Genomic analysis reveals two dominant strains of in Austria and Hungary with distinct multidrug resistance profiles.

(ORT) is an emerging pathogen that poses significant challenges to commercial poultry production due to respiratory and systemic infections, leading to economic losses. Increasing multidrug resistance further complicates effective treatment strategies. Here, we present the first large-scale genomic analysis of ORT isolates from Europe, focusing on 94 newly sequenced isolates from Austria and Hungary. Phylogenetic analysis revealed two dominant clades, O1 and O2, which included isolates from both regions, indicating a lack of geographic segregation. Both clades display high genetic similarity, indicating that each represents a single strain. Notably, Clade O1 appears to be specific to Europe and exhibits higher resistance to several antibiotics compared to Clade O2. Genome-wide association studies and database screening identified resistance genes linked to beta-lactam antibiotics (/) and tylosin (/). Despite the widespread presence of and , these genes did not consistently predict tetracycline resistance. Furthermore, we identified the insertion sequences IS and IS as key mobile elements mediating the co-localization of and / resistance genes within ORT isolates of Clade O1. These findings enhance our understanding of ORT's genetic diversity, strain distribution, and resistance mechanisms, providing a basis for improved monitoring, vaccine development, and targeted interventions. The limited genetic variability observed in our data set underscores the challenges of identifying resistance genes and highlights the need for novel approaches to fully elucidate resistance mechanisms.IMPORTANCE (ORT) is a bacterial pathogen that causes significant respiratory and systemic disease in poultry, leading to economic losses worldwide. By sequencing and comparing 94 isolates from Austria and Hungary, we discovered two major ORT strains circulating in these regions. One strain is unique to Europe and carries multiple antibiotic resistance genes on a mobile element, underscoring the need for careful antibiotic use. This study is the first large-scale genomic analysis of European ORT isolates, revealing how these strains spread and evolve over time. Our findings offer valuable insights for improving diagnostic methods, guiding vaccine development, and designing targeted strategies to manage ORT infections in commercial poultry flocks.