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• 145 ARG subtypes including high-risk ARGs were detected in the receiving waters. • ARG subtypes substantially decreased in receiving waters of PFAAs point source. • ARG profiles were significantly related to PFCAs and PFSAs. • Short-chain PFAAs had similar impacts on ARG profiles compared to long-chain PFAAs. The ecological risks posed by perfluoroalkyl acids (PFAAs) to the aquatic environment have recently been of great concern. However, little information was available on the impact of PFAAs on antibiotic resistance genes (ARGs) profiles. In this study, the receiving river of the largest fluoropolymer production facility in China was selected to investigate the effects of PFAAs on ARGs profiles. The highest PFAAs concentration for water samples near the industrial effluent discharge point was 310.9 μg/L, which was thousands times of higher than the average concentration collected at upstream sites. Perfluorooctanoic acid accounted for more than 67.2 % of ∑PFAAs concentration in water samples collected at the downstream sites, followed by perfluorohexanoic acid (3.6 %–15.9 %). 145 ARG subtypes including high-risk ARGs were detected by metagenomic technology. The results indicated that the discharge of PFAA-containing effluents had a significant impact on the abundance and diversity of ARGs in receiving waters, and PFAAs and water quality parameters (e.g., pH, NH 3 N, COD Mn , TP) could largely affect ARG profiles. Specifically, short-chain PFAAs had similar impacts on ARG profiles compared to the restricted long-chain PFAAs. This study confirmed the potential effects of PFAAs on ARGs in aquatic environment and provided more insights into the ecological risk raised by PFAAs. [Display omitted] [ABSTRACT FROM AUTHOR]

• First study on antibiotic resistome of human feces in bioelectrochemical system. • Bioelectrochemical toilets enhanced the onsite bioremediation of fecal sludge. • Electroactive bacteria enriched in bioelectrochemical toilets electrode biofilms. • Bioelectrochemical toilets reduced the transmission of antibiotic resistance genes. Effective management of fecal sludge (FS) is essential for preventing environmental and public health risks. Developing safe and efficient FS treatment technology is crucial for reducing the health risks of onsite sanitation systems. In this study, bioelectrochemical toilets (BETs) were developed to treat FS onsite. Compared with the open-circuit BETs (OC-BETs), BETs exhibited higher removal efficiencies for total organic carbon, total nitrogen, and total phosphorus. Specifically, the enhancements in removal efficiencies were 18.82 ± 1.73 %, 7.28 ± 0.32 %, and 11.41 ± 0.05 % for urine, and 19.28 ± 4.08 %, 21.65 ± 1.23 %, and 24.68 ± 0.95 % for feces, respectively. Microbiome analysis indicated that the dominant populations were affiliated with electroactive bacteria (Desulfuromonas and Pseudomonas) in the electrode biofilm of BETs. The species co-occurrence network showed that the electrode biofilm microbiome in BETs had more complex correlations than that in OC-BETs, suggesting that a weak electrical current enhanced the microbiome stability. The relative abundance of antibiotic resistance genes in BETs and OC-BETs reduced by 59.85 ± 1.32 % and 53.01 ± 2.81 % compared with the initial FS, respectively. These findings indicate that BETs are an alternative system for enhancing onsite treatment of fecal sludge and provide a theoretical foundation for the implementation of BETs. [Display omitted] [ABSTRACT FROM AUTHOR]

• Manure-borne ARGs and bacR removal assessed in experimental streams.. • The rate at which bacR was removed from the water column differed from tetW removal. • A stochastic model showed that streambed interactions govern ARG removal in streams. • The type of ARG and stream geomorphology alters the downstream transport of ARGs. Antibiotic resistance is a serious global health issue, resulting in at least 1.2 million deaths in 2019. The environment is a potentially important reservoir of antibiotic resistance; however, the fate of Antibiotic Resistance Genes (ARGs) in the environment remains poorly characterized. One important environmental source of ARGs is manure used as a soil amendment. ARGs from manure may then enter nearby flowing waterbodies, where the factors governing their downstream transport remain unknown. To address this, we conducted experiments by spiking cattle manure in an artificial stream to estimate removal rates (k ; m−1) for three ARGs (mefA, tetQ , and tetW) and a ruminant fecal marker (bacR). We then used a Stochastic Mobile-Immobile Model (SMIM) to separate the overall removal into two components , r s , and r h , corresponding to immobilizations in the surface (i.e., water column) and subsurface (i.e., streambed), respectively. Finally, we applied the SMIM across four model streams to predict the downstream travel distance of ARGs and bacR. Our results showed measurable removal for all targets in all experimental replicates (n = 3) and no differences were found in the removal rates among replicates for any target (ANCOVA; p > 0.05). We found that the removal of bacR was significantly lower than tetW (p < 0.05) and slightly lower than mefA (p = 0.088), while tetQ removal was slightly different from tetW 's (p = 0.072). We also found that r h values were orders of magnitude larger than r s for ARGs and bacR (t -test; p < 0.05). These findings suggest that ARGs and bacR are being removed from the water column through immobilization reactions occurring in the streambed. Additionally, we predicted that the 90 % removal (or D90) of targets occurs within the first 500 m in all model streams except in a slow-flow pastoral stream, which required 1400 m of downstream transport for 90 % removal. Our findings and model stand out as promising tools to predict the fate of ARGs in streams and will contribute to improving and managing agricultural practices that employ animal manure. [Display omitted] [ABSTRACT FROM AUTHOR]

The pervasive presence of microplastics (MPs) in aquatic systems facilitates the transmission of antibiotic resistance genes (ARGs), thereby posing risks to ecosystems and human well-being. However, owing to variations in environmental backgrounds and the limited scope of research subjects, studies on ARGs in MPs lack unified conclusions, particularly regarding whether different types of MPs selectively promote ARG enrichment. Analysing large-scale datasets can better encompass broad spatiotemporal scales and diverse samples, facilitating a more extensive exploration of the complex ecological relationships between MPs and ARGs. The present study integrated existing metagenomic datasets to conduct a comprehensive risk assessment and comparative analysis of resistance groups across various MPs. In addition, we endeavoured to elucidate potential associations between ARGs and bacterial taxa, as well as MP structural features, using machine learning (ML) methods. The findings of our research highlight the pivotal role of MP type in shaping plastispheres, accounting for 9.56 % of the biotic variation (Adonis index) and explaining 18.59 % of the ARG variance. Compared to conventional MPs, biodegradable MPs, such as polyhydroxyalkanoates (PHA) and polylactic acid (PLA), exhibit lower species uniformity and diversity but pose a higher risk of ARG occurrence. These ML approaches effectively forecasted ARG abundance by using the bacterial taxa and molecular structure descriptors (MDs) of MPs (average R 2 tra = 0.882, R 2 test = 0.759). Feature analysis showed that MDs associated with lipophilicity, solubility, toxicity, and surface potential significantly influenced the relative abundance of ARGs in the plastispheres. The interpretable multiple linear regression (MLR) model, particularly notable, elucidated a linear relationship between bacterial genera and ARGs, offering promise for identifying potential ARG hosts. This study offers novel insights into ARG dynamics and ecological risks within aquatic plastispheres, highlighting the importance of comprehensive MP monitoring initiatives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

• ARB can achieve >5-log reduction in the E-UV/Cl 2 process. • No photoreactivation of ARB occurred after the E-UV/Cl 2 treatment. • Intracellular ARGs can achieve >4-log reduction in the E-UV/Cl 2 process. • AFM analysis shows low risk of horizontal gene transfer after the E-UV/Cl 2 treatment. • ARG removal was associated to •OH, Cl 2 ‒•, and ClO• exposures. The UV/chlorine (UV/Cl 2) process is a developing advanced oxidation process and can efficiently remove antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). However, the transportation and storage of chlorine solutions limit the application of the UV/Cl 2 process, especially for decentralized water treatment. To overcome the limitation, an electrochemically driven UV/Cl 2 process (E-UV/Cl 2) where Cl 2 can be electrochemically produced in situ from anodic oxidation of chloride (Cl−) ubiquitously present in various water matrices was evaluated in this study. >5-log inactivation of the ARB (E. coli) was achieved within 5 s of the E-UV/Cl 2 process, and no photoreactivation of the ARB was observed after the treatment. In addition to the ARB, intracellular and extracellular ARGs (tet A, sul 1, sul 2, and erm B) could be effectively degraded (e.g., log(C 0 /C) > 4 for i-ARGs) within 5 min of the E-UV/Cl 2 process. Atomic force microscopy showed that the most of the i-ARGs were interrupted into short fragments (< 30 nm) during the E-UV/Cl 2 process, which can thus effectively prevent the self-repair of i-ARGs and the horizontal gene transfer. Modelling results showed that the abatement efficiencies of i-ARG correlated positively with the exposures of •OH, Cl 2 −•, and ClO• during the E-UV/Cl 2 process. Due to the short treatment time (5 min) required for ARB and ARG removal, insignificant concentrations of trihalomethanes (THMs) were generated during of the E-UV/Cl 2 process, and the energy consumption (E EO) of ARG removal was ∼0.20‒0.27 kWh/m3-log, which is generally comparable to that of the UV/Cl 2 process (0.18–0.23 kWh/m3-log). These results demonstrate that the E-UV/Cl 2 process can provide a feasible and attractive alternative to the UV/Cl 2 process for ARB and ARG removal in decentralized water treatment system. [Display omitted] [ABSTRACT FROM AUTHOR]

• The virome in activated sludge treating antibiotic production wastewater were revealed. • The contribution of transduction to the HGT of ARGs appears to be relatively limited. • Folate-related AMGs were the most prevalent AMGs. • Some new potential virus-host assignments were discovered. • Antibiotics has not driven viral communities into either lytic or lysogenic cycles. Viruses are the most abundant yet understudied members that may influence microbial metabolism in activated sludge treating antibiotic production wastewater. This study comprehensively investigated virome community characteristics under the selection pressure of nine types and different concentrations of antibiotics using a metagenomics approach. Of the 15,514 total viral operational taxonomic units (tOTUs) recovered, only 37.5 % were annotated. Antibiotics altered the original viral community structure in activated sludge. The proportion of some pathogenic viral families, including Herpesviridae_like , increased significantly in reactors treating erythromycin production wastewater. In total, 16.5 % of the tOTUs were associated with two or more hosts. tOTUs rarely carried antibiotic resistance genes (ARGs), and the ARG types in the tOTUs did not match the ARGs carried by the bacterial hosts. This suggests that transduction contributes little to the horizontal ARG transfer. Auxiliary metabolic genes (AMGs) were prevalent in tOTUs, and those involved in folate biosynthesis were particularly abundant, indicating their potential to mitigate antibiotic-induced host damage. This study provides comprehensive insights into the virome community in activated sludge treating antibiotic production wastewater and sheds light on the potential role of viral AMGs in mitigating antibiotic-induced stress. [ABSTRACT FROM AUTHOR]

• SPMs are hotspots for ARGs and the HGB act as supercarriers of ARGs. • Anoxic microenvironments on SPMs provide ideal habitats for HGB and other anaerobes. • SPMs facilitate synergy between HGB and denitrifiers for propagation of ARGs. Human gut antibiotic resistome widely occur in anoxic environments characterized by high density of bacterial cells and frequent transmission of antibiotic resistance genes (ARGs). Such resistome is greatly diluted, degraded, and restrained in the aerobic habitats within most natural rivers (regarded as "terrestrial guts") connecting continents and the oceans. Here we implemented a large-scale monitoring campaign extending 5,200 km along the Yellow River, and provide the first integral biogeographic pattern for both ARGs and their hosts. We identified plentiful ARGs (24 types and 809 subtypes) and their hosts (24 phyla and 757 MAGs) in three media (water, suspended particulate matter (SPM), and sediment). Unexpectedly, we found diverse human gut bacteria (HGB) acting as supercarriers of ARGs in this oxygen-rich river. We further discovered that numerous microhabitats were created within stratified biofilms that surround SPMs, particularly regarding the aggregation of anaerobic HGB. These microhabitats provide numerous ideal sinks for anaerobic bacteria and facilitate horizontal transfer of ARGs within the stratified biofilms, Furthermore, the stratification of biofilms surrounding SPMs has facilitated synergy between human gut flora and denitrifiers for propagation of ARGs in the anoxic atmospheres, leading to high occurrence of human gut antibiotic resistome. SPMs play active roles in the dynamic interactions of river water and sediment, thus accelerating the evolution of riverine resistome and transmission of human gut antibiotic resistome. This study revealed the special contribution of SPMs to the propagation of ARGs, and highlighted the necessity of making alternative strategies for sustainable management of large rivers with hyper-concentrated sediment-laden flows. [Display omitted] [ABSTRACT FROM AUTHOR]

• Fe2+/Ca(ClO) 2 conditioning simultaneously promoted sludge dewatering and ARG removal. • Fe2+/Ca(ClO) 2 conditioning was even better than Fenton treatment to eliminate ARGs. • HOCl could penetrate the cell and induce direct oxidation of intracellular ARGs. • ARG removal was achieved by reducing potential hosts and related metabolic pathways. • Fe2+/Ca(ClO) 2 conditioning oxidized intracellular ARGs without bacterial cell lysis. Pre-acidification has been shown to be crucial in attenuating antibiotic resistance genes (ARGs) during the conditioning of sewage sludge. However, it is of great significance to develop alternative conditioning approaches that can effectively eliminate sludge-borne ARGs without relying on pre-acidification. This is due to the high investment costs and operational complexities associated with sludge pre-acidification. In this study, the effects of Fe2+/Ca(ClO) 2 conditioning treatment on the enhancement of sludge dewaterability and the removal of ARGs were compared with other conditioning technologies. The dose effect and the associated mechanisms were also investigated. The findings revealed that Fe2+/Ca(ClO) 2 conditioning treatment had the highest potential, even surpassing Fenton treatment with pre-acidification, in terms of eliminating the total ARGs. Moreover, the effectiveness of the treatment was found to be dose-dependent. This study also identified that the •OH radical reacted with extracellular polymeric substance (EPS) and extracellular ARGs, and the HOCl, the production of which was positively correlated with the dose of Fe2+/Ca(ClO) 2 , could infiltrate the EPS layer and diffuse into the cell of sludge flocs, inducing the oxidation of intracellular ARGs. Furthermore, this study observed a significant decrease in the predicted hosts of ARGs and MGEs in sludge conditioned with Fe2+/Ca(ClO) 2 , accompanied by a significant downregulation of metabolic pathways associated with ARG propagation, thereby contributing to the attenuation of sludge-borne ARGs. Based on these findings, it can be concluded that Fe2+/Ca(ClO) 2 conditioning treatment holds great potential for the removal of sludge-borne ARGs while also enhancing sludge dewaterability, which mainly relies on the intracellular oxidation by HOCl. [Display omitted] [ABSTRACT FROM AUTHOR]

• The high abundance of core ARGs was found in the effluents of WWTPs. • MGE profile had a shift from influents to effluents in WWTPs. • MGEs associated with phage-related processes strongly related to ARGs in effluents. • A strong relationship was found between vir_ARGs and core ARGs in effluents. Antibiotic resistance poses a significant threat to global health, and the microbe-rich activated sludge environment may contribute to the dissemination of antibiotic resistance genes (ARGs). ARGs spread across various bacterial populations via multiple dissemination routes, including horizontal gene transfer mediated by bacteriophages (phages). However, the potential role of phages in spreading ARGs in wastewater treatment systems remains unclear. This study characterized the core resistome, mobile genetic elements (MGEs), and virus-associated ARGs (vir_ARGs) in influents (Inf) and effluents (Eff) samples from nine WWTPs in eastern China. The abundance of ARGs in the Inf samples was higher than that in the Eff samples. A total of 21 core ARGs were identified, accounting for 38.70 %-83.70 % of the different samples. There was an increase in MGEs associated with phage-related processes from influents to effluents (from 12.68 % to 21.10 %). These MGEs showed strong correlations in relative abundance and composition with the core ARGs in the Eff samples. Across the Inf and Eff samples, 58 unique vir_ARGs were detected, with the Eff samples exhibiting higher diversity of vir_ARGs than the Inf samples. Statistical analyses indicated a robust relationship between core ARG profile, MGEs associated with phage-related processes, and vir_ARG composition in the Eff samples. Additionally, the co-occurrence of MGEs and ARGs in viral genomes was observed, ranging from 22.73 % to 68.75 %. This co-occurrence may exacerbate the persistence and spread of ARGs within WWTPs. The findings present new information on the changes in core ARGs, MGEs, and phage-associated ARGs from influents to effluents in WWTPs and provide new insights into the role of phage-associated ARGs in these systems. [Display omitted] [ABSTRACT FROM AUTHOR]

• Anthropogenic sources contributed to ARGs prevalence (averagely 37%) in tap water. • Sulfonamide-, rifamycin- and tetracycline-ARGs were mostly anthropogenic in origin. • Anthropogenic contribution to 17 identified mobile ARGs was higher than other ARGs. • Mobile ARGs frequently carried by diverse pathogens pose threats to public health. • sul 1, sul 2 and aad A are indicators for anthropogenic pollution in drinking water. Although the presence of antibiotic resistance genes (ARGs) in drinking water and their potential horizontal gene transfer to pathogenic microbes are known to pose a threat to human health, their pollution levels and potential anthropogenic sources are poorly understood. In this study, broad-spectrum ARG profiling combined with machine-learning-based source classification SourceTracker was performed to investigate the pollution sources of ARGs in household drinking water collected from 95 households in 47 cities of eight countries/regions. In total, 451 ARG subtypes belonging to 19 ARG types were detected with total abundance in individual samples ranging from 1.4 × 10−4 to 1.5 × 10° copies per cell. Source tracking analysis revealed that many ARGs were highly contributed by anthropogenic sources (37.1%), mainly wastewater treatment plants. The regions with the highest detected ARG contribution from wastewater (∼84.3%) used recycled water as drinking water, indicating the need for better ARG control strategies to ensure safe water quality in these regions. Among ARG types, sulfonamide, rifamycin and tetracycline resistance genes were mostly anthropogenic in origin. The contributions of anthropogenic sources to the 20 core ARGs detected in all of the studied countries/regions varied from 36.6% to 84.1%. Moreover, the anthropogenic contribution of 17 potential mobile ARGs identified in drinking water was significantly higher than other ARGs, and metagenomic assembly revealed that these mobile ARGs were carried by diverse potential pathogens. These results indicate that human activities have exacerbated the constant input and transmission of ARGs in drinking water. Our further risk classification framework revealed three ARGs (sul 1, sul 2 and aad A) that pose the highest risk to public health given their high prevalence, anthropogenic sources and mobility, facilitating accurate monitoring and control of anthropogenic pollution in drinking water. [Display omitted] [ABSTRACT FROM AUTHOR]

A literature-based, manually-curated database of PCR primers for the detection of antibiotic resistance genes in various environments was constructed (LCPDb-ARG; lcpdb.ddg.biol.uw.edu.pl and lcpdb.ddlemb.com). Currently, this database is comprised of 607 PCR primer pairs designed for the amplification of various genes conferring resistance to antibiotics representing 10 classes of antimicrobial agents. Three parameters were assigned for each primer pair: specificity, efficacy and taxonomic efficacy. These parameters were evaluated using a novel bioinformatic tool, UniPriVal, developed for this study. UniPriVal was used to validate each primer pair against various databases, including the Bacterial Antimicrobial Resistance Reference Gene Database (BARRGDB) and those of the National Center of Biotechnology Information (NCBI). Primer pairs specific for each antibiotic resistance gene were ranked based on their model success metric value. To validate the utility and correctness of the information collected in the LCPDb-ARG, selected primer pairs were tested in bioinformatic and experimental PCR surveys. To our knowledge, this is the first database designed to facilitate PCR monitoring of the occurrence and diversity of antibiotic resistance genes in environmental and clinical samples. The internal validation system of this user-friendly application enables the quantified ranking of PCR primer pairs, which assists selection of the best primers for each application. Image 1 • A database (LCPDb-ARG) facilitates PCR screening of antibiotic resistance genes • LCPDb-ARG contains 607 PCR primer pairs for antibiotic resistance genes screening • Specificity, efficacy and taxonomic efficacy parameters were assigned for primers • Model success metric was used for the validation of overall primers usefulness • All primers for each ARG are ranked based on the value of the model success metric [ABSTRACT FROM AUTHOR]

Microbial fuel cell (MFC) is regarded as a promising alternative for enhancing the removal of antibiotic pollutants. In this study, oxytetracycline served as an electron donor in the anode chamber of MFCs, and after continuous operation for 330 days, the efficiency of removal of 10 mg/L oxytetracycline in MFCs increased to 99.00% in 78 h, whereas removal efficiency of only 58.26% was achieved in microbial controls. Compared to microbial controls, higher ATP concentration and persistent electrical stimulation mainly contributed to bioelectrochemical reactions more rapidly to enhance oxytetracycline removal in MFCs. In addition, the analysis of bacterial communities revealed that Eubacterium spp.—as the main functional bacterial genus responsible for oxytetracycline biodegradation—flourished starting from merely 0.00%–91.69% ± 0.27% (mean ± SD) in MFCs. High-throughput quantitative PCR showed that the normalized copy numbers of total antibiotic resistance genes (ARGs) and mobile genetic elements in MFCs were 1.7364 and 0.0065 copies/cell respectively, which were markedly lower than those in the microbial controls. Furthermore, there was no significant correlation between oxytetracycline concentration in the influent and abundance of ARGs in effluent from MFCs. Nevertheless, Tp614 , a transposase gene, was found to be enriched in both MFCs and microbial reactors, suggesting that it may be a common challenge for different biological processes for wastewater treatment. This study therefore showed a lower probability of upregulation and transmission of ARGs in MFCs when compared to a traditional anaerobic microbial treatment. [ABSTRACT FROM AUTHOR]

Activated sludge has been identified as a potential significant source of antibiotic resistance genes (ARGs) to the environment. Anaerobic digestion is extensively used for sludge stabilization and resource recovery, and represents a crucial process for controlling the dissemination of ARGs prior to land application of digested sludge. The objective of this study is to investigate the effect of zero valent iron (Fe 0 ) on the attenuation of seven representative tetracycline resistance genes ( tet , tet (A), tet (C), tet (G), tet (M), tet (O), tet (W), and tet (X)), and the integrase gene intI1 during thermophilic anaerobic co-digestion of waste sludge and kitchen waste. Significant decrease ( P < 0.05) in the quantities of tet (except tet (W)) and intI1 genes was observed at Fe 0 dosage of 5 g/L, whereas no significant differences ( P > 0.05) were found for all gene targets between digesters with Fe 0 dosages of 5 and 60 g/L. A first-order kinetic model favorably described the trends in concentrations of tet and intI1 gene targets during thermophilic anaerobic digestion with or without Fe 0 . Notably, tet genes encoding different resistance mechanisms behaved distinctly in anaerobic digesters, although addition of Fe 0 could enhance their reduction. The overall results of this research suggest that thermophilic anaerobic digestion with Fe 0 can be a potential alternative technology for the attenuation of tet and intI1 genes in waste sludge. [ABSTRACT FROM AUTHOR]

• Agricultural fungicides facilitated horizontal transfer of ARGs via conjugation. • The promoting conjugation of ARGs depended on specific fungicide and donor/recipient. • Chlorothalonil exposure increased SOS response and cell membrane permeability. • Azoxystrobin and carbendazim enhanced the expression of conjugation-related genes. Co-pollution of soil with pesticide residues and antibiotic resistance genes (ARGs) is increasing due to the substantial usage of pesticides and organic fertilizers in greenhouse-based agricultural production. Non-antibiotic stresses, including those from agricultural fungicides, are potential co-selectors for the horizontal transfer of ARGs, but the underlying mechanism remains unclear. Intragenus and intergenus conjugative transfer systems of the antibiotic resistant plasmid RP4 were established to examine conjugative transfer frequency under stress from four widely used fungicides: triadimefon, chlorothalonil, azoxystrobin, and carbendazim. The mechanisms were elucidated at the cellular and molecular levels using transmission electron microscopy, flow cytometry, RT-qPCR, and RNA-seq techniques. The conjugative transfer frequency of plasmid RP4 between Escherichia coli strains increased with the rising exposure concentrations of chlorothalonil, azoxystrobin, and carbendazim, but was suppressed between E. coli and Pseudomonas putida by a high fungicide concentration (10 µg/mL). Triadimefon did not significantly affect conjugative transfer frequency. Exploration of the underlying mechanisms revealed that: (i) chlorothalonil exposure mainly promoted generation of intracellular reactive oxygen species, stimulated the SOS response, and increased cell membrane permeability, while (ii) azoxystrobin and carbendazim primarily enhanced expression of conjugation-related genes on the plasmid. These findings reveal the fungicide-triggered mechanisms associated with plasmid conjugation and highlight the potential role of non-bactericidal pesticides on the dissemination of ARGs. [Display omitted] [ABSTRACT FROM AUTHOR]

• Environmental pollution of ARGs has been a public health concern. • As carrier, integrons promote the prosperity and dissemination of ARGs. • Review of mobile ARGs mediated by integrons on evolutionary, distribution & risk assessment. • Risk assessments can be realized by an integrative methodology framework. • Diverse ARGs carried by integrons with broad-spectrum hosts needs surveillance. Environmental pollution of antibiotic resistance genes (ARGs) has been a great public concern. Integrons, as mobile genetic elements, with versatile gene acquisition systems facilitate the horizontal gene transfer (HGT) and pollution disseminations of ARGs. However, little is understood about the characteristics of ARGs mediated by integrons, which hampers our monitoring and control of the mobile antimicrobial resistance risks. To address these issues, we reviewed 3,322 publications concerning detection methods and pipeline, ARG diversity and evolutionary progress, environmental and geographical distribution, bacterial hosts, gene cassettes arrangements, and based on which to identify ARGs with high risk levels mediated by integrons. Diverse ARGs of 516 subtypes attributed to 12 types were capable of being carried by integrons, with 62 core ARG subtypes prevalent in pollution source, natural and human-related environments. Hosts of ARG-carrying integrons reached 271 bacterial species, most frequently carried by opportunistic pathogens Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Moreover, the observed emergence of ARGs together with their multiple arrangements indicated the accumulation of ARGs mediated by integrons, and thus pose increasing HGT risks under modern selective agents. With the concerns of public health, we urgently call for a better monitoring and control of these high-risk ARGs. Our identified Risk Rank I ARGs (aac A7, bla OXA10, cat B3, cat B8, dfr A5) with high mobility, reviewed key trends and noteworthy advancements, and proposed future directions could be reference and guidance for standard formulation. [Display omitted] [ABSTRACT FROM AUTHOR]

This study aimed to reveal how antibiotic resistance genes (ARGs) and their horizontal and vertical transfer-related items (mobilome and bacterial hosts) respond to the transformation of anaerobic digestion (AD) from mesophilic to thermophilic using one-step temperature increase. The resistomes and mobilomes of mesophilic and thermophilic sludge were investigated using metagenome sequencing, and the changes in 24 representative ARGs belonging to three categories, class 1 integron and bacterial genera during the transition period were further followed using quantitative PCR and 454-pyrosequencing. After the temperature increase, resistome abundance in the digested sludge decreased from 125.97 ppm (day 0, mesophilic) to 50.65 ppm (day 57, thermophilic) with the reduction of most ARG types except for the aminoglycoside resistance genes. Thermophilic sludge also had a smaller mobilome, including plasmids, insertion sequences and integrons, than that of mesophilic sludge, suggesting the lower horizontal transfer potential of ARGs under thermophilic conditions. On the other hand, the total abundance of 18 bacterial genera, which were suggested as the possible hosts for 13 ARGs through network analysis, decreased from 23.27% in mesophilic sludge to 11.92% in thermophilic sludge, indicating fewer hosts for the vertical expansion of ARGs after the increase in temperature. These results indicate that the better reduction of resistome abundance by thermophilic AD might be associated with the decrease of both the horizontal and vertical transferability of ARGs. [ABSTRACT FROM AUTHOR]

• An oxidoreductase capO confers chloramphenicols resistances in bacteria. • capO catalyzes a four-electron double oxidation reaction on the C 3 -OH group. • Association with type IV secretion system confers potential mobility of capO. • Chloramphenicols pollution is the crucial driving factor for capO development. Expanding knowledge about new types of antibiotic resistance genes is of great significance in dealing with the global antibiotic resistance crisis. Herein, a novel oxidoreductase capO was discovered to be responsible for oxidative inactivation of chloramphenicol and thiamphenicol. The antibiotic resistance mechanism was comprehensively deciphered using multi-omics and multiscale computational approaches. A 66,383 bp DNA fragment carrying capO was shared among four chloramphenicol-resistant strains, and the co-occurrence of capO with a mobile genetic element cluster revealed its potential mobility among different taxa. Metagenomic analysis of 772 datasets indicated that chloramphenicol was the crucial driving factor for the development and accumulation of capO in activated sludge bioreactors treating antibiotic production wastewater. Therefore, we should pay sufficient attention to its possible prevalence and transfer to pathogens, especially in some hotspot environments contaminated with high concentrations of chloramphenicols. This finding significantly expands our knowledge boundary about chloramphenicols resistance mechanisms. [Display omitted] [ABSTRACT FROM AUTHOR]

• AOB was a recipient of RP4 plasmid in SBR and pure AOB. • Nitrite generation of pure AOB was decreased by RP4. • Ammonia removal was significantly inhibited by RP4 whether in SBR and pure AOB. • The expression of ammonia oxidation genes related to in AOB was suppressed by RP4. Antibiotic resistance genes (ARGs) have become an important public health concern. Particularly, although several ARGs have been identified in wastewater treatment plants (WWTPs), very few studies have characterized their impacts on reactor performance. Therefore, our study sought to investigate the effect of a representative conjugative transfer plasmid (RP4) encoding multidrug resistance genes on ammonia oxidation. To achieve this, we established sequencing batch reactors (SBRs) and a conjugation model with E. coli donor strains carrying the RP4 plasmid and a typical ammonia-oxidating (AOB) bacterial strain (Nitrosomonas europaea ATCC 25978) as a recipient to investigate the effect of conjugative transfer of plasmid RP4 on AOB. Our findings demonstrated that the RP4 plasmid carried by the donor strains could be transferred to AOB in the SBR and to Nitrosomonas europaea ATCC 25978. In SBR treated with donor strains carrying the RP4 plasmid, ammonia removal efficiency continuously decreased to 71%. Once the RP4 plasmid entered N. europaea ATCC 25978 in the conjugation model, ammonia removal was significantly inhibited and nitrite generation was decreased. Furthermore, the expression of several functional genes related to ammonia oxidation in AOB was suppressed following the transfer of the RP4 plasmid, including amoA, amoC, hao, nirK , and norB. In contrast, the cytL gene encoding cytochrome P460 was upregulated. These results demonstrated the ecological risk of ARGs in WWTPs, and therefore measures must be taken to avoid their transfer. [Display omitted] [ABSTRACT FROM AUTHOR]

• Thousands of underexplored viruses were recovered from wastewater treatment plants. • Viruses encode genes for the removal of contaminants like starches and celluloses. • Viruses disseminate genes for resistance to multiple drugs like fluoroquinilone. Activated sludge of wastewater treatment plants harbors a very high diversity of both microorganisms and viruses, wherein the latter control microbial dynamics and metabolisms by infection and lysis of cells. However, it remains poorly understood how viruses impact the biochemical processes of activated sludge, for example in terms of treatment efficiency and pollutant removal. Using metagenomic and metatranscriptomic deep sequencing, the present study recovered thousands of viral sequences from activated sludge samples of three conventional wastewater treatment plants. Gene-sharing network indicated that most of viruses could not be assigned to known viral genera, implying activated sludge as an underexplored reservoir for new viruses and viral diversity. In silico predictions of virus-host linkages demonstrated that infected microbial hosts, mostly belonging to bacteria, were transcriptionally active and able to hydrolyze polymers including starches, celluloses, and proteins. Some viruses encode auxiliary metabolic genes (AMGs) involved in carbon, nitrogen, and sulfur cycling, and antibiotic resistance genes (ARGs) for resistance to multiple drugs. The virus-encoded AMGs may enhance the biodegradation of contaminants like starches and celluloses, suggesting a positive role for viruses in strengthening the performance of activated sludge. However, ARGs would be disseminated to different microorganisms using viruses as gene shuttles, demonstrating the possibility for viruses to facilitate the spread of antibiotic resistance in the environment. Collectively, this study highlights the mixed blessing of viruses in wastewater treatment plants, and deciphers how they manipulate the biochemical processes in the activated sludge, with implications for both environmental protection and ecosystem security. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: Extracellular DNA exists in the environment and can be taken up by competent bacterial cells, leading to horizontal gene transfer. The persistence of extracellular plasmid and plant DNA in water microcosms was monitored in this study. Water samples were two groundwater (GW1 and GW2) and one river water (RW) samples. Three treatments included: (1) intact, (2) 0.22μm filter-sterilized, and (3) autoclaved water. DNA from a plasmid (pNS1) and a transgenic Bt (Bacillus thuringiensis) corn line, both carrying a neomycin phosphotransferase gene (nptII gene) conferring kanamycin and neomycin resistance, was inoculated into the microcosms at 0.4 and 0.8μg/ml, respectively. By monitoring its ability to transform a competent Pseudomonas stutzeri strain harboring plasmid pMR7 (P. stutzeri pMR7), plasmid DNA was degraded to undetectable levels in the intact and/or filter-sterilized water treatments within 48–96h in GW1, GW2, and RW. Meanwhile, plasmid DNA persisted in the autoclaved treatment throughout the entire incubation period. For plant DNA, a highly sensitive real-time PCR method using SYBR Green I was developed to monitor the degradation dynamics of the nptII gene carried by the transgenic corn line in the microcosms. The results showed that the concentration of plant DNA was reduced by two orders of magnitude (from 0.8–0.008μg/ml) within 96h in the intact and filter-sterilized treatments of GW1, GW2, and RW, in contrast to its persistence in the autoclaved treatment. In addition, no kanamycin resistant (KmR) transformants were detected from in situ transformation of P. stutzeri pMR7 with plasmid pNS1 DNA. [Copyright &y& Elsevier]

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]

This study assessed the influence of substrate type on pollutants removal, antibiotic resistance gene (ARG) fate and bacterial community evolution in up-flow microbial fuel cell constructed wetlands (UCW-MFC) with graphite and Mn ore electrode substrates. Better COD removal and higher bacterial community diversity and electricity generation performance were achieved in Mn ore constructed UCW-MFC (Mn). However, the lower concentration of sulfadiazine (SDZ) and the total abundances of ARGs were obtained in the effluent in the graphite constructed UCW-MFC (s), which may be related to higher graphite adsorption and filter capacity. Notably, both reactors can remove more than 97.8% of ciprofloxacin. In addition, significant negative correlations were observed between SDZ, COD concentration, ARG abundances and bacterial a-diversity indices. The LEfse analysis revealed significantly different bacterial communities due to the substrate differences in the two reactors, and Geobacter , a typical model electro-active bacteria (EAB), was greatly enriched on the anode of UCW-MFC (Mn). In contrast, the relative abundance of methanogens (Methanosaeta) was inhibited. PICRUSt analysis results further demonstrated that the abundance of extracellular electron transfer related functional genes was increased, but the methanogen function genes and multiple antibiotic resistance genes in UCW-MFC (Mn) anode were reduced. Redundancy analyses indicated that substrate type, antibiotic accumulation and bacterial community were the main factors affecting ARGs. Moreover, the potential ARG hosts and the co-occurrence of ARGs and intI1 were revealed by network analysis. Image 1 • The electro-active bacteria Geobacter was greatly enriched on Mn ore anode. • Methanogen functional genes predicted by PICRUSt reduced on Mn ore anode. • Higher COD removal and electricity generation performance was obtained in UCW-MFC (Mn). • The migration of ARGs and their hosts from water phase to substrate reduced the ARGs. [ABSTRACT FROM AUTHOR]

Microplastics have been found to be ubiquitous in freshwater ecosystems, providing a novel substrate for biofilm formation. Here, we incubated biofilm on microplastics and two natural substrates (rock and leaf) under a controlled environment to investigate the differences of microbial community structure, antibiotic resistance gene (ARG) profiles, and ARG microbial hosts between biofilms on three types of substrates. Results from high-throughput sequencing of 16S rRNA gene revealed that microplastic biofilm had a distinctive community structure. Network analyses suggested that microplastic biofilm possessed the highest node connected community, but with lower average path length, network diameter and modularity compared with biofilm on two natural particles. Metagenomic analyses further revealed microplastic biofilm with broad-spectrum and distinctive resistome. Specifically, according to taxonomic annotation of ARG microbial hosts, two opportunisitic human pathogens (Pseudomonas monteilii , Pseudomonas mendocina) and one plant pathogen (Pseudomonas syringae) were detected only in the microplastic biofilm, but not in biofilms formed on natural substrates. Our findings suggest that microplastic is a novel microbial niche and may serve as a vector for ARGs and pathogens to new environment in river water, generating freshwater environmental risk and exerting adverse impacts on human health. Image 1 • Microplastic biofilm has unique microbial community structure. • Distinctive antibiotic resistance genes were detected in the microplastic biofilm. • Opportunistic human pathogens were enriched in microplastic biofilm. [ABSTRACT FROM AUTHOR]

Reuse of municipal wastewater is a growing global trend, but currently there is lack of consensus regarding the potential dissemination of antibiotic resistance elements by treated wastewater irrigation. We tracked intI 1, a proxy for anthropogenic pollution, and an assemblage of antibiotic resistance genes associated with mobile elements and/or wastewater (bla GES , bla OXA2 , bla OXA10 , bla TEM , bla CTX-M-32 and qnr S) in treated wastewater effluents, effluent stabilization reservoirs, and along irrigation water-soil-crop continua in experimental lysimeters and large-scale commercial fields. While several of the targeted antibiotic resistance genes were profuse in effluents, there was almost no correlation between gene abundance in irrigation water and those detected in soil, and no evidence of systematic gene transfer to irrigated soil or crops. In contrast, soil intI 1 abundance correlated strongly to irrigation water levels in lysimeters and sandy field soils, but this was not the case for clay-rich soils or for most of the analyzed crops, suggesting that intI 1 may not always be a reliable marker for tracking the impact of treated wastewater irrigation. We hypothesize that "ecological boundaries" expedited by biotic and abiotic factors constrain dissemination of antibiotic resistance elements, and assert that a more holistic perception of these factors is crucial for understanding and managing antibiotic resistance dissemination. Image 1 • We targeted antibiotic resistance genes (ARGs) in effluents and irrigated fields. • bla OXA and bla GES genes were strongly linked to treated wastewater effluents. • Effluent storage in reservoirs substantially reduced ARG abundance. • intI 1 distribution in irrigated soil was only partially linked to water quality. • Ecological barriers seem to restrict ARG dissemination to irrigated soil and crops. [ABSTRACT FROM AUTHOR]