Application of multiplex PCR-based nanopore sequencing for rapid detection and surveillance of antimicrobial-resistant Mycoplasma bovis in Japanese dairy cattle

Mycoplasma bovis causes mastitis and respiratory disease, leading to economic losses in the dairy industry. Conventional culture and antimicrobial susceptibility testing require approximately 2 weeks, promoting empirical antimicrobial use and emergence of resistant strains, highlighting the need for rapid detection methods. Resistance-associated mutations have been detected in rrl and rrs (for macrolides, lincosamides, tetracyclines, and phenicols) and gyrA and parC (for fluoroquinolones), providing targets for a rapid prediction. We developed a multiplex PCR-based nanopore sequencing method capable of simultaneously analyzing up to 24 samples per run to detect mutations in rrl , rrs , gyrA , and parC , and infer the antimicrobial susceptibility of M. bovis within 6 h. Using 329 isolates from cattle collected in Japan (2013–2016 and 2023–2025), we linked genotypes to the minimum inhibitory concentrations of seven antimicrobials and predicted the susceptibility of all isolates. A clear association was observed between specific mutations and susceptibility to multiple antimicrobials, suggesting that susceptibility can be predicted based on target gene sequence. Subsequently, the temporal trends in the prevalence of resistance-associated mutations were examined annually. High-level macrolide- and tetracycline-associated mutations ( rrl A2059/A2060, rrs G1058) and dual gyrA-parC mutations increased markedly in recent isolates, with frequencies rising from 1.8% to 43.3%, 0.9% to 41.5%, and 59.8% to 79.8%, respectively, during 2013–2016 and 2023–2025. This nanopore-based platform enables rapid genetics-based antimicrobial guidance and simultaneous analysis of up to 24 samples on a low-cost flow cell, providing a practical tool for clinical decision-making and surveillance of emerging multidrug-resistant M. bovis in dairy cattle.

IMPORTANCE

Mycoplasma bovis is a major cause of bovine mastitis and respiratory disease, leading to substantial economic losses and increased antimicrobial use in the dairy industry. Because conventional culture and susceptibility testing are time-consuming, empirical treatment is common, often resulting in therapeutic failure and the emergence of multidrug-resistant strains. This study developed a rapid diagnostic platform using multiplex PCR and nanopore sequencing to predict antimicrobial susceptibility within only 6 h. Our analysis of 329 Japanese isolates reveals a significant temporal increase in mutations associated with resistance to macrolides, tetracyclines, and fluoroquinolones between 2013–2016 and 2023–2025. This cost-effective method enables simultaneous analysis of multiple samples, providing a practical tool for evidence-based clinical decision-making. These findings highlight the urgent need for molecular surveillance to monitor and control the spread of highly resistant M. bovis lineages in global cattle populations.