Mycoplasma bovis point mutations as genetic markers for antimicrobial resistance revealed by Genome Wide Association Study (lecture, 12 min.)

Mycoplasma bovis is an important primary pathogen in cattle industry and results in many health and welfare problems. Current control mainly depends on antimicrobial therapy because clear insights in transmission dynamics and a registered vaccination strategy is lacking in Europe. Routine Antimicrobial Susceptibility Testing (AST) is usually not performed mainly because of its extended time (2 weeks), the lack of standardisation & clinical breakpoints. Hence, the aim of this study was the implementation of a first Genome Wide Association Study (GWAS) approach to reveal genetic point mutations associated with resistance phenotypes in Belgian M. bovis isolates using nanopore sequencing to speed up the AST process. High quality and complete genomes of 100 M. bovis field strains were obtained from nanopore long-reads using a custom-trained Bonito basecalling model (Vereecke et al., 2020). All genomes were classified into wild type and non-wild type populations based on their phenotypes for relevant antimicrobial drugs. Subsequently, a k-mer based GWAS was performed to link genotypes with phenotypes. This highlighted potential genetic markers for macrolides (gamithromycin, tylosin) in the 23S rRNA gene & L22 protein and for enrofloxacin in the GyrA and ParC protein. In addition, we observed a previously identified mutation in the 23S rRNA allele, associating with the tilmicosin resistance phenotypes, and two new 16S rRNA mutations associated with gentamicin resistance. In conclusion, this study shows the potential of quick high quality nanopore whole genome sequencing and GWAS to evaluate phenotypic Epidemiological cutoff thresholds and its implementation in quicker identification of acquired resistance in M. bovis field strains.