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907100.9901RAC: Repository of Antibiotic resistance Cassettes. Antibiotic resistance in bacteria is often due to acquisition of resistance genes associated with different mobile genetic elements. In Gram-negative bacteria, many resistance genes are found as part of small mobile genetic elements called gene cassettes, generally found integrated into larger elements called integrons. Integrons carrying antibiotic resistance gene cassettes are often associated with mobile elements and here are designated 'mobile resistance integrons' (MRIs). More than one cassette can be inserted in the same integron to create arrays that contribute to the spread of multi-resistance. In many sequences in databases such as GenBank, only the genes within cassettes, rather than whole cassettes, are annotated and the same gene/cassette may be given different names in different entries, hampering analysis. We have developed the Repository of Antibiotic resistance Cassettes (RAC) website to provide an archive of gene cassettes that includes alternative gene names from multiple nomenclature systems and allows the community to contribute new cassettes. RAC also offers an additional function that allows users to submit sequences containing cassettes or arrays for annotation using the automatic annotation system Attacca. Attacca recognizes features (gene cassettes, integron regions) and identifies cassette arrays as patterns of features and can also distinguish minor cassette variants that may encode different resistance phenotypes (aacA4 cassettes and bla cassettes-encoding β-lactamases). Gaps in annotations are manually reviewed and those found to correspond to novel cassettes are assigned unique names. While there are other websites dedicated to integrons or antibiotic resistance genes, none includes a complete list of antibiotic resistance gene cassettes in MRI or offers consistent annotation and appropriate naming of all of these cassettes in submitted sequences. RAC thus provides a unique resource for researchers, which should reduce confusion and improve the quality of annotations of gene cassettes in integrons associated with antibiotic resistance. DATABASE URL: http://www2.chi.unsw.edu.au/rac.201122140215
907410.9900BacAnt: A Combination Annotation Server for Bacterial DNA Sequences to Identify Antibiotic Resistance Genes, Integrons, and Transposable Elements. Whole genome sequencing (WGS) of bacteria has become a routine method in diagnostic laboratories. One of the clinically most useful advantages of WGS is the ability to predict antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in bacterial sequences. This allows comprehensive investigations of such genetic features but can also be used for epidemiological studies. A plethora of software programs have been developed for the detailed annotation of bacterial DNA sequences, such as rapid annotation using subsystem technology (RAST), Resfinder, ISfinder, INTEGRALL and The Transposon Registry. Unfortunately, to this day, a reliable annotation tool of the combination of ARGs and MGEs is not available, and the generation of genbank files requires much manual input. Here, we present a new webserver which allows the annotation of ARGs, integrons and transposable elements at the same time. The pipeline generates genbank files automatically, which are compatible with Easyfig for comparative genomic analysis. Our BacAnt code and standalone software package are available at https://github.com/xthua/bacant with an accompanying web application at http://bacant.net.202134367079
812720.9898Microbial Multitrophic Communities Drive the Variation of Antibiotic Resistome in the Gut of Soil Woodlice (Crustacea: Isopoda). Multitrophic communities inhabit in soil faunal gut, including bacteria, fungi, and protists, which have been considered a hidden reservoir for antibiotic resistance genes (ARGs). However, there is a dearth of research focusing on the relationships between ARGs and multitrophic communities in the gut of soil faunas. Here, we studied the contribution of multitrophic communities to variations of ARGs in the soil woodlouse gut. The results revealed diverse and abundant ARGs in the woodlouse gut. Network analysis further exhibited strong connections between key ecological module members and ARGs, suggesting that multitrophic communities in the keystone ecological cluster may play a pivotal role in the variation of ARGs in the woodlouse gut. Moreover, long-term application of sewage sludge significantly altered the woodlice gut resistome and interkingdom communities. The variation portioning analysis indicated that the fungal community has a greater contribution to variations of ARGs than bacterial and protistan communities in the woodlice gut after long-term application of sewage sludge. Together, our results showed that changes in gut microbiota associated with agricultural practices (e.g., sewage sludge application) can largely alter the gut interkingdom network in ecologically relevant soil animals, with implications for antibiotic resistance, which advances our understanding of the microecological drivers of ARGs in terrestrial ecosystem.202235876241
737330.9898Distributional Pattern of Bacteria, Protists, and Diatoms in Ocean according to Water Depth in the Northern South China Sea. Ocean microbiomes provide insightful details about the condition of water and the global impact of marine ecosystems. A fine-scale analysis of ocean microbes may shed light on the dynamics and function of the ocean microbiome community. In this study, we evaluated the changes in the community and function of marine bacteria, protists, and diatoms corresponding to different ocean depths using next-generation sequencing methods. We found that diatoms displayed a potential water-depth pattern in species richness (alpha diversity) and community composition (beta diversity). However, for bacteria and protists, there was no significant relationship between water depth and species richness. This may be related to the biological characteristics of diatoms. The photosynthesis of diatoms and their distribution may be associated with the fluctuating light regime in the underwater climate. Moreover, salinity displayed negative effects on the abundance of some diatom and bacterial groups, which indicates that salinity may be one of the factors restricting ocean microorganism diversity. In addition, compared to the global ocean microbiome composition, function, and antibiotic resistance genes, a water depth pattern due to the fine-scale region was not observed in this study. IMPORTANCE Fine-scale analysis of ocean microbes provides insights into the dynamics and functions of the ocean microbiome community. Here, using amplicon and metagenome sequencing methods, we found that diatoms in the northern South China Sea displayed a potential water-depth pattern in species richness and community composition, which may be related to their biological characteristics. The potential effects of the differences in geographic sites mainly occurred in the diatom and bacterial communities. Moreover, given the correlation between the environmental factors and relative abundance of antibiotic resistance genes (ARGs), the study of ocean ARG distribution patterns should integrate the potential effects of environmental factors.202236222702
769640.9898Noise reduction strategies in metagenomic chromosome confirmation capture to link antibiotic resistance genes to microbial hosts. The gut microbiota is a reservoir for antimicrobial resistance genes (ARGs). With current sequencing methods, it is difficult to assign ARGs to their microbial hosts, particularly if these ARGs are located on plasmids. Metagenomic chromosome conformation capture approaches (meta3C and Hi-C) have recently been developed to link bacterial genes to phylogenetic markers, thus potentially allowing the assignment of ARGs to their hosts on a microbiome-wide scale. Here, we generated a meta3C dataset of a human stool sample and used previously published meta3C and Hi-C datasets to investigate bacterial hosts of ARGs in the human gut microbiome. Sequence reads mapping to repetitive elements were found to cause problematic noise in, and may importantly skew interpretation of, meta3C and Hi-C data. We provide a strategy to improve the signal-to-noise ratio by discarding reads that map to insertion sequence elements and to the end of contigs. We also show the importance of using spike-in controls to quantify whether the cross-linking step in meta3C and Hi-C protocols has been successful. After filtering to remove artefactual links, 87 ARGs were assigned to their bacterial hosts across all datasets, including 27 ARGs in the meta3C dataset we generated. We show that commensal gut bacteria are an important reservoir for ARGs, with genes coding for aminoglycoside and tetracycline resistance being widespread in anaerobic commensals of the human gut.202337272920
767450.9898Insights into gut microbiomes in stem cell transplantation by comprehensive shotgun long-read sequencing. The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members is limited using 16S rDNA sequencing. Here, we developed a pipeline enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using our pipeline we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, characterized by Bacteroides spp. /Phocaeicola spp., mixed composition and Enterococcus abundances. We revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples. In the course of HSCT, bacterial strains were stable or newly acquired. Our results demonstrate the disruptive potential of alloHSCTon the gut microbiome and pave the way for future comprehensive microbiome studies based on long-read metagenomics.202438374282
638860.9897A Metagenome from a Steam Vent in Los Azufres Geothermal Field Shows an Abundance of Thermoplasmatales archaea and Bacteria from the Phyla Actinomycetota and Pseudomonadota. Los Azufres National Park is a geothermal field that has a wide number of thermal manifestations; nevertheless, the microbial communities in many of these environments remain unknown. In this study, a metagenome from a sediment sample from Los Azufres National Park was sequenced. In this metagenome, we found that the microbial diversity corresponds to bacteria (Actinomycetota, Pseudomonadota), archaea (Thermoplasmatales and Candidatus Micrarchaeota and Candidatus Parvarchaeota), eukarya (Cyanidiaceae), and viruses (Fussellovirus and Caudoviricetes). The functional annotation showed genes related to the carbon fixation pathway, sulfur metabolism, genes involved in heat and cold shock, and heavy-metal resistance. From the sediment, it was possible to recover two metagenome-assembled genomes from Ferrimicrobium and Cuniculiplasma. Our results showed that there are a large number of microorganisms in Los Azufres that deserve to be studied.202337504286
774070.9897Diversity, functions, and antibiotic resistance genes of bacteria and fungi are examined in the bamboo plant phyllosphere that serve as food for the giant pandas. The phyllosphere of bamboo is rich in microorganisms that can disrupt the intestinal microbiota of the giant pandas that consume them, potentially leading to their death. In the present study, the abundance, diversity, biological functions (e.g., KEGG and CAZyme), and antibiotic resistance genes (ARGs) of bacteria and fungi in two bamboo species phyllosphere (Chimonobambusa szechuanensis, CS; Bashania fangiana, BF) in Daxiangling Nature Reserve (an important part of the Giant Panda National Park) were investigated respectively by amplicon sequencing of the whole 16S rRNA and ITS1-ITS2 genes on PacBio Sequel and whole-metagenome shotgun sequencing on Illumina NovaSeq 6000 platform. The results suggested that there were respectively 18 bacterial and 34 fungi biomarkers between the phyllosphere of the two species of bamboo. Beta diversity of bacteria and fungi communities exited between the two bamboos according to the (un)weighted UniFrac distance matrix. Moreover, the functional analysis showed that the largest relative abundance was found in the genes related to metabolism and global and overview maps. Glycoside hydrolases (GHs) and glycosyl transferases (GTs) have a higher abundance in two bamboo phyllospheres. Co-occurrence network modeling suggested that bacteria and fungi communities in CS phyllosphere employed a much more complex metabolic network than that in BF, and the abundance of multidrug, tetracycline, and glycopeptide resistance genes was higher and closely correlated with other ARGs. This study references the basis for protecting bamboo resources foraged by wild giant pandas and predicts the risk of antibiotic resistance in bamboo phyllosphere bacterial and fungal microbiota in the Giant Panda National Park, China.202539168909
322780.9896Geographic pattern of antibiotic resistance genes in the metagenomes of the giant panda. The rise in infections by antibiotic-resistant bacteria poses a serious public health problem worldwide. The gut microbiome of animals is a reservoir for antibiotic resistance genes (ARGs). However, the correlation between the gut microbiome of wild animals and ARGs remains controversial. Here, based on the metagenomes of giant pandas (including three wild populations from the Qinling, Qionglai and Xiaoxiangling Mountains, and two major captive populations from Yaan and Chengdu), we investigated the potential correlation between the constitution of the gut microbiome and the composition of ARGs across the different geographic locations and living environments. We found that the types of ARGs were correlated with gut microbiome composition. The NMDS cluster analysis using Jaccard distance of the ARGs composition of the gut microbiome of wild giant pandas displayed a difference based on geographic location. Captivity also had an effect on the differences in ARGs composition. Furthermore, we found that the Qinling population exhibited profound dissimilarities of both gut microbiome composition and ARGs (the highest proportion of Clostridium and vancomycin resistance genes) when compared to the other wild and captive populations studies, which was supported by previous giant panda whole-genome sequencing analysis. In this study, we provide an example of a potential consensus pattern regarding host population genetics, symbiotic gut microbiome and ARGs. We revealed that habitat isolation impacts the ARG structure in the gut microbiome of mammals. Therefore, the difference in ARG composition between giant panda populations will provide some basic information for their conservation and management, especially for captive populations.202132812361
765790.9896Comparison of Fecal Antimicrobial Resistance Genes in Captive and Wild Asian Elephants. The Asian elephant (Elephas maximus) is a flagship species of tropical rainforests, and it has generated much concern. In this case, the gut bacterial communities of captive and wild Asian elephants are particularly noteworthy. We aim to compare the differences in bacterial diversity and antibiotic resistance gene (ARG) subtypes in fecal samples of Asian elephants from different habitats, which may affect host health. Analyses reveal that differences in the dominant species of gut bacteria between captive and wild Asian elephants may result in significant differences in ARGs. Network analysis of bacterial communities in captive Asian elephants has identified potentially pathogenic species. Many negative correlations in network analysis suggest that different food sources may lead to differences in bacterial communities and ARGs. Results also indicate that the ARG levels in local captive breeding of Asian elephants are close to those of the wild type. However, we found that local captive elephants carry fewer ARG types than their wild counterparts. This study reveals the profile and relationship between bacterial communities and ARGs in different sources of Asian elephant feces, providing primary data for captive breeding and rescuing wild Asian elephants.202337237762
7646100.9895Assessment of Bacterial Community and Other Microorganism Along the Lam Takhong Watercourse, Nakhon Ratchasima, Thailand. Lam Takhong, a vital watercourse in Nakhon Ratchasima province, Thailand, supports agricultural, recreational, and urban activities. Originating in a national park, it flows through urban areas before discharging into a dam and running off via the sluice gate. While water quality monitoring is routine, microbial community data have never been reported. This study assesses the microorganism diversity and functional genes in Lam Takhong watercourse using a shotgun sequencing metagenomics approach. Water samples were collected from the upstream, midstream, and downstream sections. The midstream area exhibited the highest abundance of fecal coliform bacteria, plankton, and benthos, suggesting elevated pollution levels. Genes related to metabolism, particularly carbohydrate and amino acid pathways, were predominant. Proteobacteria was the most abundant phylum found in the water, with Limnohabitans as the dominant planktonic bacteria. Bacteria such as Staphylococcus, Mycobacterium, Escherichia, Pseudomonas, Enterococcus, Neisseria, Streptomyces, and Salmonella were detected, along with antibiotic resistance genes, raising public health concerns. These findings emphasize the need for microbial monitoring in the Lam Takhong to determine the potential water quality bioindicator and prevent potential disease spread through the water system.202540244481
7437110.9895Bacteriophages vehiculate a high amount of antibiotic resistance determinants of bacterial origin in the Orne River ecosystem. Aquatic environments are important dissemination routes of antibiotic resistance genes (ARGs) from and to pathogenic bacteria. Nevertheless, in these complex matrices, identifying and characterizing the driving microbial actors and ARG dissemination mechanisms they are involved in remain difficult. We here explored the distribution/compartmentalization of a panel of ARGs and mobile genetic elements (MGEs) in bacteria and bacteriophages collected in the water, suspended material and surface sediments from the Orne River ecosystem (France). By using a new bacteriophage DNA extraction method, we showed that, when packaging bacterial DNA, bacteriophages rather encapsidate both ARGs and MGEs than 16S rRNA genes, i.e. chromosomal fragments. We also show that the bacteria and bacteriophage capsid contents in ARGs/MGEs were similarly influenced by seasonality but that the distribution of ARGs/MGEs between the river physical compartments (water vs. suspended mater vs. sediment) is more impacted when these markers were carried by bacteria. These demonstrations will likely modify our understanding of the formation and fate of transducing viral particles in the environment. Consequently, they will also likely modify our estimations of the relative frequencies of the different horizontal gene transfer mechanisms in disseminating antibiotic resistance by reinforcing the roles played by environmental bacteriophages and transduction.202235672875
3220120.9895Metabolically-active bacteria in reclaimed water and ponds revealed using bromodeoxyuridine DNA labeling coupled with 16S rRNA and shotgun sequencing. Understanding the complex microbiota of agricultural irrigation water is vital to multiple sectors of sustainable agriculture and public health. To date, microbiome characterization methods have provided comprehensive profiles of aquatic microbiotas, but have not described which taxa are likely metabolically-active. Here, we combined 5‑bromo‑2'-deoxyuridine (BrdU) labeling with 16S rRNA and shotgun sequencing to identify metabolically-active bacteria in reclaimed and agricultural pond water samples (n = 28) recovered from the Mid-Atlantic United States between March 2017 and January 2018. BrdU-treated samples were significantly less diverse (alpha diversity) compared to non-BrdU-treated samples. The most abundant taxa in the metabolically-active fraction of water samples (BrdU-treated samples) were unclassified Actinobacteria, Flavobacterium spp., Pseudomonas spp. and Aeromonas spp. Interestingly, we also observed that antimicrobial resistance and virulence gene profiles seemed to be more diverse and more abundant in non-BrdU-treated water samples compared to BrdU-treated samples. These findings raise the possibility that these genes may be associated more with relic (inactive) DNA present in the tested water types rather than viable, metabolically-active microorganisms. Our study demonstrates that the coupled use of BrdU labeling and sequencing can enhance understanding of the metabolically-active fraction of bacterial communities in alternative irrigation water sources. Agricultural pond and reclaimed waters are vital to the future of sustainable agriculture, and thus, the full understanding of the pathogenic potential of these waters is important to guide mitigation strategies that ensure appropriate water quality for intended purposes.202032726735
7476130.9895Bacterial phylogeny structures soil resistomes across habitats. Ancient and diverse antibiotic resistance genes (ARGs) have previously been identified from soil, including genes identical to those in human pathogens. Despite the apparent overlap between soil and clinical resistomes, factors influencing ARG composition in soil and their movement between genomes and habitats remain largely unknown. General metagenome functions often correlate with the underlying structure of bacterial communities. However, ARGs are proposed to be highly mobile, prompting speculation that resistomes may not correlate with phylogenetic signatures or ecological divisions. To investigate these relationships, we performed functional metagenomic selections for resistance to 18 antibiotics from 18 agricultural and grassland soils. The 2,895 ARGs we discovered were mostly new, and represent all major resistance mechanisms. We demonstrate that distinct soil types harbour distinct resistomes, and that the addition of nitrogen fertilizer strongly influenced soil ARG content. Resistome composition also correlated with microbial phylogenetic and taxonomic structure, both across and within soil types. Consistent with this strong correlation, mobility elements (genes responsible for horizontal gene transfer between bacteria such as transposases and integrases) syntenic with ARGs were rare in soil by comparison with sequenced pathogens, suggesting that ARGs may not transfer between soil bacteria as readily as is observed between human pathogens. Together, our results indicate that bacterial community composition is the primary determinant of soil ARG content, challenging previous hypotheses that horizontal gene transfer effectively decouples resistomes from phylogeny.201424847883
7487140.9894Impact of cyanobacterial bloom on microbiomes of freshwater lakes. Cyanobacterial blooms are harmful because of their cyanotoxins production. It occurs due to the eutrophication of freshwater reserves. Nagpur city has three lakes which serves as public water resource and are affected by algal bloom events. Metagenomic approach was used for the exploration of taxonomic, catabolic, and resistome profile of these lakes. Taxonomic profiling indicated prevalence of cyanobacterial species like Microcystis, Anabaena, Trichodesmium, Microcoleus, and Nodularia. These bacteria are well known for their association with algal bloom and microcystin production. High Performance Liquid Chromatography (HPLC) detected the presence of microcystin toxin and concentration was relatively higher in Ambazari lakewater. In addition, the presence of several antibiotic resistance genes like vancomycin, beta-lactamase, methicillin, and fluoroquinolones were observed. Genes conferring metal resistance such as copper, cadmium, zinc were also mined indicating presence of metal resistant microorganisms. The study suggests that lake water contaminated with Microcystis (algal bloom) harbors complex microbial community having diverse catabolic and resistome profiles, which negatively affect the ecosystems services.202134785623
3776150.9894FARME DB: a functional antibiotic resistance element database. Antibiotic resistance (AR) is a major global public health threat but few resources exist that catalog AR genes outside of a clinical context. Current AR sequence databases are assembled almost exclusively from genomic sequences derived from clinical bacterial isolates and thus do not include many microbial sequences derived from environmental samples that confer resistance in functional metagenomic studies. These environmental metagenomic sequences often show little or no similarity to AR sequences from clinical isolates using standard classification criteria. In addition, existing AR databases provide no information about flanking sequences containing regulatory or mobile genetic elements. To help address this issue, we created an annotated database of DNA and protein sequences derived exclusively from environmental metagenomic sequences showing AR in laboratory experiments. Our Functional Antibiotic Resistant Metagenomic Element (FARME) database is a compilation of publically available DNA sequences and predicted protein sequences conferring AR as well as regulatory elements, mobile genetic elements and predicted proteins flanking antibiotic resistant genes. FARME is the first database to focus on functional metagenomic AR gene elements and provides a resource to better understand AR in the 99% of bacteria which cannot be cultured and the relationship between environmental AR sequences and antibiotic resistant genes derived from cultured isolates.Database URL: http://staff.washington.edu/jwallace/farme.201728077567
8714160.9894Tales from the tomb: the microbial ecology of exposed rock surfaces. Although a broad diversity of eukaryotic and bacterial taxa reside on rock surfaces where they can influence the weathering of rocks and minerals, these communities and their contributions to mineral weathering remain poorly resolved. To build a more comprehensive understanding of the diversity, ecology and potential functional attributes of microbial communities living on rock, we sampled 149 tombstones across three continents and analysed their bacterial and eukaryotic communities via marker gene and shotgun metagenomic sequencing. We found that geographic location and climate were important factors structuring the composition of these communities. Moreover, the tombstone-associated microbial communities varied as a function of rock type, with granite and limestone tombstones from the same cemeteries harbouring taxonomically distinct microbial communities. The granite and limestone-associated communities also had distinct functional attributes, with granite-associated bacteria having more genes linked to acid tolerance and chemotaxis, while bacteria on limestone were more likely to be lichen associated and have genes involved in photosynthesis and radiation resistance. Together these results indicate that rock-dwelling microbes exhibit adaptations to survive the stresses of the rock surface, differ based on location, climate and rock type, and seem pre-disposed to different ecological strategies (symbiotic versus free-living lifestyles) depending on the rock type.201829235707
6794170.9894Beyond cyanotoxins: increased Legionella, antibiotic resistance genes in western Lake Erie water and disinfection-byproducts in their finished water. BACKGROUND: Western Lake Erie is suffering from harmful cyanobacterial blooms, primarily toxic Microcystis spp., affecting the ecosystem, water safety, and the regional economy. Continued bloom occurrence has raised concerns about public health implications. However, there has been no investigation regarding the potential increase of Legionella and antibiotic resistance genes in source water, and disinfection byproducts in municipal treated drinking water caused by these bloom events. METHODS: Over 2 years, source water (total n = 118) and finished water (total n = 118) samples were collected from drinking water plants situated in western Lake Erie (bloom site) and central Lake Erie (control site). Bloom-related parameters were determined, such as microcystin (MC), toxic Microcystis, total organic carbon, N, and P. Disinfection byproducts (DBPs) [total trihalomethanes (THMs) and haloacetic acids (HAAs)] were assessed in finished water. Genetic markers for Legionella, antibiotic resistance genes, and mobile genetic elements were quantified in source and finished waters. RESULTS: Significantly higher levels of MC-producing Microcystis were observed in the western Lake Erie site compared to the control site. Analysis of DBPs revealed significantly elevated THMs concentrations at the bloom site, while HAAs concentrations remained similar between the two sites. Legionella spp. levels were significantly higher in the bloom site, showing a significant relationship with total cyanobacteria. Abundance of ARGs (tetQ and sul1) and mobile genetic elements (MGEs) were also significantly higher at the bloom site. DISCUSSION: Although overall abundance decreased in finished water, relative abundance of ARGs and MGE among total bacteria increased after treatment, particularly at the bloom site. The findings underscore the need for ongoing efforts to mitigate bloom frequency and intensity in the lake. Moreover, optimizing water treatment processes during bloom episodes is crucial to maintain water quality. The associations observed between bloom conditions, ARGs, and Legionella, necessitate future investigations into the potential enhancement of antibiotic-resistant bacteria and Legionella spp. due to blooms, both in lake environments and drinking water distribution systems.202337700867
5127180.9894ResFinderFG v2.0: a database of antibiotic resistance genes obtained by functional metagenomics. Metagenomics can be used to monitor the spread of antibiotic resistance genes (ARGs). ARGs found in databases such as ResFinder and CARD primarily originate from culturable and pathogenic bacteria, while ARGs from non-culturable and non-pathogenic bacteria remain understudied. Functional metagenomics is based on phenotypic gene selection and can identify ARGs from non-culturable bacteria with a potentially low identity shared with known ARGs. In 2016, the ResFinderFG v1.0 database was created to collect ARGs from functional metagenomics studies. Here, we present the second version of the database, ResFinderFG v2.0, which is available on the Center of Genomic Epidemiology web server (https://cge.food.dtu.dk/services/ResFinderFG/). It comprises 3913 ARGs identified by functional metagenomics from 50 carefully curated datasets. We assessed its potential to detect ARGs in comparison to other popular databases in gut, soil and water (marine + freshwater) Global Microbial Gene Catalogues (https://gmgc.embl.de). ResFinderFG v2.0 allowed for the detection of ARGs that were not detected using other databases. These included ARGs conferring resistance to beta-lactams, cycline, phenicol, glycopeptide/cycloserine and trimethoprim/sulfonamide. Thus, ResFinderFG v2.0 can be used to identify ARGs differing from those found in conventional databases and therefore improve the description of resistomes.202337207327
9079190.9894Review, Evaluation, and Directions for Gene-Targeted Assembly for Ecological Analyses of Metagenomes. Shotgun metagenomics has greatly advanced our understanding of microbial communities over the last decade. Metagenomic analyses often include assembly and genome binning, computationally daunting tasks especially for big data from complex environments such as soil and sediments. In many studies, however, only a subset of genes and pathways involved in specific functions are of interest; thus, it is not necessary to attempt global assembly. In addition, methods that target genes can be computationally more efficient and produce more accurate assembly by leveraging rich databases, especially for those genes that are of broad interest such as those involved in biogeochemical cycles, biodegradation, and antibiotic resistance or used as phylogenetic markers. Here, we review six gene-targeted assemblers with unique algorithms for extracting and/or assembling targeted genes: Xander, MegaGTA, SAT-Assembler, HMM-GRASPx, GenSeed-HMM, and MEGAN. We tested these tools using two datasets with known genomes, a synthetic community of artificial reads derived from the genomes of 17 bacteria, shotgun sequence data from a mock community with 48 bacteria and 16 archaea genomes, and a large soil shotgun metagenomic dataset. We compared assemblies of a universal single copy gene (rplB) and two N cycle genes (nifH and nirK). We measured their computational efficiency, sensitivity, specificity, and chimera rate and found Xander and MegaGTA, which both use a probabilistic graph structure to model the genes, have the best overall performance with all three datasets, although MEGAN, a reference matching assembler, had better sensitivity with synthetic and mock community members chosen from its reference collection. Also, Xander and MegaGTA are the only tools that include post-assembly scripts tuned for common molecular ecology and diversity analyses. Additionally, we provide a mathematical model for estimating the probability of assembling targeted genes in a metagenome for estimating required sequencing depth.201931749830