Microbial Genomics最新文献

{"title":"Refining <i>Salinivibrio</i> pangenome dynamics and biotechnological potential through comparative analysis.","authors":"Crystal E Young, Harrison O'Sullivan, Hussain Alattas, Ravi Tiwari, Andrew Macrae, Daniel V Murphy, Colin Scott","doi":"10.1099/mgen.0.001786","DOIUrl":"10.1099/mgen.0.001786","url":null,"abstract":"<p><p>Current understanding of genomic diversity within the halophilic genus <i>Salinivibrio</i> relies predominantly on draft genomes, with only seven complete genomes among the 62 publicly available. Previous pangenome analysis suggested a closed genomic structure while concluding that <i>Salinivibrio</i> lacks polyhydroxyalkanoate (PHA) degradation capacity despite possessing biosynthesis genes. Here, we present eight complete <i>Salinivibrio</i> genomes from Pearse Lakes (Rottnest Island, Western Australia) generated using Oxford Nanopore long-read sequencing, alongside re-analysis of 38 high-quality public genomes (≥90% completeness and ≤5% contamination cut-off). Pangenome analysis revealed a more open structure than previously reported, with a core genome comprising 25% of total gene clusters and an accessory genome accounting for 71%. Panstripe analysis demonstrated significant temporal signal in gene gain and loss events associated with phylogenetic branch length (core: <i>P</i>=1.72×10⁻⁴; tip: <i>P</i>=2.64×10⁻¹⁴). All 46 genomes contained complete PHA biosynthesis operons (<i>phaB-phaA-phaP-phaC</i>) with high sequence conservation under strong purifying selection (Z=30.30, <i>P</i><0.001). In a genome that readily gains and loses genes, this conservation indicates that PHA synthesis is a maintained pathway, which is difficult to reconcile with a previous report that <i>Salinivibrio</i> lacks PHA degradation capacity. We therefore searched the genomes by Hidden Markov Model-based homology rather than standard annotation and identified seven putative depolymerases that form a single accessory cluster in 15% of strains, all previously annotated as 3-oxoadipate enol-lactonase-2. These candidates retained all catalytic residues characteristic of active depolymerases but are divergent from reference PHA depolymerases which could explain why annotation missed them. They remain putative and require biochemical confirmation. Both the expanded pangenome and these candidates emerged from standardized homology-based re-analysis, showing that annotation-dependent approaches can overlook genomic diversity and divergent enzyme families in non-model organisms. Together, these results establish <i>Salinivibrio</i> as a genomically dynamic genus with potential for halophilic bioplastic production.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13384114/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148520687","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":"Mapping potential pathogen profiling in cetacean blow: comparative insights from sequencing technologies.","authors":"Johann Frederick Jaitner, Nicola Gambardella, Luís Afonso, Raul Valente, Maria Paola Tomasino, Ana Mafalda Correia, Massimiliano Rosso, Filipe Alves, Catarina Magalhães","doi":"10.1099/mgen.0.001773","DOIUrl":"10.1099/mgen.0.001773","url":null,"abstract":"<p><p>Cetaceans play a critical role in marine ecosystems and function as sentinel species for detecting environmental perturbations, underscoring the importance of assessing their health for effective marine conservation. This study employed 16S rRNA gene sequencing to characterize the prokaryotic communities present in exhaled breath condensate (EBC) samples from cetaceans, utilizing both short-read (Illumina) and long-read (PacBio) sequencing platforms. Putative pathogenic taxa were identified using the Multiple Bacterial Pathogen Detection (MBPD) database. Substantial differences in microbial community composition were observed between sequencing approaches. The PacBio platform yielded 2,373 amplicon sequence variants (ASVs) spanning 30 bacterial phyla, with 614 ASVs identified as potential pathogens. In contrast, the Illumina dataset generated 350 ASVs across 17 phyla, of which 46 were flagged as potentially pathogenic. Discrepancies were also evident in diversity metrics: PacBio-derived profiles exhibited higher alpha diversity and produced beta diversity clustering patterns that corresponded with sample metadata, while Illumina-based profiles did not reveal meaningful clustering. Distinct EBC microbial signatures were identified for <i>Globicephala macrorhynchus</i> and <i>Delphinus delphis</i>, with clear differences from the surrounding seawater microbiota. These findings support the use of EBC as a non-invasive and informative tool for respiratory microbiome analysis in marine mammals. Notably, this study provides the first characterization of the respiratory microbiota in <i>D. delphis</i>, offering a valuable methodological baseline for future research into host-microbiome interactions, health assessment and putative pathogen monitoring in free-ranging cetacean populations, using non-invasive approaches.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13344881/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148405196","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":"<i>In silico</i> and experimental assessment of the 16S-ITS-23S operon as a genetic marker for high-resolution bacterial species identification.","authors":"Ana C Yamakawa, Matthew Davies, Hyun S Gweon","doi":"10.1099/mgen.0.001793","DOIUrl":"10.1099/mgen.0.001793","url":null,"abstract":"<p><p>Accurate species-level identification of bacteria is crucial for public health surveillance, but standard methods like 16S rRNA hypervariable region (HVR) sequencing often lack sufficient resolution. This study systematically evaluates the taxonomic resolving power of the full 16S-ITS-23S ribosomal RNA (<i>rrn</i>) operon, enabled by high-accuracy long-read technology, compared to the full-length 16S gene and HVRs. A dual-validation framework was employed, combining an <i>in silico</i> analysis of a curated reference database with an experimental validation using rat faecal samples. In silico, the <i>rrn</i> operon demonstrated unequivocally superior accuracy across the full range of tested thresholds for both nucleotide mismatches and pairwise identity, consistently maintaining the highest proportion of monospecies clusters (i.e. clusters containing a single species). For instance, at a relaxed threshold of 30 mismatches, the operon maintained 96.8% monospecies clusters, compared to 80.5% for the full-length 16S gene and <66% for all HVRs. This theoretical advantage was confirmed experimentally through three parallel sequencing strategies: operon sequencing enabled species-level taxonomic assignment for 75.2% of unique sequences, significantly outperforming full-length 16S (62.1%) and dramatically surpassing the standard V4 HVR approach (18.5%). An internal bias-control analysis confirmed that these differences were due to the superior information content of the operon marker itself. Our findings provide robust evidence that high-accuracy long-read sequencing of the <i>rrn</i> operon is a superior method for culture-free bacterial surveillance, offering a new gold standard for high-resolution taxonomic profiling in complex environmental and host-associated samples.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13399229/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579181","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":"Genomic analysis of intrahospital transmission of carbapenem-resistant Gram-negative bacteria: a multicentre study in Japan.","authors":"Yasufumi Matsumura, Yohei Doi, Kayoko Hayakawa, Takehiro Hashimoto, Yusuke Tsuda, Sho Saito, Masahiro Suzuki, Aki Sakurai, Koh Shinohara, Keiichiro Mori, Kohei Uemura, Shinya Tsuzuki, Takashi Matono, Naoya Itoh, Ryota Hase, Hideaki Kato, Momoko Mawatari, Takeya Tsutsumi, Go Yamamoto, Yasuhiro Tsuchido, Masaki Yamamoto, Miki Nagao","doi":"10.1099/mgen.0.001770","DOIUrl":"10.1099/mgen.0.001770","url":null,"abstract":"<p><p>We aimed to identify genomically defined strain transmission events of carbapenem-resistant Gram-negative bacteria (CRGNB) in hospitals, estimate species-specific transmission rates and identify risk factors for transmission. The study included 708 patients from a Japanese prospective multicentre cohort study, the MultiDrug-Resistant organisms clinical research network, from whom at least one CRGNB isolate was collected between 2019 and 2024 (750 isolates in total). Clinical data were retrieved from electronic medical records, and microbiological analyses were performed at a central reference laboratory. Transmission events were determined via whole-genome sequencing data and single-nucleotide polymorphism-based genomic clustering. We identified 37 genomic clusters associated with intrahospital transmission and 1 associated with interhospital transmission. Ninety-four patients (13%) carried at least one isolate involved in intrahospital transmission, and each transmission event involved two to six patients. Patients with <i>Acinetobacter</i> spp. (60% involved in intrahospital transmission), <i>Enterobacter cloacae</i> complex (23%) and <i>Klebsiella oxytoca</i> complex (59%) were significantly associated with intrahospital transmission and were considered high-risk species groups. Patients with <i>Enterobacterales</i> isolates carrying acquired carbapenemase genes had higher intrahospital transmission rates relative to those without (31% vs. 2%). In multivariable analysis, independent risk factors for patients with intrahospital transmission were overlap in ward and time with other CRGNB-positive patients, carriage of high-risk species groups and acquired carbapenemase genes. In this Japanese multicentre cohort, intrahospital transmission of CRGNB was associated with both microbiological characteristics and patient- and healthcare-related factors, underscoring the need for targeted surveillance and infection prevention strategies.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13340626/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148396902","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":"<i>Salmonella</i> bloodstream infection and concurrent intestinal colonization in children with severe acute malnutrition in Niger.","authors":"Kirsty Sands, Raúl Campillo, Ian Boostrom, Giulia Lai, Aditya Kumar Lankapalli, Kate Cook, Brekhna Hassan, Edward Ar Portal, Mei Li, Lim S Jones, Tassiou Elhadji Ibrahim, Idi Kalla, Nathan Sayinzonga-Makombe, Sheila Isanaka, Rupa Kanapathipillai, Christopher Mambula, Isabelle Mouniaman, Céline Langendorf, Timothy R Walsh, Owen Brad Spiller","doi":"10.1099/mgen.0.001788","DOIUrl":"10.1099/mgen.0.001788","url":null,"abstract":"<p><p>Children with severe acute malnutrition (SAM) are at high risk of invasive infection. In sub-Saharan Africa, invasive non-typhoidal <i>Salmonella enterica</i> (iNTS) serovars are a leading cause of paediatric bloodstream infection. However, low-resource settings lack genomic data linking intestinal carriage with concurrent BSI in children presenting with SAM.We conducted a longitudinal cohort study among children aged 0-59 months admitted for inpatient management of complicated SAM at the Madarounfa Intensive Nutritional Rehabilitation Centre, Maradi, Niger (2016-2017). Blood cultures and rectal swabs were obtained on admission (and during hospitalization if symptoms worsened). Genomic population structure of <i>S. enterica</i> serovars Enteritidis and Typhimurium was compared using phylogeny and clustering approaches. Among 1,371 enrolled children, 87 <i>Salmonella</i> isolates were recovered from 83 BSI episodes in 80 children. Invasive isolates belonged to a few globally circulating lineages, whereas carriage-only isolates demonstrated more heterogeneous genotypes. Minimum inhibitory concentrations were determined for 58 viable BSI isolates: susceptibility was retained to third-generation cephalosporins and carbapenems, while resistance to ampicillin (95%) and amoxicillin/clavulanate (90%) was common.A nested case-control analysis of rectal swabs from 232 children (58 iNTS BSI cases; 174 non-BSI controls) was performed to test the association between gut carriage and BSI. <i>Salmonella</i> was detected in 44/58 (76%) cases vs. 7/174 (4%) controls, yielding a relative risk of 18.86 (CI 95% 9.00-39.53; <i>χ</i>²=126.7; <i>P</i><0.0001). Clustering analyses confirmed close genomic relatedness within patient pairs and indicated circulation of a limited number of endemic clones. Among controls with faecal <i>Salmonella</i>, serovars were diverse and distinct from invasive lineages.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13384236/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148520712","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":"Lineage-aware comparison of extended-spectrum <i>β</i>-lactamase-producing <i>Escherichia coli</i> from unweaned dairy calves and human references reveals host-structured plasmidomes and co-selection.","authors":"Mykhailo Savin, Jason Jeremia Hayer, Nico T Mutters, Tim Erler, Sophie Simon, Lea Griesdorn, Julia Steinhoff-Wagner, Jens A Hammerl, Céline Heinemann, Alexander J Probst","doi":"10.1099/mgen.0.001783","DOIUrl":"10.1099/mgen.0.001783","url":null,"abstract":"<p><p>Antimicrobial resistance in <i>Escherichia coli</i> is shaped not only by resistance genes themselves but also by their chromosomal or plasmid localization and co-occurrence with biocide/metal resistance genes (BMRGs), virulence-associated genes and mobile genetic elements. We applied chromosome- and plasmid-resolved genomics to 109 extended-spectrum <i>β</i>-lactamase-producing <i>E. coli</i> isolates from unweaned dairy calves (<i>n</i>=484) in Germany and compared them with 479 human-associated reference genomes. Calf isolates were polyclonal and dominated by phylogroups A and B1. Resistance was predominantly plasmid-borne: 41% of isolates carried antibiotic resistance genes (ARGs) exclusively on plasmids, whereas only 4.6% carried ARGs exclusively on chromosomes. The chromosomal-versus-plasmid distribution of acquired ARGs differed significantly across phylogroups (<i>P</i><0.05) and sequence types (all <i>P</i><0.01). Conjugative plasmids accounted for 94.6% of plasmid-borne ARG occurrences and carried significantly more ARGs than mobilizable plasmids (<i>P</i>=3.66×10^-42). ARG and BMRG counts were strongly correlated at the plasmid level (ρ=0.574, <i>P</i>=8.0×10^-41), and class 1 integrons marked enriched multidrug plasmids with increased ARGs (<i>P</i>=6.22×10^-34) and BMRGs (<i>P</i>=3.00×10^-29). At the isolate level, calf isolates carried more acquired ARGs in unadjusted comparisons, but this host-associated difference was largely explained by population structure. At the plasmid level, however, host-associated differences persisted after adjustment: human plasmids carried more ARGs (IRR 1.66, <i>P</i>=0.0017) and showed a strong host×mobility interaction (IRR 4.61, <i>P</i>=4.9×10-8), stronger ARG-BMRG coupling and a higher prevalence of integrons. These findings show that antimicrobial resistance ecology in <i>E. coli</i> is shaped not only by which resistance genes are present, but by where they are located, what they are linked to and how readily their genomic carriers can disseminate.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13379246/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148471354","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":"Novel transposon Tn<i>8026</i> contributes to the global spread of transmissible linezolid resistance in <i>Enterococcus</i> via a linear plasmid.","authors":"Michael B Hall, Yiyang Xue, Tricia S E Lee, Ella Herring, Jocelyn Hume, Ryan R Wick, Timothy Kidd, Naomi Runnegar, Patrick N A Harris, Bianca Graves, Leah W Roberts","doi":"10.1099/mgen.0.001803","DOIUrl":"10.1099/mgen.0.001803","url":null,"abstract":"<p><p>Linezolid is a critical last-resort antimicrobial for multidrug-resistant <i>Enterococcus faecium</i>, particularly against vancomycin-resistant lineages where therapeutic options are severely limited. While resistance has historically arisen through <i>de novo</i> chromosomal mutations, the global emergence of transferable resistance mechanisms threatens to render more infections untreatable. Here, we characterize a recent (2023-2024) hospital-associated outbreak of linezolid-resistant <i>E. faecium</i> in Queensland, Australia. Although the cohort comprised a variety of sequence types, the outbreak was primarily driven by the clonal expansion of an ST80 lineage carrying the plasmid-borne <i>poxtA</i> gene. Standard short-read genomic surveillance failed to resolve the genetic context of the resistance determinant. However, long-read sequencing revealed that <i>poxtA</i> was carried within a novel transposon, Tn<i>8026</i>, situated on a linear plasmid. Structural analysis defined Tn<i>8026</i> as a unique element flanked by IS<i>1678</i> and the novel insertion sequence IS<i>Efa26</i>. Furthermore, we identified an instance of Tn<i>8026</i> integration into the chromosome, providing functional evidence of its mobility and capacity for stabilization within the genome. Global genomic screening demonstrated that Tn<i>8026</i> significantly predates the local outbreak, identified in a historical Norwegian isolate from 2012, indicating a long-standing yet unrecognized global reservoir. Phylogenomic analysis provided strong evidence that the linear plasmid was imported from the Indian subcontinent, initiating a chain of silent dissemination in eastern Australia where the lineage circulated undetected prior to clinical recognition. Crucially, we also confirmed the presence of the linear plasmid in <i>Enterococcus gallinarum</i>, demonstrating its capacity to mobilize transmissible linezolid resistance across enterococcal species boundaries. These findings emphasize the need for detailed long-read-based surveillance of mobile genetic elements, with a particular focus on identifying linear plasmids that are often overlooked.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13417996/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148605046","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":"A microbial mirage: when microbiome metrics may obscure ecological meaning.","authors":"Jake M Robinson, Linda Guentas, Martin F Breed","doi":"10.1099/mgen.0.001777","DOIUrl":"10.1099/mgen.0.001777","url":null,"abstract":"<p><p>Metrics such as alpha diversity, inferred functional potential and network complexity have become standard metrics in microbiome research. While they offer convenient ways to summarize complex data, these metrics may sometimes obscure more than they reveal. Alpha diversity, for example, measures richness and evenness. However, two samples may exhibit identical diversity scores, yet one could be dominated by beneficial taxa and the other by pathogens. Similarly, the presence of genes associated with particular functions does not guarantee that those functions are expressed or ecologically relevant under given conditions. Functional inference is also limited by database bias and often lacks empirical validation. Likewise, correlation-based network analyses can produce spurious associations driven by shared environmental covariates, sequencing depth or batch effects. These issues are routinely encountered in genomic workflows - from 16S/ITS amplicon surveys to shotgun metagenomics, genome-resolved metagenomics and gene-centric network analyses - where apparently 'clean' summary metrics can mask very different ecological realities. Here, we use simple, domain-relevant examples to illustrate how over-reliance on these metrics can lead to misinterpretation. Rather than rejecting these approaches, we outline when they are most informative, when they require caution and what complementary analyses can strengthen ecological inference. We propose a practical framework based on four questions: what exactly is being summarized, at what biological level, under which ecological conditions and with what form of validation? While acknowledging their value, we argue for greater critical scrutiny in their application and interpretation, and advocate for approaches that prioritize functional validation, temporal resolution and systems thinking to support more meaningful ecological insight.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148405182","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":"Performance of mechanically sheared DNA in multiplexed Oxford Nanopore sequencing for <i>Salmonella</i> Typhi genomic surveillance.","authors":"Hsu Thinzar Maung, Masamoto Morita, Su Myat Han, Hiroyuki Mishima, Chisa Koga, Jan Wendzl C Evangelista, Mark John Girasol, Shuichi Suzuki, Koh-Ichiro Yoshiura, Toshio Kodama, Chris Smith, Hirotaka Hiyoshi","doi":"10.1099/mgen.0.001805","DOIUrl":"10.1099/mgen.0.001805","url":null,"abstract":"<p><p>Pathogen genomic surveillance is essential for monitoring high-risk lineages and antimicrobial resistance in <i>Salmonella enterica</i> serovar Typhi (<i>S</i>. Typhi), yet implementing whole-genome sequencing in sentinel hospital laboratories remains challenging. We evaluated whether mechanically sheared DNA, paired with high accuracy or super accuracy (SUP) basecalling, enables reliable Oxford Nanopore Technologies (ONT)-only sequencing for multiplexed <i>S</i>. Typhi genomic surveillance. Genomic DNA from three benchmark strains (Ty2, Ty42 and Ty43) was either mechanically sheared to ~15 kb or left unsheared and sequenced in a controlled 6-plex run. The optimized workflow was subsequently evaluated in a 24-plex field run that included 3 benchmark strains and 21 clinical isolates from a sentinel hospital in Manila, Philippines. Both runs were performed using the ONT Ligation Sequencing Kit with Native Barcoding 24 V14 (SQK-NBD114.24) and R10.4.1 flow cells (FLO-MIN114). Assembly metrics, single-nucleotide variation (SNV) concordance, core-genome MLST and downstream functional outputs were compared across DNA preparations and basecalling modes. In the 6-plex run, sheared libraries consistently achieved >1,000× pre-filtered coverage across all strains, whereas unsheared libraries showed highly variable depths and reduced stability after downsampling. When paired with SUP basecalling, sheared datasets produced assemblies comparable to Illumina and hybrid references across both structural accuracy and functional outputs. In the 24-plex run, all samples achieved >100× coverage, and 18 of the 22 retained <i>S</i>. Typhi genomes (81.8%) yielded fully circularized assemblies after exclusion of 2 non-<i>S</i>. Typhi isolates. Serotype prediction, genotyping and <i>in silico</i> antimicrobial resistance predictions remained concordant with the corresponding 6-plex benchmark results. These findings demonstrate that mechanical DNA shearing improves coverage uniformity under multiplexed conditions and, together with SUP basecalling, supports ONT-only <i>S</i>. Typhi genome reconstruction for routine genomic surveillance and broad phylogenetic contextualization. However, for fine-scale transmission analysis and high-resolution SNV interpretation, short-read or hybrid sequencing remains important. Overall, this approach provides a scalable framework for high-multiplex <i>S</i>. Typhi genomic surveillance in sentinel laboratory settings.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13405487/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148605148","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":"Benchmarking DNA extraction protocols across use cases for culture-independent Nanopore metagenomics.","authors":"Tara Signorelli, Matthew Walker, James Robertson, Kaye Quizon, Yongxiang Zhang, Aleisha R Reimer, Shannon H C Eagle","doi":"10.1099/mgen.0.001738","DOIUrl":"10.1099/mgen.0.001738","url":null,"abstract":"<p><p>Oxford Nanopore Technologies (ONT) sequencing offers several advantages for metagenomics, including long reads, rapid turnaround, low upfront cost, scalability and portability. However, for ONT metagenomics, DNA yield, quality and integrity are important considerations when selecting an extraction method. Many metagenomic extraction methods use harsh lysis conditions to extract a wide range of species and provide an accurate community composition, but these conditions can compromise DNA fragment length. Therefore, extraction methods for ONT metagenomics must balance DNA shearing and recovery with representative community lysis. We systematically evaluated DNA extraction methods for ONT metagenomic sequencing using a use case-oriented framework. Among nearly 50 extraction methods screened, 7 were selected for detailed comparison based on suitability for metagenomics, variation in methodology, availability, cost and processing time: Norgen BioTek Corp's Stool DNA Isolation (NG), Zymo Research's ZymoBIOMICS Quick-DNA HMW MagBead (ZMG), Qiagen's DNeasy Blood and Tissue (QBT), Macherey-Nagel's NucleoMag DNA Microbiome (MN), Zymo Research's ZymoBIOMICS DNA Mini Prep (ZMI), Qiagen's DNeasy PowerSoil/QIAamp PowerFecal Pro (PS) and Qiagen's QIAamp Fast DNA Stool Mini (QIA). Methods were tested using Zymo Research's ZymoBIOMICS Microbial Community Standard (MCS), a matrix-free mock community with known composition. DNA extracts were sequenced on an ONT PromethION using the Rapid Barcoding Kit, except QIA due to insufficient DNA yield. Metrics for the method, DNA extracts, sequencing and genomes were evaluated, revealing trade-offs between methods. The two magnetic bead methods, MN and ZMG, produced the highest mean read length N50 values (13.9 and 16.5 kb, respectively) but showed apparent community compositions skewed towards Gram-negative bacteria. In contrast, ZMI and PS maintained a community composition close to expected, with reduced mean read length N50 values (4.5 vs. 7.5 kb). Performance across various metrics is presented in the context of the following use cases: maximizing genome coverage and assembly completeness, preserving composition accuracy, targeting specific species and limiting required resources (equipment, time or budget). The metrics and use case considerations presented offer practical guidance for informed selection of DNA extraction methods for ONT metagenomics. For accurate community composition, ZMI or PS are recommended, while PS and ZMG perform best at maximizing genome coverage and assembly completeness. NG and QBT may be the most economical options, though performance trade-offs were observed. Finally, PS may be the preferred method for time-sensitive diagnostic or field applications.</p>","PeriodicalId":18487,"journal":{"name":"Microbial Genomics","volume":"12 7","pages":""},"PeriodicalIF":4.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13543651/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148448116","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}