Effects of different assembly strategies on gene annotation in activated sludge. - Related Documents




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769901.0000Effects of different assembly strategies on gene annotation in activated sludge. Activated sludge comprises diverse bacteria, fungi, and other microorganisms, featuring a rich repertoire of genes involved in antibiotic resistance, pollutant degradation, and elemental cycling. In this regard, hybrid assembly technology can revolutionize metagenomics by detecting greater gene diversity in environmental samples. Nonetheless, the optimal utilization and comparability of genomic information between hybrid assembly and short- or long-read technology remain unclear. To address this gap, we compared the performance of the hybrid assembly, short- and long-read technologies, abundance and diversity of annotated genes, and taxonomic diversity by analysing 46, 161, and 45 activated sludge metagenomic datasets, respectively. The results revealed that hybrid assembly technology exhibited the best performance, generating the most contiguous and longest contigs but with a lower proportion of high-quality metagenome-assembled genomes than short-read technology. Compared with short- or long-read technologies, hybrid assembly technology can detect a greater diversity of microbiota and antibiotic resistance genes, as well as a wider range of potential hosts. However, this approach may yield lower gene abundance and pathogen detection. Our study revealed the specific advantages and disadvantages of hybrid assembly and short- and long-read applications in wastewater treatment plants, and our approach could serve as a blueprint to be extended to terrestrial environments.202438734289
770010.9999Rapid identification of antibiotic resistance gene hosts by prescreening ARG-like reads. Effective risk assessment and control of environmental antibiotic resistance depend on comprehensive information about antibiotic resistance genes (ARGs) and their microbial hosts. Advances in sequencing technologies and bioinformatics have enabled the identification of ARG hosts using metagenome-assembled contigs and genomes. However, these approaches often suffer from information loss and require extensive computational resources. Here we introduce a bioinformatic strategy that identifies ARG hosts by prescreening ARG-like reads (ALRs) directly from total metagenomic datasets. This ALR-based method offers several advantages: (1) it enables the detection of low-abundance ARG hosts with higher accuracy in complex environments; (2) it establishes a direct relationship between the abundance of ARGs and their hosts; and (3) it reduces computation time by approximately 44-96% compared to strategies relying on assembled contigs and genomes. We applied our ALR-based strategy alongside two traditional methods to investigate a typical human-impacted environment. The results were consistent across all methods, revealing that ARGs are predominantly carried by Gammaproteobacteria and Bacilli, and their distribution patterns may indicate the impact of wastewater discharge on coastal resistome. Our strategy provides rapid and accurate identification of antibiotic-resistant bacteria, offering valuable insights for the high-throughput surveillance of environmental antibiotic resistance. This study further expands our knowledge of ARG-related risk management in future.202540059905
733820.9997Sensitivity and consistency of long- and short-read metagenomics and epicPCR for the detection of antibiotic resistance genes and their bacterial hosts in wastewater. Wastewater surveillance is a powerful tool to assess the risks associated with antibiotic resistance in communities. One challenge is selecting which analytical tool to deploy to measure risk indicators, such as antibiotic resistance genes (ARGs) and their respective bacterial hosts. Although metagenomics is frequently used for analyzing ARGs, few studies have compared the performance of long-read and short-read metagenomics in identifying which bacteria harbor ARGs in wastewater. Furthermore, for ARG host detection, untargeted metagenomics has not been compared to targeted methods such as epicPCR. Here, we 1) evaluated long-read and short-read metagenomics as well as epicPCR for detecting ARG hosts in wastewater, and 2) investigated the host range of ARGs across the wastewater treatment plant (WWTP) to evaluate host proliferation. Results highlighted long-read revealed a wider range of ARG hosts compared to short-read metagenomics. Nonetheless, the ARG host range detected by long-read metagenomics only represented a subset of the hosts detected by epicPCR. The ARG-host linkages across the influent and effluent of the WWTP were characterized. Results showed the ARG-host phylum linkages were relatively consistent across the WWTP, whereas new ARG-host species linkages appeared in the WWTP effluent. The ARG-host linkages of several clinically relevant species found in the effluent were identified.202438490149
771430.9997Functional traits and health implications of the global household drinking-water microbiome retrieved using an integrative genome-centric approach. The biological safety of drinking water plays a crucial role in public health protection. However, research on the drinking water microbiome remains in its infancy, especially little is known about the potentially pathogenic bacteria in and functional characteristics of the microbiome in household tap water that people are directly exposed to. In this study, we used a genomic-centric approach to construct a genetic catalogue of the drinking water microbiome by analysing 116 metagenomic datasets of household tap water worldwide, spanning nine countries/regions on five continents. We reconstructed 859 high-quality metagenome-assembled genomes (MAGs) spanning 27 bacterial and 2 archaeal phyla, and found that the core MAGs belonging to the phylum Proteobacteria encoded the highest metabolic functional diversity of the 33 key complete metabolic modules. In particular, we found that two core MAGs of Brevibacillus and Methylomona encoded genes for methane metabolism, which may support the growth of heterotrophic organisms observed in the oligotrophic ecosystem. Four MAGs of complete ammonia oxidation (comammox) Nitrospira were identified and functional metabolic analysis suggested these may enable mixotrophic growth and encode genes for reactive oxygen stress defence and arsenite reduction that could aid survival in the environment of oligotrophic drinking water systems. Four MAGs were annotated as potentially pathogenic bacteria (PPB) and thus represented a possible public health concern. They belonged to the genera Acinetobacter (n = 3) and Mycobacterium (n = 1), with a total relative abundance of 1.06 % in all samples. The genomes of PPB A. junii and A. ursingii were discovered to contain antibiotic resistance genes and mobile genetic elements that could contribute to antimicrobial dissemination in drinking water. Further network analysis suggested that symbiotic microbes which support the growth of pathogenic bacteria can be targets for future surveillance and removal.202438183799
769840.9997Detecting horizontal gene transfer with metagenomics co-barcoding sequencing. Horizontal gene transfer (HGT) is the process through which genetic information is transferred between different genomes and that played a crucial role in bacterial evolution. HGT can enable bacteria to rapidly acquire antibiotic resistance and bacteria that have acquired resistance is spreading within the microbiome. Conventional methods of characterizing HGT patterns include short-read metagenomic sequencing (short-reads mNGS), long-read sequencing, and single-cell sequencing. These approaches present several limitations, such as short-read fragments, high amounts of input DNA, and sequencing costs, respectively. Here, we attempt to circumvent present limitations to detect HGT by developing a metagenomics co-barcode sequencing workflow (MECOS) and applying it to the human and mouse gut microbiomes. In addition to that, we have over 10-fold increased contig length compared to short-reads mNGS; we also obtained exceeding 30 million paired reads with co-barcode information. Applying the novel bioinformatic pipeline, we integrated this co-barcoding information and the context information from long reads, and observed over 50-fold HGT events after we corrected the potential wrong HGT events. Specifically, we detected approximately 3,000 HGT blocks in individual samples, encompassing ~6,000 genes and ~100 taxonomic groups, including loci conferring tetracycline resistance through ribosomal protection. MECOS provides a valuable tool for investigating HGT and advance our understanding on the evolution of natural microbial communities within hosts.IMPORTANCEIn this study, to better identify horizontal gene transfer (HGT) in individual samples, we introduce a new co-barcoding sequencing system called metagenomics co-barcoding sequencing (MECOS), which has three significant improvements: (i) long DNA fragment extraction, (ii) a special transposome insertion, (iii) hybridization of DNA to barcode beads, and (4) an integrated bioinformatic pipeline. Using our approach, we have over 10-fold increased contig length compared to short-reads mNGS, and observed over 50-fold HGT events after we corrected the potential wrong HGT events. Our results indicate the presence of approximately 3,000 HGT blocks, involving roughly 6,000 genes and 100 taxonomic groups in individual samples. Notably, these HGT events are predominantly enriched in genes that confer tetracycline resistance via ribosomal protection. MECOS is a useful tool for investigating HGT and the evolution of natural microbial communities within hosts, thereby advancing our understanding of microbial ecology and evolution.202438315121
965350.9997Evaluating the mobility potential of antibiotic resistance genes in environmental resistomes without metagenomics. Antibiotic resistance genes are ubiquitous in the environment. However, only a fraction of them are mobile and able to spread to pathogenic bacteria. Until now, studying the mobility of antibiotic resistance genes in environmental resistomes has been challenging due to inadequate sensitivity and difficulties in contig assembly of metagenome based methods. We developed a new cost and labor efficient method based on Inverse PCR and long read sequencing for studying mobility potential of environmental resistance genes. We applied Inverse PCR on sediment samples and identified 79 different MGE clusters associated with the studied resistance genes, including novel mobile genetic elements, co-selected resistance genes and a new putative antibiotic resistance gene. The results show that the method can be used in antibiotic resistance early warning systems. In comparison to metagenomics, Inverse PCR was markedly more sensitive and provided more data on resistance gene mobility and co-selected resistances.201627767072
748060.9997Genetic compatibility and ecological connectivity drive the dissemination of antibiotic resistance genes. The dissemination of mobile antibiotic resistance genes (ARGs) via horizontal gene transfer is a significant threat to public health globally. The flow of ARGs into and between pathogens, however, remains poorly understood, limiting our ability to develop strategies for managing the antibiotic resistance crisis. Therefore, we aim to identify genetic and ecological factors that are fundamental for successful horizontal ARG transfer. We used a phylogenetic method to identify instances of horizontal ARG transfer in ~1 million bacterial genomes. This data was then integrated with >20,000 metagenomes representing animal, human, soil, water, and wastewater microbiomes to develop random forest models that can reliably predict horizontal ARG transfer between bacteria. Our results suggest that genetic incompatibility, measured as nucleotide composition dissimilarity, negatively influences the likelihood of transfer of ARGs between evolutionarily divergent bacteria. Conversely, environmental co-occurrence increases the likelihood, especially in humans and wastewater, in which several environment-specific dissemination patterns are observed. This study provides data-driven ways to predict the spread of ARGs and provides insights into the mechanisms governing this evolutionary process.202540090954
325270.9997Exploring phylogenetic diversity of antibiotic resistance genes in activated sludge: A host and genomic location perspective. Antibiotic resistance has emerged as a significant global public health issue. The environmental behaviors of antibiotic resistance genes (ARGs), such as their persistence and horizontal transfer, have been extensively investigated. However, the genetic diversity characteristics of ARGs remain underexplored, which limits a comprehensive analysis of their roles in the environment. In this study, we examined the genetic diversity of ARGs in activated sludge from 44 wastewater treatment plants in five countries. Most ARGs detected in activated sludge possessed multiple variants, with a median of 48. The number of variants of gd-ARGs varied among different resistance mechanisms and ARG types. The number of potential variants of ARGs was strongly correlated with host diversity. Pseudomonas spp. and Klebsiella pneumoniae, identified as pathogenic bacteria, harbored multiple ARGs and had the most variants. Most ARG subtypes on plasmids and chromosomes showed divergent evolution. Molecular docking of AdeH proteins revealed that genomic location affects tetracycline binding energy. The findings underscore the intricate interplay between genetic variation and environmental adaptation in ARGs, offering a novel perspective on the spread of antibiotic resistance.202540216056
325680.9997Co-localization of antibiotic resistance genes is widespread in the infant gut microbiome and associates with an immature gut microbial composition. BACKGROUND: In environmental bacteria, the selective advantage of antibiotic resistance genes (ARGs) can be increased through co-localization with genes such as other ARGs, biocide resistance genes, metal resistance genes, and virulence genes (VGs). The gut microbiome of infants has been shown to contain numerous ARGs, however, co-localization related to ARGs is unknown during early life despite frequent exposures to biocides and metals from an early age. RESULTS: We conducted a comprehensive analysis of genetic co-localization of resistance genes in a cohort of 662 Danish children and examined the association between such co-localization and environmental factors as well as gut microbial maturation. Our study showed that co-localization of ARGs with other resistance and virulence genes is common in the early gut microbiome and is associated with gut bacteria that are indicative of low maturity. Statistical models showed that co-localization occurred mainly in the phylum Proteobacteria independent of high ARG content and contig length. We evaluated the stochasticity of co-localization occurrence using enrichment scores. The most common forms of co-localization involved tetracycline and fluoroquinolone resistance genes, and, on plasmids, co-localization predominantly occurred in the form of class 1 integrons. Antibiotic use caused a short-term increase in mobile ARGs, while non-mobile ARGs showed no significant change. Finally, we found that a high abundance of VGs was associated with low gut microbial maturity and that VGs showed even higher potential for mobility than ARGs. CONCLUSIONS: We found that the phenomenon of co-localization between ARGs and other resistance and VGs was prevalent in the gut at the beginning of life. It reveals the diversity that sustains antibiotic resistance and therefore indirectly emphasizes the need to apply caution in the use of antimicrobial agents in clinical practice, animal husbandry, and daily life to mitigate the escalation of resistance. Video Abstract.202438730321
346090.9997Bioprospecting for β-lactam resistance genes using a metagenomics-guided strategy. 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.201728584911
7475100.9997A Metagenomic Investigation of Spatial and Temporal Changes in Sewage Microbiomes across a University Campus. Wastewater microbial communities are not static and can vary significantly across time and space, but this variation and the factors driving the observed spatiotemporal variation often remain undetermined. We used a shotgun metagenomic approach to investigate changes in wastewater microbial communities across 17 locations in a sewer network, with samples collected from each location over a 3-week period. Fecal material-derived bacteria constituted a relatively small fraction of the taxa found in the collected samples, highlighting the importance of environmental sources to the sewage microbiome. The prokaryotic communities were highly variable in composition depending on the location within the sampling network, and this spatial variation was most strongly associated with location-specific differences in sewage pH. However, we also observed substantial temporal variation in the composition of the prokaryotic communities at individual locations. This temporal variation was asynchronous across sampling locations, emphasizing the importance of independently considering both spatial and temporal variation when assessing the wastewater microbiome. The spatiotemporal patterns in viral community composition closely tracked those of the prokaryotic communities, allowing us to putatively identify the bacterial hosts of some of the dominant viruses in these systems. Finally, we found that antibiotic resistance gene profiles also exhibit a high degree of spatiotemporal variability, with most of these genes unlikely to be derived from fecal bacteria. Together, these results emphasize the dynamic nature of the wastewater microbiome, the challenges associated with studying these systems, and the utility of metagenomic approaches for building a multifaceted understanding of these microbial communities and their functional attributes. IMPORTANCE Sewage systems harbor extensive microbial diversity, including microbes derived from both human and environmental sources. Studies of the sewage microbiome are useful for monitoring public health and the health of our infrastructure, but the sewage microbiome can be highly variable in ways that are often unresolved. We sequenced DNA recovered from wastewater samples collected over a 3-week period at 17 locations in a single sewer system to determine how these communities vary across time and space. Most of the wastewater bacteria, and the antibiotic resistance genes they harbor, were not derived from human feces, but human usage patterns did impact how the amounts and types of bacteria and bacterial genes we found in these systems varied over time. Likewise, the wastewater communities, including both bacteria and their viruses, varied depending on location within the sewage network, highlighting the challenges and opportunities in efforts to monitor and understand the sewage microbiome.202236121163
6891110.9997Feedstock-dependent antibiotic resistance gene patterns and expression profiles in industrial scale biogas plants revealed by meta-omics technology. This study investigated antimicrobial resistance in the anaerobic digesters of two industrial-scale biogas plants processing agricultural biomass and municipal wastewater sludge. A combination of deep sequencing and genome-centric workflow was implemented for metagenomic and metatranscriptomics data analysis to comprehensively examine potential antimicrobial resistance in microbial communities. Anaerobic microbes were found to harbour numerous antibiotic resistance genes (ARGs), with 58.85% of the metagenome-assembled genomes (MAGs) harbouring antibiotic resistance. A moderately positive correlation was observed between the abundance and expression of ARGs. ARGs were located primarily on bacterial chromosomes. A higher expression of resistance genes was observed on plasmids than on chromosomes. Risk index assessment suggests that most ARGs identified posed a significant risk to human health. However, potentially pathogenic bacteria showed lower ARG expression than non-pathogenic ones, indicating that anaerobic treatment is effective against pathogenic microbes. Resistomes at the gene category level were associated with various antibiotic resistance categories, including multidrug resistance, beta-lactams, glycopeptides, peptides, and macrolide-lincosamide-streptogramin (MLS). Differential expression analysis revealed specific genes associated with potential pathogenicity, emphasizing the importance of active gene expression in assessing the risks associated with ARGs.202539461216
7486120.9997Body size: A hidden trait of the organisms that influences the distribution of antibiotic resistance genes in soil. Body size is a key life-history trait of organisms, which has important ecological functions. However, the relationship between soil antibiotic resistance gene (ARG) distribution and organisms' body size has not been systematically reported so far. Herein, the impact of organic fertilizer on the soil ARGs and organisms (bacteria, fungi, and nematode) at the aggregate level was analyzed. The results showed that the smaller the soil aggregate size, the greater the abundance of ARGs, and the larger the body size of bacteria and nematodes. Further analysis revealed significant positive correlations of ARG abundance with the body sizes of bacteria, fungi, and nematodes, respectively. Additionally, the structural equation model demonstrated that changes in soil fertility mainly regulate the ARG abundance by affecting bacterial body size. The random forest model revealed that total phosphorus was the primary soil fertility factor influencing the body size of organisms. Therefore, these findings proposed that excessive application of phosphate fertilizers could increase the risk of soil ARG transmission by increasing the body size of soil organisms. This study highlights the significance of organisms' body size in determining the distribution of soil ARGs and proposes a new disadvantage of excessive fertilization from the perspective of ARGs.202438696961
7482130.9997Prophage-encoded antibiotic resistance genes are enriched in human-impacted environments. The spread of antibiotic resistance genes (ARGs) poses a substantial threat to human health. Phage-mediated transduction could exacerbate ARG transmission. While several case studies exist, it is yet unclear to what extent phages encode and mobilize ARGs at the global scale and whether human impacts play a role in this across different habitats. Here, we combine 38,605 bacterial genomes, 1432 metagenomes, and 1186 metatranscriptomes across 12 contrasting habitats to explore the distribution of prophages and their cargo ARGs in natural and human-impacted environments. Worldwide, we observe a significant increase in the abundance, diversity, and activity of prophage-encoded ARGs in human-impacted habitats linked with relatively higher risk of past antibiotic exposure. This effect was driven by phage-encoded cargo ARGs that could be mobilized to provide increased resistance in heterologous E. coli host for a subset of analyzed strains. Our findings suggest that human activities have altered bacteria-phage interactions, enriching ARGs in prophages and making ARGs more mobile across habitats globally.202439333115
9654140.9997Studying the Association between Antibiotic Resistance Genes and Insertion Sequences in Metagenomes: Challenges and Pitfalls. Antibiotic resistance is an issue in many areas of human activity. The mobilization of antibiotic resistance genes within the bacterial community makes it difficult to study and control the phenomenon. It is known that certain insertion sequences, which are mobile genetic elements, can participate in the mobilization of antibiotic resistance genes and in the expression of these genes. However, the magnitude of the contribution of insertion sequences to the mobility of antibiotic resistance genes remains understudied. In this study, the relationships between insertion sequences and antibiotic resistance genes present in the microbiome were investigated using two public datasets. The first made it possible to analyze the effects of different antibiotics in a controlled mouse model. The second dataset came from a study of the differences between conventional and organic-raised cattle. Although it was possible to find statistically significant correlations between the insertion sequences and antibiotic resistance genes in both datasets, several challenges remain to better understand the contribution of insertion sequences to the motility of antibiotic resistance genes. Obtaining more complete and less fragmented metagenomes with long-read sequencing technologies could make it possible to understand the mechanisms favoring horizontal transfers within the microbiome with greater precision.202336671375
7481150.9997The Bacterial Mobile Resistome Transfer Network Connecting the Animal and Human Microbiomes. Horizontally acquired antibiotic resistance genes (ARGs) in bacteria are highly mobile and have been ranked as principal risk resistance determinants. However, the transfer network of the mobile resistome and the forces driving mobile ARG transfer are largely unknown. Here, we present the whole profile of the mobile resistome in 23,425 bacterial genomes and explore the effects of phylogeny and ecology on the recent transfer (≥99% nucleotide identity) of mobile ARGs. We found that mobile ARGs are mainly present in four bacterial phyla and are significantly enriched in Proteobacteria The recent mobile ARG transfer network, which comprises 703 bacterial species and 16,859 species pairs, is shaped by the bacterial phylogeny, while an ecological barrier also exists, especially when interrogating bacteria colonizing different human body sites. Phylogeny is still a driving force for the transfer of mobile ARGs between farm animals and the human gut, and, interestingly, the mobile ARGs that are shared between the human and animal gut microbiomes are also harbored by diverse human pathogens. Taking these results together, we suggest that phylogeny and ecology are complementary in shaping the bacterial mobile resistome and exert synergistic effects on the development of antibiotic resistance in human pathogens. IMPORTANCE: The development of antibiotic resistance threatens our modern medical achievements. The dissemination of antibiotic resistance can be largely attributed to the transfer of bacterial mobile antibiotic resistance genes (ARGs). Revealing the transfer network of these genes in bacteria and the forces driving the gene flow is of great importance for controlling and predicting the emergence of antibiotic resistance in the clinic. Here, by analyzing tens of thousands of bacterial genomes and millions of human and animal gut bacterial genes, we reveal that the transfer of mobile ARGs is mainly controlled by bacterial phylogeny but under ecological constraints. We also found that dozens of ARGs are transferred between the human and animal gut and human pathogens. This work demonstrates the whole profile of mobile ARGs and their transfer network in bacteria and provides further insight into the evolution and spread of antibiotic resistance in nature.201627613679
7462160.9997A global atlas of marine antibiotic resistance genes and their expression. Oceans serve as global reservoirs of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). However, little is known about the traits and expression of ARGs in response to environmental factors. We analyzed 347 metagenomes and 182 metatranscriptomes to determine the distribution, hosts, and expression of ARGs in oceans. Our study found that the diversity and abundance of ARGs varied with latitude and depth. The core marine resistome mainly conferred glycopeptide and multidrug resistance. The hosts of this resistome were mainly limited to the core marine microbiome, with phylogenetic barriers to the horizontal transfer of ARGs, transfers being more frequent within species than between species. Sixty-five percent of the marine ARGs identified were expressed. More than 90% of high-risk ARGs were more likely to be expressed. Anthropogenic activity might affect the expression of ARGs by altering nitrate and phosphate concentrations and ocean temperature. Machine-learning models predict >97% of marine ARGs will change expression by 2100. High-risk ARGs will shift to low latitudes and regions with high anthropogenic activity, such as the Pacific and Atlantic Oceans. Certain ARGs serve a dual role in antibiotic resistance and potentially participate in element cycling, along with other unknown functions. Determining whether changes in ARG expression are beneficial to ecosystems and human health is challenging without comprehensive understanding of their functions. Our study identified a core resistome in the oceans and quantified the expression of ARGs for the development of future control strategies under global change.202337604017
7479170.9997Metagenomic investigation reveals bacteriophage-mediated horizontal transfer of antibiotic resistance genes in microbial communities of an organic agricultural ecosystem. Antibiotic resistance has become a serious health concern worldwide. The potential impact of viruses, bacteriophages in particular, on spreading antibiotic resistance genes is still controversial due to the complexity of bacteriophage-bacterial interactions within diverse environments. In this study, we determined the microbiome profiles and the potential antibiotic resistance gene (ARG) transfer between bacterial and viral populations in different agricultural samples using a high-resolution analysis of the metagenomes. The results of this study provide compelling genetic evidence for ARG transfer through bacteriophage-bacteria interactions, revealing the inherent risks associated with bacteriophage-mediated ARG transfer across the agricultural microbiome.202337754684
9649180.9997Bacteria of the order Burkholderiales are original environmental hosts of type II trimethoprim resistance genes (dfrB). It is consensus that clinically relevant antibiotic resistance genes have their origin in environmental bacteria, including the large pool of primarily benign species. Yet, for the vast majority of acquired antibiotic resistance genes, the original environmental host(s) has not been identified to date. Closing this knowledge gap could improve our understanding of how antimicrobial resistance proliferates in the bacterial domain and shed light on the crucial step of initial resistance gene mobilization in particular. Here, we combine information from publicly available long- and short-read environmental metagenomes as well as whole-genome sequences to identify the original environmental hosts of dfrB, a family of genes conferring resistance to trimethoprim. Although this gene family stands in the shadow of the more widespread, structurally different dfrA, it has recently gained attention through the discovery of several new members. Based on the genetic context of dfrB observed in long-read metagenomes, we predicted bacteria of the order Burkholderiales to function as original environmental hosts of the predominant gene variants in both soil and freshwater. The predictions were independently confirmed by whole-genome datasets and statistical correlations between dfrB abundance and taxonomic composition of environmental bacterial communities. Our study suggests that Burkholderiales in general and the family Comamonadaceae in particular represent environmental origins of dfrB genes, some of which now contribute to the acquired resistome of facultative pathogens. We propose that our workflow centered on long-read environmental metagenomes allows for the identification of the original hosts of further clinically relevant antibiotic resistance genes.202439658215
3339190.9997Examining the taxonomic distribution of tetracycline resistance in a wastewater plant. Microbial communities serve as reservoirs of antibiotic resistance genes (ARGs) and facilitate the dissemination of these genes to bacteria that infect humans. Relatively little is known about the taxonomic distribution of bacteria harboring ARGs in these reservoirs and the avenues of transmission due to the technical hurdles associated with characterizing the contents of complex microbial populations and the assignment of genes to particular genomes. Focusing on the array of tetracycline resistance (Tc(r)) genes in the primary and secondary phases of wastewater treatment, 17 of the 22 assayed Tc(r) genes were detected in at least one sample. We then applied emulsion, paired isolation, and concatenation PCR (epicPCR) to link tetracycline resistance genes to specific bacterial hosts. Whereas Tc(r) genes tend to vary in their distributions among bacterial taxa according to their modes of action, there were numerous instances in which a particular Tc(r) gene was associated with a host that was distantly related to all other bacteria bearing the same gene, including several hosts not previously identified. Tc(r) genes are far less host-restricted than previously assumed, indicating that complex microbial communities serve as settings where ARGs are spread among divergent bacterial phyla.202438317688