Genotypic - Phenotypic Discordance of Antimicrobial Resistance in U.S. Ornithobacterium rhinotracheale Field Isolates

• Integrated phenotypic and genomic AMR profiling of Ornithobacterium rhinotracheale • Resistance phenotypes are poorly explained by known AMR genes, highlighting database gaps. • Multidrug resistance spans clades and sequence types, indicating broad dissemination. • Results highlight a need for phenotypic testing and improved AMR surveillance in poultry health Ornithobacterium rhinotracheale is a gram-negative respiratory pathogen of poultry that causes production losses in turkeys and chickens. Antibiotic therapy remains common, yet resistance patterns vary by strain and region, complicating control and raising concerns about the dissemination of antimicrobial resistance (AMR). This study aimed to characterize and compare phenotypic and genomic AMR in 28 O. rhinotracheale field isolates using broth microdilution against 11 antimicrobials and whole-genome screening for resistance determinants. All isolates were resistant to colistin, nalidixic acid, imipenem, and chloramphenicol; resistance to gentamicin and kanamycin was nearly universal, whereas all isolates were susceptible to tylosin. Susceptibility to ampicillin, amoxicillin, cefoxitin, and tetracycline was variable, and every isolate met the definition of multidrug resistant. Genotypically, only two antimicrobial resistance genes were detected in all isolates, tetQ (tetracycline) and blaORR/blaORR-1 (carbapenems). These genotyping results explained imipenem resistance but not the broader phenotypes, including inconsistent tetracycline resistance outcomes despite tetQ carriage. Phylogenomics grouped the isolates into four clades spanning ST1, ST9, and ST35, with extensive multi-drug resistance (MDR) observed across clades and sequence types. This pronounced genotype–phenotype discordance highlights critical gaps in current AMR gene catalogs for understudied pathogens such as O. rhinotracheale and underscore the need for expanded surveillance to inform treatment decisions and antimicrobial stewardship in poultry production systems.