Streptococcus ruminantium-associated sheep mastitis outbreak detected in Italy is distinct from bovine isolates

Streptococcus ruminantium is the causative agent of several bovine and ovine diseases, however reports are uncommon and application of whole genome sequencing to identify is rare. We report for the frst time, a severe ovine mastitis outbreak caused by S. ruminantium in Italy, 2022. S. ruminantium was isolated from 12 adult lactating ewes with difuse nodules in the mammary parenchyma and predominantly serous and clotted milk. All outbreak isolates, along with fve additional historical Italian isolates (between 2011 and 2017), were genomically characterised and then analysed in the context of all publicly available S. ruminantium genomes. Antimicrobial susceptibility testing was performed to determine the MICs of 16 antibiotics. The results showed that all isolates were susceptible to all antimicrobials tested except kanamycin. Single Nucleotide Variant analysis confrmed this as a clonal outbreak across 10 sheep (≤15 SNVs), while the two others were colonised by more distantly related clones (≤53 pairwise SNVs), indicating the presence of multiple infecting lineages. The fve historical S. ruminantium isolates were comprised of genetically-distant singletons (between 1259 and 5430 pairwise SNVs to 2022 outbreak isolates). Ovine isolates were found to be genetically distinct to bovine isolates, forming monophyletic groups. Bovine isolates were similarly made up of singleton clones in all but two isolates. Taken together, our genomic analysis using all globally available genomes is consistent with general opportunistic pathogenesis of S. ruminantium. We encourage future genomic surveillance eforts to facilitate outbreak detection, as well as improve our understanding of this poorlyunderstood, multi-host, zoonotic pathogen. Keywords Sheep, ovine, mastitis, outbreak, Streptococcus ruminantium, antimicrobial susceptibility, whole-genome sequencing