have potentially different sorption behaviour due to the differe nces in extracellular polymeric substances (EPS) and have different biodegradation patterns due to the differences in biomass composition among size fractions. Therefore, we investigated the capacity of AGS to remove OMPs via sorption and biodegradation by performing controlled batch experiments in the lab. We separated the sludge into different sizes in order to understand the role of different sludge composition on OMP removal.

We observed notable sorption (>40% removal) for 10 of the 24 OMPs tested in our study (Figure 1). The 10 OMPs include 3 fluoroquinolones (norfloxacin, ofloxacin, and ciprofloxacin), 3 macrolides (clarithromycin, azithromycin, and erythromycin), 2 beta-blockers (propranolol and atenolol), tetracycline, and citalopram. We noted that all of these 10 compounds are ionizable, with 6 positively charged compounds and 4 zwitterionic compounds at pH 7. Considering that sludge biomass typically has a negative charge, this seems to indicate electrostatic interactions between the 10 OMPs and th e sludge. Larger fractions contributed more to sorption than smaller granules and flocs, as expressed per unit of biomass. Specifically, the normalized Kd¬ for large fractions was up to 100%larger than for small fractions, suggesting that larger fractions contribute more to sorption in real AGS systems than smaller fractions and flocs. However, sorption kinetics were most likely retarded by diffusion limitations in large granules. Biodegradation was observed for a number of compounds. Overall, this study elucidated the roles of sorption and biodegradation in the removal of OMPs in AGS WWTPs, showing that these processes are size fraction dependent.

The first therapeutic use of antimicrobial agents initiated their endless arms race with antimicrobial resistance (AMR). Although the genes encoding antimicrobial resistance are ancient and ubiquitous in various environmental compartments, including aquatic environments, over eight decades of exposure to selective pressure has changed the way antimicrobial resistance genes (ARGs) emerge and transmit among the three One Health sectors (that is, the intersected sectors of humans, animals and the environment). The dissemination of ARGs has been facilitated by the widespread use of antimicrobials, along with direct and secondary pollution pathways. Current global consensus dictates that AMR should be addressed under a One Health framework. AMR National Action Plans have frequently been formulated. However, the capacity for implementation is not ready in most countries, especially in low- and middle-income regions. This is in part due to the substantial challenges in documenting and controlling cross-sector AMR connectivity. Here we describe the past and current status of AMR, emphasizing the contribution of connectivity to global AMR burden. We discuss connectivity at ecological, microbial and genetic levels; propose an approach based on genomics and metagenomics to assess connectivity; and finally advocate for cross-sector studies to better understand AMR connectivity and mitigate dissemination. We believe that such harmonized connectivity studies will facilitate coordinated actions and investments across sectors and regions to scale up AMR management globally.

Each year, an estimated 7·7 million deaths are attributed to bacterial infections, of which 4.95 million are associated with drug-resistant pathogens, and 1·27 million are caused by bacterial pathogens resistant to the antibiotics available. Access to effective antibiotics when indicated prolongs life, reduces disability, reduces health-care expenses, and enables access to other life-saving medical innovations. Antimicrobial resistance undoes these benefits and is a major barrier to attainment of the Sustainable Development Goals, including targets for newborn survival, progress on healthy ageing, and alleviation of poverty. Adverse consequences from antimicrobial resistance are seen across the human life course in both health-care-associated and community-associated infections, as well as in animals and the food chain. The small set of effective antibiotics has narrowed, especially in resource-poor settings, and people who are very young, very old, and severely ill are particularly susceptible to resistant infections. This paper, the first in a Series on the challenge of antimicrobial resistance, considers the global scope of the problem and how it should be measured. Robust and actionable data are needed to drive changes and inform effective interventions to contain resistance. Surveillance must cover all geographical regions, minimise biases towards hospital-derived data, and include non-human niches.

Aerobic granular sludge (AGS) is gaining popularity as an alternative to activated sludge for wastewater treatment. However, little information is available on AGS regarding the removal of organic micropollutants (OMPs) through sorption. In this study, the sorption behavior of 24 OMPs at environmentally relevant concentrations (1 μg/L) was investigated in six sludge fractions of varying sizes (>4 mm, 2–4 mm, 1–2 mm, 0.6–1 mm, 0.2–0.6 mm, and <0.2 mm) from a full-scale AGS reactor using batch experiments. Sorption was significant (removal efficiency >40 %) for 10 OMPs, including 4 zwitterionic and 6 positively charged pharmaceuticals, indicating the importance of electrostatic interaction for OMP sorption in AGS systems. Larger granules exhibited a higher sorption coefficient and capacity than smaller AGS fractions, probably due to increased extracellular polymeric substance content for larger granules. Equilibrium OMP sorption was only reached after 72 h in granules larger than 2 mm, indicating an effect of longer diffusion distance for OMPs into larger granules. Additionally, compared to activated sludge, AGS demonstrates a similar or even slightly higher sorption capacity for 10 OMPs at 1 μg/L. Overall, this study is the first to investigate the sorption behavior of six AGS size fractions for OMPs at environmentally relevant concentrations (1 μg/L) and propose the possible roles of different-sized sludge in OMP sorption in the full-scale AGS reactor.

Source-separated gray water reclamation using nanofiltration as an advanced post-treatment option has received substantial interest in meeting the growing water demand. During reclamation, membrane integrity is crucial to ensure the water’s safety. This study evaluated several chemical and novel microbial indicators as indirect membrane integrity-monitoring methods for hollow fiber nanofiltration membranes in reclamation schemes. Under normal conditions, high retention of divalent ions and organic matter and near-complete removal of Escherichia coli (E. coli) were observed. Limited removal of the antibiotic gene (ARG) tetO was observed due to low feed concentrations and a higher detection limit (LOD). While 16S rRNA and ARG sul1 were not limited by their LODs, lower removals were observed, most likely due to free-floating DNA passing through the membranes. A broken fiber in a pilot-scale module reduced organic matter and microorganism removal substantially, while flux and ion rejection remained similar. Predictions made using the observed results and a previously proposed model allowed for the evaluation of the selected methods in upscaled reclamation systems. Based on these results, it was concluded that microorganisms could be employed as indicators in indirect membrane integrity-monitoring methods in large-scale reclamation schemes, while UV_254nm absorbance (used in organic matter determination) could be a viable solution in pilot-scale systems.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health"framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.