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As antibiotic resistance continues to spread globally, there is growing interest in the potential to limit the spread of antibiotic resistance genes (ARGs) from wastewater sources. In particular, operational conditions during sludge digestion may serve to discourage selection of resistant bacteria, reduce horizontal transfer of ARGs, and aid in hydrolysis of DNA. This study applied metagenomic analysis to examine the removal efficiency of ARGs through thermophilic and mesophilic anaerobic digestion using bench-scale reactors. Although the relative abundance of various ARGs shifted from influent to effluent sludge, there was no measureable change in the abundance of total ARGs or their diversity in either the thermophilic or mesophilic treatment. Among the 35 major ARG subtypes detected in feed sludge, substantial reductions (removal efficiency >90 %) of 8 and 13 ARGs were achieved by thermophilic and mesophilic digestion, respectively. However, resistance genes of aadA, macB, and sul1 were enriched during the thermophilic anaerobic digestion, while resistance genes of erythromycin esterase type I, sul1, and tetM were enriched during the mesophilic anaerobic digestion. Efflux pump remained to be the major antibiotic resistance mechanism in sludge samples, but the portion of ARGs encoding resistance via target modification increased in the anaerobically digested sludge relative to the feed. Metagenomic analysis provided insight into the potential for anaerobic digestion to mitigate a broad array of ARGs. [ABSTRACT FROM AUTHOR]

We developed a fast method to construct local sub-databases from the NCBI-nr database for the quick similarity search and annotation of huge metagenomic datasets based on BLAST-MEGAN approach. A three-step sub-database annotation pipeline (SAP) was further proposed to conduct the annotation in a much more time-efficient way which required far less computational capacity than the direct NCBI-nr database BLAST-MEGAN approach. The 1st BLAST of SAP was conducted using the original metagenomic dataset against the constructed sub-database for a quick screening of candidate target sequences. Then, the candidate target sequences identified in the 1st BLAST were subjected to the 2nd BLAST against the whole NCBI-nr database. The BLAST results were finally annotated using MEGAN to filter out those mistakenly selected sequences in the 1st BLAST to guarantee the accuracy of the results. Based on the tests conducted in this study, SAP achieved a speedup of ∼150–385 times at the BLAST e-value of 1e–5, compared to the direct BLAST against NCBI-nr database. The annotation results of SAP are exactly in agreement with those of the direct NCBI-nr database BLAST-MEGAN approach, which is very time-consuming and computationally intensive. Selecting rigorous thresholds (e.g. e-value of 1e–10) would further accelerate SAP process. The SAP pipeline may also be coupled with novel similarity search tools (e.g. RAPsearch) other than BLAST to achieve even faster annotation of huge metagenomic datasets. Above all, this sub-database construction method and SAP pipeline provides a new time-efficient and convenient annotation similarity search strategy for laboratories without access to high performance computing facilities. SAP also offers a solution to high performance computing facilities for the processing of more similarity search tasks. [ABSTRACT FROM AUTHOR]

Sensitive detection of Mycobacterium tuberculosis (TB) in small percentages in metagenomic samples is essential for microbial classification and drug resistance prediction. However, traditional methods, such as bacterial culture and microscopy, are time-consuming and sometimes have limited TB detection sensitivity. Oxford nanopore technologies (ONT) MinION sequencing allows rapid and simple sample preparation for sequencing. Its recently developed adaptive sequencing selects reads from targets while allowing real-time base-calling to achieve sequence enrichment or depletion during sequencing. Another common enrichment method is PCR amplification of the target TB genes. In this study, we compared both methods using ONT MinION sequencing for TB detection and variant calling in metagenomic samples using both simulation runs and those with synthetic and patient samples. We found that both methods effectively enrich TB reads from a high percentage of human (95%) and other microbial DNA. Adaptive sequencing with readfish and UNCALLDE achieved a 3.9-fold and 2.2-fold enrichment compared to the control run. We provide a simple automatic analysis framework to support the detection of TB for clinical use, openly available at https://github.com/HKU-BAL/ONT-TB-NF. Depending on the patient's medical condition and sample type, we recommend users evaluate and optimize their workflow for different clinical specimens to improve the detection limit. [ABSTRACT FROM AUTHOR]

The fast development of next generation sequencing (NGS) has dramatically increased the application of metagenomics in various aspects. Functional annotation is a major step in the metagenomics studies. Fast annotation of functional genes has been a challenge because of the deluge of NGS data and expanding databases. A hybrid annotation pipeline proposed previously for taxonomic assignments was evaluated in this study for metagenomic sequences annotation of specific functional genes, such as antibiotic resistance genes, arsenic resistance genes and key genes in nitrogen metabolism. The hybrid approach using UBLAST and BLASTX is 44–177 times faster than direct BLASTX in the annotation using the small protein database for the specific functional genes, with the cost of missing a small portion (<1.8%) of target sequences compared with direct BLASTX hits. Different from direct BLASTX, the time required for specific functional genes annotation using the hybrid annotation pipeline depends on the abundance for the target genes. Thus this hybrid annotation pipeline is more suitable in specific functional genes annotation than in comprehensive functional genes annotation. [ABSTRACT FROM AUTHOR]

In the present study, a newly developed metagenomic analysis approach was applied to investigate the abundance and diversity of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in aquaculture farm sediments, activated sludge, biofilm, anaerobic digestion sludge, and river water. BLASTX analysis against the Comprehensive Antibiotic Resistance Database was conducted for the metagenomic sequence data of each sample and then the ARG-like sequences were sorted based on structured sub-database using customized scripts. The results showed that freshwater fishpond sediment had the highest abundance (196 ppm), and anaerobic digestion sludge possessed the highest diversity (133 subtypes) of ARGs among the samples in this study. Significantly, rifampin resistance genes were universal in all the diverse samples and consistently accounted for 26.9~38.6 % of the total annotated ARG sequences. Furthermore, a significant linear correlation ( R = 0.924) was found between diversities (number of subtypes) of ARGs and diversities of plasmids in diverse samples. This work provided a wide spectrum scan of ARGs and MGEs in different environments and revealed the prevalence of rifampin resistance genes and the strong correlation between ARG diversity and plasmid diversity for the first time. [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]

• 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]

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]

Abstract The benefits of extensive artificial sweeteners use come at a cost of their ubiquitous occurrence in the aquatic environment. Biodegradation is crucial for the removal of artificial sweeteners in the environment, yet comprehensive characterizations of the degradation consortia that degrade these compounds have not been initiated. Here, we performed metagenomic analysis of microbial communities fulfilling complete mineralization of two typical artificial sweeteners, i.e. saccharin and cyclamate. Genome-resolved metagenomics enabled the recovery and metabolic characterization of total 23 population genomes from 8 phyla in the two consortia, most of which represented novel species. The saccharin-degrading consortia was notably dominated by a betaproteobacterial genome from the family Rhodocyclaceae , accounting for 15.5% of total sequences. For the cyclamate enrichment, 28.1% of the total sequences were assigned to three similarly abundant Alphaproteobacteria population genomes belonging to the family Sphingomonadaceae and Methylobacteriaceae. The metabolic potential of these population genomes were examined to potentially identify the roles of these populations in biodegradation of artificial sweeteners, and focusing on the energy and nutrient metabolisms. Graphical abstract Unlabelled Image Highlights • The first metagenomic study on artificial sweeteners' biodegradation. • A rapid UPLC-MS/MS detection method was established for saccharin and cyclamate. • 23 binned genomes represented the major saccharin/cyclamate-degrading consortia. • Dominant organisms in saccharin/cyclamate-degrading were markedly different. • A metabolic model of degrading consortia was constructed. [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]

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]

Summary: The human gut microbiota is an important reservoir of antibiotic resistance genes (ARGs). A metagenomic approach and network analysis were used to establish a comprehensive antibiotic resistome catalog and to obtain co‐occurrence patterns between ARGs and microbial taxa in fecal samples from 180 healthy individuals from 11 different countries. In total, 507 ARG subtypes belonging to 20 ARG types were detected with abundances ranging from 7.12 × 10−7 to 2.72 × 10−1 copy of ARG/copy of 16S‐rRNA gene. Tetracycline, multidrug, macrolide‐lincosamide‐streptogramin, bacitracin, vancomycin, beta‐lactam and aminoglycoside resistance genes were the top seven most abundant ARG types. The multidrug ABC transporter, aadE, bacA, acrB, tetM, tetW, vanR and vanS were shared by all 180 individuals, suggesting their common occurrence in the human gut. Compared to populations from the other 10 countries, the Chinese population harboured the most abundant ARGs. Moreover, LEfSe analysis suggested that the MLS resistance type and its subtype ‘ermF’ were representative ARGs of the Chinese population. Antibiotic inactivation, antibiotic target alteration and antibiotic efflux were the dominant resistance mechanism categories in all populations. Procrustes analysis revealed that microbial phylogeny structured the antibiotic resistome. Co‐occurrence patterns obtained via network analysis implied that 12 species might be potential hosts of 58 ARG subtypes. [ABSTRACT FROM AUTHOR]

During the past years, antibiotic-resistant bacteria (ARB) leading for the spreading of antibiotic resistance genes (ARGs) became a global problem, especially multidrug-resistant (MDR) bacteria are considered the prime culprit of antibiotic resistance. However, the correlation between the antibiotic-resistant phenotype and the ARG profiles remains poorly understood. In the present study, metagenomic functional screening and metagenomic analysis of coliforms were combined to explore the phenotype and genotype of the ARBs from municipal sewage. Our results showed that the ARG co-occurrence was widespread in the municipal sewage. The present study also highlighted the high abundance of ARGs from antibiotic resistance coliforms especially the MDR coliforms with ARG level of 33.8 ± 4.2 copies per cell. The ARG profiles and the antibiotic resistance phenotypes of the isolated antibiotic resistant coliforms were also correlated and indicated that the resistance to the related antibiotic (ampicillin, kanamycin, erythromycin, chloramphenicol, and tetracycline) was mostly contributed by the ARGs belonging to the subtypes of β-lactamase, aminoglycoside 3-phosphotransferase, phosphotransferase type 2, chloramphenicol acetyltransferase, tetA, etc. [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]

Emergence of new antibiotic resistance bacteria poses a serious threat to human health, which is largely attributed to the evolution and spread of antibiotic resistance genes (ARGs). In this work, a metagenomics-guided strategy consisting of metagenomic analysis and function validation was proposed for rapidly identifying novel ARGs from hot spots of ARG dissemination, such as wastewater treatment plants (WWTPs) and animal feces. We used an antibiotic resistance gene database to annotate 76 putative β-lactam resistance genes from the metagenomes of sludge and chicken feces. Among these 76 candidate genes, 25 target genes that shared 40~70% amino acid identity to known β-lactamases were cloned by PCR from the metagenomes. Their resistances to four β-lactam antibiotics were further demonstrated. Furthermore, the validated ARGs were used as the reference sequences to identify novel ARGs in eight environmental samples, suggesting the necessity of re-examining the profiles of ARGs in environmental samples using the validated novel ARG sequences. This metagenomics-guided pipeline does not rely on the activity of ARGs during the initial screening process and may specifically select novel ARG sequences for function validation, which make it suitable for the high-throughput screening of novel ARGs from environmental metagenomes. [ABSTRACT FROM AUTHOR]

[Display omitted] • Sucralose can be completely degraded by the enriched consortia within 4 days. • Five biotransformation products were detected and a degradation pathway was proposed. • The enrichments were characterized by combining Illumina and Nanopore technologies. • The function of sucralose biodegradation was driven by novel species. Sucralose has been regarded as an emerging pollutant with growing concerns owing to its environmental persistence and potential risks to ecosystems and human health. However, limited information is currently available regarding its biodegradability and degradation pathway in the environment. In this study, complete and efficient sucralose biodegradation was achieved by enriched consortia seeded with activated sludge. In the enrichments with sucralose as the sole carbon source, 73 % of the total organic carbon was removed with a maximum degradation rate of 3.87 mg sucralose/g VSS·h−1, coupling with the release of three chloride ions of sucralose. Additionally, five biotransformation products, namely TP-409N, TP-373N, TP-357N, TP-455N, and TP-393N, were determined by UPLC-QTOF-MS, and an aerobic sucralose-degrading pathway was proposed. Then, Illumina and Nanopore sequencing were employed to provide a genome-centric resolution of microbial communities, demonstrating that the enriched consortia were dominated by Proteobacteria , Bacteroidota , Chloroflexota , and Planctomycetota. At the species level, over half of metagenome-assembled genomes were potentially affiliated with new lineages, implying that the function of sucralose biodegradation was driven by some novel species (at the genus level). Combined with the network analysis, species from UBA11579 and Polyangiaceae were suspected to be involved in the biodegradation of sucralose. This research evidenced the bacterial biodegradability of sucralose and first demonstrated the sucralose-biodegrading pathway and microbial communities, providing novel insights into sucralose biodegradation in the environment. [ABSTRACT FROM AUTHOR]

Background: Methanogenic biodegradation of aromatic compounds depends on syntrophic metabolism. However, metabolic enzymes and pathways of uncultured microorganisms and their ecological interactions with methanogenic consortia are unknown because of their resistance to isolation and limited genomic information. Results: Genome-resolved metagenomics approaches were used to reconstruct and dissect 23 prokaryotic genomes from 37 and 20 °C methanogenic phenol-degrading reactors. Comparative genomic evidence suggests that temperature difference leads to the colonization of two distinct cooperative sub-communities that can respire sulfate/sulfite/sulfur or nitrate/nitrite compounds and compete for uptake of methanogenic substrates (e.g., acetate and hydrogen). This competition may differentiate methanogenesis. The uncultured ε-Proteobacterium G1, whose close relatives have broad ecological niches including the deep-sea vents, aquifers, sediment, limestone caves, spring, and anaerobic digesters, is implicated as a Sulfurovum-like facultative anaerobic diazotroph with metabolic versatility and remarkable environmental adaptability. We provide first genomic evidence for butyrate, alcohol, and carbohydrate utilization by a Chloroflexi T78 clade bacterium, and phenol carboxylation and assimilatory sulfite reduction in a Cryptanaerobacter bacterium. Conclusion: Genome-resolved metagenomics enriches our view on the differentiation of microbial community composition, metabolic pathways, and ecological interactions in temperature-differentiated methanogenic phenol-degrading bioreactors. These findings suggest optimization strategies for methanogenesis on phenol, such as temperature control, protection from light, feed desulfurization, and hydrogen sulfide removal from bioreactors. Moreover, decoding genome-borne properties (e.g., antibiotic, arsenic, and heavy metal resistance) of uncultured bacteria help to bring up alternative schemes to isolate them. [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]

Digested residues from biogas plants are often used as biofertilizers for agricultural crops cultivation. The antibiotic resistance genes (ARGs) in digested residues pose a high risk to public health due to their potential spread to the disease-causing microorganisms and thus reduce the susceptibility of disease-causing microorganisms to antibiotics in medical treatment. A high-throughput sequencing (HTS)-based metagenomic approach was used in the present study to investigate the variations of ARGs in full-scale biogas reactors and the correlations of ARGs with microbial communities and metal resistance genes (MRGs). The total abundance of ARGs in all the samples varied from 7 × 10-3 to 1.08 × 10-1 copy of ARG/copy of 16S-rRNA gene, and the samples obtained from thermophilic biogas reactors had a lower total abundance of ARGs, indicating the superiority of thermophilic anaerobic digestion for ARGs removal. ARGs in all the samples were composed of 175 ARG subtypes; however, only 7 ARG subtypes were shared by all the samples. Principal component analysis and canonical correspondence analysis clustered the samples into three groups (samples from manure-based mesophilic reactors, manure-based thermophilic reactors, and sludge-based mesophilic reactors), and substrate, temperature, and hydraulic retention time (HRT) as well as volatile fatty acids (VFAs) were identified as crucial environmental variables affecting the ARGs compositions. Procrustes analysis revealed microbial community composition was the determinant of ARGs composition in biogas reactors, and there was also a significant correlation between ARGs composition and MRGs composition. Network analysis further revealed the co-occurrence of ARGs with specific microorganisms and MRGs. [ABSTRACT FROM AUTHOR]

Acid sulfate soil (ASS) is sensitive to redox fluctuations induced by climate change and human activities. Oxidation of sulfur and sulfide in ASS leads to the release of acid and consequently metals, posing severe hazards to coastal environment, while microbial contribution and response to oxidation is poorly understood. Here we used metagenomic sequencing to delineate the shift in microbial community structures and functional genes in ASS upon exposure to aerobic conditions. Aerobic incubation resulted in significant shifts in microbial communities in both topsoil and parent material. Archaea decreased significantly in the parent material after aerobic incubation. The relative abundance of sulfur cycling genes in the parent material layer was significantly higher than those in the topsoil, and multiple sulfide oxidation genes increased after aerobic oxidation. Metagenomic assembly enabled construction of eight key draft genomes from ASS. Three of them (GS3, GS6 and GT3) are novel strains of Thermoplasmatales , Acidothermales ( Acidothermus ) and Acidimicrobiales , respectively. Functional gene annotation of these population genomes revealed a dominance of sulfur cycling genes and acid tolerant genes. These findings highlight the microbial response to environmental change and identify the ecological adaptation and survival strategies of microbial communities in acid sulfate soils. [ABSTRACT FROM AUTHOR]

The effectiveness and treatment conditions of FeCl- and AlCl-coagulated municipal sewage sludge from chemically enhanced primary treatment (CEPT) using anaerobic digestion (AD) and the structure of microbial community were investigated. The results based on 297 measurements under different operational conditions demonstrate good average AD performance of CEPT sludge, that is, percent volatile solid reduction of 58 %, specific biogas production (or biogas yield) of 0.92 m/kg volatile solids (VS) destroyed, and methane content of 65.4 %. FeCl dosing, organic loading rate, temperature, and hydraulic retention time all significantly affected AD performance. FeCl dosing greatly improved specific methane production (methane yield) by 38-54 % and significantly reduced hydrogen sulfide (HS) content in biogas (from up to 13,250 to <200 ppm), contributing to higher methane recovery and simplified biogas cleaning for power generation. Metagenomic analysis suggested that anaerobic digesters of both CEPT sludge and combined primary and secondary sludge were dominated by Bacteroidetes, Proteobacteria, Firmicutes, Actinobacteria, Thermotogae, and Chloroflexi. However, Methanomicrobia methanogens were better enriched in the anaerobic digesters of CEPT sludge than in the combined sludge. Further, different sources of CEPT sludge with various chemical properties nurtured shared and unique microbial community composition. Combined, this study supports AD as an efficient technology for CEPT sludge treatment and poses first insights into the microbial community structure. [ABSTRACT FROM AUTHOR]

Biofilms are widely used in wastewater treatment for their particular enhancement of nitrogen removal and other significant advantages. In this study, the diversity and potential functions of nitrogen removal bacteria in suspended activated sludge (AS) and biofilm of a full-scale hybrid reactor were uncovered by metagenomes (∼34 Gb), coupled with PCR-based 454 reads (>33 K reads). The results indicated that the diversity and abundance of nitrifiers and denitrifiers in biofilm did not surpass that in AS, while more nitrification and denitrification genes were indeed found in biofilm than AS, suggesting that the increased nitrogen removal ability by applying biofilm might be attributed to the enhancement of removal efficiency, rather than the biomass accumulation of nitrogen removal bacteria. The gene annotation and phylogenetic analysis results revealed that AS and biofilm samples consisted of 6.0 % and 9.4 % of novel functional genes for nitrogen removal and 18 % and 30 % of new Nitrospira species for nitrite-oxidizing bacteria, respectively. Moreover, the identification of Nitrospira-like amoA genes provided metagenomic evidence for the presence of complete ammonia oxidizer (comammox) with the functional potential to perform the complete oxidation of ammonia to nitrate. These findings have significant implications in expanding our knowledge of the biological nitrogen transformations in wastewater treatment. [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]

• 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]

Improvement on the bioconversion of cellulosic biomass depends much on the expanded knowledge on the underlying microbial structure and the relevant genetic information. In this study, metagenomic analysis was applied to characterize an enriched mesophilic cellulose-converting consortium, to explore its cellulose-hydrolyzing genes, and to discern genes involved in methanogenesis. Cellulose conversion efficiency of the mesophilic consortium enriched in this study was around 70 %. Apart from methane, acetate was the major fermentation product in the liquid phase, while propionate and butyrate were also detected at relatively high concentrations. With the intention to uncover the biological factors that might shape the varying cellulose conversion efficiency at different temperatures, results of this mesophilic consortium were then compared with that of a previously reported thermophilic cellulose-converting consortium. It was found that the mesophilic consortium harbored a larger pool of putative carbohydrate-active genes, with 813 of them in 54 GH modules and 607 genes in 13 CBM modules. Methanobacteriaceae and Methanosaetaceae were the two methanogen families identified, with a preponderance of the hydrogenotrophic Methanobacteriaceae. In contrast to its relatively high diversity and high abundance of carbohydrate-active genes, the abundance of genes involved in the methane metabolism was comparatively lower in the mesophilic consortium. A biological enhancement on the methanogenic process might serve as an effective option for the improvement of the cellulose bioconversion at mesophilic temperature. [ABSTRACT FROM AUTHOR]

A metagenomic approach and network analysis was used to investigate the wide-spectrum profiles of antibiotic resistance genes (ARGs) and their co-occurrence patterns in 50 samples from 10 typical environments. In total, 260 ARG subtypes belonging to 18 ARG types were detected with an abundance range of 5.4 × 10−6-2.2 × 10−1 copy of ARG per copy of 16S-rRNA gene. The trend of the total ARG abundances in environments matched well with the levels of anthropogenic impacts on these environments. From the less impacted environments to the seriously impacted environments, the total ARG abundances increased up to three orders of magnitude, that is, from 3.2 × 10−3 to 3.1 × 100 copy of ARG per copy of 16S-rRNA gene. The abundant ARGs were associated with aminoglycoside, bacitracin, β-lactam, chloramphenicol, macrolide-lincosamide-streptogramin, quinolone, sulphonamide and tetracycline, in agreement with the antibiotics extensively used in human medicine or veterinary medicine/promoters. The widespread occurrences and abundance variation trend of vancomycin resistance genes in different environments might imply the spread of vancomycin resistance genes because of the selective pressure resulting from vancomycin use. The simultaneous enrichment of 12 ARG types in adult chicken faeces suggests the coselection of multiple ARGs in this production system. Non-metric multidimensional scaling analysis revealed that samples belonging to the same environment generally possessed similar ARG compositions. Based on the co-occurrence pattern revealed by network analysis, tetM and aminoglycoside resistance protein, the hubs of the ARG network, are proposed to be indicators to quantitatively estimate the abundance of 23 other co-occurring ARG subtypes by power functions. [ABSTRACT FROM AUTHOR]

Although dairy manure is widely applied to land, it is relatively understudied compared to other livestock as a potential source of antibiotic resistance genes (ARGs) to the environment and ultimately to human pathogens. Ceftiofur, the most widely used antibiotic used in U.S. dairy cows, is a 3rd generation cephalosporin, a critically important class of antibiotics to human health. The objective of this study was to evaluate the effect of typical ceftiofur antibiotic treatment on the prevalence of ARGs in the fecal microbiome of dairy cows using a metagenomics approach. β-lactam ARGs were found to be elevated in feces from Holstein cows administered ceftiofur (n = 3) relative to control cows (n = 3). However, total numbers of ARGs across all classes were not measurably affected by ceftiofur treatment, likely because of dominance of unaffected tetracycline ARGs in the metagenomics libraries. Functional analysis via MG-RAST further revealed that ceftiofur treatment resulted in increases in gene sequences associated with “phages, prophages, transposable elements, and plasmids”, suggesting that this treatment also enriched the ability to horizontally transfer ARGs. Additional functional shifts were noted with ceftiofur treatment (e.g., increase in genes associated with stress, chemotaxis, and resistance to toxic compounds; decrease in genes associated with metabolism of aromatic compounds and cell division and cell cycle), along with measureable taxonomic shifts (increase in Bacterioidia and decrease in Actinobacteria). This study demonstrates that ceftiofur has a broad, measureable and immediate effect on the cow fecal metagenome. Given the importance of 3rd generation cephalospirins to human medicine, their continued use in dairy cattle should be carefully considered and waste treatment strategies to slow ARG dissemination from dairy cattle manure should be explored. [ABSTRACT FROM AUTHOR]

In this study, we utilized the Illumina high-throughput metagenomic approach to investigate diversity and abundance of both microbial community and copper resistance genes (CuRGs) in activated sludge (AS) which was enriched under copper selective stress up to 800 mg/L. The raw datasets (~3.5 Gb for each sample, i.e., the copper-enriched AS and the control AS) were merged and normalized for the BLAST analyses against the SILVA SSU rRNA gene database and self-constructed copper resistance protein database (CuRD). Also, the raw metagenomic sequences were assembled into contigs and analyzed based on Open Reading Frames (ORFs) to identify potentially novel copper resistance genes. Among the different resistance systems for copper detoxification under the high copper stress condition, the Cus system was the most enriched system. The results also indicated that genes encoding multi-copper oxidase played a more important role than those encoding efflux proteins. More significantly, several potentially novel copper resistance ORFs were identified by Pfam search and phylogenic analysis. This study demonstrated a new understanding of microbial-mediated copper resistance under high copper stress using high-throughput shotgun sequencing technique. [ABSTRACT FROM AUTHOR]

Here, shotgun metagenomic sequencing was conducted to reveal the hydrogen-oxidizing autotrophic-denitrifying metabolism in an enriched Thauera-dominated consortium. A draft genome named Thauera R4 of over 90 % completeness (3.8 Mb) was retrieved mainly by a coverage-defined binning method from 3.5 Gb paired-end Illumina reads. We identified 1,263 genes (accounting for 33 % of total genes in the finished genome of Thauera aminoaromatica MZ1T) with average nucleotide identity of 87.6 % shared between Thauera R4 and T. aminoaromatica MZ1T. Although Thauera R4 and T. aminoaromatica shared quite similar nitrogen metabolism and a high nucleotide similarity (98.8 %) in their 16S ribosomal RNA genes, they showed different functional potentials in several important environmentally relevant processes. Unlike T. aminoaromatica MZ1T, Thauera R4 carries an operon of [NiFe]-hydrogenase (EC 1.12.99.6) catalyzing molecular hydrogen oxidation in nitrate-rich solution. Moreover, Thauera R4 is a mixtrophic bacterium possessing key enzymes for autotrophic CO-fixation and heterotrophic acetate assimilation metabolism. This Thauera R4 bin provides another genetic reference to better understand the niches of Thauera and demonstrates a model pipeline to reveal functional profiles and reconstruct novel and dominant genomes from a simplified mixed culture in environmental studies. [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]

This study applied Illumina high-throughput sequencing to explore the microbial communities and functions in anaerobic digestion sludge (ADS) from two wastewater treatment plants based on a metagenomic view. Taxonomic analysis using SILVA SSU database indicated that Proteobacteria (9.52-13.50 %), Bacteroidetes (7.18 %-10.65 %) and Firmicutes (7.53 %-9.46 %) were the most abundant phyla in the ADS. Differences of microbial communities between the two types of ADS were identified. Genera of Methanosaeta and Methanosarcina were the major methanogens. Functional analysis by SEED subsystems showed that the basic metabolic functions of metagenomes in the four ADS samples had no significant difference among them, but they were different from other microbial communities from activated sludge, human faeces, ocean and soil. Abundances of genes in methanogenesis pathway were also quantified using a methanogenesis genes database extracted from KEGG. Results showed that acetotrophic was the major methanogenic pathway in the anaerobic sludge digestion. [ABSTRACT FROM AUTHOR]

Abstract: The abundance and diversity of biodegradation genes (BDGs) and potential degradation pathways of dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohexane (HCH), and atrazine (ATZ) in freshwater and marine sediments were investigated by metagenomic analysis using 6 datasets (16Gb in total). The datasets were derived using Illumina high-throughput sequencing and were based on BLAST against self-established databases of BDGs, DDT degradation genes (DDGs), HCH degradation genes (HDGs), and ATZ degradation genes (ADGs). The results showed that the abundance and diversity of BDGs, DDGs, HDGs, and ADGs varied with sample source and locations. The lip and mnp genes, which encode for peroxidase, and the carA gene, which encodes for laccase, were detected as the dominant genes for degradation of organic pollutants. The hdt, hdg, and atzB genes, which encode for hydratase, dehalogenase, and ethylaminohydrolase, were found to be the most abundant genes involved in DDT, HCH, and ATZ degradation, respectively. The identified 69 genera capable of degrading organic pollutants were mostly affiliated with Proteobacteria (49.3%) and Actinobacteria (21.7%). Four genera, including Plesiocystis, Anaerolinea, Jannaschia, and Mycobacterium, were the major biodegradation populations in all sediments. In this study, the nearly complete biodegradation pathways of DDT and ATZ were found, and the partial degradation pathway of HCH was detected in all sediments. [Copyright &y& Elsevier]

Metagenomic technique was employed to characterize the seasonal dynamics of activated sludge ( AS) communities in a municipal wastewater treatment plant ( WWTP) over 4 years. The results indicated that contrary to E ukaryota (mainly R otifera and N ematoda), abundances of B acteria and A rchaea (mainly E uryarchaeota) were significantly higher in winter than summer. Two-way analysis of variance and canonical correspondence analysis revealed that many functionally important genera followed strong seasonal variation patterns driven by temperature and salinity gradients; among them, two nitrifying bacteria, N itrospira and N itrosomonas, displayed much higher abundances in summer, whereas phosphate-removing genus T etrasphaera, denitrifier P aracoccus and potential human faecal bacteria, i.e. B ifidobacterium, D orea and R uminococcus, showed significantly higher abundances in winter. Particularly, occurrence of dual variation patterns beyond explanation merely by seasonality indicated that multivariables (e.g. dissolved oxygen, sludge retention time, nutrients) participated in shaping AS community structure. However, S EED subsystems annotation showed that functional categories in AS showed no significant difference between summer and winter, indicating that compared with its microbial components, the functional profiles of AS were much more stable. Taken together, our study provides novel insights into the microbial community variations in AS and discloses their correlations with influential factors in WWTPs. [ABSTRACT FROM AUTHOR]

Arsenic contamination in a water system is an urgent environmental problem that has caused severe endemic arsenicosis in south and southeast Asian countries. It is well known that microbially mediated arsenic metabolism can enhance arsenic mobility, bioavailability, and toxicity. Here, for the first time, we applied the Illumina high-throughput metagenomic and metatranscriptomic approaches to study the distribution, diversity, abundance, and expression of microbial arsenic metabolism genes in activated sludge and five coastal sediments. An average depth of ~2.6 Gb clean data for each sample was finally obtained for BLAST analysis after quality filtration and normalization. The results revealed that: (1) highly diverse arsenic metabolism-like genes were found and the overall abundance varied from 0.18 % to 0.26 % in the six metagenomic data sets; (2) arsenic metabolism-like genes were expressed with extremely low levels or no expression at all in activated sludge; and (3) the distribution, diversity, and abundance of aioA-like, arrA-like, arsB-like, ACR3-like, and arsM-like genes varied significantly in the six surveyed environments. This study provided a novel perspective on understanding the ecology of arsenic metabolism in different water environments using high-throughput sequencing technique. [ABSTRACT FROM AUTHOR]

Abstract: The abundance, diversity, and distribution of biodegradation genes (BDGs) and phenol degradation genes (PDGs) in activated sludge (AS) from two wastewater treatment plants (WWTPs) at different sampling times were assessed by metagenomic analysis using a total of 15 datasets derived from Illumina high-throughput sequencing and BLAST comparisons to BDGs and PDGs databases. The results showed that the abundance (0.015–0.030%) and diversity of BDGs in AS varied with the WWTP and the sampling times. The p450 and pmo genes were the most abundant genes in the BDGs and PDGs subgroups, respectively. MG-RAST analysis revealed that 87 detected bacterial genera potentially capable of degrading pollutants were mostly affiliated with Proteobacteria (59.8%), Bacteroidetes (17.2%), and Actinobacteria (9.2%). Mycobacterium, belonging to Actinobacteria, was found to be the most abundant genus (23.4%). This method could be used to monitor an AS’s biodegradation ability for organic pollutants and to evaluate its wastewater treatment efficiency. [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]

• 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]

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]

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]