Federico Cunha ^1* Yuting Zhai ² Segundo Casaro ¹ Kristi L. Jones ¹ Modesto Hernandez ¹ Rafael S. Bisinotto ¹ Subhashinie Kariyawasam ³ Mary B. Brown ⁴ Ashley Phillips ⁵ Kwangcheol C. Jeong ² Klibs N. Galvao ^6*

• ¹ Department of Large Animal Clinical Sciences, University of Florida, Gainesville, United States
• ² Department of Animal Sciences and Emerging Pathogens Institute, University of Florida, Gainesville, United States
• ³ Department of Comparative, Diagnostic, and Population Medicine, University of Florida, Gainesville, United States
• ⁴ Department of Infectious Diseases and Immunology, University of Florida, Gainesville, United States
• ⁵ Athens Veterinary Diagnostic Laboratory, University of Georgia, Athens, Georgia, United States
• ⁶ Department of Large Animal Clinical Sciences and D. H. Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, United States

Helcococcus ovis (H. ovis) is an opportunistic bacterial pathogen of a wide range of animal hosts including domestic ruminants, swine, avians, and humans. In this study, we sequenced the genomes of 35 Helcococcus sp. clinical isolates from the uterus of dairy cows and explored their antimicrobial resistance and biochemical phenotypes in vitro. Phylogenetic and average nucleotide identity analyses classified four Helcococcus isolates within a cryptic clade representing an undescribed species, for which we propose the name Helcococcus bovis sp. nov. By establishing this new species clade, we also resolve the longstanding question of the classification of the Tongji strain responsible for a confirmed human conjunctival infection. This strain did not neatly fit into H. ovis and is instead a member of H. bovis . We applied whole genome comparative analyses to explore the pangenome, resistome, virulome, and taxonomic diversity of the remaining 31 H. ovis isolates. An overwhelming 97% of H. ovis strains (30 out of 31) harbor mobile tetracycline resistance genes and displayed significantly increased minimum inhibitory concentrations of tetracyclines in vitro. The high prevalence of mobile tetracycline resistance genes makes H. ovis a significant antimicrobial resistance gene reservoir in our food chain. Finally, the phylogenetic distribution of co-occurring high-virulence determinant genes of H. ovis across unlinked and distant loci highlights an instance of convergent gene loss in the species. In summary, this study showed that mobile genetic element-mediated tetracycline resistance is widespread in H. ovis, and that there is evidence of co-occurring virulence factors across clades suggesting convergent gene loss in the species. Finally, we introduced a novel Helcococcus species closely related to H. ovis, called H. bovis sp. nov., which has been reported to cause infection in humans.. Mobile genetic element-mediated tetracycline resistance is widespread in H. ovis . Co-occurring virulence factors across clades suggest convergent gene loss in the species . Helcococcus bovis is a novel species closely related to Helcococcus ovis that has been reported to cause infection in humans