Microbial Genomics最新文献

{"title":"Genomic structure of <i>stx2i</i> genes and prophages determined in Shiga toxin-producing <i>Escherichia coli</i> isolated from food, animal and human samples in Germany.","authors":"Maria Borowiak, Carlus Deneke, Christina Lang, Andre Göhler, Angelika Fruth, Elisabeth Schuh, Michaela Projahn","doi":"10.1099/mgen.0.001841","DOIUrl":"10.1099/mgen.0.001841","url":null,"abstract":"<p><p>Shiga toxin-producing <i>Escherichia coli</i> (STEC) are important foodborne pathogens and a threat to human health. Their main virulence factor, the Shiga toxin (Stx), is encoded in two subunit genes (<i>stxA</i> and <i>stxB</i>) and occurs in various subtypes. Here, we report the occurrence of strains harbouring the new subtype <i>stx2i</i>. Isolates from food (<i>n</i>=8), animal (<i>n</i>=3) and human (<i>n</i>=2) samples were subjected to whole-genome sequencing for detailed molecular analyses. All strains harboured an identical <i>stx2i</i> variant independent of the genoserotype and differed from the first detected variant in 2016. In five of the strains, inactive <i>stx</i> operons were determined due to a transposon insertion in the <i>stxA2i</i> subunit gene. <i>Stx</i> phages and insertion sites were comparable between the strains of the same genoserotype. As the <i>stx2i</i> variant was also identified in a human clinical sample, we developed a specific PCR, as there are possible cross-reactions with other typing methods currently used in routine diagnostics. In total, our data show that STEC strains of this specific <i>stx2i</i> variant occur in various matrices of food, animals and humans and that there might be an underdetected number of strains due to insufficient typing methods.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13641118/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149081359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative genomics approaches to identify genomic regions associated with the antimicrobial activity of <i>Pseudomonas protegens</i> PBL3.","authors":"Shilu Dahal, Chia Sin Liew, Jean-Jack Riethoven, Clemencia M Rojas","doi":"10.1099/mgen.0.001827","DOIUrl":"10.1099/mgen.0.001827","url":null,"abstract":"<p><p>The environmental bacterium <i>Pseudomonas protegens</i> PBL3 has antagonistic activity against the plant pathogenic bacterium <i>Burkholderia glumae</i>, an important pathogen in rice. The antimicrobial activity of <i>P. protegens</i> PBL3 was found in the bacteria-free secreted fraction (secretome), but the specific molecules, as well as the genetic basis of that activity, have not been identified. In this study, we integrated genomic information with antimicrobial assays on <i>P. protegens</i> PBL3 and additional six <i>Pseudomonas</i> spp. strains, to identify putative genomic regions in <i>P. protegens</i> PBL3 associated with antimicrobial activity. We hypothesized that <i>Pseudomonas</i> spp. strains with antimicrobial activity against <i>B. glumae</i> have conserved genes with <i>P. protegens</i> PBL3 that are absent in strains lacking activity. Comparative genomics analyses with anvi'o and progressiveMauve, and using <i>P. protegens</i> PBL3 as the reference genome, revealed 188 genes uniquely present in antimicrobial-producing strains. Seven of those genes were annotated as biosynthetic gene clusters predicted to encode secondary metabolites; additional genes were grouped into 25 contiguous clusters with functions annotated as secretion, signal transduction, regulation, transport/efflux, carbohydrate metabolism and one with an additional uncharacterized function. Altogether, this study uncovered a complex and multi-functional network of candidate genes, suggesting that the antimicrobial activity in <i>P. protegens</i> PBL3 is not limited to biosynthetic pathways but also involves additional regulatory, metabolic and export modules to synthesize and deploy antimicrobials.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13541209/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148887893","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"'Epidemiology of <i>L</i> <i>egionella</i>: Genome-bAsed Typing' (el_gato) - a new bioinformatic tool for identifying sequence-based types of <i>Legionella pneumophila</i> from whole-genome sequencing data.","authors":"Alan J Collins, Dev Mashruwala, Vasanta Chivukula, Natalia A Kozak-Muiznieks, Lavanya Rishishwar, Emily T Norris, Melisa J Willby, Jennafer A P Hamlin, Will A Overholt","doi":"10.1099/mgen.0.001822","DOIUrl":"10.1099/mgen.0.001822","url":null,"abstract":"<p><p>Sequence-based typing (SBT) via Sanger sequencing has been the standard for describing <i>Legionella pneumophila</i> relatedness for two decades. SBT involves sequencing seven loci, identifying alleles using the United Kingdom Health Security Agency database and inferring the corresponding sequence type (ST). While similar SBT approaches for other organisms can be easily adapted to whole-genome sequencing (WGS), <i>L. pneumophila</i> presents two challenges for this adaptation: multiple copies of one locus (<i>mompS</i>) and extensive heterogeneity in a second locus (<i>neuA/neuAh</i>). Although several computational methods have been proposed to address these issues, a WGS-based replacement with equal resolution to traditional SBT has been elusive. To address this gap, we developed el_gato (<b>E</b>pidemiology of <i>Legionella</i>: <b>G</b>enome-b<b>A</b>sed <b>T</b>yping; https://github.com/CDCgov/el_gato), which offers several advantages over existing methods: (1) a novel approach for resolving multiple <i>mompS</i> alleles identified in the same isolate, (2) the ability to capture diverse <i>neuA/neuAh</i> alleles, (3) fast single-threaded execution with an average of ~27 s per sample, (4) easy installation via Bioconda or Docker/Singularity and (5) an updated database as of May 2026. el_gato works with either paired-end short reads or genome assemblies, performing more accurately with paired-end short reads at least 250 bp in length. We compared el_gato against two other <i>in silico</i> SBT tools ('mompS', hereafter referred to as the mompS tool and 'legsta') using a dataset of 441 isolates with STs previously determined by Sanger sequencing. el_gato correctly identified the ST for 98.9% of the test isolates, compared to 95.2% for the mompS tool and 42.2% for legsta, demonstrating a significant improvement compared to the mompS tool (adjusted <i>P</i>=2.48×10^-3) and legsta (adjusted <i>P</i>=9.90×10^-55) in ST identification. Furthermore, el_gato's determination of ST was not significantly different from Sanger sequencing (adjusted <i>P</i>=1.00). In summary, el_gato improves <i>in silico</i> SBT and, given its performance, is poised to support the public health community.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13581270/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148977148","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The (un)successful global spread of <i>Acinetobacter baumannii</i> ST3.","authors":"Eradah Abu Sabah, Margaret M C Lam, Francois Lebreton, Patrick T McGann, Mehrad Hamidian","doi":"10.1099/mgen.0.001819","DOIUrl":"10.1099/mgen.0.001819","url":null,"abstract":"<p><p><i>Acinetobacter baumannii</i> sequence type 3 (ST3) has been reported in multiple regions but has not achieved the global dominance of major clones such as ST1 and ST2. Here, we analysed 383 ST3 genomes, including 205 isolates from the Multidrug-Resistant Organism Repository and Surveillance Network (MRSN) and 178 publicly available genomes, to investigate the population structure, resistance gene repertoire and evolutionary dynamics of this lineage. Phylogenetic analysis revealed two major clades with multiple subclades, with 82% of genomes linked to the Middle East, highlighting this region as the likely origin or reservoir. Despite detection across 12 countries, ST3 remains relatively uncommon globally, except in Israel where it is locally abundant. Pan-genome analysis revealed a core genome comprising ~42% of total gene content, comparable to other major sequence types. Extensive recombination was observed, including large chromosomal segments, a total of ~350 kb and ~1.2 Mb shared with ST1 and ST2, respectively, as well as recurrent exchanges at the ISAba1-<i>ampC</i> locus. A chromosomal resistance island, designated AbST3GRI, was identified in nearly all genomes and exhibited structural diversity driven by IS<i>26</i>. A total of 132 genomes carried carbapenem resistance genes, predominantly <i>bla</i> _OXA-23. However, ST3 genomes contain a relatively limited repertoire of resistance genes compared to globally dominant clones. Despite retaining most key virulence determinants, ST3 has not undergone widespread global expansion. Our findings suggest that, although genetically dynamic and capable of acquiring resistance, the lack of extensive antibiotic resistance gene repertoire may have limited the global success of this lineage.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13583062/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148977192","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Host-strain compatibility shapes host transcriptional responses in macrophage <i>in vitro</i> infection models of <i>Mycobacterium tuberculosis</i>.","authors":"Paula Ruiz-Rodriguez, Marta Caballer-Gual, Guillem Santamaria, Hellen Hiza, Mireia Coscollá","doi":"10.1099/mgen.0.001826","DOIUrl":"10.1099/mgen.0.001826","url":null,"abstract":"<p><p>Differences in host distribution are observed across <i>Mycobacterium tuberculosis</i> complex (MTBC) lineages, but investigating the molecular basis of host compatibility <i>in vivo</i> is challenging. Here, we establish an epidemiology-guided <i>in vitro</i> framework to operationalize 'host compatibility' as a theoretical parameter that can be approximated experimentally using macrophage infection readouts. We infected two widely used but biologically distinct models, human THP-1-derived macrophages (THP-1m) and bovine macrophages (BoMac), with four MTBC human and animal strains. We quantified infected-cell frequency, intracellular bacterial burden dynamics, cell death and host transcriptome by RNA-seq. Across both models, infection phenotypes were concordant with the <i>a priori</i> epidemiology-informed reference pairings: the most compatible strain showed a higher late intracellular bacterial burden in its respective host, and the corresponding transcriptomes were enriched for replication, repair and cell-cycle signatures, whereas the opposite pairings showed lower bacterial burdens and stronger enrichment for endosomal, antigen-presentation and immune-signalling pathways in each cell type. Given the role of phospholipase C and virulence, we assessed <i>plc</i> expression and performed pharmacological inhibition experiments. These analyses revealed lineage- and macrophage-context-dependent expression patterns and reduced intracellular burden and cytotoxicity in THP-1m but not in BoMac. This framework links MTBC strain diversity to macrophage infection phenotypes and prioritizes candidate pathways potentially involved in bacterial-host interactions.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600463/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Parallel evolution of chimeric genes in microbial evolution experiments.","authors":"Rohan Maddamsetti, Mehmet Orman","doi":"10.1099/mgen.0.001850","DOIUrl":"10.1099/mgen.0.001850","url":null,"abstract":"<p><p>Recombination can generate new or improved proteins by merging pieces of pre-existing genes into a new whole. Here, when we reanalysed genomic data from several laboratory evolution experiments with <i>Escherichia coli</i>, evidence for the convergent evolution of chimeric genes became apparent. In these experiments, a pair of paralogous genes was recombined into a single hybrid copy by large genomic deletions. In one case, the excision of a cryptic e14 prophage occurred in 6 out of 8 replicates of a 22-day fluoroquinolone-resistance evolution experiment. These parallel prophage excisions recombined an <i>icd</i> isocitrate dehydrogenase gene with a homologous <i>icdC</i> C-terminal fragment pseudogene. In the other case, convergent 23 kbp deletions recombined <i>cpsG</i> paralogs across replicate populations of the Lenski long-term evolution experiment with <i>E. coli</i>, generating a chimeric phosphomannomutase. Together, these results indicate that chimeric genes evolve rapidly in bacteria and are more common than previously believed because they are easily missed by standard genomic analyses. Experiments are needed to determine whether these chimeric proteins are adaptive or are merely by-products of their associated genomic deletions.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13626634/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149081365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expanded detection of canine enteric viruses in UK dogs with diarrhoea.","authors":"Jack Pilgrim, Aleksandra Rzeszutek, Edward Cunningham-Oakes, Shirley Bonner, Larry Roberts, Alistair C Darby, Alan D Radford","doi":"10.1099/mgen.0.001799","DOIUrl":"10.1099/mgen.0.001799","url":null,"abstract":"<p><p>Canine enteric viruses are an important cause of gastrointestinal disease in pet dogs worldwide. Routine diagnosis often relies on pathogen-specific PCR assays, which may fail to detect some viruses, particularly neglected pathogens or genetically divergent variants of established threats. This limits both clinical characterization of affected patients and broader understanding of disease ecology. To address these limitations, we applied metagenomics and a viral discovery bioinformatics pipeline to faecal samples from diarrhoeic dogs in the UK that had been submitted routinely for PCR-based diagnostic testing. Across 80 dogs, we identified 12 viruses known to infect canids, 9 of which have not previously been reported in UK dogs. Among these, several taxa with prior associations to gastrointestinal disease were identified, including canine sapovirus and canine minute virus. By contrast, for other viruses newly detected in the UK, including bufavirus and rotavirus C, clinical relevance in dogs remains unclear. Notably, an identified protoparvovirus fell within the same species as human-canine-associated parvovirus 1, a recently described lineage detected in both canine and human oropharyngeal samples. We also identified a canine parvovirus 2 strain that clustered with a predominantly wildlife-associated lineage, consistent with occasional exposure at the domestic-wildlife interface rather than established circulation in dogs. These two detections illustrate how genome-level surveillance can help prioritize viruses for targeted investigation of host range and transmission context. Overall, these data broaden the catalogue of viruses associated with diarrhoeic dogs in the UK and support periodic review of diagnostic targets informed by viral metagenomic surveillance, while highlighting the need for controlled studies to assess causality and clinical relevance.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13552968/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148905932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative essentialome analysis of six <i>Pectobacteriaceae</i> strains using the TNSEEK pipeline identifies conserved and strain-specific fitness determinants.","authors":"Julie Baltenneck, Loïc Couderc, Jacques Pédron, Guillemette Marot, Areski Flissi, Hélène Touzet, Erwan Gueguen, Marie-Anne Barny, Guy Condemine","doi":"10.1099/mgen.0.001762","DOIUrl":"10.1099/mgen.0.001762","url":null,"abstract":"<p><p>Transposon sequencing (Tn-seq) is a powerful technique for defining the essential genes required for bacterial survival. However, gene essentiality can vary significantly across taxonomic levels, and comparing large Tn-seq datasets from multiple strains presents considerable analytical challenges. To address this, we developed TNSEEK, a fully automated bioinformatics pipeline for the systematic and comparative analysis of Tn-seq experiments. We applied TNSEEK to analyse newly generated data for six soft rot <i>Pectobacteriaceae</i> strains, encompassing species from the <i>Dickeya</i> and <i>Pectobacterium</i> genera, grown in a rich medium. This approach identified a core essentialome of 225 genes, primarily involved in fundamental cellular maintenance, conserved across all 6 strains, a set comparable in size to that of the neighbouring <i>Enterobacteriaceae</i> family. Only a few genus-specific essential genes were found, highlighting interesting distinct metabolic capabilities between <i>Dickeya</i> and <i>Pectobacterium</i> genera. In striking contrast, we discovered a large variable essentialome comprising 181 strain-specific genes, many of which are of unknown function. A portion of these strain-specific essential genes are components of defence systems and prophage genomic regions. The unexpected essentiality of selected components of these modules is consistent with cellular dependency on cognate toxic, restriction or immunity functions encoded by defence-associated loci under the tested growth condition. Furthermore, a comparison with the <i>Escherichia coli</i> essentialome demonstrates that discrepancies in gene essentiality can often be attributed to differences in growth conditions, particularly temperature, as well as variations in genetic redundancy. In conclusion, the TNSEEK pipeline provides a reproducible framework for comparative analysis of mariner/Himar1 Tn-seq datasets across multiple strains.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13566846/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148945216","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unravelling the genomic and functional arsenal of <i>Bacilli</i> endophytes from plants with different lifestyles and their antimicrobial potential.","authors":"Nikolaos P Arapitsas, Christos A Christakis, Savvas Paragkamian, Stefanos Soultatos, Franziska Reden, Chrysianna Psarologaki, Emmanouil Avramakis, Alexandros Stamatakis, Emmanouil A Markakis, Panagiotis F Sarris","doi":"10.1099/mgen.0.001801","DOIUrl":"https://doi.org/10.1099/mgen.0.001801","url":null,"abstract":"<p><p>Endophytic microbiomes of crop wild relatives (CWRs) adapted to extreme environments, such as halophytes, are promising sources of plant-beneficial bacteria and secondary metabolites for sustainable food production. Here, we analysed 25 <i>Bacilli</i> isolates obtained from CWRs, halophytes and other plant species in Crete, Greece. Using a hybrid Illumina-PacBio sequencing approach, we generated high-quality genomes and performed comparative genomics, phylogenetic and pangenome analyses, complemented by <i>in vitro</i> assays. We identified 312 biosynthetic gene clusters (BGCs), nearly 60% of which showed no similarity to known clusters, revealing extensive unexplored biosynthetic potential. These unique BGCs may constitute an adaptive feature enabling endophytic <i>Bacilli</i> to colonize and interact with host plants. The isolates spanned diverse genera (<i>Bacillus</i>, <i>Paenibacillus</i>, <i>Peribacillus</i>, <i>Neobacillus</i>, <i>Cytobacillus</i> and <i>Rossellomorea</i>), including three novel species. Phenotypic assays of our isolates demonstrated high salinity tolerance (up to 17.5% wt/v NaCl) and strong antagonism against major bacterial and fungal phytopathogens. Genome mining further revealed a broad array of putatively plant-beneficial traits related to growth promotion, stress adaptation, host interaction and inhibition of pathogens. Together, these findings show that <i>Bacilli</i> endophytes from wild and halophytic plants possess exceptional phylogenetic novelty, functional diversity and biosynthetic capacity, providing new genomic and ecological insights into <i>Bacilli</i> associated with plants inhabiting extreme environments.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149039702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nanopore metagenomic sequencing links clinically relevant resistance determinants to pathogens.","authors":"Harika Ürel, Ela Sauerborn, Michael Biggel, Friedemann Gebhardt, Ebenezer Foster-Nyarko, Silvio D Brugger, Rhys T White, Søren Heidelbach, Mads Albertsen, Francis Muchaamba, Tim Reska, Marc J A Stevens, Roger Stephan, Richard Fetherston, Lara Urban","doi":"10.1099/mgen.0.001834","DOIUrl":"10.1099/mgen.0.001834","url":null,"abstract":"<p><p>Culture-independent metagenomics enables the detection of plasmid-encoded antimicrobial resistance (AMR) genes directly from clinical samples; however, the clinical significance of these genes depends on their bacterial host and genomic context, which metagenomics cannot fully infer. Nanopore sequencing technology intrinsically encodes epigenetic modifications such as methylation, which can be leveraged for plasmid-host associations from metagenomic data. Existing methods rely on the recovery of metagenome-assembled genomes (MAGs), which can introduce bias towards abundant taxa and leave clinically relevant, low-abundance pathogens unassociated. To address this limitation, we extended methylation-based plasmid-host association from the MAG level to individual assembly contigs and sequencing reads. The Contig- and Unassembled-read-based Pathogen Identification and Delineation (CUPID) pipeline implements the calculation of contig and read similarity scores, which compare weighted mean methylation rates across motifs genetically shared between any contig or read pair. We validated this approach on a mock metagenomic community composed of ten carbapenem-resistant <i>Enterobacterales</i> isolates, where we achieved 93.8% accuracy at the contig level and 100% at the read level for carbapenemase plasmid-host associations. When applied to metagenomic and quasimetagenomic data of 16 patient rectal swabs collected during routine hospital surveillance, our approach assigned every detected plasmid-encoded carbapenemase to its correct bacterial host at the contig level, using matched culture-based diagnostics and whole-genome sequencing as the ground truth. Read-level analysis identified additional associations that were missed at the contig level, including a multi-host plasmid confirmed by established diagnostics. These findings demonstrate a pathway from rapid AMR gene detection using metagenomics to actionable surveillance for infection prevention, transmission tracing and outbreak investigation.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 9","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13592730/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149017282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}