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769800.9977Detecting 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
908010.9977Comparison of de-novo assembly tools for plasmid metagenome analysis. BACKGROUND: With the advent of next-generation sequencing techniques, culture-independent metagenome approaches have now made it possible to predict possible presence of genes in the environmental bacteria most of which may be non-cultivable. Short reads obtained from the deep sequencing can be assembled into long contigs some of which include plasmids. Plasmids are the circular double stranded DNA in bacteria and known as one of the major carriers of antibiotic resistance genes. OBJECTIVE: Metagenomic analyses, especially focused on plasmids, could help us predict dissemination mechanisms of antibiotic resistance genes in the environment. However, with the availability of a myriad of metagenomic assemblers, the selection of the most appropriate metagenome assembler for the plasmid metagenome study might be challenging. Therefore, in this study, we compared five open source assemblers to suggest most effective way of plasmid metagenome analysis. METHODS: IDBA-UD, MEGAHIT, SPAdes, SOAPdenovo2, and Velvet are compared for conducting plasmid metagenome analyses using two water samples. RESULTS: Our results clearly showed that abundance and types of antibiotic resistance genes on plasmids varied depending on the selection of assembly tools. IDBA-UD and MEGAHIT demonstrated the overall best assembly statistics with high N50 values with higher portion of longer contigs. CONCLUSION: These two assemblers also detected more diverse plasmids. Among the two, MEGAHIT showed more memory efficient assembly, therefore we suggest that the use of MEGAHIT for plasmid metagenome analysis may offer more diverse plasmids with less computer resource required. Here, we also summarized a fundamental plasmid metagenome work flow, especially for antibiotic resistance gene investigation.201931187446
906920.9976Pdif-mediated antibiotic resistance genes transfer in bacteria identified by pdifFinder. Modules consisting of antibiotic resistance genes (ARGs) flanked by inverted repeat Xer-specific recombination sites were thought to be mobile genetic elements that promote horizontal transmission. Less frequently, the presence of mobile modules in plasmids, which facilitate a pdif-mediated ARGs transfer, has been reported. Here, numerous ARGs and toxin-antitoxin genes have been found in pdif site pairs. However, the mechanisms underlying this apparent genetic mobility is currently not understood, and the studies relating to pdif-mediated ARGs transfer onto most bacterial genera are lacking. We developed the web server pdifFinder based on an algorithm called PdifSM that allows the prediction of diverse pdif-ARGs modules in bacterial genomes. Using test set consisting of almost 32 thousand plasmids from 717 species, PdifSM identified 481 plasmids from various bacteria containing pdif sites with ARGs. We found 28-bp-long elements from different genera with clear base preferences. The data we obtained indicate that XerCD-dif site-specific recombination mechanism may have evolutionary adapted to facilitate the pdif-mediated ARGs transfer. Through multiple sequence alignment and evolutionary analyses of duplicated pdif-ARGs modules, we discovered that pdif sites allow an interspecies transfer of ARGs but also across different genera. Mutations in pdif sites generate diverse arrays of modules which mediate multidrug-resistance, as these contain variable numbers of diverse ARGs, insertion sequences and other functional genes. The identification of pdif-ARGs modules and studies focused on the mechanism of ARGs co-transfer will help us to understand and possibly allow controlling the spread of MDR bacteria in clinical settings. The pdifFinder code, standalone software package and description with tutorials are available at https://github.com/mjshao06/pdifFinder.202336470841
466530.9976A comprehensive survey of integron-associated genes present in metagenomes. BACKGROUND: Integrons are genomic elements that mediate horizontal gene transfer by inserting and removing genetic material using site-specific recombination. Integrons are commonly found in bacterial genomes, where they maintain a large and diverse set of genes that plays an important role in adaptation and evolution. Previous studies have started to characterize the wide range of biological functions present in integrons. However, the efforts have so far mainly been limited to genomes from cultivable bacteria and amplicons generated by PCR, thus targeting only a small part of the total integron diversity. Metagenomic data, generated by direct sequencing of environmental and clinical samples, provides a more holistic and unbiased analysis of integron-associated genes. However, the fragmented nature of metagenomic data has previously made such analysis highly challenging. RESULTS: Here, we present a systematic survey of integron-associated genes in metagenomic data. The analysis was based on a newly developed computational method where integron-associated genes were identified by detecting their associated recombination sites. By processing contiguous sequences assembled from more than 10 terabases of metagenomic data, we were able to identify 13,397 unique integron-associated genes. Metagenomes from marine microbial communities had the highest occurrence of integron-associated genes with levels more than 100-fold higher than in the human microbiome. The identified genes had a large functional diversity spanning over several functional classes. Genes associated with defense mechanisms and mobility facilitators were most overrepresented and more than five times as common in integrons compared to other bacterial genes. As many as two thirds of the genes were found to encode proteins of unknown function. Less than 1% of the genes were associated with antibiotic resistance, of which several were novel, previously undescribed, resistance gene variants. CONCLUSIONS: Our results highlight the large functional diversity maintained by integrons present in unculturable bacteria and significantly expands the number of described integron-associated genes.202032689930
996340.9975Hitherto-Unnoticed Self-Transmissible Plasmids Widely Distributed among Different Environments in Japan. Various conjugative plasmids were obtained by exogenous plasmid capture, biparental mating, and/or triparental mating methods from different environmental samples in Japan. Based on phylogenetic analyses of their whole-nucleotide sequences, new IncP/P-1 plasmids that could be classified into novel subgroups were obtained. Mini-replicons of the plasmids were constructed, and each of them was incompatible with at least one of the IncP/P-1 plasmids, although they showed diverse iteron sequences in their oriV regions. There were two large clades of IncP/P-1 plasmids, clade I and II. Plasmids in clade I and II included antibiotic resistance genes. Notably, nucleotide compositions of newly found plasmids exhibited different tendencies compared with those of the previously well-studied IncP/P-1 plasmids. Indeed, the host range of plasmids of clade II was different from that of clade I. Although few PromA plasmids have been reported, the number of plasmids belonging to PromAβ, and -γ subgroups detected in this study was close to that of IncP/P-1 plasmids. The host ranges of PromAγ and PromAδ plasmids were broad and transferred to different and distinct classes of Proteobacteria. Interestingly, PromA plasmids and many IncP/P-1 plasmids do not carry any accessory genes. These findings indicate the presence of "hitherto-unnoticed" conjugative plasmids, including IncP/P-1 or PromA derivative ones in nature. These plasmids would have important roles in the exchange of various genes, including antibiotic resistance genes, among different bacteria in nature. IMPORTANCE Plasmids are known to spread among different bacteria. However, which plasmids spread among environmental samples and in which environments they are present is still poorly understood. This study showed that unidentified conjugative plasmids were present in various environments. Different novel IncP/P-1 plasmids were found, whose host ranges were different from those of known plasmids, showing wide diversity of IncP/P-1 plasmids. PromA plasmids, exhibiting a broad host range, were diversified into several subgroups and widely distributed in varied environments. These findings are important for understanding how bacteria naturally exchange their genes, including antibiotic resistance genes, a growing threat to human health worldwide.202236069618
964550.9975Horizontal Gene Transfers in prokaryotes show differential preferences for metabolic and translational genes. BACKGROUND: Horizontal gene transfer (HGT) is an important process, which contributes in bacterial pathogenesis and drug resistance. A number of methods have been proposed for detection of horizontal gene transfer. One successful approach to the detection of HGT events is due to Novichkov et al. (J. Bacteriology 186, 6575-85), who rely on comparing phylogenetic distances within a gene family with genomic distances of the source organisms. Building on their approach, we introduce outlier detection in the correlation between those two sets of distances. This approach is designed to detect horizontal transfers of core set of genes present in many bacteria. The principle behind method allows detection of xenologous gene displacements as well as acquisition of novel genes. RESULTS: Simulations indicated that our method performs better than Novichkov et al's original approach. The approach very efficiently identified HGT between distantly related bacteria and also a limited number of gene transfers between closely related bacteria. In combination with sequence similarity and likelihood tests, it yields a measure robust enough to derive a set of 171 genes deemed likely to have been horizontally transferred. Further analysis of these 171 established horizontal transfer events gave interesting insights in the direction of transfer. CONCLUSION: The majority of transfers between archaea and bacteria have occurred in the direction from bacteria to archaea rather than the other way round. Genes transferred between the archaea and bacteria are mostly metabolic genes. On the other hand, genes transferred within the bacterial phyla are mainly involved in translation.200919134215
964660.9975Effect of the environment on horizontal gene transfer between bacteria and archaea. BACKGROUND: Horizontal gene transfer, the transfer and incorporation of genetic material between different species of organisms, has an important but poorly quantified role in the adaptation of microbes to their environment. Previous work has shown that genome size and the number of horizontally transferred genes are strongly correlated. Here we consider how genome size confuses the quantification of horizontal gene transfer because the number of genes an organism accumulates over time depends on its evolutionary history and ecological context (e.g., the nutrient regime for which it is adapted). RESULTS: We investigated horizontal gene transfer between archaea and bacteria by first counting reciprocal BLAST hits among 448 bacterial and 57 archaeal genomes to find shared genes. Then we used the DarkHorse algorithm, a probability-based, lineage-weighted method (Podell & Gaasterland, 2007), to identify potential horizontally transferred genes among these shared genes. By removing the effect of genome size in the bacteria, we have identified bacteria with unusually large numbers of shared genes with archaea for their genome size. Interestingly, archaea and bacteria that live in anaerobic and/or high temperature conditions are more likely to share unusually large numbers of genes. However, high salt was not found to significantly affect the numbers of shared genes. Numbers of shared (genome size-corrected, reciprocal BLAST hits) and transferred genes (identified by DarkHorse) were strongly correlated. Thus archaea and bacteria that live in anaerobic and/or high temperature conditions are more likely to share horizontally transferred genes. These horizontally transferred genes are over-represented by genes involved in energy conversion as well as the transport and metabolism of inorganic ions and amino acids. CONCLUSIONS: Anaerobic and thermophilic bacteria share unusually large numbers of genes with archaea. This is mainly due to horizontal gene transfer of genes from the archaea to the bacteria. In general, these transfers are from archaea that live in similar oxygen and temperature conditions as the bacteria that receive the genes. Potential hotspots of horizontal gene transfer between archaea and bacteria include hot springs, marine sediments, and oil wells. Cold spots for horizontal transfer included dilute, aerobic, mesophilic environments such as marine and freshwater surface waters.201728975058
466670.9975Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes. Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses.IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.201930808697
965080.9975Plasmid-Encoded Traits Vary across Environments. Plasmids are key mobile genetic elements in bacterial evolution and ecology as they allow the rapid adaptation of bacteria under selective environmental changes. However, the genetic information associated with plasmids is usually considered separately from information about their environmental origin. To broadly understand what kinds of traits may become mobilized by plasmids in different environments, we analyzed the properties and accessory traits of 9,725 unique plasmid sequences from a publicly available database with known bacterial hosts and isolation sources. Although most plasmid research focuses on resistance traits, such genes made up <1% of the total genetic information carried by plasmids. Similar to traits encoded on the bacterial chromosome, plasmid accessory trait compositions (including general Clusters of Orthologous Genes [COG] functions, resistance genes, and carbon and nitrogen genes) varied across seven broadly defined environment types (human, animal, wastewater, plant, soil, marine, and freshwater). Despite their potential for horizontal gene transfer, plasmid traits strongly varied with their host's taxonomic assignment. However, the trait differences across environments of broad COG categories could not be entirely explained by plasmid host taxonomy, suggesting that environmental selection acts on the plasmid traits themselves. Finally, some plasmid traits and environments (e.g., resistance genes in human-related environments) were more often associated with mobilizable plasmids (those having at least one detected relaxase) than others. Overall, these findings underscore the high level of diversity of traits encoded by plasmids and provide a baseline to investigate the potential of plasmids to serve as reservoirs of adaptive traits for microbial communities. IMPORTANCE Plasmids are well known for their role in the transmission of antibiotic resistance-conferring genes. Beyond human and clinical settings, however, they disseminate many other types of genes, including those that contribute to microbially driven ecosystem processes. In this study, we identified the distribution of traits genetically encoded by plasmids isolated from seven broadly categorized environments. We find that plasmid trait content varied with both bacterial host taxonomy and environment and that, on average, half of the plasmids were potentially mobilizable. As anthropogenic activities impact ecosystems and the climate, investigating and identifying the mechanisms of how microbial communities can adapt will be imperative for predicting the impacts on ecosystem functioning.202336629415
378290.9975CRISPR spacers acquired from plasmids primarily target backbone genes, making them valuable for predicting potential hosts and host range. In recent years, there has been a surge in metagenomic studies focused on identifying plasmids in environmental samples. Although these studies have unearthed numerous novel plasmids, enriching our understanding of their environmental roles, a significant gap remains: the scarcity of information regarding the bacterial hosts of these newly discovered plasmids. Furthermore, even when plasmids are identified within bacterial isolates, the reported host is typically limited to the original isolate, with no insights into alternative hosts or the plasmid's potential host range. Given that plasmids depend on hosts for their existence, investigating plasmids without the knowledge of potential hosts offers only a partial perspective. This study introduces a method for identifying potential hosts and host ranges for plasmids through alignment with CRISPR spacers. To validate the method, we compared the PLSDB plasmids database with the CRISPR spacers database, yielding host predictions for 46% of the plasmids. When compared with reported hosts, our predictions achieved 84% concordance at the family level and 99% concordance at the phylum level. Moreover, the method frequently identified multiple potential hosts for a plasmid, thereby enabling predictions of alternative hosts and the host range. Notably, we found that CRISPR spacers predominantly target plasmid backbone genes while sparing functional genes, such as those linked to antibiotic resistance, aligning with our hypothesis that CRISPR spacers are acquired from plasmid-specific regions rather than insertion elements from diverse sources. Finally, we illustrate the network of connections among different bacterial taxa through plasmids, revealing potential pathways for horizontal gene transfer.IMPORTANCEPlasmids are notorious for their role in distributing antibiotic resistance genes, but they may also carry and distribute other environmentally important genes. Since plasmids are not free-living entities and rely on host bacteria for survival and propagation, predicting their hosts is essential. This study presents a method for predicting potential hosts for plasmids and offers insights into the potential paths for spreading functional genes between different bacteria. Understanding plasmid-host relationships is crucial for comprehending the ecological and clinical impact of plasmids and implications for various biological processes.202439508585
9866100.9975Integrons in Xanthomonas: a source of species genome diversity. Integrons are best known for assembling antibiotic resistance genes in clinical bacteria. They capture genes by using integrase-mediated site-specific recombination of mobile gene cassettes. Integrons also occur in the chromosomes of many bacteria, notably beta- and gamma-Proteobacteria. In a survey of Xanthomonas, integrons were found in all 32 strains representing 12 pathovars of two species. Their chromosomal location was downstream from the acid dehydratase gene, ilvD, suggesting that an integron was present at this site in the ancestral xanthomonad. There was considerable sequence and structural diversity among the extant integrons. The majority of integrase genes were predicted to be inactivated by frameshifts, stop codons, or large deletions, suggesting that the associated gene cassettes can no longer be mobilized. In support, groups of strains with the same deletions or stop codons/frameshifts in their integrase gene usually contained identical arrays of gene cassettes. In general, strains within individual pathovars had identical cassettes, and these exhibited no similarity to cassettes detected in other pathovars. The variety and characteristics of contemporary gene cassettes suggests that the ancestral integron had access to a diverse pool of these mobile elements, and that their genes originated outside the Xanthomonas genome. Subsequent inactivation of the integrase gene in particular lineages has largely fixed the gene cassette arrays in particular pathovars during their differentiation and specialization into ecological niches. The acquisition of diverse gene cassettes by different lineages within Xanthomonas has contributed to the species-genome diversity of the genus. The role of gene cassettes in survival on plant surfaces is currently unknown.200515755815
9867110.9975Mosaic plasmids are abundant and unevenly distributed across prokaryotic taxa. Mosaic plasmids, plasmids composed of genetic elements from distinct sources, are associated with the spread of antibiotic resistance genes. Transposons are considered the primary mechanism for mosaic plasmid formation, though other mechanisms have been observed in specific instances. The frequency with which mosaic plasmids have been described suggests they may play an important role in plasmid population dynamics. Our survey of the confirmed plasmid sequences available from complete and draft genomes in the RefSeq database shows that 46% of them fit a strict definition of mosaic. Mosaic plasmids are also not evenly distributed over the taxa represented in the database. Plasmids from some genera, including Piscirickettsia and Yersinia, are almost all mosaic, while plasmids from other genera, including Borrelia, are rarely mosaic. While some mosaic plasmids share identical regions with hundreds of others, the median mosaic plasmid only shares with 8 other plasmids. When considering only plasmids from finished genomes (51.6% of the total), mosaic plasmids have significantly higher proportions of transposase and antibiotic resistance genes. Conversely, only 56.6% of mosaic fragments (DNA fragments shared between mosaic plasmids) contain a recognizable transposase gene, and only 1.2% of mosaic fragments are flanked by inverted repeats. Mosaic fragments associated with the IS26 transposase gene are 3.8-fold more abundant than any other sequence shared between mosaic plasmids in the database, though this is at least partly due to overrepresentation of Enterobacteriaceae plasmids. Mosaic plasmids are a complicated trait of some plasmid populations, only partly explained by transposition. Though antibiotic resistance genes led to the identification of many mosaic plasmids, mosaic plasmids are a broad phenomenon encompassing many more traits than just antibiotic resistance. Further research will be required to determine the influence of ecology, host repair mechanisms, conjugation, and plasmid host range on the formation and influence of mosaic plasmids. AUTHOR SUMMARY: Plasmids are extrachromosomal genetic entities that are found in many prokaryotes. They serve as flexible storage for genes, and individual cells can make substantial changes to their characteristics by acquiring, losing, or modifying a plasmid. In some pathogenic bacteria, such as Escherichia coli, antibiotic resistance genes are known to spread primarily on plasmids. By analyzing a database of 8592 plasmid sequences we determined that many of these plasmids have exchanged genes with each other, becoming mosaics of genes from different sources. We next separated these plasmids into groups based on the organism they were isolated from and found that different groups had different fractions of mosaic plasmids. This result was unexpected and suggests that the mechanisms and selective pressures causing mosaic plasmids do not occur evenly over all species. It also suggests that plasmids may provide different levels of potential variation to different species. This work uncovers a previously unrecognized pattern in plasmids across prokaryotes, that could lead to new insights into the evolutionary role that plasmids play.201930797764
4355120.9975An expectation-maximization algorithm for estimating proportions of deletions among bacterial populations with application to study antibiotic resistance gene transfer in Enterococcus faecalis. The emergence of antibiotic resistance in bacteria limits the availability of antibiotic choices for treatment and infection control, thereby representing a major threat to human health. The de novo mutation of bacterial genomes is an essential mechanism by which bacteria acquire antibiotic resistance. Previously, deletion mutations within bacterial immune systems, ranging from dozens to thousands of base pairs (bps) in length, have been associated with the spread of antibiotic resistance. Most current methods for evaluating genomic structural variations (SVs) have concentrated on detecting them, rather than estimating the proportions of populations that carry distinct SVs. A better understanding of the distribution of mutations and subpopulations dynamics in bacterial populations is needed to appreciate antibiotic resistance evolution and movement of resistance genes through populations. Here, we propose a statistical model to estimate the proportions of genomic deletions in a mixed population based on Expectation-Maximization (EM) algorithms and next-generation sequencing (NGS) data. The method integrates both insert size and split-read mapping information to iteratively update estimated distributions. The proposed method was evaluated with three simulations that demonstrated the production of accurate estimations. The proposed method was then applied to investigate the horizontal transfers of antibiotic resistance genes in concert with changes in the CRISPR-Cas system of E. faecalis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42995-022-00144-z.202336744155
3779130.9974The transfer of antibiotic resistance genes between evolutionarily distant bacteria. Infections from antibiotic-resistant bacteria threaten human health globally. Resistance is often caused by mobile antibiotic resistance genes (ARGs) shared horizontally between bacterial genomes. Many ARGs originate from environmental and commensal bacteria and are transferred between divergent bacterial hosts before they reach pathogens. This process remains, however, poorly understood, which complicates the development of countermeasures that reduce the spread of ARGs. In this study, we aimed to systematically analyze the ARGs transferred between the most evolutionarily distant bacteria, defined here based on their phylum. We implemented an algorithm that identified inter-phylum transfers (IPTs) by combining ARG-specific phylogenetic trees with the taxonomy of the bacterial hosts. From the analysis of almost 1 million ARGs identified in >400,000 bacterial genomes, we identified 661 IPTs, which included transfers between all major bacterial phyla. The frequency of IPTs varies substantially between ARG classes and was highest for the aminoglycoside resistance gene AAC(3), while the levels for beta-lactamases were generally lower. ARGs involved in IPTs also differed between phyla, where, for example, tetracycline ARGs were commonly transferred between Firmicutes and Proteobacteria, but rarely between Actinobacteria and Proteobacteria. The results, furthermore, show that conjugative systems are seldom shared between bacterial phyla, suggesting that other mechanisms drive the dissemination of ARGs between divergent hosts. We also show that bacterial genomes involved in IPTs of ARGs are either over- or underrepresented in specific environments. These IPTs were also found to be more recent compared to transfers associated with bacteria isolated from water, soil, and sediment. While macrolide and tetracycline ARGs involved in IPTs almost always were >95% identical between phyla, corresponding β-lactamases showed a median identity of <60%. We conclude that inter-phylum transfer is recurrent, and our results offer new insights into how ARGs are disseminated between evolutionarily distant bacteria. IMPORTANCE: Antibiotic-resistant infections pose a growing threat to global health. This study reveals how genes conferring antibiotic resistance can move between bacteria that belong to different phyla lineages previously thought to be too evolutionarily distant for frequent gene exchange. By analyzing nearly 1 million resistance genes from over 400,000 bacterial genomes, the researchers uncovered hundreds of inter-phylum transfer events, exposing surprising patterns in how different classes of resistance genes spread. The findings highlight that conjugative systems are less common than expected in cross-phyla transfers and suggest that alternative mechanisms may play key roles. This new understanding of how resistance genes leap between vastly different bacterial groups can inform strategies to slow the emergence of drug-resistant infections, aiding in the development of more effective public health interventions.202540459279
4661140.9974Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities. Integrons are microbial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the performance of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class one integrons are collecting and concentrating resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.202235298369
9860150.9974Insights and inferences about integron evolution from genomic data. BACKGROUND: Integrons are mechanisms that facilitate horizontal gene transfer, allowing bacteria to integrate and express foreign DNA. These are important in the exchange of antibiotic resistance determinants, but can also transfer a diverse suite of genes unrelated to pathogenicity. Here, we provide a systematic analysis of the distribution and diversity of integron intI genes and integron-containing bacteria. RESULTS: We found integrons in 103 different pathogenic and non-pathogenic bacteria, in six major phyla. Integrons were widely scattered, and their presence was not confined to specific clades within bacterial orders. Nearly 1/3 of the intI genes that we identified were pseudogenes, containing either an internal stop codon or a frameshift mutation that would render the protein product non-functional. Additionally, 20% of bacteria contained more than one integrase gene. dN/dS ratios revealed mutational hotspots in clades of Vibrio and Shewanella intI genes. Finally, we characterized the gene cassettes associated with integrons in Methylobacillus flagellatus KT and Dechloromonas aromatica RCB, and found a heavy metal efflux gene as well as genes involved in protein folding and stability. CONCLUSION: Our analysis suggests that the present distribution of integrons is due to multiple losses and gene transfer events. While, in some cases, the ability to integrate and excise foreign DNA may be selectively advantageous, the gain, loss, or rearrangment of gene cassettes could also be deleterious, selecting against functional integrases. Thus, such a high fraction of pseudogenes may suggest that the selective impact of integrons on genomes is variable, oscillating between beneficial and deleterious, possibly depending on environmental conditions.200818513439
3773160.9974Understanding the rapid spread of antimicrobial resistance genes mediated by IS26. Insertion sequences (ISs) promote the transmission of antimicrobial resistance genes (ARGs) across bacterial populations. However, their contributions and dynamics during the transmission of resistance remain unclear. In this study, we selected IS26 as a representative transposable element to decipher the relationship between ISs and ARGs and to investigate their transfer features and transmission trends. We retrieved 2656  translocatable  IS 26 -bounded  units with  ARGs (tIS26-bUs-ARGs) in complete bacterial genomes from the NCBI RefSeq database. In total, 124 ARGs spanning 12 classes of antibiotics were detected, and the average contribution rate of IS26 to these genes was 41.2%. We found that  IS 26 -bounded  units (IS26-bUs) mediated extensive ARG dissemination within the bacteria of the Gammaproteobacteria class, showing strong transfer potential between strains, species, and even phyla. The IS26-bUs expanded in bacterial populations over time, and their temporal expansion trend was significantly correlated with antibiotic usage. This wide dissemination could be due to the nonspecific target site preference of IS26. Finally, we experimentally confirmed that the introduction of a single copy of IS26 could lead to the formation of a composite transposon mediating the transmission of "passenger" genes. These observations extend our knowledge of the IS26 and provide new insights into the mediating role of ISs in the dissemination of antibiotic resistance.202438827508
3778170.9974ggMOB: Elucidation of genomic conjugative features and associated cargo genes across bacterial genera using genus-genus mobilization networks. Horizontal gene transfer mediated by conjugation is considered an important evolutionary mechanism of bacteria. It allows organisms to quickly evolve new phenotypic properties including antimicrobial resistance (AMR) and virulence. The frequency of conjugation-mediated cargo gene exchange has not yet been comprehensively studied within and between bacterial taxa. We developed a frequency-based network of genus-genus conjugation features and candidate cargo genes from whole-genome sequence data of over 180,000 bacterial genomes, representing 1,345 genera. Using our method, which we refer to as ggMOB, we revealed that over half of the bacterial genomes contained one or more known conjugation features that matched exactly to at least one other genome. Moreover, the proportion of genomes containing these conjugation features varied substantially by genus and conjugation feature. These results and the genus-level network structure can be viewed interactively in the ggMOB interface, which allows for user-defined filtering of conjugation features and candidate cargo genes. Using the network data, we observed that the ratio of AMR gene representation in conjugative versus non-conjugative genomes exceeded 5:1, confirming that conjugation is a critical force for AMR spread across genera. Finally, we demonstrated that clustering genomes by conjugation profile sometimes correlated well with classical phylogenetic structuring; but that in some cases the clustering was highly discordant, suggesting that the importance of the accessory genome in driving bacterial evolution may be highly variable across both time and taxonomy. These results can advance scientific understanding of bacterial evolution, and can be used as a starting point for probing genus-genus gene exchange within complex microbial communities that include unculturable bacteria. ggMOB is publicly available under the GNU licence at https://ruiz-hci-lab.github.io/ggMOB/.202236568361
9848180.9974Cargo Genes of Tn7-Like Transposons Comprise an Enormous Diversity of Defense Systems, Mobile Genetic Elements, and Antibiotic Resistance Genes. Transposition is a major mechanism of horizontal gene mobility in prokaryotes. However, exploration of the genes mobilized by transposons (cargo) is hampered by the difficulty in delineating integrated transposons from their surrounding genetic context. Here, we present a computational approach that allowed us to identify the boundaries of 6,549 Tn7-like transposons. We found that 96% of these transposons carry at least one cargo gene. Delineation of distinct communities in a gene-sharing network demonstrates how transposons function as a conduit of genes between phylogenetically distant hosts. Comparative analysis of the cargo genes reveals significant enrichment of mobile genetic elements (MGEs) nested within Tn7-like transposons, such as insertion sequences and toxin-antitoxin modules, and of genes involved in recombination, anti-MGE defense, and antibiotic resistance. More unexpectedly, cargo also includes genes encoding central carbon metabolism enzymes. Twenty-two Tn7-like transposons carry both an anti-MGE defense system and antibiotic resistance genes, illustrating how bacteria can overcome these combined pressures upon acquisition of a single transposon. This work substantially expands the distribution of Tn7-like transposons, defines their evolutionary relationships, and provides a large-scale functional classification of prokaryotic genes mobilized by transposition. IMPORTANCE Transposons are major vehicles of horizontal gene transfer that, in addition to genes directly involved in transposition, carry cargo genes. However, characterization of these genes is hampered by the difficulty of identification of transposon boundaries. We developed a computational approach for detecting transposon ends and applied it to perform a comprehensive census of the cargo genes of Tn7-like transposons, a large class of bacterial mobile genetic elements (MGE), many of which employ a unique, CRISPR-mediated mechanism of site-specific transposition. The cargo genes encompass a striking diversity of MGE, defense, and antibiotic resistance systems. Unexpectedly, we also identified cargo genes encoding metabolic enzymes. Thus, Tn7-like transposons mobilize a vast repertoire of genes that can have multiple effects on the host bacteria.202134872347
9960190.9974Integrons, transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies. Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.202337655671