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PubMed2026

Conference report: the third Bacterial Genome Sequencing Pan-European Network conference.

The third Bacterial Genome Sequencing Pan-European Network conference, held in Engelberg, Switzerland (12-15 January 2026), brought together experts from six European countries to discuss the implementation of bacterial genome sequencing in clinical microbiology and public health. Key themes included regulatory frameworks (In Vitro Diagnostic Regulation, General Data Protection Regulation), standardization, quality control, data sharing, economic evaluation, and the integration of artificial intelligence and long-read sequencing into diagnostic workflows. Across presentations, panel discussions, and workshops, participants emphasized that successful implementation of genome sequencing requires more than technical capacity: it depends on robust validation, sustainable funding, interoperable data standards, ethical governance, and interdisciplinary collaboration. The meeting highlighted that sequencing should remain question-driven and clinically meaningful, balancing cost, turnaround time, and public health impact. Overall, the conference reinforced the need for coordinated European efforts to advance responsible, standardized, and sustainable genomic surveillance and diagnostics.

bacterial genome sequencing↗
PubMed2026

A tiled amplicon protocol for culture-free whole-genome sequencing of M. tuberculosis from clinical specimens.

Whole-genome sequencing of Mycobacterium tuberculosis can be a valuable tool for TB surveillance and treatment, providing insights into transmission patterns and comprehensive drug susceptibility testing. However, the slow growth of M. tuberculosis means traditional culture-based sequencing methods can take weeks to return results, which has limited the widespread adoption of these techniques and limited their use in clinical decision-making. Tiled amplicon sequencing is a fast, reliable, and cost-effective method of whole-genome sequencing that can be done directly on clinical specimens and has been implemented at scale in academic and public health laboratories across the world; it was the cornerstone of SARS-CoV-2 sequencing and has been adapted for a wide range of viral pathogens. However, similar methods are not yet available for far larger bacterial genomes. Extending this approach to M. tuberculosis would significantly reduce the cost, labor, and turnaround time for whole-genome sequencing. We designed a tiled amplicon panel consisting of 5,128 primers that covers the entire M. tuberculosis genome, the largest tiled amplicon sequencing panel we are aware of to date. Applying our amplicon panels to clinical samples of sputum, we show the ability to recover whole-genome bacterial sequences without the need for culture. The resulting sequence data can be used to determine M. tuberculosis lineage and reliably identify markers of drug resistance. Using this approach in clinical settings could reduce the time needed for comprehensive drug susceptibility testing from weeks to days and enable genomic epidemiology to be performed at scale, even in resource-limited settings.IMPORTANCEWe have developed and tested an amplicon panel, TB-seq, for the priority pathogen Mycobacterium tuberculosis, demonstrating recovery of near-full genomes directly from patient sputum, including mixed and low-concentration samples. This approach significantly reduces the turnaround time for this slow-growing bacterium while maintaining high accuracy in detecting clinically relevant mutations, including those associated with drug resistance. Given the global burden of tuberculosis and the critical need for faster diagnostic solutions, we believe our method has the potential to improve clinical decision-making and public health strategies.

Mycobacterium tuberculosis↗
PubMed2026

Applications of transposon-insertion sequencing for understanding bacterial physiology.

Transposon-insertion sequencing (Tn-seq) couples transposon mutagenesis with next-generation sequencing to identify the transposon insertion site for thousands of mutants in parallel. It is a powerful technology with a myriad of uses beyond the identification of essential genes required for a cell to grow and divide. Tn-seq is particularly useful as a high-throughput method to assign function to function-unknown genes, which have increased steadily with the abundance of newly sequenced bacterial genomes. Tn-seq has now been adapted for use in over 100 bacterial species. Here, we summarize the applications of Tn-seq for querying bacterial physiology and discuss some of the possible applications for the future.

DNA Transposable Elements↗
PubMed2025

Autocycler: long-read consensus assembly for bacterial genomes.

MOTIVATION: Long-read sequencing enables complete bacterial genome assemblies, but individual assemblers are imperfect and often produce sequence-level and structural errors. Consensus assembly using Trycycler can improve accuracy, but its lack of automation limits scalability. There is a need for an automated method to generate high-quality consensus bacterial genome assemblies from long-read data. RESULTS: We present Autocycler, a command-line tool for generating accurate bacterial genome assemblies by combining multiple alternative long-read assemblies of the same genome. Without requiring user input, Autocycler builds a compacted De Bruijn graph from the input assemblies, clusters and filters contigs, trims overlaps, and resolves consensus sequences by selecting the most common variant at each locus. It also supports manual curation when desired, allowing users to refine assemblies in challenging or important cases. In our evaluation using Oxford Nanopore Technologies reads from five bacterial isolates, Autocycler outperformed individual assemblers, automated pipelines, and other consensus tools, producing assemblies with lower error rates and improved structural accuracy. AVAILABILITY AND IMPLEMENTATION: Autocycler is implemented in Rust, open-source, and freely available at github.com/rrwick/Autocycler. It runs on Linux and macOS and is extensively documented.

Genome, Bacterial↗
PubMed2026

Draft genome sequences of four bacterial isolates from the Indian Fort Nature Preserve in Geneseo, NY.

We report the genome sequences of four bacterial strains, Bacillus subtilis, Bacillus pumilus, Pseudomonas kermanshahensis, and Kocuria rhizophila, isolated from soil or plant material from Geneseo, NY. Bacterial strains were selected based on preliminary, qualitative screening for antimicrobial production via zones of clearing and/or inhibition against lab strains.

antimicrobial production↗
PubMed2026

Draft genome sequences of 14 bacterial isolates from the rhizosphere of bioenergy sorghum.

We report the draft genome sequences of a collection of 14 bacterial isolates obtained from the rhizosphere soil of bioenergy sorghum (Sorghum bicolor [L.] Moench). These isolates represent the genera Acidovorax, Nocardioides, Agrobacterium, Peribacillus, Caulobacter, Pseudomonas, Rhizobium, Sphingomonas, Priestia, Dyadobacter, Roseomonas, and Bacillus.

bacterial isolates↗
PubMed2026

Genome sequences of four bacterial strains isolated from the phyllosphere of Mangifera indica trees in the polluted tropical city of Medellín, Colombia.

Complete and draft genome sequences of four phyllosphere-associated bacterial strains (Microbacterium radiodurans, Brachybacterium rhamnosum, Sphingomonas citri, and Curtobacterium sp.) isolated from Mangifera indica leaves in polluted Medellín, Colombia, are presented. These resources enable future studies on plant-microbe interactions and phyllosphere microbial mediation of atmospheric pollutants under urban stress.

Mangifera indica↗
PubMed2025

Emergence of clade 3 emm89 group A Streptococcus in Queensland, Australia.

The emergence of a new clade of emm89 group A Streptococcus (GAS) (clade 3) has been described in several countries. Strains in this clade have been reported to have genomic characteristics that lead to increased expression of virulence factors and may confer a selective advantage over previous emm89 strains. To investigate whether clade 3 GAS is present in the emm89 GAS population of Queensland, Australia, all emm89 GAS isolates received by the Queensland Public Health Microbiology Reference Laboratory since emm typing began in the early 2000s underwent genomic sequencing and analysis. Analysis of sequences from 293 emm89 GAS isolates demonstrated the presence of distinct genomic groups in the Queensland emm89 GAS population. Unlike emm89 GAS populations described in the UK and USA, which were mostly ST101 and ST407, there were a relatively high number of ST142 and ST812 strains in the Queensland emm89 GAS population. However, the majority of Queensland isolates belonged to clade 3, with 80% (n=233) of emm89 GAS isolated from 2006 onwards belonging to this clade. All Queensland clade 3 isolates had the reported genomic features associated with higher virulence potential including increased streptolysin O production and an acapsular phenotype. Clade 3, which has emerged to become the dominant clade of emm89 GAS in Europe and the USA, is now also the dominant clade in Queensland.

Streptococcus pyogenes↗
PubMed2024

Widespread detoxifying NO reductases impart a distinct isotopic fingerprint on N2O under anoxia.

Nitrous oxide (N2O), a potent greenhouse gas, can be generated by multiple biological and abiotic processes in diverse contexts. Accurately tracking the dominant sources of N2O has the potential to improve our understanding of N2O fluxes from soils as well as inform the diagnosis of human infections. Isotopic "Site Preference" (SP) values have been used toward this end, as bacterial and fungal nitric oxide reductases (NORs) produce N2O with different isotopic fingerprints, spanning a large range. Here, we show that flavohemoglobin (Fhp), a hitherto biogeochemically neglected yet widely distributed detoxifying bacterial NO reductase, imparts a distinct SP value onto N2O under anoxic conditions (~+10‰) that correlates with typical environmental N2O SP measurements. Using Pseudomonas aeruginosa as a model organism, we generated strains that only contained Fhp or the dissimilatory NOR, finding that in vivo N2O SP values imparted by these enzymes differ by over 10‰. Depending on the cellular physiological state, the ratio of Fhp:NOR varies significantly in wild-type cells and controls the net N2O SP biosignature: When cells grow anaerobically under denitrifying conditions, NOR dominates; when cells experience rapid, increased nitric oxide concentrations under anoxic conditions but are not growing, Fhp dominates. Other bacteria that only make Fhp generate similar N2O SP biosignatures to those measured from our P. aeruginosa Fhp-only strain. Fhp homologs in sequenced bacterial genomes currently exceed NOR homologs by nearly a factor of four. Accordingly, we suggest a different framework to guide the attribution of N2O biological sources in nature and disease.

Nitrous Oxide↗
PubMed2026

Plasmid-mediated dissemination of blaKPC-3 and multidrug resistance genes among different species of Klebsiella.

Carbapenem resistance is a serious threat to public health because carbapenems are used as last-resort antibiotics. Carbapenem resistance gene KPC (Klebsiella pneumoniae carbapenemase) inactivates a broad range of β-lactam substrates. In this manuscript, we examined intra-host transmission of blaKPC-3 via interspecies gene transfer. Two carbapenem-resistant Klebsiella pneumoniae isolates and one Klebsiella michiganensis isolate were identified from two patients. Genetic relations of these isolates were investigated with whole-genome sequencing (WGS). Hybrid assembly of bacterial genomes showed the three isolates carried plasmids that harbor common antimicrobial resistance (AMR) gene clusters that confer multidrug-class resistance, including carbapenems. Our results suggest that AMR gene clusters are disseminated across the species as fragments rather than as complete, intact plasmids.IMPORTANCEAn antimicrobial resistance gene cluster encompassing multiple drug classes on plasmids could lead a drug-susceptible pathogen to gain multidrug resistance. Interspecies gene transfer enables K. michiganensis to become multidrug-resistant through the acquisition of clustered, plasmid-encoded resistance genes spanning multiple antibiotic classes.

Plasmids↗
PubMed2025

A Step-by-Step Guide to Sequencing and Assembly of Complete Bacterial Genomes Using the Oxford Nanopore MinION.

The Oxford Nanopore (ONT) MinION enables sequencing of longer DNA/RNA fragments compared to other sequencers, such as Illumina, etc. This nanopore method provides distinct advantages for generating complete genome assemblies from microorganisms. Specifically, the R9.4 flow cells used for MinION sequencing have much lower error rates compared with earlier versions of the ONT platform. Coupled with base calling using Dorado software, higher-quality long reads can now be generated for complete bacterial genome assembly. In this chapter, we describe a detailed MinION method to assemble a complete genome from a microorganism, polish the final assembly, and evaluate the genome quality using various software tools. Because of the low cost for MinION sequencing, this platform could be an asset for virtually any laboratory interested in generating complete genomes from microorganisms.

Genome, Bacterial↗
PubMed1979

Deoxyribonucleic acid sequence homologies among bacterial insertion sequence elements and genomes of various organisms.

Plasmid and phage deoxyribonucleic acid (DNA) harboring bacterial insertion sequence (IS) elements IS1, IS2, and IS5 were characterized and used as probes to detect homologous sequences in various procaryotic and eucaryotic genomes. The hybridization method used permits the detection of sequences partially homologous to the elements. Hybridization of the IS-containing probes to each other revealed a region of limited homology between IS1 and IS2. Homologous sequences were then detected by computer analysis of the published IS1 and IS2 nucleotide sequences. The homologous sequence contains a tandemly repeated tetranucleotide sequence which resembles the repeated sequence at the hot spot for spontaneous mutations in the lacI gene (P. J. Farabaugh, U. Schmeissner, M. Hofer, and J. Miller, J. Mol. Biol. 126:847-863, 1978). Homology between the IS elements and various genomes was determined by hybridizing labeled DNA containing IS1, IS2, and IS5 sequences to Southern blots of chromosomal DNA cleaved with restriction endonucleases. IS1 and IS5 appear limited to the enteric bacteria, whereas IS2 sequences can also be detected in Pseudomonas putida, Pseudomonas aeruginosa, and Serratia marcescens. Bacteria which appear not to possess extrachromosomal elements, e.g., Caulobacter crescentus, did not show homology with any insertion sequences tested. In addition, sequences homologous to IS1, IS2, or IS5 were not detected in Saccharomyces cerevisiae, Dictyostelium discoideum, or calf thymus DNA.

Bacteria↗
PubMed2025

Using Chromosome Conformation Capture Combined with Deep Sequencing (Hi-C) to Study Genome Organization in Bacteria.

Genome organization is fundamental to all living organisms. Long DNA molecules are organized in hierarchical orders to be accommodated into eukaryotic nuclei or bacterial cells, which are thousands of folds shorter. Over the past two decades, chromosome conformation capture (3C) techniques substantially advanced our understanding of genome folding inside cells. 3C involves crosslinking and proximity ligation, and quantifies the physical contacts between two DNA regions within the genome. Coupled with high-throughput sequencing, 3C-seq and Hi-C techniques detect genome-wide DNA interactions, providing a comprehensive view of global genome organization. Here, we describe a detailed method to prepare Hi-C libraries using Bacillus subtilis, which includes procedures of crosslinking chromatin, digesting the crosslinked genome, labeling DNA ends with biotin, ligating DNA, and preparing the DNA library for sequencing using an Illumina platform.

High-Throughput Nucleotide Sequencing↗
PubMed2025

Investigating genetic, antigenic, and structural diversity in the Neisseria gonorrhoeae outer membrane protein, PorB: implications for vaccine design.

UNLABELLED: Vaccines targeting Neisseria gonorrhoeae are needed to reduce disease burden and help address the problem of antimicrobial resistance, with an understanding of relationships between gonococcal genetics and molecules influencing diversity, infection, and the immune response essential for developing effective vaccine formulations. Whole-genome sequence data can be used to investigate these relationships among thousands of gonococcal isolates, allowing the study of antigenic diversity on a population scale. Such analyses typically examine antigenic diversity occurring in complete protein sequences, generating mean diversity indices and phylogenetic analyses that can inform on vaccine potential; however, to detect and measure the immune responses elicited, epitope characterization within an antigen helps guide vaccine formulations, with epitopes commonly located in surface-exposed regions of a protein. Here, we analyzed the genetic diversity of the major gonococcal antigen, PorB, in WGS from 22,227 N. gonorrhoeae isolates. We characterized the diversity of all eight surface-exposed outer membrane loops, or variable regions (VRs), and generated a PorB VR subtyping scheme to facilitate the global and temporal detection of circulating PorB subtypes. These analyses identified the presence of dominant VR combinations that persisted over time, indicative of (i) epistatic interactions between VRs and (ii) positive selection. Strain-specific, anti-PorB IgG responses directed toward distinct VR subtypes were detected in sera obtained from participants vaccinated with 4CMenB. The deconstruction of PorB into each surface-exposed loop provides a powerful approach for evaluating vaccine candidates: the methods used here allow immunodominant regions to be detected, which is invaluable for further vaccine investigations. IMPORTANCE: In the context of rising global gonorrhea cases, the development of vaccines becomes a priority; however, N. gonorrhoeae antigenic diversity and its ability to evade the immune system complicate vaccine development. This study characterizes the genetic diversity of the outer membrane protein, PorB, a key component of the outer membrane and a major gonococcal antigen. Using genomics and machine-learning techniques, this research identified dominant PorB variants that drive the immune response, proposing potential vaccine candidates and improving our understanding of the evolutionary forces maintaining genome structure and biological fitness. Understanding these processes is crucial for designing vaccines that effectively target N. gonorrhoeae and combat the spread of multidrug-resistant gonococci.

Neisseria gonorrhoeae↗
PubMed2025

Novel, rapid, and reliable typing of vancomycin-resistant Enterococcus faecium CC17/ST80 strains using MALDI-TOF MS.

Vancomycin-resistant Enterococcus faecium (VREfm) is an important nosocomial pathogen. The recent emergence of the highly virulent clonal complex 17 (CC17) is posing a challenge for both therapeutic interventions and hospital infection control measures. Hence, prompt discrimination of CC17 VREfm from unrelated and less-virulent VREfm strains is essential for preventing its spread in hospitals and beyond. Between January 2022 and November 2024, 340 VREfm primary isolates have been identified in our lab and underwent genotyping by pulsed-field gel electrophoresis (PFGE) to survey a potential outbreak in the Tyrol region. In addition, whole-genome sequencing (WGS) was performed on a selected subset (n = 40). To curtail the lengthy time-to-result (TTR) of these methods, a novel typing protocol using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was established, validated, and optimized for rapid sample processing. PFGE and WGS showed that 61.2% of isolates (n = 208) belonged to a specific VREfm cluster identified as CC17 sequence type (ST) 80 vanA VREfm. A comprehensive MALDI-TOF MS analysis identified a distinct peak pattern specific to this lineage. This phenotypic characterization was used as a novel typing method with excellent performance (sensitivity: 1.00 [0.98-1.00], specificity: 0.89 [0.70-0.97]) and demonstrated a short TTR of 1 day after the cultural growth of VREfm. A rapid and novel MALDI-TOF MS-based typing approach for a specific CC17/ST80 vanA VREfm cluster was developed and enabled real-life application in routine diagnostics to assure accurate infection prevention and control measures. Future outbreak investigations may benefit from adopting this cost- and labor-efficient approach.IMPORTANCEThis study addresses the urgent need for faster ways to detect problematic hospital bacteria. A highly transmissible strain of Enterococcus faecium (CC17) has been spreading in healthcare settings, making infections harder to treat and control. Traditional methods to identify and track outbreaks are accurate but slow and resource-intensive, delaying critical infection control actions. By developing and validating a new method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the researchers demonstrated that this strain can be identified quickly, reliably, and at lower cost. Importantly, the new approach delivers results within a day, compared to the lengthy turnaround times of existing methods. This rapid detection tool provides hospitals with a practical solution to respond to outbreaks more effectively, prevent further spread, and protect vulnerable patients. The findings highlight a valuable step forward in strengthening hospital infection control and improving patient safety.

Enterococcus faecium↗
PubMed2024

Comprehensive analyses of a large human gut Bacteroidales culture collection reveal species- and strain-level diversity and evolution.

Species of the Bacteroidales order are among the most abundant and stable bacterial members of the human gut microbiome, with diverse impacts on human health. We cultured and sequenced the genomes of 408 Bacteroidales isolates from healthy human donors representing nine genera and 35 species and performed comparative genomic, gene-specific, metabolomic, and horizontal gene transfer analyses. Families, genera, and species could be grouped based on many distinctive features. We also observed extensive DNA transfer between diverse families, allowing for shared traits and strain evolution. Inter- and intra-species diversity is also apparent in the metabolomic profiling studies. This highly characterized and diverse Bacteroidales culture collection with strain-resolved genomic and metabolomic analyses represents a valuable resource to facilitate informed selection of strains for microbiome reconstitution.

Humans↗
PubMed2026

Whole genome sequencing analysis and functional characterization of Lacticaseibacillus rhamnosus HP-B1083.

Lacticaseibacillus rhamnosus is an important strain for the biotransformation of natural products, and its crude extract exhibits biotransformation effect on glycosidic compounds such as baicalin. To further explore the potential of this strain, particularly given its previously demonstrated high-efficiency β-glucuronidase activity for baicalin conversion, whole-genome sequencing and functional annotation of Lacticaseibacillus rhamnosus HP-B1083 were performed in this study, and its acid tolerance, bile salt tolerance, short-term heat resistance and antibacterial activity were evaluated. The results showed that the strain possessed a circular chromosome with a full length of 3,090,505 bp and a GC content of 46.69%. Gene annotation revealed that the genome contained 2941 coding sequences (CDS) and 112 non-coding RNA genes, including 60 tRNA genes, 1 tmRNA gene, 36 misc_RNA genes and 15 rRNA genes. The functional annotations further reveal that this genome is rich in genes related to carbohydrate metabolism, hydrolases, and transferases, which is highly consistent with its phenotypic characteristics in glycoside transformation and the synthesis of antibacterial substances. In addition, acid tolerance, bile salt tolerance and short-term heat resistance experiments verified that HP-B1083 had acid resistance, bile salt resistance and short-term heat resistance. Antibacterial activity tests confirmed that HP-B1083 produced inhibition zone diameters over 10 mm against common foodborne pathogenic bacteria such as Escherichia coli and Bacillus cereus. Therefore, Lacticaseibacillus rhamnosus HP-B1083 has important application prospects in the development of functional foods, preparation of enzyme preparations and pharmaceutical industry.

Whole Genome Sequencing↗