Background. Mycoplasma pneumoniae (MP) is a common causative pathogen of community-acquired pneumonia in children, with clinical presentations ranging in severity. Early stratification and timely intervention are essential for improving patient outcomes. However, a major clinical challenge lies in the limited ability to accurately distinguish between mild and severe cases based solely on early clinical indicators.Methods. This prospective real-world study investigated the differences in microbiome and metabolomics between mild and severe MP pneumonia (MPP) in children. Bronchoalveolar lavage fluid samples were collected from 153 children and subjected to metagenomic sequencing and non-targeted metabolomic analysis. Meanwhile, to enhance early diagnostic accuracy, this study developed a machine learning classification model and validated it using a third-party validation set.Results. The results revealed significant alterations in the abundance of specific bacterial communities in the severe group, most notably the coexistence of MP and Alphainfluenzavirus influenzae, which may contribute to disease exacerbation through synergistic pathogenic mechanisms. Furthermore, the macrolide resistant rate of MP in the severe group exceeded 80%, emphasizing the importance of appropriate antibiotic selection. Metabolomic analysis showed a significant enrichment of metabolites related to cellular energy metabolism and immune regulation in severe cases. The model demonstrated exceptional predictive performance, achieving an area under the curve ranging from 0.909 to 0.991, which significantly outperformed conventional clinical stratification methods.Conclusions. These findings elucidate the distinct pathophysiological mechanisms underlying both mild and severe MP infections and provide a promising framework for improving early diagnosis and personalized treatment strategies in paediatric MPP.

Aromatic hydrocarbons are widespread environmental compounds whose microbial degradation is essential for detoxification, yet the genomic distribution and evolutionary stability of many catabolic pathways remain poorly understood. The phenylacetic acid (PAA) hybrid pathway integrates aerobic and anaerobic features and represents a central route for aromatic compound degradation; however, its prevalence, evolutionary conservation and regulatory organization across prokaryotes have not been comprehensively reassessed.Here, we investigated the global distribution, structural conservation and evolutionary dynamics of the PAA pathway across prokaryotic genomes. We performed a large-scale comparative genomic analysis of 6,536 bacterial and archaeal genomes, leveraging functionally annotated genomes to enable consistent identification of pathway components, integrating protein architecture profiling, genomic context organization, phylogenetics, motif and structural analyses of phenylacetate-CoA ligase (PAL) and cophylogenetic inference. Using stringent criteria, we identified 853 high-confidence PAL predictions across 825 genomes (≈12%), revealing substantial previously unrecognized PAA metabolic potential. Structural analyses confirmed the conservation of key catalytic features, supporting functional annotation of newly identified PALs. Genomic context analyses revealed extensive pathway remodelling across lineages, yet a conserved core of local regulatory elements mediated by PaaX, PaaR and PaaY was maintained. Notably, PaaX and PaaR were, for the first time, predicted to co-occur within the same genomic context organization in several groups. In contrast, archaeal genomes predominantly encode incomplete PAA configurations, suggesting functional divergence and/or pathway fragmentation.Together, these results demonstrate that the PAA pathway is more widespread and evolutionarily dynamic than previously appreciated, combining deep enzymatic conservation with flexible genomic organization. Finally, the conservation of the core epoxidase subunits PaaA and PaaC across PAL-encoding genomes supports their use as robust genomic markers of PAA pathway potential.

Human milk glycan (HMG) metabolism, especially by bifidobacteria, is crucial for infant gut colonization and healthy microbiome development. Bifidobacterial species and even strains are highly variable in their ability and in their enzymatic repertoire for HMG metabolism. The enzymes involved in HMG metabolism often have many non-HMG-related homologues, necessitating fine-grained annotation for accurate assessment of bifidobacterial HMG metabolic capabilities. However, current annotation tools provide only broad glycoside hydrolase (GH) (sub)family classifications. Here, we present bifidoAnnotator, a tool for fine-grained annotation and visualization of bifidobacterial GH genes involved in HMG utilization. bifidoAnnotator leverages MMseqs2 (Many-against-Many sequence searching) to map protein sequences against a manually curated database of over 22,000 bifidobacterial GH proteins, organized into 13 families and 108 functional clusters, each assigned a validation status (i.e. experimentally validated, putative or hypothetical). The tool performs hierarchical annotation at family and cluster levels, identifying consistently annotated protein variants rather than just broad family assignments, and generates publication-ready heatmaps for comparative analysis. Benchmarking on a gold standard dataset demonstrated that bifidoAnnotator has superior performance (95.9% precision, 100% recall) compared with six established tools and is an order of magnitude faster than the most accurate competitor. bifidoAnnotator's superior performance and computational efficiency represent a meaningful advance in high-throughput genomic annotation workflows, enabling detailed characterization of strain-level functional diversity in bifidobacterial HMG metabolism.

Viral non-structural proteins are key mediators of host-virus interplay, including RNA modification dynamics. The function of the transmissible gastroenteritis virus (TGEV) gene 7, which encodes a non-structural protein, remains poorly understood. Using Oxford Nanopore direct RNA sequencing, we explored the host and viral RNA landscapes modulated by TGEV gene 7 in Swine testis cells. Deletion of the TGEV gene 7 halved viral RNA replication yet significantly increased m6A modification levels on both the viral genome and host mRNAs. This epitranscriptomic rewiring was accompanied by reciprocal shifts in the m6A regulators FTO (eraser) and RBM15 (writer). Despite comparable bulk transcriptome changes, gene 7 deletion introduced additional differentially expressed genes, showing stronger enrichment of antiviral and chemokine pathways, indicating heightened innate immunity. PolyA analysis uncovered a gene 7-dependent extension of viral, but not host, polyA tails. These findings highlight RNA-modification machinery as a potential target for coronavirus control and provide a framework for vaccine strategies exploiting gene 7 attenuation.

Background. Klebsiella pneumoniae (Kpn) is an important cause of healthcare-associated infections (HAIs). In low- and middle-income countries, HAI due to Kpn disproportionately affects neonates. In this study, we investigated the genomic changes that occurred during long-term circulation of a Kpn sequence type (ST) 39 clone, causing a disproportionate number of infections on the neonatal ward at a tertiary healthcare facility in Malawi in 2017.Methods. We analysed whole-genome sequences of Kpn ST39 collected from Queen Elizabeth Central Hospital over a 20-year period, including the generation of several high-quality hybrid genomes. We compared virulence markers, antibiotic resistance determinants and mobile genetic elements, focusing on variable regions between strains from the outbreak clone in 2017 and genomes from other co-occurring ST39 lineages.Results. We identified eight variable genomic regions that demonstrate the plasticity of Kpn within ST, including the role of bacteriophages in shaping the genome of ST39.Conclusions. The analysed Kpn ST39 lineages have a highly variable genome capable of incorporating large genomic regions during prolonged hospital circulation, which may offer a selective advantage in hospital environments and provide resistance to antimicrobial agents.

Bodo is a cosmopolitan genus of free-living bacterivorous single-celled flagellates in the class Kinetoplastea. Genus Bodo is considered the closest free-living lineage to the parasitic lineages Trypanosoma and Leishmania, the causative agents of the human diseases sleeping sickness, Chagas disease and leishmaniasis. Currently, a single genome exists for the one formally described species in the genus, Bodo saltans. Previous studies on B. saltans have shown that it is dependent on an endosymbiotic bacterium from the order Holosporales, ‘Candidatus Bodocaedibacter vickermanii’. Using single-cell sequencing, we isolated, sequenced and assembled genomes for seven uncultured Bodo spp. cells from a freshwater sample from Royal Leamington Spa, UK. Using comparative genomics, we show that these seven cells represent three potentially novel Bodo species exhibiting unexpected levels of diversity at the genome level. Our results indicate that small subunit ribosomal DNA sequencing, often used to classify Bodo flagellates, is insufficient for determining species delimitation in this genus. In addition, we recovered a Holosporales bacterium genome from all seven Bodo spp. cells. Surprisingly, these seven endosymbionts also represent three novel species, congruent with the phylogeny of the host and exhibiting lineage-specific adaptations. This diversity and host–symbiont association would be indistinguishable in routinely used metabarcoding or bulk sequencing pipelines, thus demonstrating the power of single-cell sequencing to reveal diversity within lineages of microbial eukaryotes.

Whole bacterial genome sequence reconstruction using Oxford Nanopore Technologies ('Nanopore') long-read-only sequencing may offer a lower-cost, higher-throughput alternative for pathogen surveillance to 'hybrid' assembly with recent improvements in Nanopore sequencing accuracy. We evaluated the accuracy, including plasmid reconstruction, of Nanopore long-read-only genome assemblies of Enterobacterales. We sequenced 92 genomes from clinical Enterobacterales isolates, collected in England under a national surveillance programme, with long-read Nanopore (R10.4.1, Dorado v5.0.0 super-high-accuracy basecalled) and short-read Illumina (NovaSeq) sequencing approaches. Genomes were assembled using three long-read-only (Flye, Hybracter long and Autocycler) and three hybrid assemblers (Hybracter hybrid, Unicycler normal and bold). Three polishing modalities (Medaka v2 with subsampled or un-subsampled long-reads; Polypolish+Pypolca with short-reads) were investigated. Autocycler circularised the most chromosomes [87/92 (95%)]. Plasmid sequence reconstruction was comparable among all assemblers except Flye, all recovering 90-96% of plasmids, although the 'ground truth' was uncertain. Flye performed worse than other assemblers on almost all metrics. Autocycler+Medaka (un-subsampled long-reads) was the most accurate long-read-only assembler/polisher combination, comparable to hybrid assemblies [median 0 (IQR: 0-0) single nucleotide variants (SNVs) and 0 (IQR: 0-1) insertions/deletions (indels) per genome; median quality value/Q score 100 (IQR: 64-100)], with only 4/92 genome sequences having >10 SNVs/indels. Medaka polishing with un-subsampled long-reads resulted in small improvements in indels, but not SNVs for both Flye and Autocycler assemblies. Seven-locus multi-locus sequence type, antimicrobial resistance, virulence and stress gene annotation was equivalent across assembler/polisher combinations. Nanopore long-read-only bacterial genome assembly with Autocycler combined with Medaka polishing (using un-subsampled reads) is similarly accurate and possibly more complete than hybrid assemblies, representing a viable alternative for incorporating high-quality genomic data, including plasmids, into Enterobacterales surveillance.

Global dissemination of emergent multidrug-resistant (MDR) Salmonella Infantis (ESI) is of great public health concern. ESI exhibits increased virulence and MDR phenotypes, features conferred by a conjugative megaplasmid (pESI-like). In Mexico, the potential circulation of ESI clones has been overlooked. This study assessed the structure, diversity, genomic features and transmission dynamics of Salmonella Infantis isolates (n=191) from cattle, pigs, chickens, humans, surface waters and the environment from surveys conducted by research laboratories and government agencies in the 2008–2024 timeframe. Three genomic approaches were implemented: blast analysis, SNP-based phylogenetic analysis and ancestral state reconstruction analysis. The phylogeny divided the population into two divergent sublineages, based on the presence/absence of pESI-like plasmids. The apparently recent and massive acquisition of these plasmids had a profound impact at the population level, changing the proportion of isolates with MDR genotypes from 0 to 68%. Although ESI was mostly associated with chickens, it was also present in surface waters. In fact, our ancestral state reconstruction analysis showed that ESI likely evolved from surface water ancestors within poultry production environments. Most locally circulating ESI clones (73%) harbour resistance factors to 6–8 antibiotic classes, including extended-spectrum cephalosporins (blaCTX-M-65) and fluoroquinolones (gyrA D87Y mutation), a feature shared with global ESI variants that we coined as CTX-M-gyrA6-8. Control measures are urgently needed to prevent further dissemination of ESI, as well as the systematic surveillance of CTX-M-gyrA6-8 clones, particularly in poultry, to reduce the risk of human exposure to this potentially deadly pathogen.

The growing resistance of Enterobacterales to last-resort antibiotics such as carbapenems puts a significant burden on healthcare systems, also due to plasmids driving a rapid spread of carbapenem resistance. We here evaluate the use of long-read nanopore sequencing to investigate carbapenem resistance dynamics and the role of plasmid transfers and environmental reservoirs in the hospital setting. Over 13 months, routine clinical diagnostics identified recurring isolates of carbapenem-resistant Citrobacter species carrying Klebsiella pneumoniae carbapenemases (KPCs) and/or OXA-48-like carbapenemases from patient screening and hospital drain samples. While routine diagnostic approaches provided limited insights into the carbapenem resistance dynamics, we show that near-complete de novo assembly of chromosomes and plasmids by long-read nanopore sequencing allowed for high-resolution strain identification, plasmid profiling, and antibiotic resistance gene detection. Notably, genomically nearly indistinguishable Citrobacter freundii of the high-risk sequence type ST91 genomes were recovered from screening samples collected in the same hospital room 1 year apart. We further provide evidence of a KPC-2-encoding IncN plasmid that is likely to have spread across bacterial species and between patient and drain isolates, which emphasizes the role of contaminated drains in the persistence and dissemination of antimicrobial resistance within the hospital environment. Overall, this study demonstrates the value of long-read nanopore sequencing for uncovering the complex dynamics of carbapenem resistance spread and persistence in the hospital setting and its potential implications for infection prevention and control.