Sessions

Otitis externa is a common disease among dogs that is often difficult to treat and not always successfully resolved. The reason for the therapeutic failure lies in the challenge of eliminating primary causes and contributing factors, anatomical conformations, and the difficulty of accurately identifying the causative pathogens (bacteria, fungi). Classical microbial diagnostic methods (culturing, PCR) may be unsuitable for detecting certain pathogens, especially in polymicrobial infections. The consequence can be a recurrent or chronic end-stage otitis externa, treatable only by an invasive surgical procedure (Total Ear Canal Ablation, Lateral, Bulla Osteotomy, TECA/LBO).

Metagenomic sequencing, especially with the nanopore sequencing platform of Oxford Nanopore Technologies (ONT), enables a rapid, comprehensive, real time analysis of the microbiome, including detailed genetic features (virulence and antimicrobial resistance genes). In the present study, we analysed a sample from the right external auditory canal of an 11-year-old Yorkshire Terrier suffering from chronic otitis externa. The sample was taken before TECA/LBO surgery. During routine culture, Pasteurella multocida and mixed anaerobic flora were detected. Following shotgun sequencing and bioinformatic analysis of the metagenome, the presence of Lawsonella clevelandensis was confirmed. Assembly of the reads derived from the bacterium resulted in a single circular chromosome of 1,909,339 bp, containing 1,727 predicted genes, effectively representing the complete bacterial genome. This Gram-positive, anaerobic bacteria was first discovered in 2016 from human abscesses. Due to its fastidious nature and slow growth, it is frequently missed in routine examinations. To our knowledge, a metagenomic assembly of animal origin has not been reported until now. We identified a single antimicrobial resistance gene (EXO-1, beta-lactamase family) and detected no virulence factor genes. Pan-genome analysis revealed high genetic diversity, with the animal-derived strain distinguished from human isolates by its unique gene profile.

Our results highlight the advantages of nanopore sequencing-based rapid metagenomic diagnostics for difficult-to-culture bacteria. Furthermore, they contribute to the expanding body of knowledge on this often-overlooked pathogen. Further research is essential to clarify the exact role of the pathogen, but the case presented here demonstrates that metagenomic approaches may become an indispensable tool in future veterinary diagnostics.