Identification of genes and mutations involved in antimicrobial low susceptibility in <i>Mycoplasma bovirhinis</i> isolates from Japan and development of a genetic method for antimicrobial susceptibility discrimination

ABSTRACT Mycoplasma bovirhinis ( Mycoplasmopsis bovirhinis ) is a commensal bacterium that infects the bovine respiratory tract and poses a risk for calf pneumonia. This study aimed to develop a simple and rapid genetic method for assessing antimicrobial susceptibility by identifying genes and mutations associated with decreased susceptibility through genomic analysis of isolate exhibiting low antimicrobial susceptibility and by directly detecting these genetic determinants. As a result, phage-related regions encoding homologous genes—namely, aminoglycoside 6-adenylyltransferase, aminoglycoside 3′-phosphotransferase, and a tetracycline ribosomal protection protein—associated with decreased susceptibility to aminoglycosides or tetracyclines were identified. Furthermore, point mutations associated with decreased susceptibility to macrolides, lincomycins, and fluoroquinolones were identified in the 23S ribosomal RNA gene ( rrl ), DNA gyrase gene ( gyrA ), DNA topoisomerase IV gene ( parC ), and its homologous gene ( parC ′). Field strains harboring these determinants and mutations have been widely detected in Japan since 2001. These genes and mutations can be easily and rapidly detected by PCR, DNA sequencing, and melting curve analysis using hybridization probes, making it possible to identify effective antimicrobial agents prior to antimicrobial treatment, which was not possible with conventional, time-consuming, culture-based antimicrobial susceptibility testing for mycoplasmas. This approach is expected to improve cure rates and reduce the risk of using ineffective antibiotics. IMPORTANCE Antimicrobial susceptibility testing for mycoplasma using conventional culture methods takes a long time due to the slow culture rate. This has been a major problem in selecting effective antibacterial agents for antimicrobial therapy. Therefore, we identified the genes and mutations involved in decreased susceptibility and developed a genetic susceptibility determination method that can directly detect them. This method makes it possible to quickly and easily detect the antimicrobial susceptibility characteristics of mycoplasmas, which have been difficult to understand until now. In recent years, the emergence and spread of low-susceptibility field strains of various bovine mycoplasmas has been confirmed, raising concerns about this situation. Utilizing this method is expected to improve cure rates and reduce the use of antimicrobial agents.