Mycoplasma bovis is a major and primary bovine pathogen causing respiratory and reproductive disorders, mastitis and arthritis. Due to its persistent nature it is difficult to combat infections on farms. No effective vaccine is able to prevent M. bovis infection, leaving antimicrobials as the only first-line treatment. Nevertheless, therapy with antimicrobials is rarely efficient since increased resistance to many commercially available antimicrobials has been reported. An accurate diagnosis including antimicrobial resistance profiling is required to combat infections with working antibiotics, but this cannot be achieved fast enough with classical diagnostics. Here we developed a nanopore-based workflow allowing M. bovis species typing and Antimicrobial Resistance (AMR) Profiling applicable in point-of-care settings in control of M. bovis infections. Our new diagnostic tool was verified with 100 field strains of M. bovis for which whole genome sequencing and MIC testing was performed for practice-relevant antibiotics. Besides whole genome species typing, Single Nucleotide Polymorphism (SNP) analysis was performed to associate strain-specific genetic markers with its phenotypic AMR antibiogram. Raw fast5 outputs ranged from 5.4 Gb up to 17.2 Gb, with an average N50 of 5.5 ± 1.3 Kb per run with 11 M. bovis strains. Furthermore, including the M. bovis PG45 type strain within every run as internal control, inter-run accuracy reached up to 99.95% sequence identity. Since computation time presents a new bottleneck in this new workflow, we exploited a GPU-based bioinformatics pipeline speeding up full bioinformatics analysis to be completed within 10 hours for 11 M. bovis genomes. This new M. bovis diagnostics pipeline delivers a high accurate species identification along with an accurate genotypic antibiogram. This will be accelerated even further to facilitate proper antimicrobial therapy selection for the rapid control of bovine mycoplasmosis
