The environmental bacterium Pseudomonas protegens PBL3 has antagonistic activity against the plant pathogenic bacterium Burkholderia glumae, an important pathogen in rice. The antimicrobial activity of P. protegens 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 P. protegens PBL3 and additional six Pseudomonas spp. strains, to identify putative genomic regions in P. protegens PBL3 associated with antimicrobial activity. We hypothesized that Pseudomonas spp. strains with antimicrobial activity against B. glumae have conserved genes with P. protegens PBL3 that are absent in strains lacking activity. Comparative genomics analyses with anvi'o and progressiveMauve, and using P. protegens 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 P. protegens PBL3 is not limited to biosynthetic pathways but also involves additional regulatory, metabolic and export modules to synthesize and deploy antimicrobials.
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.
Cobalt influences the methanol metabolism of Desulfofundulus kuznetsovii TPOSR, specifically by modulating the activity of one of its alcohol dehydrogenases (ADH), Adh1. However, the effects of cobalt on the broader proteome of strain TPOSR, as well as the utilization of alcohols besides methanol, remain unexplored. Here, proteomic analyses of strain TPOSR grown with and without cobalt on different alcohol substrates show that cobalt starvation impacts multiple cellular processes, including cobalamin biosynthesis, iron-sulphur cluster assembly and, most prominently, energy metabolism as indicated by altered abundances of hydrogenases and NAD(P)-dependent oxidoreductases. Despite the presence of six ADH-encoding genes in the genome, Adh1 is the dominant ADH during growth not only on methanol but also on several primary alcohols and diols (ethanol, 1-propanol, 1,2-propanediol, 1,3-propanediol, butanol, pentanol and heptanol). Enzymatic assays with purified Adh1 confirm activity with these substrates, except 1,3-propanediol, and show no activity toward secondary alcohols (2-propanol and 2-butanol). Comparative proteomics analyses of other sulphate-reducing microorganisms (SRMs), namely Desulfofundulus australicum and Solidesulfovibrio carbinolicus, further indicate that methanol and ethanol oxidation in SRMs is mediated by a single ADH/AOR pair. Together, these findings highlight the central role of cobalt in alcohol metabolism in strain TPOSR and identify conserved ADH/AOR enzymes as promising candidates for biotechnological applications.
The human-restricted enteric pathogen Salmonella enterica serovar Typhi ( S . Typhi) is the causative agent of the life-threatening typhoid fever. Although S . Typhi incidence is relatively low in the USA, routine surveillance of S . Typhi is critical to track the emergence and spread of high-risk lineages in non-endemic areas. In this study, we analysed 151 genomes of S . Typhi isolates from patients who were clinically confirmed with typhoid fever across New York State between 2016 and 2023. We used the GenoTyphi classification scheme and identified established multidrug-resistant and extensively drug-resistant lineages. We detected the presence of the globally widespread genotype 4.3.1 (haplotype 58) and its derivative 4.3.1.1.P1, which recently emerged in Pakistan, as well as the Bangladesh-restricted lineages 3.3.2.Bd1 and 3.3.2.Bd2 in our dataset. Ten mutations and 14 acquired genes associated with antimicrobial resistance (AMR) were present across the entire population, with 86.8% of the genomes possessing at least one of these AMR determinants. The gyrA S83F mutation conferring quinolone and triclosan resistance was the most frequently detected (94 genomes). Combinations of dfrA7+catA1 (resistance to trimethoprim and chloramphenicol, respectively) and sul2+aph(3″)-Ib+aph(6)-Id (resistance to sulphonamide and aminoglycosides, respectively) co-occurred frequently and were associated with IncQ and IncY plasmid replicons. Phylogenetic contextualization against a global dataset of 1,643 genomes from 20 countries across five continents, including other parts of the USA, from the same time period showed geographic intermingling, suggesting the spread of high-risk genotypes of international origins to New York State. Altogether, these findings reveal the presence of globally dominant resistant genotypes that are likely facilitated by human travel in New York State, where typhoid fever is not endemic. Long-term genomic surveillance is critical to AMR profiling, identifying genotypic shifts in regional S . Typhi populations, monitoring transmission routes and guiding effective public health interventions.
Background. Recent outbreaks of gastrointestinal symptoms among gay, bisexual and other men who have sex with men (GBMSM) have been caused by multidrug-resistant (MDR) Shigella sonnei carrying bla CTX-M-27 or bla CTX-M-15. To date, the bla CTX-M-3 variant has not been associated with sexually transmitted shigellosis in the UK.Methods. Routine surveillance identified an outbreak S. sonnei among men in England in June 2025. Short-read sequencing data were analysed to identify antimicrobial resistance (AMR) determinants and to investigate the phylogenetic context of the outbreak. Long-read Oxford Nanopore Technology sequencing data were analysed to characterize the AMR-encoding plasmid content.Results. Of the 83 cases linked to the outbreak cluster, 91.5% were adult males and none reported recent travel outside Europe; these outbreak characteristics (specifically adult male, no travel) are consistent with transmission among European GBMSM networks (Mitchell et al. 2019, Mitchell et al. 2021). Phylogenetic analysis placed the outbreak cluster within a wider clade historically associated with travel to the Middle East. The outbreak isolates were MDR; 90.4% (n=75/83) had bla CTX-M-3 located on an IncI1B/O plasmid. This is the first report of the IncI1B/O plasmid type and bla CTX-M-3 in GBMSM-associated MDR S. sonnei in England.Conclusions. Acquisition of the IncI1B/O plasmid encoding bla CTX-M-3 represents an independent evolutionary event, separate from previously described GBMSM epidemics driven by acquisition of IncFII plasmids encoding bla CTX-M-27 or bla CTX-M-15. This study provides further evidence of the parallel emergence of bla CTX-M variants conferring resistance to the third-generation cephalosporins. Whole-genome sequencing based surveillance and routine susceptibility testing of MDR S. sonnei are essential, as this pathogen continues to diversify and spread.
Plastic waste pollution is a global issue that threatens biodiversity and human health. Current plastic waste management practices are not sufficient to keep up with increasing plastic production rates. Microorganisms have the capacity to degrade different types of bio-based and synthetic plastics through enzymatic reactions, offering an alternative solution to traditional plastic recycling techniques. A limited number of plastic-degrading enzymes have been identified, sequenced and characterized; however, studies exploring the distribution of homologues of these enzymes across habitats and microbial taxa have remained scarce. Here, we applied analytical techniques to search for genes encoding potential plastic-degrading enzymes in environmental metagenome datasets and genomes of the Genome Taxonomy Database (GTDB) to explore the geographic and taxonomic distribution patterns of plastic-degrading microorganisms. Hidden Markov Models (HMMs) were constructed from amino acid sequences of known, experimentally verified and putative plastic-degrading enzymes. The HMMs were applied to landfill, soil, river, lake and ocean metagenomes and all archaeal and bacterial genomes in the GTDB. An abundance of hits was discovered across aquatic and terrestrial metagenomes with the majority occurring in polluted rivers, polar oceans and deep ocean samples. GTDB hits were mainly consistent with known plastic-degrading microbial lineages, while also revealing potential plastic-degrading archaeal taxa. The results of this study may be able to assist in the discovery of novel plastic-degrading enzymes for application in plastic waste biodegradation solutions.
Pseudomonas aeruginosa bacteremic pneumonia carries exceptionally high mortality, yet there is a paucity of genomic characterization of the strains causing this infection. We performed hybrid sequencing (Illumina and Oxford Nanopore) of 12 non-redundant P. aeruginosa isolates from patients with severe bacteremic pneumonia admitted to the intensive care unit of a tertiary hospital between 2015 and 2023. The 12 isolates were assigned to nine distinct sequence types, suggesting that severe bacteremic pneumonia can arise from diverse P. aeruginosa lineages rather than being dominated by a single specialized or high-risk clone. In the combined dataset of our isolates and publicly available Korean P. aeruginosa genomes, type III secretion system exotoxin genotypes exoU and exoS showed a mutually exclusive and phylogenetically segregated distribution, as previously reported, with both genotypes represented among bacteremic pneumonia isolates. Carbapenemase genes were detected in only one isolate, PA22 (ST773), which harboured bla NDM-1 together with bla OXA-796 and was the only isolate displaying phenotypic carbapenem resistance and multidrug resistance. To assess the clonal relationship between bla NDM-1-positive PA22 and the carbapenemase-negative ST773 isolate PA20 and to track the evolution of PA22 resistome within a broader epidemiological context, we investigated the population structure of a global ST773 dataset. Core genome MLST-based minimum spanning trees revealed a deep bifurcation within ST773, separating bla NDM-1-positive and carbapenemase-negative lineages. Korean ST773 isolates formed two distinct clusters within the NDM-1-positive lineage, with the PA22-containing cluster phylogenetically proximal to isolates from the United States. Within the NDM-1-positive Korean cluster, bla OXA-796 was located in conserved class 1 integron gene cassette arrays that exhibit ongoing structural diversification among closely related isolates, evidenced by variable integration of IS110 elements. Our findings demonstrate that severe bacteremic pneumonia arises from phylogenetically diverse P. aeruginosa lineages and provide genomic context for the NDM-1-producing ST773 clone that is rapidly emerging in Korea.
Aspergillus welwitschiae is a widespread fungus with diverse roles as a plant mutualist, opportunistic human pathogen and industrial enzyme producer. The endophytic strain AwOcstreb1, isolated from halophytic rice (Oryza coarctata), promotes growth in commercial rice under normal and saline conditions. Despite its significance, genomic and metabolic resources for A. welwitschiae remain limited, with no complete genome information available for endophytic strains within the species. Moreover, the close relationship of this species to Aspergillus niger complicates its taxonomic resolution. We performed whole-genome and transcriptomic sequencing of AwOcstreb1 cultured on potato dextrose agar, along with -MS-based volatile metabolite profiling. Comparative analyses included simple sequence repeat (SSR), transposable element (TE; including starships) and carbohydrate-active enzyme (CAZyme) profiling across A. welwitschiae strains. Evolutionary relationships with A. niger were examined using average nucleotide identity (ANI) and orthologous gene clustering, supported by phylogenomic reconstruction. Genes for mycotoxin production and plant growth-promoting traits were also searched in this strain. The AwOcstreb1 genome is 37.7 Mb with 13,242 predicted genes, of which 66.6% were actively expressed under potato dextrose agar growth. The genome harbours 5,126 SSRs, 19,434 TEs and a CAZyme composition similar to other A. welwitschiae strains. Although established marker genes such as CaM and β-tubulin identify AwOcstreb1 as A. welwitschiae, whole-genome ANI and orthologous gene-based analyses place A. welwitschiae strains within the broader A. niger species complex, suggesting that it represents a population-level group rather than a clearly separated species, a view that is still not widely adopted. Synteny analysis showed that the AwOcstreb1 genes are highly collinear with those of A. niger. Genes involved in phosphate and zinc solubilization and siderophore biosynthesis were detected, whereas ochratoxin A biosynthetic genes were absent. Although the presence of fumonisin genes was detected, only a trace amount of the toxin was detected both in culture as well as rice grains. Among 172 strain-specific orthogroups, several encode intrinsically disordered, secreted or membrane-associated proteins that are potentially linked to endophytic lifestyle adaptations. Volatile metabolite profiling identified compounds such as 17-pentatriacontene, eicosane and octanal, each linked to known biological sources and potential functions, such as antifungal, antibacterial and anti-inflammatory activities. Several additional metabolites were also identified, whose biological roles need further investigation. This integrated omics study provides foundational insights into the endophytic potential and genomic distinctiveness of AwOcstreb1. This work opens new avenues for exploring A. welwitschiae for sustainable agriculture and fungal biology.
Plasmid-mediated colistin and tigecycline resistance threatens last-resort therapeutic options. We investigated the farm-to-fork dissemination of Escherichia coli encoding mcr-1.1 and tet(X4) in a vertically integrated broiler production system. A total of 200 samples were collected in 2024, representing four sequential production stages: broiler breeders, day-old chicks, day-30 broilers and retail meat. All positive [mcr-1.1 and/or tet(X4)] isolates underwent Illumina short-read sequencing, with long-read sequencing of three representatives. The study was enriched and analysed with mcr-1.1 and tet(X4) encoding 213 publicly available E. coli genomes from Pakistan. Of 18 isolates, tet(X4) was detected in 11 and mcr-1.1 in 7 isolates with a single breeder isolate (ST-398) co-harbouring both on separate plasmids. tet(X4) predominated breeders and day-30 broilers, whereas mcr-1.1 was distributed across all four stages. ST-1011 exhibited a pattern of clonal farm-to-fork transmission of tet(X4) via IncFIB(AP001918)-IncFII providing molecular evidence suggestive of clonal transmission. Analysis of 231 genomes across 87 sequence types (STs) revealed contrasting evolutionary trajectories: mcr-1.1 with post-mobilization stabilization marked by complete absence of ISApl1 and highly conserved IncI2 plasmid (93%), whereas tet(X4) retained active transposition within IS26-bounded elements. tet(X4) carriage was also associated with elevated resistance gene burden, driven by the IncF megaplasmids. All 18 isolates exhibited adhesion to chicken intestinal epithelial cells (CHIC-8E11), with high adhesion capacity among tet(X4)-positive isolates. This study demonstrates that mcr-1.1 and tet(X4) disseminate through poultry production via distinct mechanisms. These findings highlight the need for integrated One Health surveillance of antimicrobial resistance dissemination.
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 (rrn) 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 in silico analysis of a curated reference database with an experimental validation using rat faecal samples. In silico, the rrn 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 rrn 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.
Escherichia coli is predominantly an intestinal commensal; however, avian pathogenic E. coli (APEC) causes colibacillosis in poultry. The APEC pathotype lacks a clear genetic definition, further complicated by its opportunistic nature. To compare the genomic characteristics of avian pathogenic and commensal E. coli , isolates from diseased and healthy broiler flocks in Sweden were analysed, collected between 2022 and 2024. Clinical isolates ( n =202) were collected at necropsy from 40 flocks during colibacillosis outbreaks, and non-clinical isolates ( n =109) were obtained from litter using sock sampling in 60 unaffected flocks. Whole-genome sequencing was performed to determine sequence types (STs), serotypes, phylogroups, virulence-associated genes (VAGs) and to identify ColV plasmids. A five-gene APEC marker panel targeting plasmid-associated virulence genes ( iutA, hlyF, iss, iroN and ompT ) was used to classify isolates as APEC or non-APEC, and high-risk clones were identified according to the APECtyper scheme. Clinical isolates comprised 22 STs and 25 serotypes and were dominated (59%) by the ST23 O78:H4 clone within phylogroup C. Non-clinical isolates were more diverse (44 STs, 67 serotypes), primarily within phylogroups A (48%) and B1 (33%), with no clone predominating. Clinical isolates carried significantly more VAGs ( P <0.001). Overall, 97% of clinical isolates were identified as APEC, all of which carried a ColV plasmid. Among non-clinical isolates, 28% were APEC, of which 80% were ColV-positive. However, clinical APEC isolates carried significantly more ColV-associated virulence gene clusters than non-clinical APEC isolates ( P <0.001). Only 5% of non-APEC isolates were ColV-positive. High-risk clones were restricted to clinical APEC isolates (63%). These findings indicate that colibacillosis in Swedish broilers was largely driven by a dominant APEC clone during the study period, highlighting the need for coordinated surveillance and targeted control of high-risk clones. The presence of VAG reservoirs among isolates from unaffected flocks, together with the limitations of marker-based APEC typing, supports integrated frameworks combining lineage, VAG profiles and plasmid content for more reliable APEC identification and pathogenicity assessment.
Plasmids are extrachromosomal mobile genetic elements that can facilitate rapid bacterial adaptation by transferring genes between individuals. Whilst plasmids are known to exist in diverse habitats and encode a range of traits, most of our knowledge about plasmids comes from clinically associated antimicrobial resistance (AMR) plasmids that have already been recruited as vectors of drug resistance and have likely been shaped by strong selection for plasmid-encoded antibiotic resistance. Here, we investigated 26 plasmids from the pQBR collection - a set of large, co-existing mercury resistance environmental plasmids isolated in Pseudomonas spp. from a field in Oxfordshire in the 1990s - and explored the ability of pQBR plasmids to transfer novel chromosomally encoded traits. New whole-genome sequences for 25 plasmids confirmed that these soil-isolated plasmids are generally very large (140-588 kb), constitute at least six distinct genetic groups and have relatives in various other Pseudomonas species and habitats. Despite significant nucleotide-level divergence, Groups I (pQBR103-like, ~406 kb) and IV (pQBR57-like, ~328 kb) showed remarkable ancient similarities in synteny and gene content both with one another and with the PInc-2/IncP-2 family of plasmids known to transfer clinically significant drug resistance between Pseudomonas aeruginosa hosts. None of the pQBR plasmids sequenced to date harboured known AMR determinants, but putative phage defence systems and metal resistances were evident. Transposable elements, including the Tn5042 mercury resistance transposon, were responsible for significant structural variation within plasmid groups, consistent with a predominant role of transposons in rapidly remodelling plasmids. To experimentally test the ability of pQBR plasmids to spread new traits, we developed a novel transposon transfer assay which showed that certain Group IV pQBR plasmids were especially effective at acquiring the chromosomally encoded transposon Tn6291 and that this ability to transfer transposons was likely due to specific plasmid factors rather than generic conjugation rate. Our work presents a tractable set of sequenced plasmids suitable for exploring the evolution and dynamics of gene acquisition by pre-AMR plasmids and provides a key case study highlighting the pervasive interplay between plasmids and transposable elements that can drive microbial genome evolution.
Children with severe acute malnutrition (SAM) are at high risk of invasive infection. In sub-Saharan Africa, invasive non-typhoidal Salmonella enterica (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 S. enterica serovars Enteritidis and Typhimurium was compared using phylogeny and clustering approaches. Among 1,371 enrolled children, 87 Salmonella 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. Salmonella 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; χ²=126.7; P<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 Salmonella, serovars were diverse and distinct from invasive lineages.
Current understanding of genomic diversity within the halophilic genus Salinivibrio 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 Salinivibrio lacks polyhydroxyalkanoate (PHA) degradation capacity despite possessing biosynthesis genes. Here, we present eight complete Salinivibrio 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: P =1.72×10⁻⁴; tip: P =2.64×10⁻¹⁴). All 46 genomes contained complete PHA biosynthesis operons ( phaB-phaA-phaP-phaC ) with high sequence conservation under strong purifying selection (Z=30.30, P <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 Salinivibrio 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 Salinivibrio as a genomically dynamic genus with potential for halophilic bioplastic production.
Klebsiella pneumoniae sequence type 48 (Kp-ST48) is a globally distributed clone linked to antimicrobial resistance (AMR) yet lacks a comprehensive genomic analysis. Here, we investigated the persistence, transmission dynamics and global context of ST48 in a large tertiary hospital in Berlin, Germany. Between 2014 and 2022, 48 surveillance and 15 putative outbreak Kp-ST48 isolates were isolated in a tertiary care, multi-site hospital in Berlin, Germany. Genomic diversity was analysed by short- and long-read sequencing. Additionally, we included 223 publicly available Kp-ST48 genomes from five continents over 40 years (1982-2022) in the phylodynamic analysis. We identified two genetically distinct clades (A and B) within the global Kp-ST48 population. The global spread of Kp-ST48 was driven by clade B, which included all the genomes from the Berlin hospital. Two hospital-specific lineages (1 and 2) were identified with distinct population dynamics. Lineage 2 was transient and linked to a putative outbreak in 2019. Meanwhile, lineage 1 was first detected in 2014 and persisted for over 8 years until 2022, with multiple putative patient-to-patient and indirect transmission events identified. Carbapenem resistance determinants (ompK35/36 mutations, bla KPC, bla NDM, bla OXA-48, bla VIM) were present in 57% (n=163/286) of genomes, and up to three bla CTX-M-15 copies were found integrated into chromosomes. Although Kp-ST48 generally did not contain a high number of virulence genes, 19 genomes showed potential for AMR-hypervirulence convergence. This study reveals the endemic persistence with outbreak potentials of Kp-ST48 in a hospital over 8 years, characterized by high genome plasticity. Our results highlight the global distribution of this clone, which warrants continuous surveillance.
Antimicrobial resistance in Escherichia coli 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 β-lactamase-producing E. coli isolates from unweaned dairy calves (n=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 (P<0.05) and sequence types (all P<0.01). Conjugative plasmids accounted for 94.6% of plasmid-borne ARG occurrences and carried significantly more ARGs than mobilizable plasmids (P=3.66×10-42). ARG and BMRG counts were strongly correlated at the plasmid level (ρ=0.574, P=8.0×10-41), and class 1 integrons marked enriched multidrug plasmids with increased ARGs (P=6.22×10-34) and BMRGs (P=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, P=0.0017) and showed a strong host×mobility interaction (IRR 4.61, P=4.9×10-8), stronger ARG-BMRG coupling and a higher prevalence of integrons. These findings show that antimicrobial resistance ecology in E. coli 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.
Maternally inherited symbionts are central to arthropod biology, functioning both as mutualistic partners and as reproductive parasites. Genomic analyses provide critical insight into these interactions. Here, we sequenced, assembled and examined the genomes of Spiroplasma and Rickettsia co-infecting the lacewing Mallada desjardinsi, with the aim of elucidating the male-killing phenotype of Spiroplasma and predicting potential phenotypes for Rickettsia. In Spiroplasma, we identified a set of candidate effector genes. However, Spaid-like genes, where present, lacked the functional domains previously demonstrated to be important for male-killing. The Rickettsia genome contained two cifA/cifB gene pairs, consistent with the capacity to induce cytoplasmic incompatibility (CI). Unexpectedly, both symbiont genomes were markedly expanded relative to congenerics. We describe this pattern - that contrasts with the classical trajectory of genome reduction in symbionts - as secondary genome expansion. Expansion was driven by extensive proliferation of mobile elements: Rickettsia harboured an exceptionally high number of insertion sequences, while Spiroplasma accumulated both insertion sequences and prophage regions. Collectively, our findings indicate that the canonical Spaid-mediated male-killing mechanism is not conserved in the Spiroplasma of M. desjardinsi, while Rickettsia may induce CI. Moreover, the parallel genome expansions observed suggest that secondary expansion events may be influenced by host-associated factors rather than occurring stochastically.
Xanthomonas arboricola is a plant-associated bacterial species that comprises the pathovars pruni (Xap), juglandis (Xaj), corylina (Xac), which are of high economic concern; and the pathovars arracaciae, celebensis, fragariae and zantedeschiae, which are less relevant, meaning they have a lower economic impact, are less widely distributed, or cause less severe diseases in their host plants. Moreover, the species also includes strains without pathovar affiliation. The pathogenicity of the less economically relevant pathovars as well as the non-affiliated strains has been debated. The present study analysed all the X. arboricola genomes deposited in GenBank by March 2025, focusing on type III secretion system (T3SS), type III effectors (T3Es), cell wall-degrading enzymes (CWDE), amylases and hydrolytic activity.The average nucleotide identity analysis showed that strains belonging to the three main pathovars clustered according to their pathovar designation, harbouring both T3SS and a large T3Es repertoire. However, strains belonging to pathovars of lower economic relevance or with no pathovar affiliation were clustered into two groups, G1 and G2. Remarkably, strains belonging to these less relevant pathovars were scattered along these two groups, exhibiting notable genetic diversity. Moreover, strains in G1 generally displayed T3SS but a limited set of T3Es, whereas strains in G2 lacked a T3SS and exhibited an even more reduced T3Es repertoire. Regarding CWDE, differences in the amount and profiles of cellulolytic, hemicellulolytic and pectinolytic enzymes were observed. While Xap and Xaj possessed the highest counts of hemicellulolytic enzymes, strains in G1 and G2 harboured the highest numbers of cellulolytic and pectinolytic enzymes. Moreover, hydrolytic activities were assayed in a set of strains comprising the three main pathovars and G1 and G2 clusters. It was demonstrated that G1 and G2 strains caused soft rot in pepper fruits, while the main pathovars did not. Furthermore, all the X. arboricola strains analysed hydrolysed starch, except for Xap, which also harboured a reduced set of amylolytic enzymes. The doubtful, atypical or weak pathogenicity, the genomic diversity, the limited content of T3Es, together with the soft rot capacity, the higher counts of cellulolytic and pectinolytic enzymes and the literature, suggest that strains in G1 and G2 would probably be commensal or saprophytic strains that may behave as opportunistic pathogens under certain conditions.
Methicillin-resistant Staphylococcus aureus (MRSA) ST764, a variant of ST5, has emerged and spread in Japan and China. We investigated the genome of a Thai isolate (SATU136) and the global phylogeny of ST764 to understand its global transmission history. The complete genome of SATU136 consists of a 2.89 Mb chromosome with a type II SCCmec element and a qacB-carrying plasmid but lacks the arginine catabolic mobile element (ACME). Based on the currently available global dataset, phylogenetic and phylodynamic analyses suggest that ST764 emerged in Japan in the early 1980s and was subsequently inferred to have disseminated to China and Thailand during the 1990s and early 2000s, coinciding with a peak in its overall effective population size. After this period, transmission was inferred to have become more geographically structured, with distinct clades forming in each country. Although cross-country transmission was inferred to be limited overall, onward dissemination to non-Asian countries was also detected within the Thailand-associated lineage. In contrast, the ACME element remained confined to a single Japanese subclade, with currently no evidence of spread beyond Japan. The relatively structured geographic distribution of ST764 offers a window for early detection upon introduction to new countries, which may facilitate control of its spread.
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.
