Exploration of mobile genetic elements in the ruminal microbiome of Nellore cattle.

Metagenomics has made it feasible to elucidate the intricacies of the ruminal microbiome and its role in the differentiation of animal production phenotypes of significance. The search for mobile genetic elements (MGEs) has taken on great importance, as they play a critical role in the transfer of genetic material between organisms. Furthermore, these elements serve a dual purpose by controlling populations through lytic bacteriophages, thereby maintaining ecological equilibrium and driving the evolutionary progress of host microorganisms. In this study, we aimed to identify the association between ruminal bacteria and their MGEs in Nellore cattle using physical chromosomal links through the Hi-C method. Shotgun metagenomic sequencing and the proximity ligation method ProxiMeta were used to analyze DNA, getting 1,713,111,307 bp, which gave rise to 107 metagenome-assembled genomes from rumen samples of four Nellore cows maintained on pasture. Taxonomic analysis revealed that most of the bacterial genomes belonged to the families Lachnospiraceae, Bacteroidaceae, Ruminococcaceae, Saccharofermentanaceae, and Treponemataceae and mostly encoded pathways for central carbon and other carbohydrate metabolisms. A total of 31 associations between host bacteria and MGE were identified, including 17 links to viruses and 14 links to plasmids. Additionally, we found 12 antibiotic resistance genes. To our knowledge, this is the first study in Brazilian cattle that connect MGEs with their microbial hosts. It identifies MGEs present in the rumen of pasture-raised Nellore cattle, offering insights that could advance biotechnology for food digestion and improve ruminant performance in production systems.

The complete genome sequence of unculturable Mycoplasma faucium obtained through clinical metagenomic next-generation sequencing.

Introduction: Diagnosing Mycoplasma faucium poses challenges, and it's unclear if its rare isolation is due to infrequent occurrence or its fastidious nutritional requirements. Methods: This study analyzes the complete genome sequence of M. faucium, obtained directly from the pus of a sternum infection in a lung transplant patient using metagenomic sequencing. Results: Genome analysis revealed limited therapeutic options for the M. faucium infection, primarily susceptibility to tetracyclines. Three classes of mobile genetic elements were identified: two new insertion sequences, a new prophage (phiUMCG-1), and a species-specific variant of a mycoplasma integrative and conjugative element (MICE). Additionally, a Type I Restriction-Modification system was identified, featuring 5'-terminally truncated hsdS pseudogenes with overlapping repeats, indicating the potential for forming alternative hsdS variants through recombination. Conclusion: This study represents the first-ever acquisition of a complete circularized bacterial genome directly from a patient sample obtained from invasive infection of a primary sterile site using culture-independent, PCR-free clinical metagenomics.

The evolution of antibiotic resistance islands occurs within the framework of plasmid lineages.

Bacterial pathogens carrying multidrug resistance (MDR) plasmids are a major threat to human health. The acquisition of antibiotic resistance genes (ARGs) in plasmids is often facilitated by mobile genetic elements that copy or translocate ARGs between DNA molecules. The agglomeration of mobile elements in plasmids generates resistance islands comprising multiple ARGs. However, whether the emergence of resistance islands is restricted to specific MDR plasmid lineages remains understudied. Here we show that the agglomeration of ARGs in resistance islands is biased towards specific large plasmid lineages. Analyzing 6784 plasmids in 2441 Escherichia, Salmonella, and Klebsiella isolates, we quantify that 84% of the ARGs in MDR plasmids are found in resistance islands. We furthermore observe rapid evolution of ARG combinations in resistance islands. Most regions identified as resistance islands are shared among closely related plasmids but rarely among distantly related plasmids. Our results suggest the presence of barriers for the dissemination of ARGs between plasmid lineages, which are related to plasmid genetic properties, host range and the plasmid evolutionary history. The agglomeration of ARGs in plasmids is attributed to the workings of mobile genetic elements that operate within the framework of existing plasmid lineages.

Multidrug resistance in Salmonella isolates of swine origin: mobile genetic elements and plasmids associated with cephalosporin resistance with potential transmission to humans.

The emergence of foodborne Salmonella strains carrying antimicrobial resistance (AMR) in mobile genetic elements (MGE) is a significant public health threat in a One Health context requiring continuous surveillance. Resistance to ciprofloxacin and cephalosporins is of particular concern. Since pigs are a relevant source of foodborne Salmonella for human beings, we studied transmissible AMR genes and MGE in a collection of 83 strains showing 9 different serovars and 15 patterns of multidrug resistant (MDR) previously isolated from pigs raised in the conventional breeding system of Northern Spain. All isolates were susceptible to ciprofloxacin and three isolates carried blaCMY-2 or blaCTX-M-9 genes responsible for cefotaxime resistance. Filter mating experiments showed that the two plasmids carrying blaCTX-M-9 were conjugative while that carrying blaCMY-2 was self-transmissible by transformation. Whole-genome sequencing and comparative analyses were performed on the isolates and plasmids. The IncC plasmid pSB109, carrying blaCMY-2, was similar to one found in S. Reading from cattle, indicating potential horizontal transfer between serovars and animal sources. The IncHI2 plasmids pSH102 in S. Heidelberg and pSTM45 in S. Typhimurium ST34, carrying blaCTX-M-9, shared similar backbones and two novel "complex class 1 integrons" containing different AMR and heavy metal genes. Our findings emphasize the importance of sequencing techniques to identify emerging AMR regions in conjugative and stable plasmids from livestock production. The presence of MGE carrying clinically relevant AMR genes raises public health concerns, requiring monitoring to mitigate the emergence of bacteria carrying AMR genes and subsequent spread through animals and food.IMPORTANCEThe emergence of foodborne Salmonella strains carrying antimicrobial resistance (AMR) in mobile genetic elements (MGE) is a significant public health threat in a One Health context. Since pigs are a relevant source of foodborne Salmonella for humans, in this study, we investigate different aspects of AMR in a collection of 83 Salmonella showing nine different serovars and 15 patterns of multidrug resistant (MDR) isolated from pigs raised in the conventional breeding system. Our findings emphasize the importance of sequencing techniques to identify emerging AMR regions in conjugative and stable plasmids from livestock production. The presence of MGE carrying clinically relevant AMR genes raises public health concerns, requiring monitoring to mitigate the emergence of bacteria carrying AMR genes and subsequent spread through animals and food.

Improving the detection of integrative conjugative elements in bovine nasopharyngeal swabs using multiplex recombinase polymerase amplification.

Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) can possess integrative and conjugative elements (ICEs), a diverse group of mobile genetic elements that acquire antimicrobial resistance (AMR) genes (ARGs) and decrease the therapeutic efficacy of antimicrobial drugs. We developed a duplex recombinase polymerase amplification (RPA) assay to detect up to two variants of ICEs in these Pasteurellaceae. Whole genome sequence analysis of M. haemolytica, P. multocida, and H. somni isolates harbouring ICEs revealed the presence of tnpA or ebrB next to tet(H), a conserved ARG that is frequently detected in ICEs within BRD-associated bacteria. This real-time multiplex RPA assay targeted both ICE variants simultaneously, denoted as tetH_tnpA and tetH_ebrB, with a limit of detection (LOD) of 29 (95% CI [23, 46]) and 38 genome copies (95% CI [30, 59]), respectively. DNA was extracted from 100 deep nasopharyngeal swabs collected from feedlot cattle on arrival. Samples were tested for ICEs using a real-time multiplex RPA assay, and for M. haemolytica, P. multocida, H. somni, and Mycoplasma bovis using both culture methods and RPA. The assay provided sensitive and accurate identification of ICEs in extracted DNA, providing a useful molecular tool for timely detection of potential risk factors associated with the development of antimicrobial-resistant BRD in feedlot cattle.

Mobile genetic elements mediate the cross-media transmission of antibiotic resistance genes from pig farms and their risks.

Manure composting in traditional small-scale pig farms leads to the migration and diffusion of antibiotics and antibiotics resistance genes (ARGs) along the chain of transmission to the surrounding environment, increasing the risk of environmental resistance. Understanding the transmission patterns, driving factors, and health risks of ARGs on small-scale pig farms is important for effective control of ARGs transmission. This study was conducted on a small pig farm and its surrounding environment. The cross-media transmission of ARGs and their risks in the farming habitat were investigated using Metagenomic annotation and qPCR quantitative detection. The results indicate that ARGs in farms spread with manure pile-soil-channel sediment-mudflat sediment. Pig farm manure contributed 22.49 % of the mudflat sediment ARGs. Mobile genetic elements mediate the spread of ARGs across different media. Among them, tnpA and IS26 have the highest degree. Transmission of high-risk ARGs sul1 and tetM resulted in a 50 % and 116 % increase in host risk for sediment, respectively. This study provides a basis for farm manure management and control of the ARGs spread.

Plastiome: Plastisphere-enriched mobile resistome in aquatic environments.

Aquatic microplastics (MPs) act as reservoirs for microbial communities, fostering the formation of a mobile resistome encompassing diverse antibiotic (ARGs) and biocide/metal resistance genes (BMRGs), and mobile genetic elements (MGEs). This collective genetic repertoire, referred to as the "plastiome," can potentially perpetuate environmental antimicrobial resistance (AMR). Our study examining two Japanese rivers near Tokyo revealed that waterborne MPs are primarily composed of polyethylene and polypropylene fibers and sheets of diverse origin. Clinically important genera like Exiguobacterium and Eubacterium were notably enriched on MPs. Metagenomic analysis uncovered a 3.46-fold higher enrichment of ARGs on MPs than those in water, with multidrug resistance genes (MDRGs) and BMRGs prevailing, particularly within MPs. Specific ARG and BMRG subtypes linked to resistance to vancomycin, beta-lactams, biocides, arsenic, and mercury showed selective enrichment on MPs. Network analysis revealed intense associations between host genera with ARGs, BMRGs, and MGEs on MPs, emphasizing their role in coselection. In contrast, river water exhibited weaker associations. This study underscores the complex interactions shaping the mobile plastiome in aquatic environments and emphasizes the global imperative for research to comprehend and effectively control AMR within the One Health framework.

Pyrite from acid mine drainage promotes the removal of antibiotic resistance genes and mobile genetic elements in karst watershed with abundant calcium carbonate.

More information is needed to fully comprehend how acid mine drainage (AMD) affects the phototransformation of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in karst water and sewage-irrigated farmland soil with abundant carbonate rocks (CaCO3) due to increasing pollution of AMD formed from pyrite (FeS2). The results showed FeS2 accelerated the inactivation of ARB with an inactivation of 8.7 log. Notably, extracellular and intracellular ARGs and mobile genetic elements (MGEs) also experienced rapid degradation. Additionally, the pH of the solution buffered by CaCO3 significantly influenced the photo-inactivation of ARB. The Fe2+ in neutral solution was present in Fe(II) coordination with strong reducing potential and played a crucial role in generating •OH (7.0 µM), which caused severe damage to ARB, ARGs, and MGEs. The •OH induced by photo-Fenton of FeS2 posed pressure to ARB, promoting oxidative stress response and increasing generation of reactive oxygen species (ROS), ultimately damaging cell membranes, proteins and DNA. Moreover, FeS2 contributed to a decrease in MIC of ARB from 24 mg/L to 4 mg/L. These findings highlight the importance of AMD in influencing karst water and sewage-irrigated farmland soil ecosystems. They are also critical in advancing the utilization of FeS2 to inactivate pathogenic bacteria.

Type II and IV toxin-antitoxin systems coordinately stabilize the integrative and conjugative element of the ICESa2603 family conferring multiple drug resistance in Streptococcus suis.

Integrative and conjugative elements (ICEs) play a vital role in bacterial evolution by carrying essential genes that confer adaptive functions to the host. Despite their importance, the mechanism underlying the stable inheritance of ICEs, which is necessary for the acquisition of new traits in bacteria, remains poorly understood. Here, we identified SezAT, a type II toxin-antitoxin (TA) system, and AbiE, a type IV TA system encoded within the ICESsuHN105, coordinately promote ICE stabilization and mediate multidrug resistance in Streptococcus suis. Deletion of SezAT or AbiE did not affect the strain's antibiotic susceptibility, but their duple deletion increased susceptibility, mainly mediated by the antitoxins SezA and AbiEi. Further studies have revealed that SezA and AbiEi affect the genetic stability of ICESsuHN105 by moderating the excision and extrachromosomal copy number, consequently affecting the antibiotic resistance conferred by ICE. The DNA-binding proteins AbiEi and SezA, which bind palindromic sequences in the promoter, coordinately modulate ICE excision and extracellular copy number by binding to sequences in the origin-of-transfer (oriT) and the attL sites, respectively. Furthermore, AbiEi negatively regulates the transcription of SezAT by binding directly to its promoter, optimizing the coordinate network of SezAT and AbiE in maintaining ICESsuHN105 stability. Importantly, SezAT and AbiE are widespread and conserved in ICEs harbouring diverse drug-resistance genes, and their coordinated effects in promoting ICE stability and mediating drug resistance may be broadly applicable to other ICEs. Altogether, our study uncovers the TA system's role in maintaining the genetic stability of ICE and offers potential targets for overcoming the dissemination and evolution of drug resistance.

Geographical distribution of mobile genetic elements in microbial communities along the Yucatan coast.

Horizontal gene transfer (HGT) is a well-documented strategy used by bacteria to enhance their adaptability to challenging environmental conditions. Through HGT, a group of conserved genetic elements known as mobile genetic elements (MGEs) is disseminated within bacterial communities. MGEs offer numerous advantages to the host, increasing its fitness by acquiring new functions that help bacteria contend with adverse conditions, including exposure to heavy metal and antibiotics. This study explores MGEs within microbial communities along the Yucatan coast using a metatranscriptomics approach. Prior to this research, nothing was known about the coastal Yucatan's microbial environmental mobilome and HGT processes between these bacterial communities. This study reveals a positive correlation between MGEs and antibiotic resistance genes (ARGs) along the Yucatan coast, with higher MGEs abundance in more contaminated sites. The Proteobacteria and Firmicutes groups exhibited the highest number of MGEs. It's important to highlight that the most abundant classes of MGEs might not be the ones most strongly linked to ARGs, as observed for the recombination/repair class. This work presents the first geographical distribution of the environmental mobilome in Yucatan Peninsula mangroves.

Defence systems and horizontal gene transfer in bacteria.

Horizontal gene transfer (HGT) is a fundamental process in prokaryotic evolution, contributing significantly to diversification and adaptation. HGT is typically facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages, which often impose fitness costs on their hosts. However, a considerable number of bacterial genes are involved in defence mechanisms that limit the propagation of MGEs, suggesting they may actively restrict HGT. In our study, we investigated whether defence systems limit HGT by examining the relationship between the HGT rate and the presence of 73 defence systems across 12 bacterial species. We discovered that only six defence systems, three of which were different CRISPR-Cas subtypes, were associated with a reduced gene gain rate at the species evolution scale. Hosts of these defence systems tend to have a smaller pangenome size and fewer phage-related genes compared to genomes without these systems. This suggests that these defence mechanisms inhibit HGT by limiting prophage integration. We hypothesize that the restriction of HGT by defence systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and the fitness effect of HGT in bacterial populations.

Methicillin resistance in Staphylococcus pseudintermedius encoded within novel staphylococcal cassette chromosome mec (SCCmec) variants.

BACKGROUND: Staphylococcus pseudintermedius is a common opportunistic pathogen of companion dogs and an occasional human pathogen. Treatment is hampered by antimicrobial resistance including methicillin resistance encoded by mecA within the mobile genetic element SCCmec. OBJECTIVES: SCCmec elements are diverse, especially in non-Staphyloccocus aureus staphylococci, and novel variants are likely to be present in S. pseudintermedius. The aim was to characterize the SCCmec elements found in four canine clinical isolates of S. pseudintermedius. MATERIAL AND METHODS: Isolates were whole-genome sequenced and SCCmec elements were assembled, annotated and compared to known SCCmec types. RESULTS AND DISCUSSION: Two novel SSCmec are present in these isolates. SCCmec7017-61515 is characterized by a novel combination of a Class A mec gene complex and a type 5 ccr previously only described in composite SCCmec elements. The other three isolates share a novel composite SCCmec with features of SCCmec types IV and VI. CONCLUSIONS: S. pseudintermedius is a reservoir of novel SSCmec elements that has implications for understanding antimicrobial resistant in veterinary and human medicine.

Overlapping Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis household transmission and mobile genetic element exchange.

Streptococcus dysgalactiae subspecies equisimilis (SDSE) and Streptococcus pyogenes share skin and throat niches with extensive genomic homology and horizontal gene transfer (HGT) possibly underlying shared disease phenotypes. It is unknown if cross-species transmission interaction occurs. Here, we conduct a genomic analysis of a longitudinal household survey in remote Australian First Nations communities for patterns of cross-species transmission interaction and HGT. Collected from 4547 person-consultations, we analyse 294 SDSE and 315 S. pyogenes genomes. We find SDSE and S. pyogenes transmission intersects extensively among households and show that patterns of co-occurrence and transmission links are consistent with independent transmission without inter-species interference. We identify at least one of three near-identical cross-species mobile genetic elements (MGEs) carrying antimicrobial resistance or streptodornase virulence genes in 55 (19%) SDSE and 23 (7%) S. pyogenes isolates. These findings demonstrate co-circulation of both pathogens and HGT in communities with a high burden of streptococcal disease, supporting a need to integrate SDSE and S. pyogenes surveillance and control efforts.

Profiles of phage in global hospital wastewater: Association with microbial hosts, antibiotic resistance genes, metal resistance genes, and mobile genetic elements.

Hospital wastewater (HWW) is known to host taxonomically diverse microbial communities, yet limited information is available on the phages infecting these microorganisms. To fill this knowledge gap, we conducted an in-depth analysis using 377 publicly available HWW metagenomic datasets from 16 countries across 4 continents in the NCBI SRA database to elucidate phage-host dynamics and phage contributions to resistance gene transmission. We first assembled a metagenomic HWW phage catalog comprising 13,812 phage operational taxonomic units (pOTUs). The majority of these pOTUs belonged to the Caudoviricetes order, representing 75.29 % of this catalog. Based on the lifestyle of phages, we found that potentially virulent phages predominated in HWW. Specifically, 583 pOTUs have been predicted to have the capability to lyse 81 potentially pathogenic bacteria, suggesting the promising role of HWW phages as a viable alternative to antibiotics. Among all pOTUs, 1.56 % of pOTUs carry 108 subtypes of antibiotic resistance genes (ARGs), 0.96 % of pOTUs carry 76 subtypes of metal resistance genes (MRGs), and 0.96 % of pOTUs carry 22 subtypes of non-phage mobile genetic elements (MGEs). Predictions indicate that certain phages carrying ARGs, MRGs, and non-phage MGEs could infect bacteria hosts, even potential pathogens. This suggests that phages in HWW may contribute to the dissemination of resistance-associated genes in the environment. This meta-analysis provides the first global catalog of HWW phages, revealing their correlations with microbial hosts and pahge-associated ARGs, MRG, and non-phage MGEs. The insights gained from this research hold promise for advancing the applications of phages in medical and industrial contexts.

Mobile genetic element-based gene editing and genome engineering: Recent advances and applications.

Genome engineering has revolutionized several scientific fields, ranging from biochemistry and fundamental research to therapeutic uses and crop development. Diverse engineering toolkits have been developed and used to effectively modify the genome sequences of organisms. However, there is a lack of extensive reviews on genome engineering technologies based on mobile genetic elements (MGEs), which induce genetic diversity within host cells by changing their locations in the genome. This review provides a comprehensive update on the versatility of MGEs as powerful genome engineering tools that offers efficient solutions to challenges associated with genome engineering. MGEs, including DNA transposons, retrotransposons, retrons, and CRISPR-associated transposons, offer various advantages, such as a broad host range, genome-wide mutagenesis, efficient large-size DNA integration, multiplexing capabilities, and in situ single-stranded DNA generation. We focused on the components, mechanisms, and features of each MGE-based tool to highlight their cellular applications. Finally, we discussed the current challenges of MGE-based genome engineering and provided insights into the evolving landscape of this transformative technology. In conclusion, the combination of genome engineering with MGE demonstrates remarkable potential for addressing various challenges and advancing the field of genetic manipulation, and promises to revolutionize our ability to engineer and understand the genomes of diverse organisms.

Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.

Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.

A review focusing on mechanisms and ecological risks of enrichment and propagation of antibiotic resistance genes and mobile genetic elements by microplastic biofilms.

Microplastics (MPs) are emerging ubiquitous pollutants in aquatic environment and have received extensive global attention. In addition to the traditional studies related to the toxicity of MPs and their carrier effects, their unique surface-induced biofilm formation also increases the ecotoxicity potential of MPs from multiple perspectives. In this review, the ecological risks of MPs biofilms were summarized and assessed in detail from several aspects, including the formation and factors affecting the development of MPs biofilms, the selective enrichment and propagation mechanisms of current pollution status of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in MPs biofilms, the dominant bacterial communities in MPs biofilms, as well as the potential risks of ARGs and MGEs transferring from MPs biofilms to aquatic organisms. On this basis, this paper also put forward the inadequacy and prospects of the current research and revealed that the MGEs-mediated ARG propagation on MPs under actual environmental conditions and the ecological risk of the transmission of ARGs and MGEs to aquatic organisms and human beings are hot spots for future research. Relevant research from the perspective of MPs biofilm should be carried out as soon as possible to provide support for the ecological pollution prevention and control of MPs.

Antibiotic resistance genes and mobile genetic elements in different rivers: The link with antibiotics, microbial communities, and human activities.

Rivers as a critical sink for antibiotic resistance genes (ARGs), and the distribution and spread of ARGs are related to environmental factors, human activities, and biotic factors (e.g. mobile genetic elements (MGEs)). However, the potential link among ARGs, microbial community, and MGEs in rivers under different antibiotic concentration and human activities remains unclear. In this study, 2 urban rivers (URs), 1 rural-urban river (RUR), and 2 rural rivers (RRs) were investigated to identify the spatial-temporal variation and driving force of ARGs. The total concentration of quinolones (QNs) was 160.1-2151 ng·g-1 in URs, 23.34-1188 ng·g-1 in RUR, and 16.39-85.98 ng·g-1 in RRs. Total population (TP), gross domestic production (GDP), sewage, industrial enterprise (IE), and IEGDP appeared significantly spatial difference in URs, RUR, and RRs. In terms of ARGs, 145-161 subtypes were detected in URs, 59-61 subtypes in RURs, and 46-79 subtypes in RRs. For MGEs, 55-60 MGEs subtypes were detected in URs, 29-30 subtypes in RUR, and 29-35 subtypes in RRs. Significantly positive correlation between MGEs and ARGs were found in these rivers. More ARGs subtypes were related to MGEs in URs than those in RUR and RRs. Overall, MGEs and QNs showed significantly direct positive impact on the abundance of ARGs in all rivers, while microbial community was significantly positive impact on the ARGs abundance in URs and RUR. The ARGs abundance in URs/RUR were directly positive influenced by microbial community/MGEs/socioeconomic elements (SEs)/QNs, while those in RRs were directly positive influenced by QNs/MGEs and indirectly positive impacted by SEs. Most QNs resistance risk showed significantly positive correlation with the abundance of ARGs types. Therefore, not only need to consider the concentration of antibiotics, but also should pay more attention to SEs and MGEs in antibiotics risk management and control.

Microbial evolution through horizontal gene transfer by mobile genetic elements.

Mobile genetic elements (MGEs) are crucial for horizontal gene transfer (HGT) in bacteria and facilitate their rapid evolution and adaptation. MGEs include plasmids, integrative and conjugative elements, transposons, insertion sequences and bacteriophages. Notably, the spread of antimicrobial resistance genes (ARGs), which poses a serious threat to public health, is primarily attributable to HGT through MGEs. This mini-review aims to provide an overview of the mechanisms by which MGEs mediate HGT in microbes. Specifically, the behaviour of conjugative plasmids in different environments and conditions was discussed, and recent methodologies for tracing the dynamics of MGEs were summarised. A comprehensive understanding of the mechanisms underlying HGT and the role of MGEs in bacterial evolution and adaptation is important to develop strategies to combat the spread of ARGs.

Genome-centric analyses of 165 metagenomes show that mobile genetic elements are crucial for the transmission of antimicrobial resistance genes to pathogens in activated sludge and wastewater.

Wastewater is considered a reservoir of antimicrobial resistance genes (ARGs), where the abundant antimicrobial-resistant bacteria and mobile genetic elements facilitate horizontal gene transfer. However, the prevalence and extent of these phenomena in different taxonomic groups that inhabit wastewater are still not fully understood. Here, we determined the presence of ARGs in metagenome-assembled genomes (MAGs) and evaluated the risks of MAG-carrying ARGs in potential human pathogens. The potential of these ARGs to be transmitted horizontally or vertically was also determined. A total of 5,916 MAGs (completeness >50%, contamination <10%) were recovered, covering 68 phyla and 279 genera. MAGs were dereplicated into 1,204 genome operational taxonomic units (gOTUs) as a proxy for species ( average nucleotide identity >0.95). The dominant ARG classes detected were bacitracin, multi-drug, macrolide-lincosamide-streptogramin (MLS), glycopeptide, and aminoglycoside, and 10.26% of them were located on plasmids. The main hosts of ARGs belonged to Escherichia, Klebsiella, Acinetobacter, Gresbergeria, Mycobacterium, and Thauera. Our data showed that 253 MAGs carried virulence factor genes (VFGs) divided into 44 gOTUs, of which 45 MAGs were carriers of ARGs, indicating that potential human pathogens carried ARGs. Alarmingly, the MAG assigned as Escherichia coli contained 159 VFGs, of which 95 were located on chromosomes and 10 on plasmids. In addition to shedding light on the prevalence of ARGs in individual genomes recovered from activated sludge and wastewater, our study demonstrates a workflow that can identify antimicrobial-resistant pathogens in complex microbial communities. IMPORTANCE: Antimicrobial resistance (AMR) threatens the health of humans, animals, and natural ecosystems. In our study, an analysis of 165 metagenomes from wastewater revealed antibiotic-targeted alteration, efflux, and inactivation as the most prevalent AMR mechanisms. We identified several genera correlated with multiple ARGs, including Klebsiella, Escherichia, Acinetobacter, Nitrospira, Ottowia, Pseudomonas, and Thauera, which could have significant implications for AMR transmission. The abundance of bacA, mexL, and aph(3")-I in the genomes calls for their urgent management in wastewater. Our approach could be applied to different ecosystems to assess the risk of potential pathogens containing ARGs. Our findings highlight the importance of managing AMR in wastewater and can help design measures to reduce the transmission and evolution of AMR in these systems.