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 phageencoded 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. Impact Statement Strangles, caused by Streptococcus equi subsp. equi, is highly contagious and results in high morbidity and economic losses to the equine industry. Previous genomic analyses determined that S. equi evolved into a successful equine host-restricted pathogen due to the acquisition of several prophages and integrative conjugative elements. It also identified a potential genetic population sweep in the early twentieth century. We have combined multiple published genomic datasets and newly sequenced isolates to create the largest genomic dataset of S. equi to date. This has enabled us to shed new light on the evolutionary events that led to the successful pathogen we see infecting equines currently. Our work provides a deeper understanding of these processes and provides groundwork for further genomic analyses of contemporary S. equi. We have demonstrated that S.equi is continuing to evolve by identifying the potential acquisition of another prophage in a proportion of isolates and a second ongoing genetic population sweep.
Sara Frosth (Wed,) studied this question.