Horizontal gene transfer in plant microbiomes: integrons as hotspots for cross-species gene exchange.

Plant microbiomes play important roles in plant health and fitness. Bacterial horizontal gene transfer (HGT) can influence plant health outcomes, driving the spread of both plant growth-promoting and phytopathogenic traits. However, community dynamics, including the range of genetic elements and bacteria involved in this process are still poorly understood. Integrons are genetic elements recently shown to be abundant in plant microbiomes, and are associated with HGT across broad phylogenetic boundaries. They facilitate the spread of gene cassettes, small mobile elements that collectively confer a diverse suite of adaptive functions. Here, we analysed 5,565 plant-associated bacterial genomes to investigate the prevalence and functional diversity of integrons in this niche. We found that integrons are particularly abundant in the genomes of Pseudomonadales, Burkholderiales, and Xanthomonadales. In total, we detected nearly 9,000 gene cassettes, and found that many could be involved in plant growth promotion or phytopathogenicity, suggesting that integrons might play a role in bacterial mutualistic or pathogenic lifestyles. The rhizosphere was enriched in cassettes involved in the transport and metabolism of diverse substrates, suggesting that they may aid in adaptation to this environment, which is rich in root exudates. We also found that integrons facilitate cross-species HGT, which is particularly enhanced in the phyllosphere. This finding may provide an ideal opportunity to promote plant growth by fostering the spread of genes cassettes relevant to leaf health. Together, our findings suggest that integrons are important elements in plant microbiomes that drive HGT, and have the potential to facilitate plant host adaptation.

Exploring virus-host-environment interactions in a chemotrophic-based underground estuary.

BACKGROUND: Viruses play important roles in modulating microbial communities and influencing global biogeochemistry. There is now growing interest in characterising their ecological roles across diverse biomes. However, little is known about viral ecology in low-nutrient, chemotrophic-based environments. In such ecosystems, virus-driven manipulation of nutrient cycles might have profound impacts across trophic levels. In particular, anchialine environments, which are low-energy underground estuaries sustained by chemotrophic processes, represent ideal model systems to study novel virus-host-environment interactions. RESULTS: Here, we employ metagenomic sequencing to investigate the viral community in Bundera Sinkhole, an anchialine ecosystem rich in endemic species supported by microbial chemosynthesis. We find that the viruses are highly novel, with less than 2% representing described viruses, and are hugely abundant, making up as much as 12% of microbial intracellular DNA. These highly abundant viruses largely infect important prokaryotic taxa that drive key metabolic processes in the sinkhole. Further, the abundance of viral auxiliary metabolic genes (AMGs) involved in nucleotide and protein synthesis was strongly correlated with declines in environmental phosphate and sulphate concentrations. These AMGs encoded key enzymes needed to produce sulphur-containing amino acids, and phosphorus metabolic enzymes involved in purine and pyrimidine nucleotide synthesis. We hypothesise that this correlation is either due to selection of these AMGs under low phosphate and sulphate concentrations, highlighting the dynamic interactions between viruses, their hosts, and the environment; or, that these AMGs are driving increased viral nucleotide and protein synthesis via manipulation of host phosphorus and sulphur metabolism, consequently driving nutrient depletion in the surrounding water. CONCLUSION: This study represents the first metagenomic investigation of viruses in anchialine ecosystems, and provides new hypotheses and insights into virus-host-environment interactions in such 'dark', low-energy environments. This is particularly important since anchialine ecosystems are characterised by diverse endemic species, both in their microbial and faunal assemblages, which are primarily supported by microbial chemosynthesis. Thus, virus-host-environment interactions could have profound effects cascading through all trophic levels.

Dissecting molecular evolution of class 1 integron gene cassettes and identifying their bacterial hosts in suburban creeks via epicPCR.

OBJECTIVES: Our study aimed to sequence class 1 integrons in uncultured environmental bacterial cells in freshwater from suburban creeks and uncover the taxonomy of their bacterial hosts. We also aimed to characterize integron gene cassettes with altered DNA sequences relative to those from databases or literature and identify key signatures of their molecular evolution. METHODS: We applied a single-cell fusion PCR-based technique-emulsion, paired isolation and concatenation PCR (epicPCR)-to link class 1 integron gene cassette arrays to the phylogenetic markers of their bacterial hosts. The levels of streptomycin resistance conferred by the WT and altered aadA5 and aadA11 gene cassettes that encode aminoglycoside (3″) adenylyltransferases were experimentally quantified in an Escherichia coli host. RESULTS: Class 1 integron gene cassette arrays were detected in Alphaproteobacteria and Gammaproteobacteria hosts. A subset of three gene cassettes displayed signatures of molecular evolution, namely the gain of a regulatory 5'-untranslated region (5'-UTR), the loss of attC recombination sites between adjacent gene cassettes, and the invasion of a 5'-UTR by an IS element. Notably, our experimental testing of a novel variant of the aadA11 gene cassette demonstrated that gaining the observed 5'-UTR contributed to a 3-fold increase in the MIC of streptomycin relative to the ancestral reference gene cassette in E. coli. CONCLUSIONS: Dissecting the observed signatures of molecular evolution of class 1 integrons allowed us to explain their effects on antibiotic resistance phenotypes, while identifying their bacterial hosts enabled us to make better inferences on the likely origins of novel gene cassettes and IS that invade known gene cassettes.

Functional enrichment of integrons: Facilitators of antimicrobial resistance and niche adaptation.

Integrons are genetic elements, found among diverse bacteria and archaea, that capture and rearrange gene cassettes to rapidly generate genetic diversity and drive adaptation. Despite their broad taxonomic and geographic prevalence, and their role in microbial adaptation, the functions of gene cassettes remain poorly characterized. Here, using a combination of bioinformatic and experimental analyses, we examined the functional diversity of gene cassettes from different environments. We find that cassettes encode diverse antimicrobial resistance (AMR) determinants, including those conferring resistance to antibiotics currently in the developmental pipeline. Further, we find a subset of cassette functions is universally enriched relative to their broader metagenomes. These are largely involved in (a)biotic interactions, including AMR, phage defense, virulence, biodegradation, and stress tolerance. The remainder of functions are sample-specific, suggesting that they confer localised functions relevant to their microenvironment. Together, they comprise functional profiles different from bulk metagenomes, representing niche-adaptive components of the prokaryotic pangenome.

Stratified microbial communities in Australia's only anchialine cave are taxonomically novel and drive chemotrophic energy production via coupled nitrogen-sulphur cycling.

BACKGROUND: Anchialine environments, in which oceanic water mixes with freshwater in coastal aquifers, are characterised by stratified water columns with complex physicochemical profiles. These environments, also known as subterranean estuaries, support an abundance of endemic macro and microorganisms. There is now growing interest in characterising the metabolisms of anchialine microbial communities, which is essential for understanding how complex ecosystems are supported in extreme environments, and assessing their vulnerability to environmental change. However, the diversity of metabolic strategies that are utilised in anchialine ecosystems remains poorly understood. RESULTS: Here, we employ shotgun metagenomics to elucidate the key microorganisms and their dominant metabolisms along a physicochemical profile in Bundera Sinkhole, the only known continental subterranean estuary in the Southern Hemisphere. Genome-resolved metagenomics suggests that the communities are largely represented by novel taxonomic lineages, with 75% of metagenome-assembled genomes assigned to entirely new or uncharacterised families. These diverse and novel taxa displayed depth-dependent metabolisms, reflecting distinct phases along dissolved oxygen and salinity gradients. In particular, the communities appear to drive nutrient feedback loops involving nitrification, nitrate ammonification, and sulphate cycling. Genomic analysis of the most highly abundant members in this system suggests that an important source of chemotrophic energy is generated via the metabolic coupling of nitrogen and sulphur cycling. CONCLUSION: These findings substantially contribute to our understanding of the novel and specialised microbial communities in anchialine ecosystems, and highlight key chemosynthetic pathways that appear to be important in these energy-limited environments. Such knowledge is essential for the conservation of anchialine ecosystems, and sheds light on adaptive processes in extreme environments. Video Abstract.

Sub-inhibitory gentamicin pollution induces gentamicin resistance gene integration in class 1 integrons in the environment.

Antibiotics at sub-inhibitory concentrations are often found in the environment. Here they could impose selective pressure on bacteria, leading to the selection and dissemination of antibiotic resistance, despite being under the inhibitory threshold. The goal of this study was to evaluate the effects of sub-inhibitory concentrations of gentamicin on environmental class 1 integron cassettes in natural river microbial communities. Gentamicin at sub-inhibitory concentrations promoted the integration and selection of gentamicin resistance genes (GmRG) in class 1 integrons after only a one-day exposure. Therefore, sub-inhibitory concentrations of gentamicin induced integron rearrangements, increasing the mobilization potential of gentamicin resistance genes and potentially increasing their dissemination in the environment. This study demonstrates the effects of antibiotics at sub-inhibitory concentrations in the environment and supports concerns about antibiotics as emerging pollutants.

Plastic leachate exposure drives antibiotic resistance and virulence in marine bacterial communities.

Plastic pollution is a serious global problem, with more than 12 million tonnes of plastic waste entering the oceans every year. Plastic debris can have considerable impacts on microbial community structure and functions in marine environments, and has been associated with an enrichment in pathogenic bacteria and antimicrobial resistance (AMR) genes. However, our understanding of these impacts is largely restricted to microbial assemblages on plastic surfaces. It is therefore unclear whether these effects are driven by the surface properties of plastics, providing an additional niche for certain microbes residing in biofilms, and/or chemicals leached from plastics, the effects of which could extend to surrounding planktonic bacteria. Here, we examine the effects of polyvinyl chloride (PVC) plastic leachate exposure on the relative abundance of genes associated with bacterial pathogenicity and AMR within a seawater microcosm community. We show that PVC leachate, in the absence of plastic surfaces, drives an enrichment in AMR and virulence genes. In particular, leachate exposure significantly enriches AMR genes that confer multidrug, aminoglycoside and peptide antibiotic resistance. Additionally, enrichment of genes involved in the extracellular secretion of virulence proteins was observed among pathogens of marine organisms. This study provides the first evidence that chemicals leached from plastic particles alone can enrich genes related to microbial pathogenesis within a bacterial community, expanding our knowledge of the environmental impacts of plastic pollution with potential consequences for human and ecosystem health.

Uncovering Bacterial Hosts of Class 1 Integrons in an Urban Coastal Aquatic Environment with a Single-Cell Fusion-Polymerase Chain Reaction Technology.

Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic materials across taxa. Class 1 integrons are genetic elements that correlate strongly with anthropogenic pollution and contribute to the spread of antimicrobial resistance (AMR) genes via HGT. Despite their significance to human health, there is a shortage of robust, culture-free surveillance technologies for identifying uncultivated environmental taxa that harbor class 1 integrons. We developed a modified version of epicPCR (emulsion, paired isolation, and concatenation polymerase chain reaction (PCR)) that links class 1 integrons amplified from single bacterial cells to taxonomic markers from the same cells in emulsified aqueous droplets. Using this single-cell genomic approach and Nanopore sequencing, we successfully assigned class 1 integron gene cassette arrays containing mostly AMR genes to their hosts in coastal water samples that were affected by pollution. Our work presents the first application of epicPCR for targeting variable, multigene loci of interest. We also identified the Rhizobacter genus as novel hosts of class 1 integrons. These findings establish epicPCR as a powerful tool for linking taxa to class 1 integrons in environmental bacterial communities and offer the potential to direct mitigation efforts toward hotspots of class 1 integron-mediated dissemination of AMR.

Identification of integrons and gene cassette-associated recombination sites in bacteriophage genomes.

Bacteriophages are versatile mobile genetic elements that play key roles in driving the evolution of their bacterial hosts through horizontal gene transfer. Phages co-evolve with their bacterial hosts and have plastic genomes with extensive mosaicism. In this study, we present bioinformatic and experimental evidence that temperate and virulent (lytic) phages carry integrons, including integron-integrase genes, attC/attI recombination sites and gene cassettes. Integrons are normally found in Bacteria, where they capture, express and re-arrange mobile gene cassettes via integron-integrase activity. We demonstrate experimentally that a panel of attC sites carried in virulent phage can be recognized by the bacterial class 1 integron-integrase (IntI1) and then integrated into the paradigmatic attI1 recombination site using an attC x attI recombination assay. With an increasing number of phage genomes projected to become available, more phage-associated integrons and their components will likely be identified in the future. The discovery of integron components in bacteriophages establishes a new route for lateral transfer of these elements and their cargo genes between bacterial host cells.

Class 1 integron and related antimicrobial resistance gene dynamics along a complex freshwater system affected by different anthropogenic pressures.

The risk for human health posed by polluted aquatic environments, and especially those carrying antibiotic resistance genes (ARGs) of clinical interest, is still debated. This is because of our limited knowledge of the dynamics of antimicrobial resistance in the environment, the selection mechanisms underlying the spread of ARGs, and the ecological factors potentially favoring their return to humans. The Class 1 integron is one of the most effective platforms for the dissemination of ARGs. In this study we investigated a freshwater system consisting of a lake-river-lake continuum, determining the abundance of class 1 integrons and their associated ARGs by a modulated metagenomic approach. Bacterial abundance and community composition were used to identify the potential carriers of class 1 integrons and their associated ARGs over a period of six months. Class 1 integrons and their ARG cargoes were significantly more abundant in riverine sampling sites receiving treated wastewater. Further, class 1 integrons carried ARGs ranked at the highest risk for human health (e.g., catB genes), in particular, genes encoding resistance to aminoglycosides. Genera of potential pathogens, such as Pseudomonas and Escherichia-Shigella, were correlated with class 1 integrons. The lake-river-lake system demonstrated a clear relationship between the integrase gene of class 1 integrons (intI1) and anthropogenic impact, but also a strong environmental filtering that favored the elimination of intI1 once the human derived stressors were reduced. Overall, the results of this study underline the role class 1 integrons as proxy of anthropogenic pollution and suggest this genetic platform as an important driver of aminoglycoside resistance genes, including high risk ARGs, of potential concern for human health.

Discovery of integrons in Archaea: Platforms for cross-domain gene transfer.

Horizontal gene transfer between different domains of life is increasingly being recognized as an important evolutionary driver, with the potential to increase the pace of biochemical innovation and environmental adaptation. However, the mechanisms underlying the recruitment of exogenous genes from foreign domains are mostly unknown. Integrons are a family of genetic elements that facilitate this process within Bacteria. However, they have not been reported outside Bacteria, and thus their potential role in cross-domain gene transfer has not been investigated. Here, we discover that integrons are also present in 75 archaeal metagenome-assembled genomes from nine phyla, and are particularly enriched among Asgard archaea. Furthermore, we provide experimental evidence that integrons can facilitate the recruitment of archaeal genes by bacteria. Our findings establish a previously unknown mechanism of cross-domain gene transfer whereby bacteria can incorporate archaeal genes from their surrounding environment via integron activity. These findings have important implications for prokaryotic ecology and evolution.

Methods 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.

New perspectives on mobile genetic elements: a paradigm shift for managing the antibiotic resistance crisis.

Mobile genetic elements (MGEs) are primary facilitators in the global spread of antibiotic resistance. Here, we present novel ecological and evolutionary perspectives to understand and manage these elements: as selfish entities that exhibit biological individuality, as pollutants that replicate and as invasive species that thrive under human impact. Importantly, each viewpoint suggests new means to control their activity and spread. When seen as biological individuals, MGEs can be regarded as therapeutic targets in their own right. We highlight promising conjugation-inhibiting compounds that could be administered alongside antibiotic treatment. Viewed as pollutants, sewage treatment methods could be modified to efficiently remove antimicrobials and the resistance genes that they select. Finally, by recognizing the invasive characteristics of MGEs, we might apply strategies developed for the management of invasive species. These include environmental restoration to reduce antimicrobial selection, early detection to help inform appropriate antibiotic usage, and biocontrol strategies that target MGEs, constituting precision antimicrobials. These actions, which embody the One Health approach, target different characteristics of MGEs that are pertinent at the cellular, community, landscape and global levels. The strategies could act on multiple fronts and, together, might provide a more fruitful means to combat the global resistance crisis. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

The Natural History of Integrons.

Integrons were first identified because of their central role in assembling and disseminating antibiotic resistance genes in commensal and pathogenic bacteria. However, these clinically relevant integrons represent only a small proportion of integron diversity. Integrons are now known to be ancient genetic elements that are hotspots for genomic diversity, helping to generate adaptive phenotypes. This perspective examines the diversity, functions, and activities of integrons within both natural and clinical environments. We show how the fundamental properties of integrons exquisitely pre-adapted them to respond to the selection pressures imposed by the human use of antimicrobial compounds. We then follow the extraordinary increase in abundance of one class of integrons (class 1) that has resulted from its acquisition by multiple mobile genetic elements, and subsequent colonisation of diverse bacterial species, and a wide range of animal hosts. Consequently, this class of integrons has become a significant pollutant in its own right, to the extent that it can now be detected in most ecosystems. As human activities continue to drive environmental instability, integrons will likely continue to play key roles in bacterial adaptation in both natural and clinical settings. Understanding the ecological and evolutionary dynamics of integrons can help us predict and shape these outcomes that have direct relevance to human and ecosystem health.

Predicting the taxonomic and environmental sources of integron gene cassettes using structural and sequence homology of attC sites.

Integrons are bacterial genetic elements that can capture mobile gene cassettes. They are mostly known for their role in the spread of antibiotic resistance cassettes, contributing significantly to the global resistance crisis. These resistance cassettes likely originated from sedentary chromosomal integrons, having subsequently been acquired and disseminated by mobilised integrons. However, their taxonomic and environmental origins are unknown. Here, we use cassette recombination sites (attCs) to predict the origins of those resistance cassettes now spread by mobile integrons. We modelled the structure and sequence homology of 1,978 chromosomal attCs from 11 different taxa. Using these models, we show that at least 27% of resistance cassettes have attCs that are structurally conserved among one of three taxa (Xanthomonadales, Spirochaetes and Vibrionales). Indeed, we found some resistance cassettes still residing in sedentary chromosomal integrons of the predicted taxa. Further, we show that attCs cluster according to host environment rather than host phylogeny, allowing us to assign their likely environmental sources. For example, the majority of ß-lactamases and aminoglycoside acetyltransferases, the two most prevalent resistance cassettes, appear to have originated from marine environments. Together, our data represent the first evidence of the taxonomic and environmental origins of resistance cassettes spread by mobile integrons.

Erratum: Virome composition in marine fish revealed by meta-transcriptomics.

[This corrects the article DOI: 10.1093/ve/veab005.].

Virome composition in marine fish revealed by meta-transcriptomics.

Revealing the determinants of virome composition is central to placing disease emergence in a broader evolutionary context. Fish are the most species-rich group of vertebrates and so provide an ideal model system to study the factors that shape virome compositions and their evolution. We characterized the viromes of nineteen wild-caught species of marine fish using total RNA sequencing (meta-transcriptomics) combined with analyses of sequence and protein structural homology to identify divergent viruses that often evade characterization. From this, we identified twenty-five new vertebrate-associated viruses and a further twenty-two viruses likely associated with fish diet or their microbiomes. The vertebrate-associated viruses identified here included the first fish virus in the Matonaviridae (single-strand, negative-sense RNA virus). Other viruses fell within the Astroviridae, Picornaviridae, Arenaviridae, Reoviridae, Hepadnaviridae, Paramyxoviridae, Rhabdoviridae, Hantaviridae, Filoviridae, and Flaviviridae, and were sometimes phylogenetically distinct from known fish viruses. We also show how key metrics of virome composition-viral richness, abundance, and diversity-can be analysed along with host ecological and biological factors as a means to understand virus ecology. Accordingly, these data suggest that that the vertebrate-associated viromes of the fish sampled here are predominantly shaped by the phylogenetic history (i.e. taxonomic order) of their hosts, along with several biological factors including water temperature, habitat depth, community diversity and swimming behaviour. No such correlations were found for viruses associated with porifera, molluscs, arthropods, fungi, and algae, that are unlikely to replicate in fish hosts. Overall, these data indicate that fish harbour particularly large and complex viromes and the vast majority of fish viromes are undescribed.

A survey of sub-inhibitory concentrations of antibiotics in the environment.

Antibiotics are poorly metabolized, and can enter the environment via human waste streams, agricultural run-off and pharmaceutical effluent. We consequently expect to see a concentration gradient of antibiotic compounds radiating from areas of human population. Such antibiotics should be thought of as pollutants, as they can accumulate, and have biological effects. These antibiotic pollutants can increase rates of mutation and lateral transfer events, and continue to exert selection pressure even at sub-inhibitory concentrations. Here, we conducted a literature survey on environmental concentrations of antibiotics. We collated 887 data points from 40 peer-reviewed papers. We then determined whether these concentrations were biologically relevant by comparing them to their minimum selective concentrations, usually defined as between 1/4 and 1/230 of the minimum inhibitory concentration. Environmental concentrations of antibiotics surveyed often fall into this range. In general, the antibiotic concentrations recorded in aquatic and sediment samples were similar. These findings indicate that environmental concentrations of antibiotics are likely to be influencing microbial ecology, and to be driving the selection of antibiotic resistant bacteria.

A Novel Family of Acinetobacter Mega-Plasmids Are Disseminating Multi-Drug Resistance Across the Globe While Acquiring Location-Specific Accessory Genes.

Acinetobacter species are emerging as major nosocomial pathogens, aided by their ability to acquire resistance to all classes of antibiotics. A key factor leading to their multi-drug resistance phenotypes is the acquisition of a wide variety of mobile genetic elements, particularly large conjugative plasmids. Here, we characterize a family of 21 multi-drug resistance mega-plasmids in 11 different Acinetobacter species isolated from various locations across the globe. The plasmid family exhibits a highly dynamic and diverse accessory genome, including 221 antibiotic resistance genes (ARGs) that confer resistance to 13 classes of antibiotics. We show that plasmids isolated within the same geographic region are often evolutionarily divergent members of this family based on their core-genome, yet they exhibit a more similar accessory genome. Individual plasmids, therefore, can disseminate to different locations around the globe, where they then appear to acquire diverse sets of accessory genes from their local surroundings. Further, we show that plasmids from several geographic regions were enriched with location-specific functional traits. Together, our findings show that these mega-plasmids can transmit across species boundaries, have the capacity for global dissemination, can accumulate a diverse suite of location-specific accessory genes, and can confer multi-drug resistance phenotypes of significant concern for human health. We therefore highlight this previously undescribed plasmid family as a serious threat to healthcare systems worldwide. These findings also add to the growing concern that mega-plasmids are key disseminators of antibiotic resistance and require global surveillance.