Microbial Genomics

Campylobacter jejuni is a leading foodborne cause of gastroenteritis, but genomic surveillance remains uneven across Africa. A recent East Africa study combined whole-genome sequencing and antimicrobial susceptibility testing for Campylobacter isolates from humans with diarrhea in Kenya and poultry in Tanzania, showing high sequence-type diversity and substantially higher multidrug resistance in poultry. We extended this regional evidence by analyzing 1,013 publicly available C. jejuni genomes, including 718 African and 295 non-African comparator genomes, with standardized assembly, genotyping, phylogenomics, pangenome reconstruction, antimicrobial resistance, virulence, and mobile-element profiling. African genomes were geographically concentrated but genetically diverse, included globally distributed and regionally enriched lineages, and showed an open pangenome dominated by low-frequency gene families. Resistance and virulence determinants were unevenly distributed by region and lineage. These findings place African C. jejuni diversity within a global evolutionary framework and support expanded, integrated One Health genomic surveillance. Data summaryAll genome sequence data analysed in this study were retrieved from publicly accessible repositories, including the National Center for Biotechnology Information Sequence Read Archive and Assembly resources and corresponding records available through the International Nucleotide Sequence Database Collaboration where applicable. Accession identifiers, BioSample records, run accessions, country metadata and host/source information for all analysed genomes are provided in the combined Supplementary Data workbook. No new sequence data were generated. The analysis used publicly available data generated by other investigators, and the original data-generating studies are cited where appropriate. Derived analytical outputs supporting the findings are included in the manuscript and Supplementary Information. Impact statementGenomic surveillance of Campylobacter jejuni remains uneven globally, and African data are still underrepresented in many comparative analyses. This study brings together publicly available African and non-African C. jejuni genomes in a single standardized comparative framework, linking population structure, pangenome composition, antimicrobial-resistance determinants, virulence-associated loci and mobile-element profiles. The work shows that African C. jejuni diversity is not peripheral to the global population: African genomes include globally distributed sequence types, regionally enriched lineages and a large accessory-gene repertoire. By separating genomic surveillance signals from population-representative prevalence claims, the study provides a cautious framework for interpreting public genome collections from settings with unequal sampling. The findings support broader One Health genomic surveillance, improved metadata completeness and geographically balanced sequencing to better understand foodborne transmission, resistance evolution and lineage diversification in this important zoonotic pathogen.

OXA-48-like carbapenemases have been historically rare, however steady increases both locally and globally have warranted further investigation into their spread. Here we present the largest genomic analysis of blaOXA-181-producing bacteria in Australia to date, focusing on a single jurisdiction over seven years (2017 -- 2024). The initial investigation was prompted by an outbreak in 2017, where enhanced genomic surveillance in a single hospital identified 85 outbreak isolates related to an imported Escherichia coli ST38, carrying blaOXA-181 on an IncX3/colKP3 plasmid (previously reported as pOXA181). After four months of intensive infection control, the initial outbreak strain was eliminated. To confirm the outbreak plasmid was also contained, we collected all blaOXA-181-positive isolates from the same jurisdiction over subsequent years and sequenced with both Illumina and Oxford Nanopore Technologies to investigate clonal and mobile genetic element mediated spread. While continued surveillance post-2017 did not identify the same E. coli strain following the outbreak, pOXA181 plasmids were identified in >70% of surveillance isolates, with minimal genetic changes, which initially suggested local plasmid-mediated spread. Additional comparison to a global collection of pOXA181 plasmids found that epidemiologically unrelated pOXA181 plasmids were near identical, with no rearrangements and low, or no, single nucleotide polymorphisms. This suggests the mutation rate of pOXA-181 is incompatible with recent genomic transmission inference. This study highlights the current genomic epidemiology and drivers of blaOXA-181 and further demonstrates the necessity for detailed understanding of plasmid evolutionary rates to inform genomic surveillance.

Enterobacterales pathogens that encode multiple carbapenemase genes are an emerging public health threat. The genomic and plasmid context of the multi-carbapenemase producers has not yet been explored at scale. We analysed 1501 publicly-available global genomes that encoded two or more genes from the five prevalent carbapenemase families (IMP, KPC, NDM, OXA-48-like, and VIM). We observed 241 distinct allelic combinations, with NDM/OXA-48-like (25.6%), multiple KPC (25.3%), KPC/NDM (18.8%), and multiple NDM (10.9%) being the most common groups. We found that many genomes were part of high-risk lineages (48.0%). Most genomes carried carbapenemase genes on two plasmids (59.2%) or one plasmid (23.5%), and most genomes encoded different carbapenemase families (56.2%) rather than copies of the same allele (36.2%). We observed multiple mechanisms of multi-carbapenemase evolution including plasmid co-integration, convergence with virulence, and generation of novel allele diversity through duplication. Multi-carbapenemase plasmids had more AMR genes that conferred resistance to more AMR classes compared to plasmids encoding a single carbapenemase. The proportion of cross-class combinations (KPC/NDM and NDM/OXA-48-like) increased significantly over time, with NDM-5/OXA-181 (3.3-fold) and NDM-4/OXA-181 (8.9-fold) combinations enriched. These findings provide valuable insights into the genomic features of multi-carbapenemase Enterobacterales to support the development of effective surveillance programmes.

Carbapenem-resistant Escherichia coli (CREc) recovered sequentially from one patient typically retain the same carbapenemase, with escalating resistance usually attributed to porin loss combined with pre-existing {beta}-lactamase expression. We used whole-genome sequencing to characterize a clonal pair of CREc isolates, CAEC145 and CAEC155, recovered 25 days apart from a hospitalized patient with sequential urinary and bloodstream infection. Both belonged to sequence type 361 (ST361), phylogroup A, serotype O-nontypeable:H30, and were separated by only 28 core-genome SNPs, confirming clonal relatedness. Despite this, the isolates differed sharply in carbapenemase content. CAEC145 carried blaOXA-1207, a recently described OXA-48-family variant, on a conjugative IncFII(pCoo)/ColKP3 plasmid, whereas CAEC155 lacked this determinant and instead harbored blaNDM-4 on a conserved IncX3 plasmid nearly identical to pJEG027, a member of a globally disseminated IncX3 lineage. This genotypic shift tracked a clear phenotypic transition. CAEC145 remained susceptible to imipenem and meropenem while resistant to ertapenem, whereas CAEC155 showed uniform high-level resistance to all three carbapenems and to ceftazidime-avibactam. Both isolates, however, remained susceptible to imipenem-relebactam, meropenem-vaborbactam, and cefiderocol. Comparative genomics linked the blaOXA-1207 element to a {Delta}Tn6361 transposon structure also found in the original German isolates where blaOXA-1207 was first described, and a 137-genome core-genome phylogeny placed both isolates within a globally disseminated ST361 lineage carrying multiple carbapenemase classes. These findings document, to our knowledge, the first within-host succession from an OXA-48-like to an NDM-type carbapenemase during a single sequential E. coli infection, driven by plasmid-level displacement rather than in-place gene evolution, with implications for genomic surveillance and antibiotic selection.

Introduction. Carbapenemase-producing Enterobacterales (CPE) represent a major threat to hospitalised patients worldwide. The dissemination of carbapenemase genes, such as blaIMP, is frequently mediated by mobile genetic elements including plasmids. During a previously described multispecies, healthcare-associated outbreak of blaIMP-positive CPE in North West London, two unusual isolates, IMP47 (Klebsiella grimontii) and IMP76 (K. pneumoniae), recovered in 2019 from routinely collected rectal swabs of inpatients, were predicted to harbour blaIMP-1-carrying IncHI1 megaplasmids. Aims. This study aimed to determine complete genomic sequences of IMP47 and IMP76, resolve the genetic context of blaIMP-1, and assess the conjugative mobility of blaIMP-1-carrying megaplasmids. Methods. Genomic sequences of both isolates were recovered through hybrid assembly of Oxford Nanopore and Illumina sequencing reads. Complete plasmid sequences were characterised to determine replicons, conjugation machinery, and genes encoding resistance to antimicrobials or other stress factors. Integrons and transposable elements (TEs) within flanking regions of blaIMP-1 were resolved through genome annotation and search against public databases. Liquid-mating experiments were performed to assess the mobility of blaIMP-1-carrying plasmids. Results. Completed genome assemblies were generated from both isolates, confirming two blaIMP-1-carrying megaplasmids, pIMP47 (391 kbp) and pIMP76_1 (519 kbp), of the replicon type IncHI1A(pNDM-CIT)/IncHI1B(pNDM-CIT). The blaIMP-1 locus was carried by nearly identical class 1 integrons in both plasmids and a closely related IncHI1 megaplasmid pEB3_IMP1 (361 kbp) previously identified in South West England. Comparative analysis revealed conserved genetic structures linking blaIMP-1 to mercury-resistance genes and TEs Tn6025, TnAs3, IS26, and IS5075, suggesting a history of recombination and potential for TE-mediated mobilisation. Conjugation experiments confirmed transfer of pIMP76_1 into a recipient K. pneumoniae strain, resulting in acquisition of ertapenem resistance, whereas transfer of pIMP47 was not observed under the tested conditions. Conclusion. Two IMP-producing IncHI1 megaplasmids in gut-colonising Klebsiella species revealed TE-mediated blaIMP-1 mobilisation. The co-localisation of blaIMP-1 and metal-resistance genes in both plasmids highlights the potential for co-selection of blaIMP-1 in environments enriched with metal ions. Our findings underscore the importance of longitudinal genomic surveillance of carbapenemase-encoding megaplasmids in healthcare settings.

Plasmids play a significant role in shaping the pathogenic potential of Shiga toxin-producing Escherichia coli (STEC) by encoding virulence and adaptive genes that complement chromosomal determinants. While plasmid-encoded factors such as ehxA, espP, katP, and toxB are known to enhance intestinal colonization and host interaction, the diversity and evolutionary patterns of STEC plasmids remain insufficiently characterized. Most previous studies have focused on single serogroups, particularly O157, leaving cross-serogroup comparisons largely unexplored. To address this gap, we conducted an in-depth investigation of 109 complete plasmid sequences (1.5-187 kb) from the "Top Seven" STEC serogroups (O26, O45, O103, O111, O121, O145, and O157), retrieved from NCBI, to examine their structural organization, virulence composition, resistance patterns, mobility potential, and evolutionary dynamics. Our analysis revealed the dominance of F-type plasmids carrying IncFIB and IncFII replicons across serogroups, along with lineage-specific associations with major virulence genes. By examining the enterohemolysin operon across ehxA-positive plasmids, we identified a highly conserved structural framework maintained across 92.5% of analyzed sequences, with minimal variation. We further characterized antimicrobial resistance gene distribution, finding that only 11% of plasmids (12/109) carried such genes, of which 92% were multidrug-resistant. Analysis of mobility features revealed that predicted conjugative potential and MOBF-type relaxases varied considerably among serogroups. The 32 plasmids without virulence or resistance cargo carried at most colicin determinants, yet all received a predicted mobility class, with conjugative machinery confined to plasmids above 37 kb. At the plasmid level, serogroups O26/O103 are closely related and carry relatively high-risk virulence profiles, while the O121/O145 group exhibits moderate virulence gene inventories. The O45/O111 group is further distinguished by its transfer features, while O157 strains form a distinct clade with the broadest array of plasmid-encoded high-risk virulence genes.

Background: Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the leading causes of foodborne gastroenteritis worldwide and an increasingly important driver of antimicrobial-resistant infections. Despite its public health importance, genomic surveillance of S. Enteritidis remains sparse across the Middle East, where integrated One Health studies linking human, food, and animal reservoirs are limited. This lack of regional genomic data has hindered understanding of the population structure, transmission dynamics, and dissemination of antimicrobial-resistant lineages. We therefore performed a large-scale One Health genomic investigation of S. Enteritidis in the Kingdom of Saudi Arabia, integrating human, food, poultry, and global genomic datasets to characterize its evolutionary history, transmission dynamics, and antimicrobial resistance. Methods: A total of 220 Salmonella enterica serovar Enteritidis (S. Enteritidis) isolates obtained in Saudi Arabia from human clinical infections and food products between 2020 and 2023 underwent whole-genome sequencing. To position these isolates within a global evolutionary context, they were integrated with all publicly available S. Enteritidis genomes, yielding a comparative dataset of 397 genomes. A comprehensive genomic analysis framework was employed, encompassing population genomic, phylogenetic, and phylodynamic analyses to define population structure, infer evolutionary history, and investigate transmission dynamics. In addition, the genomic determinants of antimicrobial resistance and virulence were characterized, with particular emphasis on plasmid architecture. Results: Isolates from Saudi Arabia belonged predominantly to a single globally distributed epidemiological cluster of S. Enteritidis and were embedded within globally circulating transmission networks rather than forming a distinct endemic population. Within this cluster, the Saudi isolates were distributed across seven Bayesian Analysis of Population Structure (BAPS) groups, representing distinct genomic sub-clones. Multiple lineages contained closely related isolates from Europe, North America, Oceania, and the Middle East, indicating repeated international introductions and extensive global connectivity. Human and food isolates were highly intermixed throughout the phylogeny, with food-associated isolates frequently occupying central positions within transmission networks, consistent with food reservoirs acting as dissemination hubs linking local and international transmission. Phylodynamic analyses indicated that the dominant Saudi-associated lineages emerged within the past 10-15 years and underwent rapid demographic expansion following introduction into the country. Recently emerged epidemic lineages carried significantly higher antimicrobial resistance burdens than ancestral populations and were characterized by near fixation of blaTEM-1 and tetA on IncX1 plasmids, whereas clinically important ESBL genes (blaCTX-M-8 and blaSHV-12) were detected exclusively in human-associated isolates on hybrid resistance-virulence plasmids. Conclusions: The population of S. Enteritidis in Saudi Arabia is shaped by repeated introductions of globally circulating clones, transmission between food and human reservoirs, and the dissemination of resistance plasmids across lineages. The emergence of successful epidemic clones is linked to plasmid-mediated acquisition of antimicrobial resistance determinants. These findings emphasize the importance of integrated One Health surveillance strategies for controlling foodborne transmission and monitoring the emergence of antimicrobial-resistant epidemic lineages.

The global dissemination of antimicrobial resistance is increasingly driven by bacterial clones combining antimicrobial resistance with enhanced virulence and environmental adaptability. Escherichia coli sequence type 131 (ST131) has historically been regarded as a major disseminator of the extended-spectrum {beta}-lactamase (ESBL) blaCTX-M-15. However, the emergence of E. coli ST1193 carrying blaCTX-M-15 may represent an ongoing shift in the epidemiology of this resistance determinant. Here, we investigated the prevalence, genomic characteristics, virulence and antimicrobial resistance potential of ST1193 in comparison with ST131. A total of 1,136 E. coli isolates were recovered from touristic and non-touristic environments, hospital-associated samples, and aircraft toilets in Guadeloupe. Isolates were whole-genome sequenced and analysed for antimicrobial resistance and virulence determinants. Additionally, publicly available genomic data comprising 1,215 blaCTX-M-15-positive ST131 and ST1193 isolates were analysed to assess temporal and geographical trends. ST1193 was significantly associated with aircraft-associated samples and exhibited a higher antimicrobial resistance gene burden than ST131, while maintaining a comparable virulence factor content. Analysis of publicly available genomes revealed similar temporal emergence patterns for blaCTX-M-15-positive ST1193 and ST131, with ST1193 showing a more recent distribution and a higher number of deposited isolates in recent years, consistent with a potential ongoing clonal replacement. Comparative genomic analysis identified numerous virulence and adaptation-associated genes shared between both sequence types, while ST1193 additionally carried distinct determinants, including components of the transmissible locus of stress tolerance. Furthermore, quinolone resistance-associated mutations were strongly linked to blaCTX-M-15 carriage, particularly among ST1193 isolates. Together, these findings identify E. coli ST1193 as an emerging high-risk clone with substantial potential for blaCTX-M-15 dissemination. Its association with aircraft-associated samples further highlights the potential role of air travel in long-distance transmission and underscores the need to reconsider current surveillance strategies focused predominantly on ST131.

Based on recent epidemiologic studies, Streptococcus agalactiae (Group B Streptococcus; GBS) sequence type (ST) 1010 is an emerging lineage now identified in multiple countries. We report the phylogenetic and genomic characteristics of a set of 55 GBS sequence type (ST) 1010 strains, as well as two newly described single-locus variants of ST1010. A core genome phylogeny suggests that ST1010 is closely related to both ST452 and the hypervirulent clonal complex (CC) 17 GBS lineage. Notably, we demonstrate that genes encoding two virulence determinants previously described as specific to CC17 GBS, the HvgA adhesin and the serine-rich repeat protein Srr2, are both present in ST1010 genomes. Srr2 is shared with members of ST452. High-level gentamicin resistance (HLGR) encoded on an IS256 mobile element, previously described in a small number of ST1010 isolates, is present in a distinct ST1010 subclade encompassing the majority of ST1010 isolates. The relationship between ST452 (serotype IV), ST1010 (serotype IV), and ST17 (serotype III) strains suggests that ST17 may have arisen from a serotype IV ancestor and later acquired the type III capsule locus. Taken together, these findings clarify the phylogenetic position of ST1010 and suggest sequential acquisition of virulence determinants and HLGR prior to its international emergence. IMPACT STATEMENTST1010 GBS has emerged internationally, with colonizing and invasive isolates described in the United States, Dominican Republic, Netherlands, and Italy. Using a core genome phylogeny and targeted detection of genomic regions, we demonstrate that ST1010 shares specific virulence determinants with the CC17 hypervirulent GBS lineage and that HLGR is confined to a specific numerically dominant subclade of ST1010. Our work spotlights the importance of future epidemiologic and genomic surveillance of ST1010 and related lineages. DATA SUMMARYPublicly available genomic data were used from three previously published studies (Laycock KM et al., McGee L et al., Khan UB et al.), as well as a set of newly sequenced GBS genomes from clinical strains originating in New York City (NYC). The corresponding accession numbers and detailed information for all strains are provided in the Table.

Background: Surveillance tells laboratories which antimicrobial resistance genes are circulating now, but not which will change next. We built a model that forecasts, for each resistance gene and year, whether the following year brings a previously unseen allele (sequence variant), called emergence, or a change in the most common allele, called turnover. We tested it separately in Klebsiella pneumoniae and Acinetobacter baumannii. Results: We used genomes annotated with AMRFinderPlus: 2,385 gene-years across 331 genes for K.pneumoniae and 1,059 across 216 for A. baumannii. To remove the effect of sequencing effort, each gene-year was sampled to 10 records and the analysis repeated 25 times. Models were trained on earlier years and tested on later ones, and on genes held out of training. On later years every model ranked genes by risk better than simple baselines in both organisms, and results for held-out genes were close. In K.pneumoniae, turnover was predicted better than emergence (best area under the ROC curve, AUROC, 0.909 versus 0.819). In A. baumannii the order reversed (emergence 0.789, turnover 0.722). The two events also relied on different features. Emergence depended mainly on how many alleles a gene had already accumulated, in both organisms. Turnover in K. pneumoniae depended mainly on how dominant the leading allele already was, and a stability index added a small gain. In A. baumannii it was spread across many weaker features. There, 56% of changes in the leading allele involved only alleles already seen, against 23% in K. pneumoniae, which may make them harder to predict. Conclusions: Changes in resistance genes can be forecast a year ahead from routine genomic surveillance data, including for genes the model has not seen. How well it works, and what it depends on, differs between organisms, so models should be built and validated for each organism, not assumed to transfer. Such models could help laboratories decide which resistance genes to monitor most closely. Keywords: antimicrobial resistance; genomic surveillance; forecasting; machine learning; allele dynamics; Klebsiella pneumoniae; Acinetobacter baumannii

Background: Plasmids are key drivers of horizontal gene transfer (HGT), enabling the dissemination of accessory traits that shape microbial adaptation and ecological interactions. In Haloarchaea-dominant members of hypersaline environments-characterizing plasmidomes remains particularly challenging because most available genomes are incomplete, leaving many contigs unassigned to either chromosomal or plasmidic origin. This limitation hampers our ability to reconstruct archaeal plasmid diversity and to evaluate how plasmid-borne genes circulate within Haloarchaea and, potentially, outside this class. Results: Here, we present HaloClassifier, the first machine-learning tool specifically designed to distinguish plasmid- and chromosome-derived contigs in haloarchaeal genomes. Unlike existing bacterial-focused approaches, HaloClassifier uniquely integrates haloarchaeal-specific genomic signatures with coding-derived features-an underused but highly informative class of predictors-and incorporates a variable-selection strategy that substantially reduces model complexity and computational cost while maintaining high predictive accuracy. When trained on simulated contigs generated from complete haloarchaeal genomes, the model achieves 96.61% accuracy on extralarge contigs (40-100 kb), increasing to 98.1% at a 0.6 classification threshold, with only 3.55% of contigs left unclassified. For small contigs (1-5 kb), the model achieves 79.51% accuracy overall and 84.42% at the 0.6 classification threshold, with 17.07% of contigs left unclassified. The model also maintains robust performance in metagenomic datasets. Conclusions: By enabling reliable plasmid identification in fragmented assemblies, HaloClassifier fills a major gap and establishes the foundation for large-scale haloarchaeal plasmidome reconstruction. This framework will support future investigations into gene mobility, ecological adaptation, and the evolutionary dynamics of plasmids and HGT in Haloarchaea.

The emergence of a dominant poultry-associated strain of Campylobacter jejuni in New Zealand coincided with a multi-year epidemic of human campylobacteriosis. This was characterised by an increase in cases to 2006, when one of the highest national case-rates in the world was recorded, followed by a rapid decline attributed to interventions in the poultry industry. The strain belonged to an internationally rare multilocus sequence type, ST474 - a member of Clonal Complex (CC) 48 - that was highly prevalent in both poultry and human cases in New Zealand but was also identified in other samples. ST474 isolates from humans, animal reservoirs and environmental water samples were collected between 2005 and 2019 and whole genome sequenced (N=443). Isolates from a 2018/9 case-control study of campylobacteriosis in New Zealand were included for genomic comparisons with other sequence types and CCs. This presented an opportunity to examine the genomic epidemiology of a single, relatively recently emerged lineage of C. jejuni, and improve our understanding of drivers of emergence and persistence in a relatively isolated ecosystem. Six subpopulations (clades) of ST474 were identified and these varied in terms of their host association, persistence, patterns of homologous recombination, and complement of mobile genetic elements, including plasmids and chromosomally integrated elements. Clade E was associated with a single poultry supplier in the initial epidemic phase and accounted for 34.5% of human cases. Clade F emerged from a declining Clade E after the peak in human cases and was also associated with poultry. The emergence of Clade F was accompanied by the loss of integrated element CJIE5 and the acquisition of multiple blocks of recombination, likely from diverse ancestral C. jejuni lineages, and the loss of the methionine biosynthesis operon metAB. In contrast, Clades A-D were detected at low frequencies in human cases over the 14-year period of surveillance and were associated with multiple hosts. This longitudinal study sheds light on the evolutionary processes that result in strain emergence and differentiation, highlighting the role of horizontal gene transfer in the evolution and adaptation of this pathogen to new niches, and the subsequent impact on public health.

The molecular epidemiology of Acinetobacter, which is almost exclusively culture-dependent, has centered mainly on clinical isolates of A. baumannii. Yet, this overlooks sources not amenable to microbial culture and many non-A. baumannii species. Here, by analysing metagenomic projects from public transport systems worldwide, we show that metagenome-assembled genomes (MAGs) are highly relevant for assessing the unrecognized diversity of Acinetobacter. We recovered 839 Acinetobacter MAGs, with 269 being high-quality MAGs. The high-quality MAGs were across more than 20 well-defined Acinetobacter species; however, five species (A. nosocomilalis, A. ursingii, A. radioresistens, A. schindleri, and A. variabilis) had the most MAGs. Pangenome and phylogenetic diversity analyses of 685 genomes plus the 269 high-quality MAGs from those 5 species showed that MAGs are an important source of both lineage and gene diversity. Also, FST analyses and genome phylogenies show that for some species there is no genetic differentiation between MAGs and clinical isolates, implying transmission between hospitals and public transport settings. In addition to finding the antibiotic resistance genes (ARGs) in MAGs, we implemented an ad hoc strategy (leveraging the use of genomes) to recover considerably more ARGs. PCoA and PERMANOVA analyses show that, whereas in A. nosocomialis and A. ursingii, the ARG profiles of MAGs and clinical genomes are different, in A. radioresistens and A. schindleri, the ARG profiles are not different. Finally, some clinically important ARGs, such as sul1 and sul2, aadA, and blaOXA23, were found in all or some of the 5 species. Our study shows how combining MAGs and regular genomes is a powerful strategy for better appraising the genomic diversity (including the resistome) of Acinetobacter. On a broader level, our study showcases the importance of MAGs for genomic surveillance of man-made environments with clear public health implications.

Clostridium botulinum Group I and Clostridium sporogenes are closely related genomic lineages for which historical toxin-based nomenclature can obscure species-level relationships. We developed BoNTax, a curated taxonomy-first framework that separates average nucleotide identity (ANI)-based species assignment from botulinum neurotoxin (BoNT) characterization while integrating assembly quality assessment, multilocus sequence typing (MLST), AMRFinderPlus-based detection of bont genes, other virulence-associated genes, and antimicrobial resistance genes into standardized outputs. Analysis of 966 genomes informed genome-size criteria and the selection of 46 representative references comprising 22 C. botulinum Group I and 24 C. sporogenes genomes. Pairwise ANI resolved these references into two distinct species clusters: within-species ANI values were >95%, whereas between-species values were <95%, supporting a 95% species-level assignment threshold. BoNTax was implemented in GalaxyTrakr as complementary FASTQ- and FASTA-input workflows that converge on a common downstream analysis. The workflow completed analysis of all 42 validation genomes. Among the 32 genomes with a reported taxonomic identity, 29 assignments were concordant and three prompted taxonomic re-evaluations. For the ten genomes reported as taxonomically unknown, BoNTax provided ANI-based assignments. Of the three discordances, BT-22100019 was assigned to C. sporogenes rather than the reported C. botulinum Group I, whereas R1125/03 and R1135/03 showed 99.96% ANI to C. caseinilyticum DSM 46279, supporting their identification as C. caseinilyticum. MLST assignments were concordant for genomes with established profiles except PNUSAB000061, for which ST183 was not assigned by the MLST database version used in this analysis. BoNT type and subtype detection was fully concordant across toxigenic genomes, including bivalent strains. Thirty-one genomes met the predefined assembly-size QC criterion, whereas 11 received QC warnings, including all eight datasets representing four known mixed cultures. BoNTax therefore provides a reproducible genomic framework for resolving closely related clostridial lineages, identifying taxonomic annotations requiring re-evaluation, and reporting clinically relevant toxin information without conflating toxin genotype with species identity.

Background. Genomic surveillance of Gram-negative pathogens is generally selective, focusing on suspected outbreaks or resistant isolates. The extent of what such programmes miss remains unknown. It has not been quantified in a large, unselected dataset. Methods. We prospectively whole-genome sequenced isolates of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa from two tertiary-care hospitals in Copenhagen and Hannover, regardless of resistance phenotype, specimen type, or ward. We analysed 25,888 genomes from 13,712 patients. Clonal clusters were defined by single-linkage clustering at [&le;]30 SNPs, based on within-patient distances and validated against inpatient movement records. Analyses were patient-based, with hospital comparisons using matched 18-month periods. Results. Clonal clusters included 14.1% of patients in Copenhagen and 16.5% in Hannover. Most clustered patients had no multidrug-resistant isolate (71.3% and 50.0%) and had no intensive care unit (ICU)-associated isolate (86.1% and 68.7%). Sequencing blood-culture isolates alone, the usual trigger for genomic investigation, would have identified only 14 of 900 clustered patients in Hannover (1.6%). Excluding colonisation screening would have missed 240 (26.7%). No targeted strategy detected more clustered patients than random sampling of the same number of isolates. Detection increased with sequencing effort without reaching a plateau. After matching periods and adjusting for ICU association, resistance, and specimen mix, clustering remained more common in Hannover (adjusted odds ratio 1.49, 95% CI 1.28-1.73), suggesting sampling explained much, but not all, of the difference. Conclusions. Most Gram-negative clusters involved non-MDR organisms on general wards and would have been missed by surveillance focused on resistance or intensive care. What is selected determines which patients can be detected; the number of isolates sequenced determines how many are found. The observed 14-16% is a lower bound. Surveillance should sample broadly and representatively, and report what was sequenced and how much.

The Enterobacter cloacae species complex (ECC) comprises a group of closely related, opportunistic Gram negative bacteria of major public health concern due to their frequent involvement in healthcare-associated infections and their capacity to acquire and disseminate multidrug resistance, including carbapenemases. Because members of this complex are often difficult to distinguish phenotypically and their taxonomic status is subject to debate, there is a need for a standardized, species-complex wide typing scheme based on whole genome sequencing (WGS) that can be applied to any species within the complex. In this study we have developed and evaluated a core genome multi-locus sequence typing (cgMLST) scheme suitable for species within the ECC. Using 3442 publicly available genomes from 27 ECC species or subspecies we developed a scheme with 1812 loci, comprising loci present in 99% of all genomes. Among the 3442 isolates in our study, 99.9% had 95% or more of the cgMLST targets, indicating that the schema is well-defined and representative for the breadth of ECC species in our study. On two independent evaluation datasets, the scheme reliably resolved epidemiologically linked isolates with 0-3 allelic differences and returned the same outbreak clusters defined previously by higher resolution core genome SNP (cgSNP) analysis. Hierarchical clustering analysis at different levels of resolution showed that the cgMLST profiles could potentially be used to differentiate between species and sub-lineages in the complex. The cgMLST schema will improve the ability of public health laboratories to perform WGS-based surveillance of ECC species.

The vaginal microbiome is associated with reproductive and sexual health, yet its viral component remains understudied. A recent vaginal genome catalogue reported that 85.8% of its species-level viral groups were absent from all five viral databases examined. Viral identification tools depend on reference sequences, either directly or through their training data. Previous benchmarks used simulated or non-vaginal datasets. How well these tools generalise to vaginal metagenomes therefore remains unclear. We benchmarked 14 tools spanning five methodological approaches. Evaluations used controlled genome fragments, viral spike-in co-assemblies and virus-like particle (VLP)-enriched co-assemblies. A multi-evidence benchmark assessed 13 vaginal shotgun assemblies (primary cohort). An independent 30-sample cohort provided external validation. geNomad ranked first or second by Matthews correlation coefficient (MCC) in every ranked evaluation. VIBRANT achieved similar MCC to geNomad on the primary assemblies. It made eight false-positive calls among 11,577 negatives but recovered only 22% of viral contigs. VIBRANT ranked first in the independent cohort. Tool rankings agreed closely between cohorts (Spearman rho = 0.952; 95% CI 0.921 - 0.960). Detection of sequences lacking reference homologues differed substantially between tools. Two phage genomes without detectable reference homologues provided a controlled novelty test. Each evaluated composition-based tool recovered at least 74% of their 1,500-bp fragments. Marker- or reference-dependent tools recovered at most 31%. A separate analysis examined 48 unconfirmed candidate viral contigs lacking detectable nucleotide or protein homologues. Jaeger flagged 41, compared with four for VIBRANT and one for geNomad. On short fragments, attention-based tools achieved 4.0-5.5 times the mean area under the precision-recall curve of feature-based tools. ViraLM accounted for most of this difference. ViraLM also had the highest recall (sensitivity) of eukaryote-infecting viruses in the evaluated panel. VirSorter2 achieved the highest MCC on VLP-enriched contigs. These results support geNomad when prioritising overall classification performance and VIBRANT when minimising false-positive phage calls is the priority. ViraLM supports short-contig and eukaryote-infecting-virus detection. VirSorter2 supports identification in VLP-enriched assemblies, and Jaeger supports screening for candidate novel phages. These findings guide tool selection for studies investigating the vaginal virome in reproductive and sexual health.

The Klebsiella oxytoca species complex (KoSC) comprises taxonomically diverse commensals and opportunistic pathogens, but its genomic diversity remains poorly characterized across Africa. We curated publicly available African KoSC data through raw-read and public-assembly routes and analyzed 163 African genomes together with 282 global comparators. Pangenome, phylogenomic, sequence-typing, surface-locus, antimicrobial-resistance, plasmid-replicon, mobile-element, biosynthetic-gene-cluster, and virulence-component analyses were integrated. The African collection comprised K. michiganensis (112/163), K. oxytoca (36/163), K. pasteurii (8/163), and K. grimontii (7/163) from 13 countries. The African pangenome contained 4,286 core, 4,505 shell, and 18,066 cloud gene families. Official PubMLST sequence types were assigned to 129/163 genomes. Four core/intrinsic antimicrobial-resistance-associated loci (ompA, oqxA, oqxB, and blaOXY) occurred in all genomes, whereas acquired resistance determinants were heterogeneous. Intact til biosynthetic gene clusters occurred in 55/163 genomes and intact leup clusters in 103/163. leup was concentrated in K. michiganensis (102/112), whereas intact til was frequent in K. oxytoca (26/36), K. pasteurii (7/8), and K. grimontii (7/7). Klebsiella-focused Virulence Factor Database screening detected at least one curated component in 112/163 genomes, but no complete curated factor; three K. michiganensis genomes carried complete mrkABCDF structural-operon candidates. These data define an African genome-resolved baseline for KoSC diversity and identify species-structured biosynthetic loci alongside heterogeneous resistance and mobilome profiles.

The diagnosis of Legionnaires' disease (LD) caused by Legionella non-pneumophila species is likely to increase with broader use of PCR targeting Legionella spp. In this context, accurate species identification in PCR-positive but culture-negative samples is essential to improve understanding of disease epidemiology and to support source attribution. Here, we presented a validated and user-friendly bioinformatic pipeline compatible with next-generation sequencing (NGS) for analyzing the hypervariable 23S-5S region, paired with a curated database encompassing all described Legionella species as of January 2026. Parameters were optimized for sensitivity and specificity using both strains and culture-positive clinical and environmental samples. We then applied the pipeline retrospectively to 92 culture-negative PCR-positive samples collected from 2023 to 2025. Legionella species were successfully assigned in 60% (55/92) of tested samples and revealed a high diversity. Co-infections were detected in clinical samples, including combinations of L. pneumophila with L. longbeachae or L. bozemanii, while environmental samples contained up to six different species. These results demonstrate that 23S-5S amplicon NGS enables species-level identification in the absence of cultured isolates, improving surveillance of non-pneumophila Legionella cases. The proposed pipeline, implemented in QIIME2 and accompanied by a publicly available database, provides a practical framework for routine molecular monitoring and outbreak investigation.

Background Antimicrobial-resistant Gram-negative bacteria pose a major threat to global health. In the WHO Bacterial Priority Pathogen List (BPPL) 2024, several species were classified as critical or high priority for research and development of new antimicrobial therapeutics and diagnostics. However, access to well-characterised and clinically relevant bacterial isolate panels remains an unmet need for development and validation. Methods We constructed five panels of Gram-negative bacterial isolates, cultured from samples originally referred to the UK Health Security Agency Antimicrobial Resistance and Healthcare-Associated Infections Reference Unit for analysis between 2014 and 2025. For each isolate, antimicrobial susceptibilities were determined against a range of clinically relevant antibiotics, including third-generation cephalosporins, carbapenems, colistin and other last-line antibiotics. WGS was performed on all isolates, and previously described resistance determinants were characterised. Results The five panels consist of 145 isolates and represent 81 sequence types across Escherichia coli (30 isolates), Klebsiella pneumoniae (30 isolates), carbapenem-resistant Acinetobacter baumannii (30 isolates), carbapenem-resistant Pseudomonas aeruginosa (30 isolates), and a mixed panel of other healthcare-associated Enterobacterales species (25 isolates). Panels are highly representative of globally relevant strains, including those currently in circulation and on the WHO BPPL 2024, and of resistance mechanisms of public health importance. Conclusion The panels provide a diverse, comprehensive set of multidrug-resistant Gram-negative bacterial isolates, representative of strains currently in circulation globally. Selected isolates carry a breadth of important resistance mechanisms and are highly relevant to the current epidemiological landscape. The isolates and associated metadata are available to industry, academia, and other laboratories for use in developing novel antimicrobial compounds and diagnostic assays.