Sewage Sludge Promotes the Accumulation of Antibiotic Resistance Genes in Tomato Xylem.

Xylem serves as a conduit linking soil to the aboveground plant parts and facilitating the upward movement of microbes into leaves and fruits. Despite this potential, the composition of the xylem microbiome and its associated risks, including antibiotic resistance, are understudied. Here, we cultivated tomatoes and analyzed their xylem sap to assess the microbiome and antibiotic resistance profiles following treatment with sewage sludge. Our findings show that xylem microbes primarily originate from soil, albeit with reduced diversity in comparison to those of their soil microbiomes. Using single-cell Raman spectroscopy coupled with D2O labeling, we detected significantly higher metabolic activity in xylem microbes than in rhizosphere soil, with 87% of xylem microbes active compared to just 36% in the soil. Additionally, xylem was pinpointed as a reservoir for antibiotic resistance genes (ARGs), with their abundance being 2.4-6.9 times higher than in rhizosphere soil. Sludge addition dramatically increased the abundance of ARGs in xylem and also increased their mobility and host pathogenicity. Xylem represents a distinct ecological niche for microbes and is a significant reservoir for ARGs. These results could be used to manage the resistome in crops and improve food safety.

Reduction in antimicrobial resistance in a watershed after closure of livestock farms.

Natural environments play a crucial role in transmission of antimicrobial resistance (AMR). Development of methods to manage antibiotic resistance genes (ARGs) in natural environments are usually limited to the laboratory or field scale, partially due to the complex dynamics of transmission between different environmental compartments. Here, we conducted a nine-year longitudinal profiling of ARGs at a watershed scale, and provide evidence that restrictions on livestock farms near water bodies significantly reduced riverine ARG abundance. Substantial reductions were revealed in the relative abundance of genes conferring resistance to aminoglycosides (42%), MLSB (36%), multidrug (55%), tetracyclines (53%), and other gene categories (59%). Additionally, improvements in water quality were observed, with distinct changes in concentrations of dissolved reactive phosphorus, ammonium, nitrite, pH, and dissolved oxygen. Antibiotic residues and other pharmaceuticals and personal care products (PPCPs) maintain at a similarly low level. Microbial source tracking demonstrates a significant decrease in swine fecal indicators, while human fecal pollution remains unchanged. These results suggest that the reduction in ARGs was due to a substantial reduction in input of antibiotic resistant bacteria and genes from animal excreta. Our findings highlight the watershed as a living laboratory for understanding the dynamics of AMR, and for evaluating the efficacy of environmental regulations, with implications for reducing environmental risks associated with AMR on a global scale.

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.

Microbial biofilms on macroalgae harbour diverse integron gene cassettes.

Integrons are genetic platforms that capture, rearrange and express mobile modules called gene cassettes. The best characterized gene cassettes encode antibiotic resistance, but the function of most integron gene cassettes remains unknown. Functional predictions suggest that many gene cassettes could encode proteins that facilitate interactions with other cells and with the extracellular environment. Because cell interactions are essential for biofilm stability, we sequenced gene cassettes from biofilms growing on the surface of the marine macroalgae Ulva australis and Sargassum linearifolium. Algal samples were obtained from coastal rock platforms around Sydney, Australia, using seawater as a control. We demonstrated that integrons in microbial biofilms did not sample genes randomly from the surrounding seawater, but harboured specific functions that potentially provided an adaptive advantage to both the bacterial cells in biofilm communities and their macroalgal host. Further, integron gene cassettes had a well-defined spatial distribution, suggesting that each bacterial biofilm acquired these genetic elements via sampling from a large but localized pool of gene cassettes. These findings suggest two forms of filtering: a selective acquisition of different integron-containing bacterial species into the distinct biofilms on Ulva and Sargassum surfaces, and a selective retention of unique populations of gene cassettes at each sampling location.

Inter-plasmid transfer of antibiotic resistance genes accelerates antibiotic resistance in bacterial pathogens.

Antimicrobial resistance is a major threat for public health. Plasmids play a critical role in the spread of antimicrobial resistance via horizontal gene transfer between bacterial species. However, it remains unclear how plasmids originally recruit and assemble various antibiotic resistance genes (ARGs). Here, we track ARG recruitment and assembly in clinically relevant plasmids by combining a systematic analysis of 2420 complete plasmid genomes and experimental validation. Results showed that ARG transfer across plasmids is prevalent, and 87% ARGs were observed to potentially transfer among various plasmids among 8229 plasmid-borne ARGs. Interestingly, recruitment and assembly of ARGs occur mostly among compatible plasmids within the same bacterial cell, with over 88% of ARG transfers occurring between compatible plasmids. Integron and insertion sequences drive the ongoing ARG acquisition by plasmids, especially in which IS26 facilitates 63.1% of ARG transfer events among plasmids. In vitro experiment validated the important role of IS26 involved in transferring gentamicin resistance gene aacC1 between compatible plasmids. Network analysis showed four beta-lactam genes (blaTEM-1, blaNDM-4, blaKPC-2, and blaSHV-1) shuffling among 1029 plasmids and 45 clinical pathogens, suggesting that clinically alarming ARGs transferred accelerate the propagation of antibiotic resistance in clinical pathogens. ARGs in plasmids are also able to transmit across clinical and environmental boundaries, in terms of the high-sequence similarities of plasmid-borne ARGs between clinical and environmental plasmids. This study demonstrated that inter-plasmid ARG transfer is a universal mechanism for plasmid to recruit various ARGs, thus advancing our understanding of the emergence of multidrug-resistant plasmids.

Effects of organic fertilizers on plant growth and the rhizosphere microbiome.

Application of organic fertilizers is an important strategy for sustainable agriculture. The biological source of organic fertilizers determines their specific functional characteristics, but few studies have systematically examined these functions or assessed their health risk to soil ecology. To fill this gap, we analyzed 16S rRNA gene amplicon sequencing data from 637 soil samples amended with plant- and animal-derived organic fertilizers (hereafter plant fertilizers and animal fertilizers). Results showed that animal fertilizers increased the diversity of soil microbiome, while plant fertilizers maintained the stability of soil microbial community. Microcosm experiments verified that plant fertilizers were beneficial to plant root development and increased carbon cycle pathways, while animal fertilizers enriched nitrogen cycle pathways. Compared with animal fertilizers, plant fertilizers harbored a lower abundance of risk factors such as antibiotic resistance genes and viruses. Consequently, plant fertilizers might be more suitable for long-term application in agriculture. This work provides a guide for organic fertilizer selection from the perspective of soil microecology and promotes sustainable development of organic agriculture.IMPORTANCEThis study provides valuable guidance for use of organic fertilizers in agricultural production from the perspective of the microbiome and ecological risk.

Glyphosate Disorders Soil Enchytraeid Gut Microbiota and Increases Its Antibiotic Resistance Risk.

Pesticides promote the stable development of intensive global agriculture. Nevertheless, their residues in the soil can cause ecological and human health risks. Glyphosate is a popular herbicide and is generally thought to be ecologically safe and nontoxic, but this conclusion has been questioned. Herein, we investigated the interaction among soil fauna (Enchytraeus crypticus) exposed to glyphosate and found that glyphosate induced oxidative stress and detoxification responses in E. crypticus and disturbed their lipid metabolism and digestive systems. We further demonstrated that glyphosate disordered the gut microbiota of E. crypticus and increased the abundance of resistance determinants with significant human health risks. Empirical tests and structural equation models were then used to confirm that glyphosate could cause E. crypticus to generate reactive oxygen species, indirectly interfering with their gut microbiota. Our study provides important implications for deciphering the mechanisms of the ecotoxicity of pesticides under the challenge of worldwide pesticide contamination.

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.

Ecology and risks of the global plastisphere as a newly expanding microbial habitat.

Plastic offers a new niche for microorganisms, the plastisphere. The ever-increasing emission of plastic waste makes it critical to understand the microbial ecology of the plastisphere and associated effects. Here, we present a global fingerprint of the plastisphere, analyzing samples collected from freshwater, seawater, and terrestrial ecosystems. The plastisphere assembles a distinct microbial community that has a clearly higher heterogeneity and a more deterministically dominated assembly compared to natural habitats. New coexistence patterns-loose and fragile networks with mostly specialist linkages among microorganisms that are rarely found in natural habitats-are seen in the plastisphere. Plastisphere microbiomes generally have a great potential to metabolize organic compounds, which could accelerate carbon turnover. Microorganisms involved in the nitrogen cycle are also altered in the plastisphere, especially in freshwater plastispheres, where a high abundance of denitrifiers may increase the release of nitrite (aquatic toxicant) and nitrous oxide (greenhouse gas). Enrichment of animal, plant, and human pathogens means that the plastisphere could become an increasingly mobile reservoir of harmful microorganisms. Our findings highlight that if the trajectory of plastic emissions is not reversed, the expanding plastisphere could pose critical planetary health challenges.

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.

Metagenomic Insight into The Global Dissemination of The Antibiotic Resistome.

The global crisis in antimicrobial resistance continues to grow. Estimating the risks of antibiotic resistance transmission across habitats is hindered by the lack of data on mobility and habitat-specificity. Metagenomic samples of 6092 are analyzed to delineate the unique core resistomes from human feces and seven other habitats. This is found that most resistance genes (≈85%) are transmitted between external habitats and human feces. This suggests that human feces are broadly representative of the global resistome and are potentially a hub for accumulating and disseminating resistance genes. The analysis found that resistance genes with ancient horizontal gene transfer (HGT) events have a higher efficiency of transfer across habitats, suggesting that HGT may be the main driver for forming unique but partly shared resistomes in all habitats. Importantly, the human fecal resistome is historically different and influenced by HGT and age. The most important routes of cross-transmission of resistance are from the atmosphere, buildings, and animals to humans. These habitats should receive more attention for future prevention of antimicrobial resistance. The study will disentangle transmission routes of resistance genes between humans and other habitats in a One Health framework and can identify strategies for controlling the ongoing dissemination and antibiotic resistance.

A 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.

Tracing the Sources and Prevalence of Class 1 Integrons, Antimicrobial Resistance, and Trace Elements Using European Honey Bees.

Surveillance of antimicrobial resistance is essential for an effective One Health response. This study explores the efficacy of European honey bees (Apis mellifera) for biomonitoring antimicrobial resistance (AMR) in urban areas. Class 1 integrons (intI1) are investigated as a universal AMR indicator, as well as associated cassette arrays and trace element contaminants at a city-wide scale. Class 1 integrons were found to be pervasive across the urban environment, occurring in 52% (75/144) of the honey bees assessed. The area of waterbodies within the honey bee's foraging radius was associated with intI1 prevalence, indicating an exposure pathway for future investigation to address. Trace element concentrations in honey bees reflected urban sources, supporting the application of this biomonitoring approach. As the first study of intI1 in honey bees, we provide insights into the environmental transfer of bacterial DNA to a keystone species and demonstrate how intI1 biomonitoring can support the surveillance of AMR.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

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.

Antimicrobial Peptides in the Global Microbiome: Biosynthetic Genes and Resistance Determinants.

Antimicrobial peptides are a promising new class of antimicrobials that could address the antibiotic resistance crisis, which poses a major threat to human health. These peptides are present in all kingdoms of life, but especially in microorganisms, having multiple origins in diverse taxa. To date, there has been no global study on the diversity of antimicrobial peptides, the hosts in which these occur, and the potential for resistance to these agents. Here, we investigated the diversity and number of antimicrobial peptides in four main habitats (aquatic, terrestrial, human, and engineered) by analyzing 52,515 metagenome-assembled genomes. The number of antimicrobial peptides was higher in the human gut microbiome than in other habitats, and most hosts of antimicrobial peptides were habitat-specific. The relative abundance of genes that confer resistance to antimicrobial peptides varied between habitats and was generally low, except for the built environment and on human skin. The horizontal transfer of potential resistance genes among these habitats was probably constrained by ecological barriers. We systematically quantified the risk of each resistance determinant to human health and found that nearly half of them pose a threat, especially those that confer resistance to multiple AMPs and polymyxin B. Our results help identify the biosynthetic potential of antimicrobial peptides in the global microbiome, further identifying peptides with a low risk of developing resistance.

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.

Ecosystem Microbiome Science.

The microbiome contributes to multiple ecosystem functions and services through its interactions with a complex environment and other organisms. To date, however, most microbiome studies have been carried out on individual hosts or particular environmental compartments. This greatly limits a comprehensive understanding of the processes and functions performed by the microbiome and its dynamics at an ecosystem level. We propose that the theory and tools of ecosystem ecology be used to investigate the connectivity of microorganisms and their interactions with the biotic and abiotic environment within entire ecosystems and to examine their contributions to ecosystem services. Impacts of natural and anthropogenic stressors on ecosystems will likely cause cascading effects on the microbiome and lead to unpredictable outcomes, such as outbreaks of emerging infectious diseases or changes in mutualistic interactions. Despite enormous advances in microbial ecology, we are yet to study microbiomes of ecosystems as a whole. Doing so would establish a new framework for microbiome study: Ecosystem Microbiome Science. The advent and application of molecular and genomic technologies, together with data science and modeling, will accelerate progress in this field.