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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., 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.)

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., 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 © 2023. Published by Elsevier Ltd.)

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., 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 © 2023. Published by Elsevier Ltd.)

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., 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., (Crown Copyright © 2023. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., 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 © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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, sul1, sul2, aadA and bacA 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)