Abstract
ABSTRACTMycoplasma bovis causes many health and welfare problems in cattle. Due to the absence of clear insights regarding transmission dynamics and the lack of a registered vaccine in Europe, control of an outbreak depends mainly on antimicrobial therapy. Unfortunately, antimicrobial susceptibility testing (AST) is usually not performed, because it is time-consuming and no standard protocol or clinical breakpoints are available. Fast identification of genetic markers associated with acquired resistance may at least partly resolve former issues. Therefore, the aims of this study were to implement a first genome-wide association study (GWAS) approach to identify genetic markers linked to antimicrobial resistance (AMR) in M. bovis using rapid long-read sequencing and to evaluate different epidemiological cutoff (ECOFF) thresholds. High-quality genomes of 100 M. bovis isolates were generated by Nanopore sequencing, and isolates were categorized as wild-type or non-wild-type isolates based on MIC testing results. Subsequently, a k-mer-based GWAS analysis was performed to link genotypes with phenotypes based on different ECOFF thresholds. This resulted in potential genetic markers for macrolides (gamithromycin and tylosin) (23S rRNA gene and 50S ribosomal unit) and enrofloxacin (GyrA and ParC). Also, for tilmicosin and the tetracyclines, previously described mutations in both 23S rRNA alleles and in one or both 16S rRNA alleles were observed. In addition, two new 16S rRNA mutations were possibly associated with gentamicin resistance. In conclusion, this study shows the potential of quick high-quality Nanopore sequencing and GWAS analysis in the evaluation of phenotypic ECOFF thresholds and the rapid identification of M. bovis strains with acquired resistance.IMPORTANCE Mycoplasma bovis is a leading cause of pneumonia but also causes other clinical signs in cattle. Since no effective vaccine is available, current M. bovis outbreak treatment relies primarily on the use of antimicrobials. However, M. bovis is naturally resistant to different antimicrobials, and acquired resistance against macrolides and fluoroquinolones is frequently described. Therefore, AST is important to provide appropriate and rapid antimicrobial treatment in the framework of AMR and to prevent the disease from spreading and/or becoming chronic. Unfortunately, phenotypic AST is time-consuming and, due to the lack of clinical breakpoints, the interpretation of AST in M. bovis is limited to the use of ECOFF values. Therefore, the objective of this study was to identify known and potentially new genetic markers linked to AMR phenotypes of M. bovis isolates, exploiting the power of a GWAS approach. For this, we used high-quality and complete Nanopore-sequenced M. bovis genomes of 100 isolates.
Highlights
Mycoplasma bovis causes many health and welfare problems in cattle
Molecular detection of antimicrobial resistance (AMR) determinants with methods based on targeted PCR was explored in the past for M. bovis [16, 17], and targeted gene sequencing for macrolide resistance has already been implemented in research and development settings for Mycoplasma pneumoniae community-acquired infections in humans [18]
The association between point mutations identified with whole-genome sequencing and phenotypic AMR have been explored in specific regions of three M. bovis strains [19] and for macrolides with a large set of isolates [15]
Summary
Mycoplasma bovis causes many health and welfare problems in cattle. Due to the absence of clear insights regarding transmission dynamics and the lack of a registered vaccine in Europe, control of an outbreak depends mainly on antimicrobial therapy. The association between point mutations identified with whole-genome sequencing and phenotypic AMR have been explored in specific regions of three M. bovis strains [19] and for macrolides with a large set of isolates [15] These targeted approaches may result in a narrowed view, and potential new genomic alterations within genes, operons, or even promoter, enhancer, and/or inhibitory regions might be overlooked [15, 20]. The aim of this study was to identify known and potentially new genetic markers linked with AMR phenotypes in a collection of 100 M. bovis isolates, exploiting the power of a GWAS approach with high-quality and complete Nanopore-sequenced M. bovis genomes
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