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Abstract: The chlorination behaviours of 12 antibiotics belonging to six classes at environmentally relevant concentrations were systematically examined under typical conditions relevant to municipal wastewater chlorination. Cefotaxime, cefalexin, ampicillin and tetracycline were completely removed under all three initial free chlorine dosages (5 mg/L, 10 mg/L, and 15 mg/L). The removal efficiencies of sulphamethoxazole, sulphadiazine, roxithromycin, anhydro-erythromycin, ofloxacin, and trimethoprim were closely correlated to the residual free chlorine concentration, and no further significant mass removal was observed after the residual free chlorine concentration decreased to less than ∼0.75 mg/L. Ammonia plays a critical role during chlorination because of its competition with antibiotics for free chlorine to form combined chlorine, which reacts slowly with these antibiotics. Except for norfloxacin and ciprofloxacin, the removal behaviours of the 10 other target antibiotics under ammonia nitrogen concentrations ranging from 2 to 15 mg/L were characterised by a rapid initial removal rate upon contact with free chlorine during the first 5 s–1 min (depending on the specific antibiotic and ammonia nitrogen concentration) and then a much slower removal rate. Free chlorine was responsible for the reaction with antibiotics during the rapid stage (first 5 s–1 min), whereas combined chlorine reacted with antibiotics in the subsequent slow stage. Combined chlorine can remove norfloxacin and ciprofloxacin at a relatively faster rate. The presence of suspended solids at 30 mg/L slightly decreased the antibiotic removal rate. The kinetic rate constants decreased by 2.1–13.9%, while the half-lives increased by 2.0–15.0% compared to those of a 0 mg/L suspended solid for the target antibiotics. [Copyright &y& Elsevier]

Abstract: The effect of pH on chlorination behaviors of 12 antibiotics, including β-lactams, sulfonamides, fluoroquinolones, tetracyclines, macrolides, and others at environmentally relevant concentrations was systematically examined in the effluent matrix of activated sludge process. The removal of most antibiotics (except cefalexin and tetracycline) significantly depended on pH in the range of 5.5–8.5. The elimination rates of ciprofloxacin, norfloxacin, anhydro-erythromycin, and roxithromycin increased while that of sulfamethoxazole decreased significantly with the increase of pH. Sulfadiazine, ofloxacin, and trimethoprim exhibited the highest reactivity with free available chlorine under the pH of 6–7, 7, and 7.5, respectively. Not only the free available chlorine species (HOCl and OCl−), but also the antibiotics species (cationic, neutral and anionic) affected the overall reaction rate. Anionic antibiotic species are usually much more reactive (1–3 orders of magnitude greater) than cationic antibiotic species toward free available chlorine. Although OCl− is a weaker oxidant than HOCl, chlorination of sulfadiazine, sulfamethoxazole, ciprofloxacin, norfloxacin, and trimethoprim with OCl− became significant at pH>7.5. The observed kinetics rate constants calculated from species-specific rate constants could accurately (0.91< R 2 <0.99) predict the antibiotic removal in chlorination of activated sludge effluent with similar DOC and ammonia concentration to this study at a given pH value. [Copyright &y& Elsevier]

Abstract: Bulking and foaming bacteria (BFB) are notorious in wastewater treatment although they are always presented in the normal activated sludge and playing certain roles other than being harmful. Previous studies using microscopy or conventional molecular methods could hardly get the full profile of the BFB in the normal activated sludge. In this study, high throughput sequencing was adopted to investigate the BFB community, which was sub-dominant in activated sludge from 14 global wastewater treatment plants. The full-length 16S rRNA gene sequences of BFB groups were collected from previous studies to build a database for local BLAST and subsequent taxonomic assignment. The total BFB percentage in each sample ranged from 1.86% to 8.99% according to the 16S rRNA gene V4 pyrotags detected at the BLAST similarity cutoff of 97%. The most abundant and frequent BFB groups are ‘Nostocoida limicola’ I and II, Mycobacterium fortuitum, Type 1863, and ‘Microthrix parvicella’. The BFB among the activated sludge samples were both biogeographically and technological distributed to some extent. An extending application was performed to evaluate and design oligonucleotides probes based on the rich information of high similar sequences. Our study also gave an exemplified case of investigation on the specific sub-dominant functional groups in complex bacterial communities revealed by high throughput sequencing. [Copyright &y& Elsevier]

Abstract: Soluble microbial products (SMP) in the wastewater treatment process not only cause fouling to the membrane, but also generate disinfection by-products (DBP) in the effluent, thus get increasing attention. In this study, SMP produced by activated sludge and isolates under different stressful conditions, i.e. starvation, salinity, heavy metals, low pH and high temperature, were characterized to investigate the effects of these conditions on the amount of SMP and their compositions. The analysis results using size exclusion chromatography (SEC), high pressure liquid chromatography (HPLC) and fluorescence excitation emission matrix (FEEM) showed that activated sludge and isolates suffered with the same stressful condition contained almost the same concentration and composition of SMP, indicating that the stressful condition instead of the microbial species played the crucial role in the production of SMP. Among of stressful conditions tested, high temperature had stimulated the production of polysaccharides and polycarboxylate-type humic acid with high hydrophilicity, which is in positive proportion to the foulants formation potential, thus should be avoided in membrane bioreactors. Low pH had promoted the generation of hydrophobic humic acid-like or protein-like organics, which had been proved as the main disinfection byproduct (DBP) precursor, thus should be avoided in the biological treatment. Starvation had less effect on SMP production as the seeding microbes had no substrates. [ABSTRACT FROM AUTHOR]

Abstract: Denitrification-based remediation has been proved as a cost-effective approach for organic removal in sediment. However, little attention has been drawn on the concomitant autotrophic denitrification process and its impacts during such treatment. In this study, a contaminated marine sediment sample was treated with nitrate in a series of experiments to characterize the autotrophic denitrification and its impacts on metal speciation. Through treatment, as the consequence of autotrophic denitrification which accounts for 73.9% of nitrate reduction, ∼98.8% acid volatile sulfide (AVS) was oxidized to sulfate, causing changes of Zn, Cu and Pb speciation in the sediment. Their oxidizable fractions decreased by 71.7%, 13% and 71% respectively while the bound-to-carbonate fractions increased by 52.0%, >700% and >40%, and the reducible fractions also increased by 276%, >280% and 140%. Thus, the relatively stable oxidizable phase of Zn, Cu and Pb was generally transferred to the more mobile bound-to-carbonate and reducible phases. According to SEM (simultaneously extracted metal) analysis, most of extractable Zn and Pb were no longer present in the form of metal sulfides after denitrification. The (Zn+Pb)/AVS ratio increased from 0.030 to 3.1. Both sequential extraction and AVS–SEM suggested a possible increase of heavy metal mobility and, thus, toxicity. Two major species responsible for autotrophic denitrification were identified to be phylogenetically related with Sulfurimonas paralvinellae and Thiohalophilus thiocyanoxidans. [Copyright &y& Elsevier]

Over 99% of dimethyl phthalate (DMP) and 93% of chemical oxygen demand (COD) were effectively removed in a continuous upflow anaerobic sludge blanket (UASB) reactor from a wastewater containing 600mg/L DMP at 8h of hydraulic retention time (HRT), corresponding to a loading rate of 3g-COD/(Ld). Each gram of sludge, expressed as volatile suspended solids (VSS), had a maximum specific methanogenic activity (SMA) of 24mg-CH4/(g-VSSd) using DMP as the sole carbon source. The sludge yield was estimated as 0.08g-VSS/g-COD. During anaerobic degradation, DMP was de-esterified, first to mono-methyl phthalate (MMP) and then to phthalate, before being de-aromatized and subsequently converted to CH4 and CO2. The maximum specific degradation rates of DMP, MMP and phthalate were 415, 88 and 36mg/(g-VSSd), respectively. Analysis based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) showed a gradual shift of microbial population with the increase of DMP loading. [Copyright &y& Elsevier]

A sequencing batch reactor (SBR) was operated in the conventional anaerobic–aerobic mode for enhanced biological phosphate removal (EBPR) using acetate as the sole substrate. Results showed that, however, the reactor was unable to remove phosphate from wastewater. The sludge containing 1.65% of phosphate did not exhibit the typical characteristics of polyphosphate-accumulating organisms (PAO) or glycogen-accumulating organisms (GAO). Phylogenetic analysis, based on 16S rDNA sequences of individual microorganisms, showed that the microbial community of this acetate-degrading sludge was closely related to Comamonas testosteroni (43.8% of total population) of β-1-proteobacteria, Zoogloea resiniphila (25.0%) of β-2-proteobacteria, and Xanthomonas maltophilia (19.8%) of γ-proteobacteria. Results of this study imply that GAO might not be the sole group of bacteria responsible to the deterioration of phosphate removal efficiency in an EBPR reactor. [Copyright &y& Elsevier]

• Sulfide treatment results in damages and chemical transformation of microplastics. • •OH radicals formed from spontaneous oxidation of sulfide are primary oxidants. • Radical oxidation reactions enable sulfide addition to surface of microplastics. • Sulfide addition can in turn facilitate radical oxidation of microplastics. • Breakdown of plastics in sulfide-rich zones may be a source of micro-/nanoplastics. Transformation of microplastics in aquatic environments and engineered systems (e.g., wastewater treatment plants) significantly affects their transport, fate and effects. Here, we present the counterintuitive finding that sulfide, a prevalent nucleophile and reductant, can result in oxidation of microplastics, in addition to sulfide addition. Treating four model microplastics (thermoplastic polyurethane, polystyrene, polyethylene terephthalate and polyethylene) with 0.1 mM sulfide in a Tris-buffer solution (pH 7.2, 25 °C) resulted in physical damages (embrittlement and cracking) and chemical transformation (increased O/C ratio and formation of C–S bonds) of the materials. Pre-aging of the microplastics with O 3 or UV treatment had varied effects on their reactivities toward sulfide, depending on the specific structural and surface chemistry properties of the polymers. Electron paramagnetic resonance and radical trapping/quenching experiments showed that sulfide underwent spontaneous oxidation to form •OH radicals, which acted as the primary oxidant to attack the carbon atoms in the polymer chains, leading to surface oxidation and chain scission. Notably, sulfide addition, verified with X-ray photoelectron spectroscopy and 13C-nuclear magnetic resonance spectroscopy analyses, likely contributed to the physicochemical transformation of microplastics together with radical oxidation in a synergistic manner. The findings unravel an important transformation route (and a potential source) of microplastics in the environment. [Display omitted] [ABSTRACT FROM AUTHOR]

• Sorbent-sorbate based hybrid ML model shows good adaptability to OP-MP sorption. • Sensitivity analyses show valid results of OP and MP feature importance. • Compared with MPs, the properties of OPs have a greater influence on sorption. • Major mechanisms are hydrophobic partitioning, H-bond formation and π-π interaction. Microplastics (MPs) are ubiquitously distributed in freshwater systems and they can determine the environmental fate of organic pollutants (OPs) via sorption interaction. However, the diverse physicochemical properties of MPs and the wide range of OP species make a deeper understanding of sorption mechanisms challenging. Traditional isotherm-based sorption models are limited in their universality since they normally only consider the nature and characteristics of either sorbents or sorbates individually. Therefore, only specific equilibrium concentrations or specific sorption isotherms can be used to predict sorption. To systematically evaluate and predict OP sorption under the influence of both MPs and OPs properties, we collected 475 sorption data from peer-reviewed publications and developed a poly-parameter-linear-free-energy-relationship-embedded machine learning method to analyze the collected sorption datasets. Models of different algorithms were compared, and the genetic algorithm and support vector machine hybrid model displayed the best prediction performance (R2 of 0.93 and root-mean-square-error of 0.07). Finally, comparison results of three feature importance analysis tools (forward step wise method, Shapley method, and global sensitivity analysis) showed that chemical properties of MPs, excess molar refraction, and hydrogen-bonding interaction of OPs contribute the most to sorption, reflecting the dominant sorption mechanisms of hydrophobic partitioning, hydrogen bond formation, and π-π interaction, respectively. This study presents a novel sorbate-sorbent-based ML model with a wide applicability to expand our capacity in understanding the complicated process and mechanism of OP sorption on MPs. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract The proliferation of antibiotic resistance genes (ARGs) in drinking water and their potential horizontal transfer to pathogenic microbes may cause failure of antibiotics. However, antimicrobial resistome monitoring in drinking water is not currently routine. The bacterial hosts of ARGs, especially small-sized microbes in drinking water, may not be effectively removed by membrane filtration disinfection and thus pose threats to human health. In the present study, using metagenomic based approach, we investigated antibiotic resistome of small-sized microbes (0.2–0.45 μm) in 20 household drinking water samples from 12 cities in Mainland China, Hong Kong and Singapore. A total of 265 ARG subtypes belonging to 17 ARG types were detected at abundances ranging from 4.0 × 10−2 to 1.0 × 100 copies/cell. Multidrug, bacitracin and aminoglycoside resistance genes are dominant, and 43 ARG subtypes were specifically carried by small-size microbes. Metagenomic assembly strategy revealed fragments of three opportunistic pathogen, i.e. Pseudomonas alcaligenes , Pseudomonas aeruginosa and Mycobacterium gordonae , carried mex W, aph (3′)-I and aac (2′)-I, respectively. Drinking water samples were classified into three groups based on the presence of ARG, pathogen and ARG-carrying pathogen. These new insights into the antibiotic resistome of small microbes in drinking water over a broad scale indicate the need for more comprehensive ARGs monitoring and surveillance of drinking water supplies. These findings, together with the perspectives and strategies proposed in this study, could support initiatives to improve drinking water safety. Graphical abstract Image 1 Highlights • Potential risks of small-sized microbial resistome in drinking water to human health. • Antibiotic resistome monitoring and host identification (pathogen/non-pathogen). • 43 ARG subtypes specifically carried by small-sized microbes. • Classification of drinking water samples based on the presence of ARG and pathogen. [ABSTRACT FROM AUTHOR]

• The latest version of Nanopore improves precision in wastewater viral surveillance. • Efficiency in fragmented viral genome detection is maximized with shorter amplicons. • As little as 0.001 Gb Nanopore data provides accurate real-time variant information. • Nanopore demonstrates utility in revealing dynamic SARS-CoV-2 patterns in Hong Kong. • Optimal cutoffs enable uncovering novel mutations and linking them with variants. Wastewater genomic sequencing stands as a pivotal complementary tool for viral surveillance in populations. While long-read Nanopore sequencing is a promising platform to provide real-time genomic data, concerns over the sequencing accuracy of the earlier Nanopore versions have somewhat restrained its widespread application in wastewater analysis. Here, we evaluate the latest improved version of Nanopore sequencing (R10.4.1), using SARS-CoV-2 as the model infectious virus, to demonstrate its effectiveness in wastewater viral monitoring. By comparing amplicon lengths of 400 bp and 1200 bp, we revealed that shorter PCR amplification is more suitable for wastewater samples due to viral genome fragmentation. Utilizing mock wastewater samples, we validated the reliability of Nanopore sequencing for variant identification by comparing it with Illumina sequencing results. The strength of Nanopore sequencing in generating real-time genomic data for providing early warning signals was also showcased, indicating that as little as 0.001 Gb of data can provide accurate results for variant prevalence. Our evaluation also identified optimal alteration frequency cutoffs (>50 %) for precise mutation profiling, achieving >99 % precision in detecting single nucleotide variants (SNVs) and insertions/deletions (indels). Monitoring two major wastewater treatment plants in Hong Kong from September 2022 to April 2023, covering over 4.5 million population, we observed a transition in dominant variants from BA.5 to XBB lineages, with XBB.1.5 being the most prevalent variants. Mutation detection also highlighted the potential of wastewater Nanopore sequencing in uncovering novel mutations and revealed links between signature mutations and specific variants. This study not only reveals the environmental implications of Nanopore sequencing in SARS-CoV-2 surveillance but also underscores its potential in broader applications including environmental health monitoring of other epidemic viruses, which could significantly enhance the field of wastewater-based epidemiology. [Display omitted] [ABSTRACT FROM AUTHOR]

• Testing protocols for DNA (mpox) and RNA (norovirus, rotavirus) viruses in wastewater were developed and validated. • Wastewater surveillance effectively tracked the diarrhea viruses' activity in the community. • The mpox virus signal was detectable in wastewater at a low prevalence area. • Four key components were provided to increase the values of wastewater surveillance network. The wastewater surveillance network successfully established for COVID-19 showed great potential to monitor other infectious viruses, such as norovirus, rotavirus and mpox virus. In this study, we established and validated detection methods for these viruses in wastewater. We developed a supernatant-based method to detect RNA viruses from wastewater samples and applied it to the monthly diarrhea viruses (norovirus genogroup I & II, and rotavirus) surveillance in wastewater treatment plants (WWTPs) at a city-wide level for 16 months. Significant correlations were observed between the diarrhea viruses concentrations in wastewater and detection rates in faecal specimens by clinical surveillance. The highest norovirus concentration in wastewater was obtained in winter, consistent with the seasonal pattern of norovirus outbreak in Hong Kong. Additionally, we established a pellet-based method to monitor DNA viruses in wastewater and detected weak signals for mpox virus in wastewater from a WWTP serving approximately 16,700 people, when the first mpox patient in Hong Kong was admitted to the hospital within the catchment area. Genomic sequencing provided confirmatory evidence for the validity of the results. Our findings emphasized the efficacy of the wastewater surveillance network in WWTPs as a cost-effective tool to track the transmission trend of diarrhea viruses and to provide sensitive detection of novel emerging viruses such as mpox virus in low-prevalence areas. The developed methods and surveillance results provide confidence for establishing robust wastewater surveillance programs to control infectious diseases in the post-pandemic era. [Display omitted] [ABSTRACT FROM AUTHOR]

• Absolute quantification benchmarked a wide-spectrum ARG removal efficiencies in AD systems. • AD systems effectively removed total ARGs. • ARG removal efficiency was significantly affected by operating parameters. • Absolute quantification revealed the fate of potential pathogenic antibiotic-resistant bacteria in AD systems. Sewage treatment works have been considered as hotspots for the dissemination of antibiotic resistance genes (ARGs). Anaerobic digestion (AD) has emerged as a promising approach for controlling the spread of ARGs while destroying biomass in sludge. Evaluating the impact of AD on ARG removal relies on the absolute quantification of ARGs. In this study, we quantified the ARG concentrations in both full-scale and lab-scale AD systems using a cellular spike-ins based absolute quantification approach. Results demonstrated that AD effectively removed 68 ± 18 %, 55 ± 12 %, and 57 ± 19 % of total ARGs in semi-continuous AD digesters, with solid retention times of 15, 20, and 25 days, respectively. The removal efficiency of total ARGs increased as the AD process progressed in the batch digesters over 40 days. A significant negative correlation was observed between digestion time and the concentrations of certain ARG types, such as beta-lactam, sulfonamide, and tetracycline. However, certain potential pathogenic antibiotic resistant bacteria (PARB) and multi-resistant high-risk ARGs-carrying populations robustly persisted throughout the AD process, regardless of the operating conditions. This study highlighted the influence of the AD process and its operating parameters on ARG removal, and revealed the broad spectrum and persistence of PARB in AD systems. These findings provided critical insights for the management of microbial hazards. [Display omitted] [ABSTRACT FROM AUTHOR]

• Global microbiome data were integrated into AMR assessment in the Pearl River. • The river had a higher AMR level than global rivers, with WWTPs and landfills as major sources. • Detection of risk rank I and emerging ARGs highlighted AMR risk in some river reaches. • Pathogen-related families were predominant ARG-carrying bacteria in river water and sediment. • Co-sharing of ARGs across taxonomic ranks implied their transfer potentials in the community. Rivers are important in spreading antimicrobial resistance (AMR). Assessing AMR risk in large rivers is challenged by large spatial scale and numerous contamination sources. Integrating river resistome data into a global framework may help addressing this difficulty. Here, we conducted an omics-based assessment of AMR in a large river (i.e. the Pearl River in China) with global microbiome data. Results showed that antibiotic resistome in river water and sediment was more diversified than that in other rivers, with contamination levels in some river reaches higher than global baselines. Discharge of WWTP effluent and landfill waste drove AMR prevalence in the river, and the resistome level was highly associated with human and animal sources. Detection of 54 risk rank I ARGs and emerging mobilizable mcr and tet(X) highlighted AMR risk in the river reaches with high human population density and livestock pollution. Florfenicol-resistant floR therein deserved priority concerns due to its high detection frequency, dissimilar phylogenetic distance, mobilizable potential, and presence in multiple pathogens. Co-sharing of ARGs across taxonomic ranks implied their transfer potentials in the community. By comparing with global genomic data, we found that Burkholderiaceae, Enterobacteriaceae, Moraxellaceae and Pseudomonadaceae were important potential ARG-carrying bacteria in the river, and WHO priority carbapenem-resistant Enterobacteriaceae, A. baumannii and P. aeruginosa should be included in future surveillance. Collectively, the findings from this study provide an omics-benchmarked assessment strategy for public risk associated with AMR in large rivers. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: For full understanding of the microbial community in the wastewater treatment bioreactors, one of the feasible and effective ways is to investigate the massive genetic information contained in the activated sludge. In this study, high-throughput pyrosequencing was applied to analyze the 16S rRNA gene of bacteria in a laboratory-scale nitrification reactor and a full-scale wastewater treatment plant. In total, 27,458 and 26,906 effective sequence reads of the 16S rRNA gene were obtained from the Reactor and the wastewater treatment plant activated sludge samples respectively. The taxonomic complexities in the two samples were compared at phylum and genus levels. According to the pyrosequencing results, even for a laboratory-scale reactor as simple as that in this study, a small size clone library is far from enough to reflect the whole profile of the bacterial community. In addition, it was found that the commonly used informatics tool “RDP classifier” may drastically assign Nitrosomonas sequences into a wrong taxonomic unit resulting in underestimation of ammonia-oxidizing bacteria in the bioreactors. In this paper the reasons for this mistakenly assignment were analyzed and correction methods were proposed. [Copyright &y& Elsevier]

• First report of the effect of aging on the formation of solute multilayers on MPs • Enhanced competition for hydrophobic sorption sites after UV irradiation • Hydrophilic cosolutes contribute to multilayer formation after UV irradiation. • Presence of biofilm and/or NOM on MD-UV-PS enhanced sorption of hydrophobic cosolutes • Formation of solute multilayers on MPs enhanced the toxicity towards microalgae Recent studies have demonstrated that weathering modifies the physicochemical properties and sorption behavior of microplastics (MPs). However, little is known about the effects of such weathering on the simultaneous sorption by MPs of different organic pollutants in multi-solute systems. In this study, the role of cosolute properties in the formation of solute multilayers with a hydrophobic primary solute (4-MBC) on pristine and various weathered polystyrene MPs (PSMPs) was examined. Three weathered PSMPs were studied namely, UV-irradiated PS (UV-PS), microbially degraded PS (MD-NPS), and UV-irradiated PS with subsequent microbial degradation (MD-UV-PS). The weathered PSMPs generally exhibited higher degree of oxygenated functionalities with less surface hydrophobicity than pristine particles. Our findings showed that the formation of solute multilayers with hydrophobic cosolutes was drastically suppressed in UV-PS due to more severe competition at hydrophobic sorption sites. Nevertheless, hydrophilic cosolutes contributed to solute multilayer formation with 4-MBC on PSMPs after UV irradiation, probably due to the stronger sorption of hydrophilic compounds to the oxidized surfaces of these particles via enhanced H-bonding. Strikingly, the sorption of 4-MBC by MD-UV-PS was notably enhanced when hydrophobic cosolutes were present. The observed synergistic sorption indicates that adhered biofilms and/or organic matter on MD-UV-PS could sorb the hydrophobic cosolute molecules, and eventually promote sorption of 4-MBC. Our further toxicity tests revealed that such solute multilayers formed on PSMPs inhibited microalgal growth. These results suggest that the fate and biological effects of MP-mediated chemical exposure could be strongly affected by weathering processes and coexistence of multiple organic contaminants in natural environments. Image, graphical abstract [ABSTRACT FROM AUTHOR]

• Choice of DNA extraction kit caused the highest variability in microbial profiling. • Targeted metagenomes could be susceptible to different sequencing run batches. • Technical variabilities may sometimes mask biological variabilities. • A complex environmental reference material (ERM) is established for standardization. • Reproducible and high confidence microbial profiles are prepared for the ERM. Breakthroughs in DNA-based technologies, especially in metagenomic sequencing, have drastically enhanced researchers' ability to explore environmental microbiome and the associated interplays within. However, as new methodologies are being actively developed for improvements in different aspects, metagenomic workflows become diversified and heterogeneous. Through a single-variable control approach, we quantified the microbial profiling variations arising from 6 common technical variables associated with metagenomic workflows for both simple and complex samples. The incurred variations were constantly the lowest in replicates of DNA isolation and DNA sequencing library construction. Different DNA extraction kits often caused the highest variation among all the tested variables. Additionally, sequencing run batch was an important source of variability for targeted platforms. As such, the development of an environmental reference material for complex environmental samples could be beneficial in benchmarking accrued non-biological variability within and between protocols and insuring reliable and reproducible sequencing outputs immediately upstream of bioinformatic analysis. To develop an environment reference material, sequencing of a well-homogenized environmental sample composed of activated sludge was performed using different pre-analytical assays in replications. In parallel, a certified mock community was processed and sequenced. Assays were ranked based on the reconstruction of the theoretical mock community profile. The reproducibility of the best-performing assay and the microbial profile of the reference material were further ascertained. We propose the adoption of our complex environmental reference material, which could reflect the degree of diversity in environmental microbiome studies, to facilitate accurate, reproducible, and comparable environmental metagenomics-based studies. [Display omitted] [ABSTRACT FROM AUTHOR]

• Two preanalytical methods (supernatant-based and pellet-based) were established for influenza A/B wastewater surveillance. • Sanger sequencing confirmed the presence of influenza A/B RNA in wastewater. • Wastewater influenza A virus concentration effectively reflected influenza activity in the community. • H1pdm virus was the dominant subtype in the community at the time of this study. • Wastewater surveillance can provide virus types and subtypes information for public health actions in the future. Wastewater surveillance is considered as a powerful tool in providing cost-effective, population-wide and near real-time surveillance results for controlling infectious diseases (i.e., SARS-CoV-2, influenza virus), complementary to clinical surveillance. To facilitate the utility of this emerging tool, we developed two preanalytical protocols (supernatant-based and pellet-based) for influenza A/B virus (IAV/IBV) wastewater surveillance and applied them to the established wastewater surveillance network for large-scale longitudinal monitoring in Hong Kong. We tested 724 wastewater samples from 24 stationary sites for weekly surveillance for 8 months and 458 wastewater samples from 11 wastewater treatment plants (WWTPs) for more frequent (three times per week) city-wide surveillance for 4 months when influenza season commenced. We found the city-wide IAV virus concentration in wastewater were associated with the detection rate and influenza-like illness plus rates (ILI+) of clinical respiratory specimens and increased significantly after the cancelling of mask mandate that was in place for COVID-19. IBV was at low detection rates and low virus concentration levels, consistent with the low detection rates observed by clinical surveillance. In addition, we conducted virus subtype identification in selected wastewater samples, and observed the H1pdm was the major circulation subtype. Moreover, the obtained virus signals were confirmed by Sanger sequencing of PCR products, suggesting the feasibility and applicability of established methods for rapid detection of influenza virus types and subtypes in wastewater surveillance. This study demonstrates the applicability of IAV/IBV wastewater surveillance to current wastewater infrastructures and it could be used as a rapid and cost-effective surveillance strategy to track virus transmission patterns in the community for timely public health actions in the future. [ABSTRACT FROM AUTHOR]

• Achieving reliable results with sequencing throughput above 0.5 Gb. • Validating performance of variant deconvolution pipeline. • Deciphering the variant prevalence through wastewater genomic sequencing. • Tracking variant trends 16 days earlier than clinical data using wastewater data. • Uncovering novel SARS-CoV-2 mutations and alleles in wastewater. Wastewater-based epidemiology (WBE) has been widely used as a complementary approach to SARS-CoV-2 clinical surveillance. Wastewater genomic sequencing could provide valuable information on the genomic diversity of SARS-CoV-2 in the surveyed population. However, reliable detection and quantification of variants or mutations remain challenging. In this study, we used mock wastewater samples created by spiking SARS-CoV-2 variant standard RNA into wastewater RNA to evaluate the impacts of sequencing throughput on various aspects such as genome coverage, mutation detection, and SARS-CoV-2 variant deconvolution. We found that wastewater datasets with sequencing throughput greater than 0.5 Gb yielded reliable results in genomic analysis. In addition, using in silico mock datasets, we evaluated the performance of the adopted pipeline for variant deconvolution. By sequencing 86 wastewater samples covering more than 6 million people over 7 months, we presented two use cases of wastewater genomic sequencing for surveying COVID-19 in Hong Kong in WBE applications, including the replacement of Delta variants by Omicron variants, and the prevalence and development trends of three Omicron sublineages. Importantly, the wastewater genomic sequencing data were able to reveal the variant trends 16 days before the clinical data did. By investigating mutations of the spike (S) gene of the SARS-CoV-2 virus, we also showed the potential of wastewater genomic sequencing in identifying novel mutations and unique alleles. Overall, our study demonstrated the crucial role of wastewater genomic surveillance in providing valuable insights into the emergence and monitoring of new SARS-CoV-2 variants and laid a solid foundation for the development of genomic analysis methodologies for WBE of other novel emerging viruses in the future. [Display omitted] [ABSTRACT FROM AUTHOR]

Treated wastewater discharged into the environment acts as a disturbance of the natural microbial communities in terms of taxonomic composition and of functional gene pool, including antibiotic resistance genes. We tested whether stochastic and heterogeneous site-specific trajectories or generalities, potentially driven by deterministic processes, control the fate of allochthonous bacteria from anthropogenic sources and the persistence of their functional traits in freshwater. Finding generalities would allow the identification of wastewater treatments that could be effective in abating determinants of antibiotic resistance. We analysed the short-term response of native bacterial communities in waters exposed to the disturbance of wastewater at different dilutions, using a metagenomic approach that revealed both microbial community composition and the scope and abundance of the resistome that can pose indirect risks to human health. We found that the taxonomic composition of the communities after the disturbance was driven by case-specific stochastic processes, whereas the resistome had a deterministic trajectory, rapidly stabilising its functional traits with higher proportions of wastewater effluents, regardless of differences in taxonomic composition, richness of antibiotic resistance genes and of bacterial taxa, phenotypic features of the bacterial communities, and type of wastewater treatment. The observed deterministic proliferation of resistomes in freshwater bodies receiving wastewater effluents, suggests that this process may contribute to the global propagation of antibiotic resistance, and thus calls for new legislations promoting alternative tertiary treatments for the wastewater reuse, and targeting bacterial functional traits and not only bacterial abundances. Image 1 • Resistome and microbiome follow different dynamics in surface waters. • Stability of the antibiotic resistome is determined by the effluent dilution rate. • Stabilization of the resistome is independent of the quality of the treatment. [ABSTRACT FROM AUTHOR]

Denitrification is the stepwise microbial reduction of nitrate or nitrite (NO 2 −) to nitrogen gas via the obligate intermediates nitric oxide (NO) and nitrous oxide (N 2 O). Substantial N 2 O accumulation has been reported in denitrifying enhanced biological phosphorus removal (EBPR) bioreactors enriched in denitrifying polyphosphate accumulating organisms (DPAOs), but little is known about underlying mechanisms for N 2 O generation, prevalence of complete versus truncated denitrification pathways, or the impact of NO 2 − feed on DPAO-enriched consortia. To address this knowledge gap, we employed genome-resolved metagenomics to investigate nitrogen transformation potential in a NO 2 − fed denitrifying EBPR bioreactor enriched in Candidatus Accumulibacter and prone to N 2 O accumulation. Our analysis yielded 41 near-complete metagenome-assembled genomes (MAGs), including two co-occurring Accumulibacter strains affiliated with clades IA and IC (the first published genome from this clade) and 39 non-PAO flanking bacterial genomes. The dominant Accumulibacter clade IA encoded genes for complete denitrification, while the lower abundance Accumulibacter clade IC harbored all denitrification genes except for a canonical respiratory NO reductase. Analysis of the 39 non-PAO MAGs revealed a high prevalence of taxa harboring an incomplete denitrification pathway. Of the 27 MAGs harboring capacity for at least one step in the denitrification pathway, 10 were putative N 2 O producers lacking N 2 O reductase, 16 were putative N 2 O reducers that lacked at least one upstream denitrification gene, and only one harbored a complete denitrification pathway. We also documented increasing abundance over the course of reactor operation of putative N 2 O producers. Our results suggest that the unusually high levels of N 2 O production observed in this Accumulibacter-enriched consortium are linked in part to the selection for non-PAO flanking microorganisms with truncated denitrification pathways. Graphical abstract Image 1 Highlights • Two Accumulibacter clades IA and IC genomes were assembled from a denitrifying EBPR system. • Type I Accumulibacter was found to have a potential advantage in denitrifying P uptake. • 39 genomes were recovered from the non-PAO flanking reactor bacterial consortia. • A high prevalence of bacterial taxa with truncated denitrification pathways was observed. • Putative N 2 O producers increased in abundance and may contribute to N 2 O formation. [ABSTRACT FROM AUTHOR]

• Resistome was most diverse in wastewater while most abundant in fecal samples. • Industrial, urban, agricultural, and natural environments were surveyed globally. • Bacterial composition was significantly correlated with ARGs across most habitats. • A microbial source tracking model regarding resistome has been developed for MST v2.0. • Emerging mobilizable ARGs: mcr and tet(X) in these habitats were benchmarked. The widely distributed antibiotic resistance genes (ARGs) were unevenly proliferated in various habitats. Great endeavors are needed to resolve the resistome features that can differentiate or connect different habitats. This study retrieved a broad spectrum of resistome profiles from 1723 metagenomes categorized into 13 habitats, encompassing industrial, urban, agricultural, and natural environments, and spanning most continents and oceans. The resistome features (ARG types, subtypes, indicator ARGs, and emerging mobilizable ARGs: mcr and tet(X)) in these habitats were benchmarked via a standardized workflow. We found that wastewater and wastewater treatment works were characterized to be reservoirs of more diverse genotypes of ARGs than any other habitats including human and livestock fecal samples, while fecal samples were with higher ARG abundance. Bacterial taxonomy composition was significantly correlated with resistome composition across most habitats. Moreover, the source-sink connectivities were disentangled by developing the resistome-based microbial attribution prediction model. Environmental surveys with standardized bioinformatic workflow proposed in this study will help comprehensively understand the transfer of ARGs in the environment, thus prioritizing the critical environments with high risks for intervention to tackle the problem of ARGs. [Display omitted] [ABSTRACT FROM AUTHOR]

• PMA could deplete relic DNA signal while preserving viable microbial composition • Absolute quantification could be accurately achieved using cellular spike-ins • High agreement between PMA-enabled viable cell absolute quantification and CFU • Improved human health risk assessment by PMA in metagenomics through QMRA • High proportions of relic DNA (avg. 40%) found in beach waters The majority of the current regulatory practices for routine monitoring of beach water quality rely on the culture-based enumeration of faecal indicator bacteria (FIB) to develop criteria for promoting the general public's health. To address the limitations of culture methods and the arguable reliability of FIB in indicating health risks, we developed a Nanopore metagenomic sequencing-based viable cell absolute quantification workflow to rapidly and accurately estimate a broad range of microbes in beach waters by a combination of propidium monoazide (PMA) and cellular spike-ins. Using the simple synthetic bacterial communities mixed with viable and heat-killed cells, we observed near-complete relic DNA removal by PMA with minimal disturbance to the composition of viable cells, demonstrating the feasibility of PMA treatment in profiling viable cells by Nanopore sequencing. On a simple mock community comprised of 15 prokaryotic species, our results showed high accordance between the expected and estimated concentrations, suggesting the accuracy of our method in absolute quantification. We then further assessed the accuracy of our method for counting viable Escherichia coli and Vibrio spp. in beach waters by comparing to culture-based method, which were also in high agreement. Furthermore, we demonstrated that 1 Gb sequences obtained within 2 h would be sufficient to quantify a species having a concentration of ≥ 10 cells/mL in beach waters. Using our viability-resolved quantification workflow to assess the microbial risk of the beach water, we conducted (1) screening-level quantitative microbial risk assessment (QMRA) to investigate human illness risk and site-specific risk patterns that might guide risk management efforts and (2) metagenomics-based resistome risk assessment to evaluate another layer of risk caused by difficult illness treatment due to antimicrobial resistance (AMR). In summary, our metagenomic workflow for the rapid absolute quantification of viable bacteria demonstrated its great potential in paving new avenues toward holistic microbial risk assessment. [Display omitted] [ABSTRACT FROM AUTHOR]

High throughput sequencing-based metagenomic analysis and network analysis were applied to investigate the broad-spectrum profiles of ARGs in landfill leachate from 12 cities in China. In total, 526 ARG subtypes belonging to 21 ARG types were detected with abundances ranging from 1.1 × 10 −6 to 2.09 × 10 −1 copy of ARG/copy of 16S rRNA gene. 68 ARG subtypes that accounted for 73.4%–93.4% of the total ARG abundances were shared by all leachate samples. The four most abundant ARGs, sul 1, sul 2, aad A and bac A can be served as ARG indicators to quantitatively predict the total abundances by linear functions (r 2  = 0.577–0.819, P  < 0.001). No distinct regional distribution pattern of the ARGs was observed among different cities in China, while the ARG compositions of the leachate were clearly distinct from those of other environmental sample types. Nearly 90% ARG subtypes in the anaerobic digestion sludge from sewage treatment plants (STPADS) were shared by the leachate and the abundances of leachate and STPADS ARGs generalists accounted for 84.5% and 87.7% of total abundances in these two types of anaerobic samples, respectively. Furthermore, Procrustes analysis suggested that microbial community composition might be the determining factor of ARG compositions in landfill leachate. ARGs within the same type or among the different types showed higher incidences of non-random co-occurrence and 17 genera might be potential hosts of multiple ARGs. This study highlighted that landfill leachate is an important reservoir of various ARGs and provided a useful reference for the surveillance and risk management of ARGs in landfill environments. [ABSTRACT FROM AUTHOR]

Large amounts of antibiotics are currently used in livestock breeding, which is the main driving factor contributing to the occurrence, spread and proliferation of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment. In this study, high-throughput sequencing based metagenomic approaches were employed to characterize the tempo-spacial changes of antibiotic resistome, bacterial community and their correlations in pig farming wastewater and its receiving river. A total of 194 ARG subtypes within 14 ARG types were detectable in all the samples, and their total relative abundance increased in the river water after receiving wastewater discharge, while decreased in the downstream river water. Network analysis showed that 25.26% ARGs within the same type or among the different types showed higher incidences of non-random co-occurrence. The wastewater discharge evidently increased bacterial diversity and induced bacterial community shift in the receiving river water. The genera of Treponema, Prevotella , Pseudomonas, Bacteroides, Oscillibacter and Acholeplasma dominated in the wastewater samples and almost disappeared in the receiving river water, but bacterial pathogens Clostridium difficile and Arcobacter butzleri still occurred in the receiving water. Correlation analysis and host analysis consistently showed that the changes in the abundances of several key genera like Prevotella and Treponema were significantly and positively correlated with the antibiotic resistome alteration. Variation partitioning analysis indicated that bacterial community played a more important role in the resistome alteration than mobile genetic elements. This study may help to understand the correlations among antibiotic resistome, microbiota and environmental conditions in the wastewater-receiving river water. [ABSTRACT FROM AUTHOR]

We designed a highly selective surface coating to achieve enhanced rejection of endocrine disrupting compounds (EDCs) by nanofiltration membranes. A commercial NF90 membrane was first coated with polydopamine (PDA) followed by in situ immobilization of silver nanoparticles (AgNPs). This PDA/AgNPs coating greatly improved EDC rejection at the expense of slight water permeability loss (4–10%). This improvement in rejection can be attributed to a combination of enhanced size exclusion and suppressed hydrophobic interaction. A resistance-in-series analysis further reveals that the coating was highly permeable to water but highly resistant to EDCs, leading to an EDC selectivity that was an order of magnitude greater than those of the bare PDA coating and the base membrane NF90. The current study provides important insights into the design of highly selective coatings for effective retention of targeted trace organic contaminants. [ABSTRACT FROM AUTHOR]

• A PEG-precipitation method was developed for high-throughput wastewater surveillance. • The PEG-precipitation method obtained a process limit of detection of 3286 copies/L. • The PEG-precipitation method could process over 100 samples by two persons per day. • The turnaround time was optimized by reducing the centrifugation and incubation time. The effective application of wastewater surveillance is dependent on testing capacity and sensitivity to obtain high spatial resolution testing results for a timely targeted public health response. To achieve this purpose, the development of rapid, high-throughput, and sensitive virus concentration methods is urgently needed. Various protocols have been developed and implemented in wastewater surveillance networks so far, however, most of them lack the ability to scale up testing capacity or cannot achieve sufficient sensitivity for detecting SARS-CoV-2 RNA at low prevalence. In the present study, using positive raw wastewater in Hong Kong, a PEG precipitation-based three-step centrifugation method was developed, including low-speed centrifugation for large particles removal and the recovery of viral nucleic acid, and medium-speed centrifugation for the concentration of viral nucleic acid. This method could process over 100 samples by two persons per day to reach the process limit of detection (PLoD) of 3286 copies/L wastewater. Additionally, it was found that the testing capacity could be further increased by decreasing incubation and centrifugation time without significantly influencing the method sensitivity. The entire procedure uses ubiquitous reagents and instruments found in most laboratories to obtain robust testing results. This high-throughput, cost-effective, and sensitive tool will promote the establishment of nearly real-time wastewater surveillance networks for valuable public health information. [Display omitted] [ABSTRACT FROM AUTHOR]

The discovery of complete ammonia oxidizer (comammox) has fundamentally upended our perception of the global nitrogen cycle. Here, we reported four metagenome assembled genomes (MAGs) of comammox Nitrospira that were retrieved from metagenome datasets of tap water in Singapore (SG-bin1 and SG-bin2), Hainan province, China (HN-bin3) and Stanford, CA, USA (ST-bin4). Genes of phylogenetically distinct ammonia monooxygenase subunit A ( amoA ) and hydroxylamine dehydrogenase ( hao ) were identified in these four MAGs. Phylogenetic analysis based on ribosomal proteins, AmoA, hao and nitrite oxidoreductase (subunits nxrA and nxrB ) sequences indicated their close relationships with published comammox Nitrospira . Canonical ammonia-oxidizing microbes (AOM) were also identified in the three tap water samples, ammonia-oxidizing bacteria (AOB) in Singapore's and Stanford's samples and ammonia-oxidizing archaea (AOA) in Hainan's sample. The comammox amoA -like sequences were also detected from some other drinking water systems, and even outnumbered the AOA and AOB amoA- like sequences. The findings of MAGs and the occurrences of AOM in different drinking water systems provided a significant clue that comammox are widely distributed in drinking water systems. [ABSTRACT FROM AUTHOR]

An alternative process for anaerobic wastewater treatment with methane recovery is to elongate the carbon chain of volatile fatty acids (VFAs) with a formation of medium chain carboxylic acids (MCCAs), e.g. n -caproic acid with higher monetary value. A potential electron donor is glycerol as a surplus byproduct from the rapid growth of waste-derived biodiesel industry. In the current approach, an industrial chemical, 1,3-propanediol (1,3-PDO) is produced from crude glycerol along with a formation of other soluble byproducts including ethanol and volatile fatty acids (VFAs), which necessitates a significant amount of energy input for separation and purification. To circumvent the energy sink requirement and upcycle both the wastewater treatment process and the biodiesel industry, it is highly beneficial to produce a valuable secondary product from the byproducts. This pioneer study reports on thermodynamic and physiological insights gained into the co-production of 1,3-PDO and caproate from glycerol. Thermodynamics analysis demonstrated that a higher pH range is more favorable when either glycerol or ethanol acting as an electron donor, whereas a high partial pressure (27% at 1 atm) and a low pH (≤5.5) are advantageous for caproate formation with hydrogen. With the glycerol-to-acetate molar ratio of 4 and pH of 7, the physiological experiments achieved a co-production of 1,3-PDO and caproate. However, the caproate yield was low and found to be kinetic-limited. Caproate formation was significantly increased by the intermediate ethanol addition with the optimal mono-caproate formation obtained at the ethanol-to-acetate molar ratio of 3. A synergistic relationship was evinced through microbial characterization, resulting in Clostridium kluyveri and some bacteria with function of converting glycerol to VFAs. This study demonstrates that sufficient ethanol produced as an intermediate is capable of enhancing caproate formation in a synergistic pathway along with 1,3-PDO. The knowledge gleaned paves new avenues for the biodiesel industry by upcycling the byproduct crude glycerol into 1,3-PDO and caproate. [ABSTRACT FROM AUTHOR]

Activated sludge (AS) process has been widely utilized for municipal sewage and industrial wastewater treatment. Zoolgoea and its related floc-forming bacteria are required for formation of AS flocs which is the key to gravitational effluent-and-sludge separation and AS recycling. However, little is known about the genetics, biochemistry and physiology of Zoogloea and its related bacteria. This report deals with the comparative genomic analyses on two Zoogloea resiniphila draft genomes and the closely related proteobacterial species commonly found in AS. In particular, the metabolic processes involved in removal of organic matters, nitrogen and phosphorus were analyzed. Furthermore, it is revealed that a large gene cluster, encoding eight glycosyltransferases and other proteins involved in biosynthesis and export of extracellular polysaccharides (EPS), was required for floc formation. One of the two asparagine synthase paralogues, associated with this EPS biosynthesis gene cluster, was required for floc formation in Zoogloea . Similar EPS biosynthesis gene cluster(s) were identified in the genome of other AS proteobacteria including polyphosphate-accumulating Candidatus Accumulibacter phosphatis (CAP) and nitrifying Nitrosopira and Nitrosomonas bacteria, but the gene composition varies interspecifically and intraspecifically. Our results indicate that floc formation of desired AS bacteria, including CAP strains, facilitate their recruitment into AS and gradual enrichment via repeated AS settling and recycling processes. [ABSTRACT FROM AUTHOR]

• Nanopore-based ARGs identified by ARGpore2 is as accurate as BLASTP-based approach. • A more reliable ARB profile was obtained from assembly-free long reads strategy. • The WWTP-Effluent ARB intrusion cause resistome enrichment in receiving water. • Plasmids and class 1 integrons facilitate the ARGs persistence and dissemination. Wastewater treatment plant (WWTP) effluent discharge could induce the resistome enrichment in the receiving water environments. However, because of the general lack of a robust antibiotic-resistant bacteria (ARB) identification method, the driving mechanism for resistome accumulation in receiving environment is unclear. Here, we took advantage of the enhanced ARBs recognition by nanopore long reads to distinguish the indigenous ARBs and the accumulation of WWTP-borne ARBs in the receiving water body of a domestic WWTP. A bioinformatic framework (named ARGpore2: https://github.com/sustc-xylab/ARGpore2) was constructed and evaluate to facilitate antibiotic resistance genes (ARGs) and ARBs identification in nanopore reads. ARGs identification by ARGpore2 showed comparable precision and recall to that of the commonly adopt BLASTP-based method, whereas the spectrum of ARBs doubled that of the assembled Illumina dataset. Totally, we identified 33 ARBs genera carrying 65 ARG subtypes in the receiving seawater, whose concentration was in general 10 times higher than clean seawater's. Notably we report a primary resistome intrusion caused by the revival of residual microbes survived from disinfection treatment. These WWTP-borne ARBs, including several animal/human enteric pathogens, contributed up to 85% of the receiving water resistome. Plasmids and class 1 integrons were reckoned as major vehicles facilitating the persistence and dissemination of ARGs. Moreover, our work demonstrated the importance of extensive carrier identification in determining the driving force of multifactor coupled resistome booming in complicated environmental conditions, thereby paving the way for establishing priority for effective ARGs mitigation strategies. [Display omitted] [ABSTRACT FROM AUTHOR]

• The distribution and activity of ammonia/nitrite oxidizers were profiled. • AOB and anammox bacteria collectively contributed to nitrogen conversion at Stage-A. • Abundant and active comammox nitrospira and AOA were observed at Stage-B. • Complex factors shaped niche differentiation and symbiotic association in the RBC. • Phages likely act as a biotic factor mediating ammonia/nitrite oxidizer populations. Although the distribution of ammonia/nitrite oxidizers had been profiled in different habitats, current understanding is still limited regarding their niche differentiation in the integrated biofilm reactors, the symbiotic associations of ammonia/nitrite oxidizers, as well as the parasitic interaction between viruses and those functional organisms involved in the nitrogen cycle. Here, the integrated metagenomics and metatranscriptomics are applied to profile the ammonia/nitrite oxidizers communities and transcriptional activities changes along the flowpath of a concatenated full-scale rotating biological contactor (RBC) (frontend Stage-A and backend Stage-B). 19 metagenome-assembled genomes (MAGs) of ammonia/nitrite oxidizers were recovered by using a hybrid assembly approach, including four ammonia-oxidizing bacteria (AOB), two ammonia-oxidizing archaea (AOA), two complete ammonia oxidation bacteria (comammox), eight nitrite-oxidizing bacteria (NOB), and three anaerobic ammonium oxidation bacteria (anammox). Diverse AOB and anammox dominated Stage-A and collectively contributed to nitrogen conversion. With the decline of ammonia concentration along the flowpath, comammox and AOA appeared and increased in relative abundance in Stage-B, accounting for 8.8% of the entire community at the end of this reactor, and their dominating role in nitrogen turnover was indicated by the high transcription activity of their corresponding function genes. Moreover, the variation in the abundance of viruses infecting ammonia and nitrite oxidizers suggests that viruses likely act as a biotic factor mediating ammonia/nitrite oxidizer populations. This study demonstrates that complex factors shaped niche differentiation and symbiotic associations of ammonia/nitrite oxidizers in the RBC and highlights the importance of RBCs as model systems for the investigation of biotic and abiotic factors affecting the composition of microbiomes. [Display omitted] [ABSTRACT FROM AUTHOR]

• Cellular spike-ins with metagenomics for microbial absolute quantification. • Maximum growth and decay rates of microbes were calculated. • Specific activities of methanogens were computed. Anaerobic digestion (AD) relies on myriads of functions performed by complex microbial communities in customized settings, thus, a comprehensive investigation on the AD microbiome is central to the fine-tuned control. Most current AD microbiome studies are based on relative abundance, which hinders the interpretation of microbes' dynamics and inter-sample comparisons. Here, we developed an absolute quantification (AQ) approach that integrated cellular spike-ins with metagenomic sequencing to elucidate microbial community variations and population dynamics in four anaerobic digesters. Using this method, 253 microbes were defined as decaying populations with decay rates ranging from -0.05 to -5.85 d−1, wherein, a population from Flavobacteriaceae family decayed at the highest rates of -3.87 to -5.85 d−1 in four digesters. Meanwhile, 25 microbes demonstrated the growing trend in the AD processes with growth rates ranging from 0.11 to 1.77 d−1, and genome-centric analysis assigned some of the populations to the functional niches of hydrolysis, short-chain fatty acids metabolism, and methane generation. Additionally, we observed that the specific activity of methanogens was lower in the prolonged digestion stage, and redundancy analysis revealed that the feedstock composition and the digestion duration were the two key parameters in governing the AD microbial compositions. [Display omitted] [ABSTRACT FROM AUTHOR]

Understanding which/how antibiotic resistance genes (ARGs) contribute to increased acquisition of resistance by pathogens in aquatic environments are challenges of profound significance. We explored the co-occurrence and removal versus enrichment of ARGs and human bacterial pathogens (HBPs) in municipal sewage sludge digesters. We combined metagenomic detection of a wide spectrum of 323 ARGs and 83 HBPs with a correlation-based statistical approach and charted a network of their co-occurrence relationships. The results indicate that most ARGs and a minor proportion of HBPs (mainly Collinsella aerofaciens, Streptococcus salivarius and Gordonia bronchialis ) could not be removed by anaerobic digestion, revealing a biological risk of post-digestion sludge in disseminating antibiotic resistance and pathogenicity. Moreover, preferential co-occurrence patterns were evident within one ARG type (e.g., multidrug, beta-lactam, and aminoglycoside) and between two different ARG types (i.e., aminoglycoside and beta-lactam), possibly implicating co-effects of antibiotic selection pressure and co-resistance on shaping antibiotic resistome in sewage sludge. Unlike beta-lactam resistance genes, ARGs of multidrug and macrolide-lincosamide-streptogramin tended to co-occur more with HBPs. Strikingly, we presented evidence that the most straightforward biological origin of an ARG-species co-occurring event is a hosting relationship. Furthermore, a significant and robust HBP-species co-occurrence correlation provides a proper scenario for nominating HBP indicators (e.g., Bifidobacterium spp. are perfect indicators of C. aerofaciens ; r = 0.92–0.99 and P-values < 0.01). Combined, this study demonstrates a creative and effective network-based metagenomic approach for exploring ARG hosts and HBP indicators and assessing ARGs acquisition by HBPs in human-impacted environments where ARGs and HBPs may co-thrive. [ABSTRACT FROM AUTHOR]

• Eight novel assays with high predictive value for eight variants. • Near real-time monitoring for emerging variants in the community. • Reliable quantification for mutation sites even in the sewage having variants mixture. • High correlation between clinical sequencing results and sewage variant signals. • A hierarchical detection workflow for flexible deployment of the method. To effectively control the ongoing outbreaks of fast-spreading SARS-CoV-2 variants, there is an urgent need to add rapid variant detection and discrimination methods to the existing sewage surveillance systems established worldwide. We designed eight assays based on allele-specific RT-qPCR for real-time allelic discrimination of eight SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, Omicron, Lambda, Mu, and Kappa) in sewage. In silico analysis of the designed assays for identifying SARS-CoV-2 variants using more than four million SARS-CoV-2 variant sequences yielded ∼100% specificity and >90% sensitivity. All assays could sensitively discriminate and quantify target variants at levels as low as 10 viral RNA copy/µL with minimal cross-reactivity to the corresponding nontarget genotypes, even for sewage samples containing mixtures of SARS-CoV-2 variants with differential abundances. Integration of this method into the routine sewage surveillance in Hong Kong successfully identified the Beta variant in a community sewage. Complete concordance was observed between the results of viral whole-genome sequencing and those of our novel assays in sewage samples that contained exclusively the Delta variant discharged by a clinically diagnosed COVID-19 patient living in a quarantine hotel. Our assays in this method also provided real-time discrimination of the newly emerging Omicron variant in sewage two days prior to clinical test results in another quarantine hotel in Hong Kong. These novel allelic discrimination assays offer a rapid, sensitive, and specific way for detecting multiple SARS-CoV-2 variants in sewage and can be directly integrated into the existing sewage surveillance systems. [Display omitted] [ABSTRACT FROM AUTHOR]

• FW:FSS ratios caused large variations of microbial community composition and populations dynamics. • Traceable microbial clustering was shaped by different FW:FSS ratios. • Methanogenic community altered its trophic groups and energy conserving strategy to ensure methane generation. • Microbial symbiosis and interspecies competition were observed in thermophilic microbiome. A self-stabilizing microbial community lays the foundation of the efficient biochemical reactions of the anaerobic digestion (AD) process. Despite extensive profiling of microbial community dynamics under varying operating parameters, the effects of food waste (FW) to feeding sewage sludge (FSS) ratios on the microbial assembly, functional traits, and syntrophic interspecies interactions in thermophilic microbial consortia remain poorly understood. Here, we investigated the long-term impacts of the FW: FSS ratio on the thermophilic AD microbiome using genome-centric metagenomics. Both the short reads (SRs) assembly, and the iterative hybrid assembly (IHA) of SRs and nanopore long reads (LRs) were used to reconstruct metagenome-assembled genomes (MAGs) and four microbial clusters were identified, demonstrating different microbial dynamics patterns in response to varying FW:FSS ratios. Cluster C1-C3 were comprised of full functional members with genetic potentials in fulfilling empirical AD biochemical reactions, wherein, syntrophic decarboxylating acetogens could interact with methanogens, and some microbes could be energized by the electron bifurcation mechanism to drive thermodynamics unfavorable reactions. We found the co-existence of both acetogenic and hydrogenotrophic methanogens in the AD microbiome, and they altered their trophic groups to scavenge the methanogenic substrates in ensuring the methane generation in digesters with different FW:FSS ratios. Another interesting observation was that two phylogenetically close Thermotogota species showed a possible strong competition on carbon source inferred by the nearly complete genetic overlap of their relevant pathways. : [Display omitted] [ABSTRACT FROM AUTHOR]

• 22,771 bacterial OTUs were observed from 110 samples covering 38 cities in China. • Spatial turnover of drinking water communities was significantly scale-dependent. • Precipitation and source river were environmental drivers to shape communities. • Household drinking water carried potential microbiological-associated health risks. Drinking water at the point of use harbors microorganisms that may pose potential risks to human health. However, the microbial diversity and health impacts of household drinking water are poorly understood, since culture-based methods only target on specific microorganisms and low biomass of drinking water hinders a high-throughput profiling. Here, we used an optimized workflow to efficiently collect microorganisms from low-biomass drinking water and performed deep sequencing of 16S rRNA genes to profile the bacterial diversity and biogeography of 110 household drinking water samples covering 38 cities of 29 provinces/regions in China, and further explored environmental drivers and potential health implications. Our analyses revealed a diverse drinking water community comprising a total of 22,771 operational taxonomic units (OTUs). The spatial turnover of drinking water communities is scale-dependent and appears to be driven largely by rainfall and water source river. The identified potential pathogenic species may have the possibility of causing health risks. Our novel insights enhance the current understanding of the diversity and biogeography of drinking water bacterial communities within a theoretical ecological framework and have further important implications for safe drinking water management and public health protection. [Display omitted] [ABSTRACT FROM AUTHOR]

• We collected time-series influent, AS and effluent for metagenomic sequencing. • Prevalence of ARGs and MGEs was examined in effluents from global cities. • An online platform integrating the resistome risk ranking scheme was developed. • ARG variants with high risk were characterized in effluents. Wastewater treatment plants (WWTPs) are regarded as critical points in disseminating antibiotic resistance genes (ARGs). In particular, the discharging effluents from WWTPs generally bring downstream catchment areas exogenous ARGs and resistant bacteria. However, there lacks a sufficient assessment of the resistome and mobilome in effluents. In this study, a consecutive monthly sampling was conducted over 13 months in three Hong Kong (HK) WWTPs for metagenomic sequencing. Prevalence information of ARGs and mobile genetic elements (MGEs) was compared with counterparts in effluents from cities of North America, South America, Europe, and Asia. Moreover, a publicly accessible platform integrating the exposure ranking scheme, which was based on the global archive of ARG abundance, and a readily implementable online pipeline was developed to benefit communication in academia and government consultancy. Results demonstrated HK WWTPs were featured high ARG removal efficiency of 2.34–2.43 log reduction rate, and effluents were ranked in moderate levels of Level 2 and Level 3 in the exposure prioritizing scheme based on total ARG abundance. Moreover, absolute quantification of temporal variations of effluent resistome disclosed distinct changes over time among varied ARG types which were associated with prevalently used antibiotics, including quinolone and sulfonamide. This reinforces the need for real-time management of WWTP systems. Notably, ARGs of anthropogenic prevalence, high mobility, and potential pathogenicity were found to be present in HK effluents, drawing attention to the necessity for improved risk management. In addition, source tracking of effluent resistome and structural equation model analysis was conducted to explore the disparity in ARG abundance and diversity in different samples. The discovery of this study and the recommendation of a comprehensive exposure assessment will facilitate decision-making in resistome management in WWTPs to reduce the ARG and antibiotic resistant bacteria (ARB) contamination in the receiving environments. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: An innovative and cost-effective method, i.e., batch stress incubation with tetracycline in combination with Cloning-Sanger sequencing and Illumina high-throughput sequencing was developed to identify tetracycline resistant bacteria (TRB) in activated sludge (AS) treating saline sewage. This method overcomes the drawbacks of culture-based approach (unrepresentative and biased results) and utilizes both the advantages of Cloning-Sanger sequencing and Illumina high-throughput sequencing, that is, long length read for correct taxonomic assignment at lower ranks and enough sequencing depth for accurate quantification of TRB communities with medium to low abundances, respectively. High precision (relative deviation ≤16.1%) was obtained for all taxon ranks with relative abundances over 0.01%. In the AS sample, TRB consisted of 13 genera with Haliea, Microbacterium and Paracoccus as dominate genera and 6 new TRB genera, i.e., Haliea, Rheinheimera, Alishewanella, Idiomarina, Pseudorhodobacter and Algoriphagus. The increase of tetG and tetA abundance might be associated with the significant increase of Pseudomonas (tetG and tetA host) in the AS after tetracycline stress incubation. tetS abundance also showed an obvious increase after 20 mg/L tetracycline treatment. This method provided a new tool to screen other antibiotic resistant bacteria, bacteria resistant to heavy metals or disinfectants in AS samples. [Copyright &y& Elsevier]

Abstract: Antibiotic resistance genes (ARGs) are increasingly a focus of concern because they pose a potential health risk. The Pearl River (PR) and Pearl River Estuary (PRE) show a distinct gradient in anthropogenic impacts, in particular associated with the use of antibiotics, from the river, to the estuary, and on to the coast. In this study, two surveys were conducted in the PR and PRE areas during the winter and summer of 2011, respectively. Seven tet genes consisting of efflux pump (tetA, tetC, and tetH) and ribosomal protection proteins (tetB, tetM, tetO, and tetW) were analyzed using the polymerase chain reaction (PCR) technique. The tet genes, with the exception of tetA and tetH, were widely detected in the PR and PRE environments. The tet genes exhibited a trend of an increase in total concentration and diversity with the degree of anthropogenic impacts from the river to the coast, indicating that riverine input was the main source of ARGs in the region. Significant correlations were observed between tet genes and antibiotic concentrations, as well as among different environmental compartments (water and sediments). The distribution patterns of tet genes were similar between the potential sources of pollution and the highly-impacted sites, but were significantly different between less-impacted sites and highly-impacted ones or pollution sources. The results suggest that ARGs and antibiotics may be released from identical sources, and transported in a similar manner in estuary/coastal environments close to sources of pollution. [Copyright &y& Elsevier]

Abstract: This study aimed to investigate the chlorination effects on microbial antibiotic resistance in a drinking water treatment plant. Biochemical identification, 16S rRNA gene cloning and metagenomic analysis consistently indicated that Proteobacteria were the main antibiotic resistant bacteria (ARB) dominating in the drinking water and chlorine disinfection greatly affected microbial community structure. After chlorination, higher proportion of the surviving bacteria was resistant to chloramphenicol, trimethoprim and cephalothin. Quantitative real-time PCRs revealed that sulI had the highest abundance among the antibiotic resistance genes (ARGs) detected in the drinking water, followed by tetA and tetG. Chlorination caused enrichment of ampC, aphA2, bla TEM-1, tetA, tetG, ermA and ermB, but sulI was considerably removed (p < 0.05). Metagenomic analysis confirmed that drinking water chlorination could concentrate various ARGs, as well as of plasmids, insertion sequences and integrons involved in horizontal transfer of the ARGs. Water pipeline transportation tended to reduce the abundance of most ARGs, but various ARB and ARGs were still present in the tap water, which deserves more public health concerns. The results highlighted prevalence of ARB and ARGs in chlorinated drinking water and this study might be technologically useful for detecting the ARGs in water environments. [Copyright &y& Elsevier]

• Canal water microbiota composition was driven by seasonality more than location. • Micro-eukaryotic community assembly was more stochastic than bacteria. • Novel local growth factor analysis was applied to evaluate population growth. • Immigrating micro-eukaryotes better acclimated to the canal water than bacteria. Large water diversion projects are important constructions for reallocation of human-essential water resources. Deciphering microbiota dynamics and assembly mechanisms underlying canal water ecosystem services especially during long-distance diversion is a prerequisite for water quality monitoring, biohazard warning and sustainable management. Using a 1432-km canal of the South-to-North Water Diversion Projects as a model system, we answer three central questions: how bacterial and micro-eukaryotic communities spatio-temporally develop, how much ecological stochasticity contributes to microbiota assembly, and which immigrating populations better survive and navigate across the canal. We applied quantitative ribosomal RNA gene sequence analyses to investigate canal water microbial communities sampled over a year, as well as null model- and neutral model-based approaches to disentangle the microbiota assembly processes. Our results showed clear microbiota dynamics in community composition driven by seasonality more than geographic location, and seasonally dependent influence of environmental parameters. Overall, bacterial community was largely shaped by deterministic processes, whereas stochasticity dominated micro-eukaryotic community assembly. We defined a local growth factor (LGF) and demonstrated its innovative use to quantitatively infer microbial proliferation, unraveling taxonomically dependent population response to local environmental selection across canal sections. Using LGF as a quantitative indicator of immigrating capacities, we also found that most micro-eukaryotic populations (82%) from the source water sustained growth in the canal and better acclimated to the hydrodynamical water environment than bacteria (67%). Taxa inferred to largely propagate include Limnohabitans sp. and Cryptophyceae , potentially contributing to water auto-purification. Combined, our work poses first and unique insights into the microbiota assembly patterns and dynamics in the world's largest water diversion canal, providing important ecological knowledge for long-term sustainable water quality maintenance in such a giant engineered system. [Display omitted] [ABSTRACT FROM AUTHOR]

• Two acesulfame-degrading isolates, affiliated to Chelatococcus, were characterized. • The genomes of two acesulfame degraders (YT9 and HY11) were sequenced. • Three biotransformation products were demonstrated by UPLC-QTOF-MS. • Illumina and Nanopore sequencing were used to characterize the 6 enriched consortia. • All enrichments were dominated by the phyla Proteobacteria and Planctomycetes. The artificial sweetener Acesulfame (ACE) has been frequently detected in wastewater treatment plants (WWTPs) and is regarded as an emerging pollutant due to its low biodegradability. However, recent observations of ACE biodegradation in WWTPs have stimulated interest in the ACE-degrading bacteria and mineralization pathways. In this study, next-generation sequencing methods, Illumina and Nanopore sequencing, were combined to explore the ACE-degrading communities enriched from the activated sludge of six municipal wastewater treatment plants. Metagenomic investigations indicated that all enrichments were similarly dominated by the phyla Proteobacteria and Planctomycetes. Notably, at the species level, four metagenome-assembled genomes (MAGs) were shared by six enriched communities with considerable abundances, indicating that they may be responsible for ACE biodegradation in the enrichments. Besides, two ACE-degrading pure strains, affiliated to the genus Chelatococcus , were isolated from the enrichment. The genomic analysis showed that these two isolates were the new species that were genetically distinct from their relatives. Two type strains, Chelatococcus asaccharovorans DSM 6462 and Chelatococcus composti DSM 101465, could not degrade ACE, implying that the ACE-degrading capability was not shared among the different species in the genus Chelatococcus. The results of the degradation experiment showed that the two isolates could use ACE as the sole carbon source and mineralize ~90% of the total organic carbon. Three biotransformation products (TP96, TP180B, and TP182B) were demonstrated by UPLC-QTOF-MS. The results of this study provide valuable insights into ACE biodegradation and its biotransformation products. [Display omitted] [ABSTRACT FROM AUTHOR]

• Antibiotics affect the dynamics of ammonia oxidizers in activated sludge • Four antibiotics (AMP, KAN, LIN, and TMP) could selectively enrich comammox • Different antibiotics select phylogenetically distant comammox Nitrospira • Comammox in wastewater treatment systems show high metabolic versatility While the ubiquitous presence of comammox in engineered systems provides the foundation of developing a novel biological nitrogen removal process, factors contributing to the comammox dynamics in engineered systems have not been well resolved. Here, we investigate the long-term effects of ten different antibiotics on microbial community dynamics in activated sludge and the results show that both types and concentrations of antibiotics affect the taxonomic composition of nitrifiers, including comammox, ammonia-oxidizing bacteria, and canonical nitrite-oxidizing bacteria. Specifically, phylogenetically different comammox Nitrospira were selectively enriched by four types of antibiotics (i.e., ampicillin, kanamycin, lincomycin, and trimethoprim). Comparative genomic analysis of the four newly identified comammox clade A Nitrospira revealed that the comammox enriched by antibiotics shared the conserved key metabolic potentials, such as carbon fixation, complete ammonia oxidation, and utilization of hydrogen as alternative electron donors, among the known comammox organisms. Comammox strains enriched in this study also encoded genes involved in formate and cyanate metabolism that were recently reported in comammox clade A organisms from wastewater treatment systems. Our findings highlight that the comammox in activated sludge ecosystems possess high metabolic versatility than previously recognized and could be selectively enriched by some antibiotics. [Display omitted] [ABSTRACT FROM AUTHOR]

Antibiotic resistance has become a global public health concern, rendering common infections untreatable. Given the widespread occurrence, increasing attention is being turned toward environmental pathways that potentially contribute to antibiotic resistance gene (ARG) dissemination outside the clinical realm. Studies during the past decade have clearly proved the increased ARG pollution trend along with gradient of anthropogenic interference, mainly through marker-ARG detection by PCR-based approaches. However, accurate source-tracking has been always confounded by various factors in previous studies, such as autochthonous ARG level, spatiotemporal variability and environmental resistome complexity, as well as inherent method limitation. The rapidly developed metagenomics profiles ARG occurrence within the sample-wide genomic context, opening a new avenue for source tracking of environmental ARG pollution. Coupling with machine-learning classification, it has been demonstrated the potential of metagenomic ARG profiles in unambiguously assigning source contribution. Through identifying indicator ARG and recovering ARG-host genomes, metagenomics-based analysis will further increase the resolution and accuracy of source tracking. In this review, challenges and progresses in source-tracking studies on environmental ARG pollution will be discussed, with specific focus on recent metagenomics-guide approaches. We propose an integrative metagenomics-based framework, in which coordinated efforts on experimental design and metagenomic analysis will assist in realizing the ultimate goal of robust source-tracking in environmental ARG pollution. Image 1 • Environmental pollution of ARGs has been a public health concern. • Quantitative source-tracking remains as a bottleneck in marker-ARG based methods. • Metagenomics-based ARG profiling can circumvent limitations in traditional methods. • Machine-learning classification is of great potential of accurate source-tracking. • Robust source tracking can be realized by an integrative metagenomic framework. [ABSTRACT FROM AUTHOR]

Co-digestion of food waste with sewage sludge is widely applied for waste stabilization and energy recovery around the world. However, the effect of solid retention time (SRT) on the microbial population dynamics, metabolism and interspecies interaction have not been fully elucidated. Here, the influence of SRTs (5-25 days) on the performance of the co-digestion system was investigated and state-of-the-art genome-centric metagenomic analysis was employed to uncover the dynamics and metabolic network of the key players underlying the well-functioned and poorly-functioned co-digestion microbial communities. The results of the microbial analyses indicated that SRT largely shaped microbial community structure by enriching the syntrophic specialist Syntrophomonas and CO 2 /H 2 (formate)-using methanogen Methanocorpusculum in the well-functioned co-digester operated at SRT of 25 days, while selecting acid-tolerant populations Lactobacillus at SRT of 5 days. The metagenome assembled genomes (MAGs) of key players, such as Syntrophomonadaceae , Methanocorpusculum, and Mesotoga , were retrieved, additionally, the syntrophic acetate oxidation plus hydrogenotrophic methanogenesis (SAO-HM) were proposed as the dominant pathway for methane production. The metabolic interaction in the co-digestion microbial consortia was profiled by assigning MAGs into functional guilds. Functional redundancy was found in the bacterial groups in hydrolysis step, and the members in these groups reduced the direct competition by niche differentiation. Image 1 • Biomarkers of well-functioned and poorly-functioned co-digesters were pinpointed. • Hydrogenotrophic methanogenesis was the dominant pathway for methane production. • This work revealed the mutual and competitive interactions in microbial community. • Niche differentiation allowed the co-existence of potentially competitive microbes. [ABSTRACT FROM AUTHOR]

Aquatic ecosystems have been increasingly threatened by anthropogenic activities, e.g., wastewater discharge and farm operation. Several methods are adopted to evaluate the effects of anthropogenic activities on biological risk in the environment, such as qPCR and amplicon next-generation sequencing. However, these methods fall short of providing genomic information of target species, which is vital for risk assessment from genomic aspect. Here, we developed a novel approach integrating metagenomic analysis and flow cytometry to identify and quantify potential pathogenic antibiotic resistant bacteria (PARB; carrying both antibiotic resistance genes (ARGs) and virulence factor genes (VFGs)) in the environment, which are of particular concern due to their infection ability and antibiotic resistance. Based on the abundance/density of PARB, we evaluated microbiological risk in a river impacted by both municipal drainage and agriculture runoff. We collected samples upstream (mountainous area) as the control. Results showed that 81.8% of dominant PARB (33) recovered using our approach were related to known pathogenic taxa. In addition, intragenomic ARGs-VFGs coexistence patterns in the dominant Pseudomonas genomes (20 out of 71 PARB) showed high similarity with the most closely related Pseudomonas genomes from the NCBI RefSeq database. These results reflect acceptable reliability of the approach for (potential) pathogen identification in environmental samples. According to the PARB density, microbiological risk in samples from the agricultural area was significantly higher than in samples from the urban area. We speculated that this was due to the higher antibiotic usage in agriculture as well as intragenomic ARGs-VFGs co-evolution under antibiotic selective pressure. This study provides an alternative approach for the identification and quantification of PARB in aquatic environments, which can be applied for microbiological risk assessment. Image 1 • Anthropogenic activities increase microbiological risks in aquatic ecosystems. • We developed a novel metagenomic-based approach to evaluate microbiological risk. • Bacteria carrying antibiotic resistance and virulence factor genes were identified. • Intragenomic coexistence pattern analysis between ARGs and VFGs was feasible. [ABSTRACT FROM AUTHOR]

Various aromatic compounds in wastewater, especially industrial wastewater, are treated by biological processes in bioreactors which are regarded as hotspots and reservoirs of antibiotic resistance genes (ARGs). Yet, little is known about the relationship between the aromatic compound degradation process and antibiotic resistance. Here, we report on the co-occurrence of ARGs and aromatic degradation genes (ADGs) in bacteria in bioreactors. We confirmed this by bioreactor experiments and bioinformatics analysis of over 10,000 publicly available bacterial genomes. We observed a significant enrichment of ARGs in bioreactors treating wastewater that contained p -aminophenol and p -nitrophenol. The potential hosts harboring ARGs and ADGs were mainly Pseudomonas , Leucobacter , Xanthobacter, Acinetobacter, and Burkholderiaceae. Genome analysis revealed that 67.6% of the publicly available bacterial genomes harboring ADGs also harbor ARGs. Over 80% of Burkholderiales , Xanthomonales , Enterobacteriaceae , Acinetobacter , Pseudomonas , and Nocardiaceae genomes harbor both ARGs and ADGs, which strongly suggests the co-occurrence of these genes. Furthermore, bacteria carrying ADGs harbored more than twice the number of ARGs than bacteria only carrying ARGs. Network analysis suggested that multidrug, beta-lactam, aminoglycoside, macrolide-lincosamide-streptogramin, and polymyxin resistance genes are the major ARGs associated with ADGs. Taken together, the presented findings improve the understanding of ARG prevalence in biological wastewater treatment plants, and highlight the potential risk of the effect of regular aromatic compounds on the selection and spread of ARGs. Image 1 • ARGs were enriched in bioreactors treating wastewater containing aromatic compounds. • Co-occurrence of ARGs and aromatic degradation genes was observed in bacteria. • Bacteria carrying aromatic degradation genes harbor more ARGs than other bacteria. • The effects of aromatic compounds on selection of ARGs are worthy of attention. [ABSTRACT FROM AUTHOR]