- Volume 11, Issue 3, 2025

Nanopore sequencing is a third-generation technology known for its portability, real-time analysis and ability to generate long reads. It has great potential for use in clinical diagnostics, but thorough validation is required to address accuracy concerns and ensure reliable and reproducible results. In this study, we automated an open-source workflow (freely available at https://gitlab.com/FLI_Bioinfo/nanobacta) for the assembly of Oxford Nanopore sequencing data and used it to investigate the reproducibility of assembly results under consistent conditions. We used a benchmark dataset of five bacterial reference strains and generated eight technical sequencing replicates of the same DNA using the Ligation and Rapid Barcoding kits together with the Flongle and MinION flow cells. We assessed reproducibility by measuring discrepancies such as substitution and insertion/deletion errors, analysing plasmid recovery results and examining genetic markers and clustering information. We compared the results of genome assemblies with and without short-read polishing. Our results show an average reproducibility accuracy of 99.999955% for nanopore-only assemblies and 99.999996% when the short reads were used for polishing. The genomic analysis results were highly reproducible for the nanopore-only assemblies without short read in the following areas: identification of genetic markers for antimicrobial resistance and virulence, classical MLST, taxonomic classification, genome completeness and contamination analysis. Interestingly, the clustering information results from the core genome SNP and core genome MLST analyses were also highly reproducible for the nanopore-only assemblies, with pairwise differences of up to two allele differences in core genome MLST and two SNPs in core genome SNP across replicates. After polishing the assemblies with short reads, the pairwise differences for cgMLST were 0 and for cgSNP were 0–1 SNP across replicates. These results highlight the advances in sequencing accuracy of nanopore data without the use of short reads.

Aromatic ring-hydroxylating dioxygenases (ARHDs) play a crucial role in the aerobic biodegradation of both natural and anthropogenic aromatic compounds. Although their ability to process contaminants is not entirely understood, it is thought to have evolved from the transformation of structurally similar secondary plant metabolites (SPMs). Hence, to investigate this connection, we tested a variety of SPMs from the monoterpene and flavonoid classes as carbon sources and transcriptional effectors of several phylogenetically distant ARHD genes involved in the degradation of aromatic pollutants. Specifically, we focused on bphA1, nahA1 and phtA1 in Rhodococcus opacus C1, whose genomic analysis is also presented hereinafter, and bphA1a, nahA1-bphA1b and etbA1ab in Rhodococcus sp. WAY2. Whilst induction was only observed with (R)-carvone for bphA1a and nahA1-bphA1b of strain WAY2, and with p-cymene for nahA1 and nahA1-bphA1b of strains C1 and WAY2, respectively, an extensive inhibition by flavonoids was observed for most of the genes in both strains. To the best of our knowledge, our study is the first to report the effect of flavonoids and monoterpenes on the transcription of nahA1, etbA1 and phtA1 genes. In addition, we show that, in contrast to pseudomonads, many flavonoids inhibit the transcription of the ARHD genes in rhodococci. Thus, our work provides a new perspective on flavonoids as the transcriptional effectors of ARHDs, highlighting the significant variability of these enzymes and the divergent responses that they elicit. Moreover, our results contribute to understanding the complex interactions between microorganisms and SPMs and provide insights into the molecular basis of a number of them.

Marine sediments are vast, underexplored habitats and represent one of the largest carbon deposits on our planet. Microbial communities drive nutrient cycling in these sediments, but the full extent of their taxonomic and metabolic diversity remains to be explored. Here, we analysed shallow coastal and deep subseafloor sediment cores from 0.01 to nearly 600 metres below the seafloor, in the Western Pacific Region. Applying metagenomics, we identified several taxonomic clusters across all samples, which mainly aligned with depth and sediment type. Inferring functional patterns provided insights into possible ecological roles of the main microbial taxa. These included Chloroflexota, the most abundant phylum across all samples, whereby the classes Dehalococcoida and Anaerolineae dominated deep-subsurface and most shallow coastal sediments, respectively. Thermoproteota and Asgardarchaeota were the most abundant phyla among Archaea, contributing to high relative abundances of Archaea reaching over 50% in some samples. We recovered high-quality metagenome-assembled genomes for all main prokaryotic lineages and proposed names for three phyla, i.e. Tangaroaeota phyl. nov. (former RBG-13-66-14), Ryujiniota phyl. nov. (former UBA6262) and Spongiamicota phyl. nov. (former UBA8248). Metabolic capabilities across all samples ranged from aerobic respiration and photosynthesis in the shallowest sediment layers to heterotrophic carbon utilization, sulphate reduction and methanogenesis in deeper anoxic sediments. We also identified taxa with the potential to be involved in nitrogen and sulphur cycling and heterotrophic carbon utilization. In summary, this study contributes to our understanding of the taxonomic and functional diversity in benthic prokaryotic communities across marine sediments in the Western Pacific Region.

Diabetes mellitus is a complex metabolic disorder and one of the fastest-growing global public health concerns. The gut microbiota is implicated in the pathophysiology of various diseases, including diabetes. This study utilized 16S rRNA metagenomic data from a volunteer citizen science initiative to investigate microbial markers associated with diabetes status (positive or negative) and type (type 1 or type 2 diabetes mellitus) using supervised machine learning (ML) models. The diversity of the microbiome varied according to diabetes status and type. Differential microbial signatures between diabetes types and negative group revealed an increased presence of Brucellaceae, Ruminococcaceae, Clostridiaceae, Micrococcaceae, Barnesiellaceae and Fusobacteriaceae in subjects with diabetes type 1, and Veillonellaceae, Streptococcaceae and the order Gammaproteobacteria in subjects with diabetes type 2. The decision tree, elastic net, random forest (RF) and support vector machine with radial kernel ML algorithms were trained to screen and type diabetes based on microbial profiles of 76 subjects with type 1 diabetes, 366 subjects with type 2 diabetes and 250 subjects without diabetes. Using the 1000 most variable features, tree-based models were the highest-performing algorithms. The RF screening models achieved the best performance, with an average area under the receiver operating characteristic curve (AUC) of 0.76, although all models lacked sensitivity. Reducing the dataset to 500 features produced an AUC of 0.77 with sensitivity increasing by 74% from 0.46 to 0.80. Model performance improved for the classification of negative-status and type 2 diabetes. Diabetes type models performed best with 500 features, but the metric performed poorly across all model iterations. ML has the potential to facilitate early diagnosis of diabetes based on microbial profiles of the gut microbiome.

Plasmids are well-known vehicles of antimicrobial resistance (AMR) gene dissemination. Through conjugation, plasmid-encoded AMR genes are spread among neighbouring bacteria, irrespective of their strain or even their species. This process is very concerning from a public health perspective, as plasmid-borne AMR gene outbreaks are often not confined to single species or strains and are therefore more difficult to fully uncover. At the moment, the impact of plasmid conjugation on within–patient plasmid diversity is not well understood. In this work, we will tackle the role of conjugation on within–patient plasmid diversity using a dataset of carbapenemase-producing Enterobacterales. The dataset of 256 sequences originates from bacterial isolates cultured from 115 English patients over 30 months. Each patient has more than one sequence, with at least one sequence carrying an OXA-48 gene, a well-known plasmid-borne carbapenemase-encoding gene. If more than one sequence carries the OXA-48 gene, they are carried in different bacterial hosts. Using a hybrid de novo–on-reference assembly pipeline, we were able to reconstruct the full OXA-48 plasmid from short read sequencing data for 232 of the 256 sequences. Of the 115 patients, 83 (72%) patients had an identical OXA-48 plasmid in two or more sequences. Only two patients carried very different (>200 SNPs) alleles of the OXA-48 plasmid, probably from separate acquisitions. Our study shows that when more than one bacterial host carrying an OXA-48 plasmid is found in a patient, it is most likely that the same plasmid has been shared via conjugation. The event of separate acquisition of different plasmids in different bacterial hosts is highly unlikely in our dataset.

Purpose. The prevalence of multidrug-resistant (MDR) Shigella sonnei is increasing globally, raising concerns for public health. In 2022, an outbreak of MDR S. sonnei was observed in Tunisia. We aimed to evaluate the genetic profile of S. sonnei isolates during the outbreak, including their clonal relationship, antimicrobial determinants and connection to international strains.

Methods. In this study, we sequenced the whole genome of 24 S. sonnei strains collected from South Tunisia between July 2022 and November 2023. Bioinformatic analysis was conducted to confirm species identification, assign sequence types, determine core genome sequence types, analyse phylogenetic relationships and identify antimicrobial resistance determinants. Phylodynamic and phylogeographic analyses were performed to trace the spatiotemporal spread of the outbreak genotype.

Results. Our investigation revealed that 23 out of 24 isolates were grouped into the HC10-20662 genotype within the 3.6.3 subclade. All isolates carried the blaCTX-M-15 gene associated with extended-spectrum beta-lactamase production, as well as the dfrA1 and qnrS1 genes, along with the D87G mutation in gyrA. Additionally, the sul2, tet(A) and mph(A) resistance genes were present in most isolates (96%, 96 and 83, respectively). Phylogeographic analysis suggested that the outbreak genotype likely spread in Europe before being introduced into Tunisia.

Conclusion. To the best of our knowledge, this is the first MDR S. sonnei outbreak in the country. The HC10-20662 genotype appears to be responsible for a multi-country outbreak, affecting both Tunisia and Europe. Continued genomic surveillance efforts, both nationally and internationally, are essential for monitoring the dynamic evolution and global spread of MDR S. sonnei.

Staphylococcus aureus (SA) is an opportunistic pathogen and human commensal that is frequently present in the upper respiratory tract, gastrointestinal tract and skin. While SA can cause diseases ranging from minor skin infections to life-threatening bacteraemia, it can also be carried asymptomatically. Indigenous individuals in the Southwest USA experience high rates of invasive SA disease. As carriage is the most significant risk factor for disease, understanding the dynamics of SA carriage, and in particular co-carriage of multiple strains, is important to develop strategies to prevent transmission in vulnerable communities. Here, we investigated SA co-carriage and intrahost evolution by sampling several colonies from multiple anatomical sites and whole-genome sequencing (WGS) on 310 SA isolates collected from 60 Indigenous adults participating in a cross-sectional carriage study. We assessed the richness and diversity of SA isolates via differences in multilocus sequence type, core-genome SNPs and genome content. Using WGS data, we identified 95 distinct SA intra-subject lineages (ISLs) among 60 participants; co-carriage was detected in 42% (25/60). Notably, two participants each carried four distinct SA ISLs. Variation in antibiotic resistance determinants among carried strains was identified among 42% (25/60) of participants. Lastly, we found unequal distribution of clonal complex by body site, suggesting that certain lineages may be adapted to specific anatomical sites. Together, these findings suggest that co-carriage may occur more frequently than previously appreciated and further our understanding of SA intrahost diversity during carriage, which has implications for surveillance activities and epidemiological investigations.

Staphylococcus pseudintermedius is the foremost cause of opportunistic canine skin and mucosal infections worldwide. Multidrug-resistant (MDR) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) lineages have disseminated globally in the last decade and present significant treatment challenges. However, little is known regarding the factors that contribute to the success of MDR lineages. In this study, we compared the genome sequence of 110 UK isolates of S. pseudintermedius with 2166 genomes of S. pseudintermedius populations from different continents. A novel core genome multi-locus typing scheme was generated to allow large-scale, rapid and detailed analysis of S. pseudintermedius phylogenies and was used to show that the S. pseudintermedius population structure is broadly segregated into an MDR population and a non-MDR population. MRSP lineages are predicted to encode certain resistance genes either chromosomally or on plasmids, and this is associated with their MLST sequence type. A comparison of lineages most frequently implicated in disease, ST-45 and ST-71, with the phylogenetically related ST-496 lineage that has a comparatively low disease rate, revealed that ST-45 and ST-71 genomes encode distinct combinations of phage-defence systems and concurrently encode a high number of intact prophages. In contrast, ST-496 genomes encode a wider array of phage defence systems and lack intact and complete prophages. These findings indicate that MRSP lineages have significant structural genomic differences and that prophage integration and differential antiviral systems correlate with the emergence of successful genotypes.

More antibiotics are administered to livestock animals than to treat human infections. Industrialization, large animal densities and early weaning mean pigs are exposed to more antibiotics than any other livestock animal. Consequently, antimicrobial resistance (AMR) is common among commensal and pathogenic bacteria. Heavy metals (HMs) are also often used as feed additives for growth promotion and infection prevention alongside antimicrobials, and increased exposure to copper, zinc and cadmium can further encourage AMR through co-selection. In this study, we sequenced an archived collection of 112 Escherichia coli isolates from pigs in Catalonia using short- and long-read sequencing methods to detect AMR and HM tolerance genes. The most common AMR genes were mdfA (84.8%), aph(3″)-Ib (52.7%), bla TEM-1B (45.6%) and aph(6)-Id (45.6%). Genes relevant to public health, such as the extended-spectrum β-lactamases (15.4%), bla CTX-M type or bla SHV, or mobile colistin resistance (mcr) genes (13.4%), such as mcr-1, were also found. HM tolerance genes were present in almost every genome but were rarely located in plasmids, and, in most cases, AMR and HM tolerance genes were not located on the same plasmids. Of the genes predicted to increase tolerance to HMs, only those with activity to mercury were co-located on plasmids alongside other AMR determinants. However, mercury is rarely used in pig farming and does not support a scenario where AMR and HM genes are co-selected. Finally, we identified the exclusive association between mcr-4 and ColE10 plasmid, which may help target interventions to curtail its spread among pig Escherichia coli.

Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a pathogen of global concern associated with invasive community-acquired infections. The combination of hypervirulence and carbapenem resistance can result in severe and difficult-to-treat infections. This retrospective study aimed to investigate the spread of hvKp sequence type 23 (ST23) in Ireland and the convergence of hypervirulent (hv) and antimicrobial resistance genotypes. Short-read sequences (PE300) for 90 K. pneumoniae ST23 isolates were generated by the Galway Reference Laboratory Services (GRLS). Isolates were from screening swabs (n=59), invasive infections (n=18), non-invasive sites (n=12) and the hospital environment (n=1). The virulence and resistance content were assessed genomically using Kleborate (v2.2.0), ABRicate (v1.0.1) and Platon (v1.6). The in vivo virulence of the isolates was assessed using a murine model. All isolates were genotypically hv with 88/90 isolates having a maximal Kleborate virulence score of 5 including carriage of key genes. Eighty-two per cent of isolates (74/90) carried a carbapenemase gene (bla OXA-48/bla OXA-181/bla NDM-1), and 42% carried resistance genes to 3 or more antimicrobial classes. Core genomic delineation revealed the isolates to be clonal with similar resistance and virulence profiles. Two distinct clusters of Irish isolates were detected consisting of 82/90 of the isolates. Isolates associated with carriage and infection demonstrated similar in vivo virulence. An established clone of hvKp ST23 is circulating within Ireland and causing both colonization and infection of patients. The lack of reliable screening methods for hvKp makes its detection and control in the healthcare setting challenging.

Background and aims. Despite the introduction of pneumococcal conjugate vaccines (PCVs), Streptococcus pneumoniae still remains an important cause of morbidity and mortality, especially among children under 5 years in sub-Saharan Africa. We sought to determine the distribution of serotypes, lineages and antimicrobial resistance of S. pneumoniae from carriage and disease among children presenting to health facilities, 5–6 years after the introduction of PCV10 in Ethiopia.

Methods. Whole-genome sequencing (WGS) was performed on 103 S. pneumoniae (86 from nasopharyngeal swabs, 4 from blood and 13 from middle ear discharge) isolated from children aged <15 years at 3 healthcare facilities in Addis Ababa, Ethiopia, from September 2016 to August 2017. Using the WGS data, serotypes were predicted, isolates were assigned to clonal complexes, global pneumococcal sequence clusters (GPSCs) were inferred and screening for alleles and mutations that confer resistance to antibiotics was performed using multiple bioinformatic pipelines.

Results. The 103 S. pneumoniae isolates were assigned to 38 serotypes (including nontypeable) and 46 different GPSCs. The most common serotype was serotype 19A. Common GPSCs were GPSC1 [14.6% (15/103), sequence type (ST) 320, serotype 19A], GPSC268 [8.7% (9/103), ST 6882 and novel STs; serotypes 16F, 11A and 35A] and GPSC10 [8.7% (9/103), STs 2013, 230 and 8804; serotype 19A]. The four invasive isolates were serotype 19A (n=2) and serotype 33C (n=2). Resistance to penicillin (>0.06 µg ml−1, CLSI meningitis cutoff) was predicted in 57% (59/103) of the isolates, and 43% (25/58) penicillin-binding protein allele combinations were predicted to be associated with penicillin resistance. Resistance mutations in folA (I100L) and/or folP (indel between fifty-sixth and sixty-seventh aa) were identified among 66% (68/103) of the isolates, whilst tetracycline (tetM) and macrolide (ermB and mefA) resistance genes were found in 46.6% (48/103), 20.4% (21/103) and 20.4% (21/103) of the isolates, respectively. Multidrug resistance (MDR) (≥3 antibiotic classes) was observed in 31.1% (32/103) of the isolates. GPSC1 and GPSC10 accounted for 46.8% (15/32) and 18.7% (6/32) of the overall MDR.

Conclusion. Five to 6 years after the introduction of PCV10 in Ethiopia, the S. pneumoniae obtained from carriage and disease among paediatric patients showed diverse serotype and pneumococcal lineages. The most common serotype identified was 19A, expressed by the MDR lineages GPSC1 and GPSC10, which is not covered by PCV10 but is included in PCV13. Continued assessment of the impact of PCV on the population structure of S. pneumoniae in Ethiopia is warranted during and after PCV13 introduction.

In the Netherlands, the 7-valent pneumococcal conjugate vaccine (PCV) was introduced to the childhood immunization programme in 2006 and replaced by the 10-valent PCV (PCV10, GSK) in 2011. To describe invasive pneumococcal disease in the era of childhood PCV vaccination on pneumococcal bacteraemia across all ages, we collected and sequenced 979 pneumococcal blood isolates from consecutive patients with pneumococcal bacteraemia in the Gelderland area, the Netherlands, between 2000 and 2020. In total, 58% of the bacteraemia cases (n=563/979) occurred in the elderly population. Compared to the pre-PCV period (2000–2005), the odds ratio for non-PCV10 bacteraemia was 17.5 (CI 9.9–31.6; P<0.001) in the late-PCV10 period, showing an overall increase in the proportion of bacteraemia cases being caused by non-vaccine serotype pneumococci (2016–2020). The increase in non-PCV10 serotypes is mainly driven by an expansion of lineage global pneumococcal sequencing cluster 3 (GPSC3) expressing serotype 8, alongside the emergence of serotype 12F that was mediated by multiple lineages (GPSC32/GPSC26/GPSC55). Both serotypes 8 and 12F were included in the latest PCV20 formulation that is licensed to be used in children and adults in Europe. Over 20 years, we observed a low prevalence of antimicrobial resistance (AMR) as predicted by genome data. There were no significant changes in AMR prevalence after vaccine introduction (P>0.05 for all comparisons). We saw a stably low prevalence of reduced penicillin susceptibility, which was observed in multiple pneumococcal lineages, with GPSC10 being the most common in the Gelderland collection, whilst GPSC1 and GPSC6 were common among the penicillin-resistant pneumococcal blood culture isolates provided by the Netherlands Reference Laboratory for Bacterial Meningitis. Comparison to global collections of GPSC10, GPSC1 and GPSC6 isolates favored the likelihood of separate introductions of penicillin-resistant isolates rather than cloncal expansion. Genomic surveillance of pneumococcal bacteraemia in this unbiased population sample in the Netherlands supports the use of higher valency PCVs, such as PCV20, especially in adults, to prevent future bacteraemia cases caused by Streptococcus pneumoniae in the Gelderland area, the Netherlands, while maintaining a low prevalence of AMR in the pneumococcal population.

Human respiratory syncytial virus (HRSV) is a common cause of lower respiratory tract infections globally, and changes in viral epidemiology have been observed in many jurisdictions following the coronavirus disease 2019 (COVID-19) pandemic. Newly licensed vaccines and monoclonal antibodies are anticipated to alleviate the burden on healthcare systems, though such interventions may exert selective pressures on viral evolution. To evaluate the diversity of HRSV in Ireland pre- and post-COVID-19 pandemic, whole-genome sequencing was performed on HRSV-A (n=123) and -B (n=110) samples collected from community and hospitalized cases, during three HRSV seasons between 2021 and 2024. Additionally, G gene sequences, from HRSV-A (n=141) and -B (n=141), collected in the 2015–2019 period were examined. Lineages were assigned by phylogenetic analyses including reference lineages. Phylogenetic trees inferred with the G gene and whole genomes were consistent. Changes in the prevalence of certain lineages post-COVID-19 reflected the impact of non-pharmaceutical interventions (NPIs) introduced to reduce severe acute respiratory syndrome coronavirus 2 transmission, with A.D.1 and A.D.5 the dominant HRSV-A lineages and B.D.E.1 the most prevalent HRSV-B lineage. Similar trends were observed in HRSV lineages circulating across Europe during this time. The emergence of a new lineage was identified as a descendant from A.D.1, with eight distinctive substitutions in proteins G, F and L. Other circulating lineages with aa substitutions were observed in the F glycoprotein, which could impact nirsevimab binding. We provide the first comprehensive analysis of HRSV genomic diversity and evolution in Ireland over the last decade and the impact of the NPIs introduced during the COVID-19 pandemic. This study provides a foundation for future public health surveillance employing pathogen genomics to enable an evidence-based assessment of the impact of pharmaceutical interventions on HRSV evolution and disease severity.

RP4, RP1, RK2 and R68 were isolated from the multidrug-resistant bacterial wound isolates in 1969 in the Birmingham Accident Hospital, Birmingham, England, and collectively called Birmingham-group IncP-1α plasmids. These plasmids have been widely used as models to study different aspects of plasmid biology, develop genetic delivery systems and design plasmid vectors. Early studies showed that these plasmids conferred the same antibiotic resistance profile, had a similar size and were undistinguishable from each other using DNA heteroduplex electron microscopy and restriction endonuclease analyses. These observations have led to the widely held assumption that they are identical, although there has been no conclusive supporting evidence. In this work, we sequenced the plasmids RP1 and RP4 from our laboratory strain collection and compared these new sequences with the plasmids RP4 and RK2 assembled from a publicly available sequencing database, showing that the RP1, RP4 and RK2 plasmids are 60 095 bp in length and identical at the nucleotide resolution. Noteworthily, the plasmid sequence is highly conserved despite having been distributed to different labs over 50 years and propagated in different bacterial hosts, strengthening the previous observation that the bacterial host adapts to the RP4/RP1/RK2 plasmid rather than the opposite. In the updated RP4/RP1/RK2 sequence, we found a fusion gene, called pecM-orf2, that was formed putatively by a genetic deletion event. By searching for pecM-orf2 in the National Center for Biotechnology Information database, we detected remnants of the RP4/RP1/RK2 plasmid that carry features of laboratory-engineered vectors in bacterial environmental isolates, either in their chromosome or as a plasmid. This suggests a leak of these plasmids from the laboratory into the environment, which may subsequently impact bacterial evolution and raises concerns about the biocontainment of engineered plasmids when being handled in laboratory settings.

Streptococcus equi subsp. equi causes the equine respiratory disease ‘strangles’, which is highly contagious, debilitating and costly to the equine industry. S. equi emerged from the ancestral Streptococcus equi subsp. zooepidemicus and continues to evolve and disseminate globally. Previous work has shown that there was a global population replacement around the beginning of the twentieth century, obscuring the early genetic events in this emergence. Here, we have used large-scale genomic analysis of S. equi and its ancestor S. zooepidemicus to identify evolutionary events, leading to the successful expansion of S. equi. One thousand two hundred one whole-genome sequences of S. equi were recovered from clinical samples or from data available in public databases. Seventy-four whole-genome sequences representative of the diversity of S. zooepidemicus were used to compare the gene content and examine the evolutionary emergence of S. equi. A dated Bayesian phylogeny was constructed, and ancestral state reconstruction was used to determine the order and timing of gene gain and loss events between the different species and between different S. equi lineages. Additionally, a newly developed framework was used to investigate the fitness of different S. equi lineages. We identified a novel S. equi lineage, comprising isolates from donkeys in Chinese farms, which diverged nearly 300 years ago, after the emergence of S. equi from S. zooepidemicus, but before the global sweep. Ancestral state reconstruction demonstrated that phage-encoded virulence factors slaA, seeL and seeM were acquired by the global S. equi after the divergence of the basal donkey lineage. We identified the equibactin locus in both S. equi populations, but not S. zooepidemicus, reinforcing its role as a key S. equi virulence mechanism involved in its initial emergence. Evidence of a further population sweep beginning in the early 2000s was detected in the UK. This clade now accounts for more than 80% of identified UK cases since 2016. Several sub-lineages demonstrated increased fitness, within which we identified the acquisition of a new, fifth prophage containing additional toxin genes. We definitively show that acquisition of the equibactin locus was a major determinant in S. equi becoming an equid-exclusive pathogen, but that other virulence factors were fixed by the population sweep at the beginning of the twentieth century. Evidence of a secondary population sweep in the UK and acquisition of further advantageous genes implies that S. equi is continuing to adapt, and therefore, continued investigations are required to determine further risks to the equine industry.