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Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention. Understanding the emergence, evolution, and transmission of antibiotic resistance genes (ARGs) is essential to combat antimicrobial resistance. Here, Munk et al. analyse ARGs in hundreds of sewage samples from 101 countries and describe regional patterns, diverse genetic environments of common ARGs, and ARG-specific transmission patterns.

Background We benchmarked the hybrid assembly approaches of MaSuRCA, SPAdes, and Unicycler for bacterial pathogens using Illumina and Oxford Nanopore sequencing by determining genome completeness and accuracy, antimicrobial resistance (AMR), virulence potential, multilocus sequence typing (MLST), phylogeny, and pan genome. Ten bacterial species (10 strains) were tested for simulated reads of both mediocre- and low-quality, whereas 11 bacterial species (12 strains) were tested for real reads. Results Unicycler performed the best for achieving contiguous genomes, closely followed by MaSuRCA, while all SPAdes assemblies were incomplete. MaSuRCA was less tolerant of low-quality long reads than SPAdes and Unicycler. The hybrid assemblies of five antimicrobial-resistant strains with simulated reads provided consistent AMR genotypes with the reference genomes. The MaSuRCA assembly of Staphylococcus aureus with real reads contained msr(A) and tet(K) , while the reference genome and SPAdes and Unicycler assemblies harbored blaZ . The AMR genotypes of the reference genomes and hybrid assemblies were consistent for the other five antimicrobial-resistant strains with real reads. The numbers of virulence genes in all hybrid assemblies were similar to those of the reference genomes, irrespective of simulated or real reads. Only one exception existed that the reference genome and hybrid assemblies of Pseudomonas aeruginosa with mediocre-quality long reads carried 241 virulence genes, whereas 184 virulence genes were identified in the hybrid assemblies of low-quality long reads. The MaSuRCA assemblies of Escherichia coli O157:H7 and Salmonella Typhimurium with mediocre-quality long reads contained 126 and 118 virulence genes, respectively, while 110 and 107 virulence genes were detected in their MaSuRCA assemblies of low-quality long reads, respectively. All approaches performed well in our MLST and phylogenetic analyses. The pan genomes of the hybrid assemblies of S. Typhimurium with mediocre-quality long reads were similar to that of the reference genome, while SPAdes and Unicycler were more tolerant of low-quality long reads than MaSuRCA for the pan-genome analysis. All approaches functioned well in the pan-genome analysis of Campylobacter jejuni with real reads. Conclusions Our research demonstrates the hybrid assembly pipeline of Unicycler as a superior approach for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.

ABSTRACT Methanotrophs are an important group of microorganisms that counteract methane emissions to the atmosphere. Methane-oxidising bacteria of the Alpha- and Gammaproteobacteria have been studied for over a century, while methanotrophs of the phylum Verrucomicrobia are a more recent discovery. Verrucomicrobial methanotrophs are extremophiles that live in very acidic geothermal ecosystems. Currently, more than a dozen strains have been isolated, belonging to the genera Methylacidiphilum and Methylacidimicrobium. Initially, these methanotrophs were thought to be metabolically confined. However, genomic analyses and physiological and biochemical experiments over the past years revealed that verrucomicrobial methanotrophs, as well as proteobacterial methanotrophs, are much more metabolically versatile than previously assumed. Several inorganic gases and other molecules present in acidic geothermal ecosystems can be utilised, such as methane, hydrogen gas, carbon dioxide, ammonium, nitrogen gas and perhaps also hydrogen sulfide. Verrucomicrobial methanotrophs could therefore represent key players in multiple volcanic nutrient cycles and in the mitigation of greenhouse gas emissions from geothermal ecosystems. Here, we summarise the current knowledge on verrucomicrobial methanotrophs with respect to their metabolic versatility and discuss the factors that determine their diversity in their natural environment. In addition, key metabolic, morphological and ecological characteristics of verrucomicrobial and proteobacterial methanotrophs are reviewed. This review discusses the metabolic versatility of verrucomicrobial methanotrophs regarding the acidic volcanic ecosystems they thrive in and a comparison is made with the canonical proteobacterial methanotrophs.

Background: Stem rot caused by Sclerotium rolfsii is a very important soil-borne disease of peanut. S. rolfsii is a necrotrophic plant pathogenic fungus with an extensive host range and worldwide distribution. It can infect peanut stems, roots, pegs and pods, leading to varied yield losses. S. rolfsii strains GP3 and ZY collected from peanut in different provinces of China exhibited a significant difference in aggressiveness on peanut plants by artificial inoculation test. In this study, de-novo genome sequencing of these two distinct strains was performed aiming to reveal the genomic basis of difference in aggressiveness. Results: Scleotium rolfsii strains GP3 and ZY, with weak and high aggressiveness on peanut plants, exhibited similar growth rate and oxalic acid production in laboratory. The genomes of S. rolfsii strains GP3 and ZY were sequenced by Pacbio long read technology and exhibited 70.51 Mb and 70.61 Mb, with contigs of 27 and 23, and encoded 17, 097 and 16,743 gene models, respectively. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires, which might be associated with aggressiveness, differed between GP3 and ZY. There were 58 and 45 unique pathogen-host interaction (PHI) genes in GP3 and ZY, respectively. The ZY strain had more carbohydrateactive enzymes (CAZymes) in its secretome than GP3, especially in the glycoside hydrolase family (GH), the carbohydrate esterase family (CBM), and the polysaccharide lyase family (PL). GP3 and ZY also had different effector candidates and putative secondary metabolite synthetic gene clusters. These results indicated that differences in PHI, secreted CAZymes, effectors and secondary metabolites may play important roles in aggressive difference between these two strains. Conclusions: The data provided a further understanding of the S. rolfsii genome. Genomic comparison provided clues to the difference in aggressiveness of S. rolfsii strains.

Background Ctenopharyngodon idella (grass carp), Mylopharyngodon piceus (black carp), Hypophthalmichthys nobilis (bighead carp), and Hypophthalmichthys molitrix (silver carp), collectively known as the four major domesticated Asian carp, are freshwater fish species from the family Cyprinidae and are widely consumed in China. Current studies on these species primarily focus on immune system regulation and the growth and development of individual species. However, in-depth genomic investigations and comprehensive comparative analysis remained limited. Methods The complete genomes of Ctenopharyngodon idella , Mylopharyngodon piceus and Hypophthalmichthys nobilis were assembled using a hybrid approach that integrated both next- and third-generation sequencing reads, followed by annotation using the MAKER2 pipeline. Based on the high-quality genomes of Ctenopharyngodon idella , Mylopharyngodon piceus Hypophthalmichthys nobilis , and Hypophthalmichthys molitrix , a comparative genomic analysis was conducted using bioinformatic tools to investigate gene family evolution in these four domesticated Asian carp species. Results High-quality genomes of Ctenopharyngodon idella, Mylopharyngodon piceus , and Hypophthalmichthys nobilis were assembled, achieving over 90% completeness. Immune-related gene families, including MHC class I and NLRC3-like genes, have undergone rapid evolution, with Ctenopharyngodon idella exhibiting significant expansion of NLRC3-like genes. Massive tandem duplication events were identified in trace amine-associated receptors (TAARs), and rapid expansion was observed in TAAR16 and TAAR29. Additionally, a novel TAAR gene cluster was identified in all four Asian carp species. Comparative genomic analysis revealed the expansion of type 1 taste receptor genes, particularly in Ctenopharyngodon idella and Mylopharyngodon piceus . Conclusion This study has successfully constructed the high-quality genomes of Ctenopharyngodon idella , Mylopharyngodon piceus , and Hypophthalmichthys nobilis . The comparative genomic analysis revealed the evolution of immune-related genes and chemosensory receptors in the four major domesticated Asian carp species. These findings suggested the enhanced immunity and sensory perception in these species, providing valuable insights into their adaptation, survival and reproduction.

Background The Trebouxiophyceae is a diverse and species-rich class within the Chlorophyta, exhibiting a wide array of lineages and remarkable variations in morphology and ecology. This group encompasses various lifestyles, including photobionts in symbiotic relationships, free-living forms, and parasitic heterotrophs lacking photosynthetic capacity. Trebouxiophycean algae have attracted considerable scientific interest due to their fundamental biological significance and their promising applications in biotechnology. This study presents a comprehensive genomic analysis of six newly sequenced strains of Trebouxiophyceae, expanding upon a foundation of 25 previously reported high-quality genomes to conduct comparative genomics and evolutionary assessments. Results Molecular phylogenetic analyses based on 18 S rDNA and single-copy orthologues confirmed the accurate identification of species. The analyzed strains exhibited variable genome sizes ranging from 2.37 Mb to 106.45 Mb, with GC content varying between 46.19% and 67.20%, and repeat content ranging from 1.67 to 19.73%. Gene family expansion and contraction analyses revealed that the subaerial species Apatococcus exhibited the most extensive expansions, while Picochlorum , along with the ancestors of the parasitic genera ( Auxenochlorella , Helicosporidium , and Prototheca ) experienced pronounced contractions. Evolutionary analyses using the branch model and branch-site model in PAML indicated that genera with the most marked gene family expansion and contraction also contained orthogroups undergoing positive selection and rapid evolution. Comparative assessments of biosynthetic gene clusters (BGCs), nitrogen transport and assimilation proteins, hexose-proton symporter-like genes ( HUP1 , HUP2 , and HUP3 ), and C 4 -related enzymes across 31 Trebouxiophyceae genomes revealed further patterns of adaptation. Coccomyxa was the only genus containing all the ten types of BGCs, while most other genera exhibited relatively fewer BGCs. The nitrate transporter and the urea active transporter were both absent in the three parasitic genera, and urease, the urease accessory proteins and arginase were nearly universally missing in all the species. All the species possessed the HUP1 , HUP2 , and HUP3 genes, except that HUP2 was absent in Prototheca and Picochlorum , and the relative abundances of the three genes varied among genera. The NAD-ME, and PCK subtypes of C 4 -related genes were widely distributed in all the samples, while the malate dehydrogenase (NADP+) was identified only in the four freshwater strains belonging to Chlorella and Coccomyxa . Conclusions Expanded gene families, along with the rapid evolution and positive selection genes, likely played important roles in environmental adaption across terrestrial and marine habitat. Conversely, genome streamlining due to widespread gene families likely contributed to the parasitic heterotrophic lifestyles. Additionally, the distribution of BGCs, nitrogen transport proteins and HUP-like genes, and the types of C 4 -related enzymes perhaps highlighted the potential of Trebouxiophyceae to adapt to complex and varied environmental conditions.

Background Bacillus has been gradually applied to human production activities as a new type of biological control agent and probiotic preparation. This study investigates a newly isolated Bacillus altitudinis G03 utilizing whole genome sequencing, comparative genome analysis and in vitro experiments to comprehensively disclose its beneficial traits. Results According to whole genome sequencing and assembly data, the genome of G03 consists of a circular chromosome containing 10 gene clusters linked to the biosynthesis of secondary metabolites, 139 CAZymes, an antibiotic resistance gene, and 2 prophage elements, which show great potential in antibacterial and antifungal properties. G03 also encodes secreted proteins involved in important biological processes such as nutrient absorption, colonization, quorum sensing, and antibiotic synthesis. In vitro experiments confirm that G03 successfully inhibits the growth of several fungi and pathogenic bacteria, hydrolyzes protein and cellulose, and is capable of forming strong adhesive biofilms. Furthermore, comparative genomics analysis reveals that G03 contains 78 unique genes associated with various functions such as bacteriocin immunity and sporulation. Selective pressure analysis indicates that the genes associated with integral components of membrane and regulation of transcription are strongly positively selected, which may shape the evolution of G03. Conclusions Collectively, these results provide a strong theoretical framework for further research and utilization of B. altitudinis G03 as a novel probiotic and biocontrol agent. Graphical Abstract

Metschnikowia pulcherrima includes strains of applied agro-food interest, particularly due to the antimicrobial activity against plant pathogens, contribution to the aroma of fermented beverages, and preliminary evidence related to probiotic activity. This biotechnological relevance sheds new light of interest on the biology of this yeast. To better understand and expand its biotechnological potential and applicability, the genomes of M. pulcherrima NRRL Y-7111  T , NRRL Y-48695, CBS 10357, and NRRL Y-48712 were sequenced, and de-novo assembled. Between 10,671 and 14,548 genes were predicted and the cooperative genomic analyses were integrated with experimental assessments relating to traits relevant for biotechnological application and safety. In silico and in vitro safety assessment revealed intermediate sensitivity for itraconazole; furthermore, variants of the genes related to pulcherrimin production and transport were found in all the genomes. Moreover, an arsenal of carbohydrate-active enzymes (CAZymes) was unravelled, and their predicted localization was investigated. This study expands the body of knowledge on M. pulcherrima , including traits relevant for defining its safety as a bioresource, which is a pivotal aspect for its possible inclusion in the European Food Safety Authority (EFSA) Qualified Presumption of Safety (QPS) list and its application in REgulated food/feed PROducts (REPRO) both in the European Union & aligned European countries. Graphical Abstract Key points • A pipeline for genomic characterisation and safety assessment of unconventional yeasts, using M. pulcherrima as a model species was developed. • M. pulcherrima strains can be considered safe and safety data can be used to develop a body of knowledge to include M. pulcherrima in EFSA QPS list. • Analysis of the predicted localization of CAZymes allowed the detection of compounds as potential biological control agents.

Background Pectobacterium carotovorum subsp. brasiliense is a broad host range bacterial pathogen, which causes blackleg of potatoes and bacterial soft rot of vegetables worldwide. Production of plant cell wall degrading enzymes is usually critical for Pectobacterium infection. However, other virulence factors and the mechanisms of genetic adaptation still need to be studied in detail. Results In this study, the complete genome of P. carotovorum subsp. brasiliense strain SX309 isolated from cucumber was compared with eight other pathogenic bacteria belonging to the Pectobacterium genus, which were isolated from various host plants. Genome comparison revealed that most virulence genes are highly conserved in the Pectobacterium strains, especially for the key virulence determinants involved in the biosynthesis of extracellular enzymes and others including the type II and III secretion systems, quorum sensing system, flagellar and chemotactic genes. Nevertheless, some variable regions of the T6SS and the CRISP-Cas immune system are unique for P. carotovorum subsp. brasiliense . Conclusions The extensive comparative genomics analysis revealed highly conserved virulence genes in the Pectobacterium strains. However, several variable regions of type VI secretion system and two subtype Cas mechanism-Cas immune systems possibly contribute to the process of Pectobacterium infection and adaptive immunity.

Background Pseudomonas syringae pv. actinidiae (Psa) is an important bacterial plant pathogen that causes severe damage to the kiwifruit industry worldwide. Three Psa strains were recently obtained from different kiwifruit orchards in Anhui Province, China. The present study mainly focused on the variations in virulence and genome characteristics of these strains based on the pathogenicity assays and comparative genomic analyses. Results Three strains were identified as biovar 3 (Psa3), along with strain QSY6 showing higher virulence than JZY2 and YXH1 in pathogenicity assays. The whole genome assembly revealed that each of the three strains had a circular chromosome and a complete plasmid. The chromosome sizes ranged from 6.5 to 6.6 Mb with a GC content of approximately 58.39 to 58.46%, and a predicted number of protein-coding sequences ranging from 5,884 to 6,019. The three strains clustered tightly with 8 Psa3 reference strains in terms of average nucleotide identity (ANI), whole-genome-based phylogenetic analysis, and pangenome analysis, while they were evolutionarily distinct from other biovars (Psa1 and Psa5). Variations were observed in the repertoire of effectors of the type III secretion system among all 15 strains. Moreover, synteny analysis of the three sequenced strains revealed eight genomic regions containing 308 genes exclusively present in the highly virulent strain QSY6. Further investigation of these genes showed that 16 virulence-related genes highlight several key factors, such as effector delivery systems (type III secretion systems) and adherence (type IV pilus), which might be crucial for the virulence of QSY6. Conclusion Three Psa strains were identified and showed variant virulence in kiwifruit plant. Complete genome sequences and comparative genomic analyses further provided a theoretical basis for the potential pathogenic factors responsible for kiwifruit bacterial canker.