Abstract

Antimicrobial resistance (AMR) in Mycoplasma bovis has been previously associated with topoisomerase and ribosomal gene mutations rather than specific resistance-conferring genes. Using whole genome sequencing (WGS) to identify potential new AMR mechanisms for M. bovis, it was found that a 2019 clinical isolate with high MIC (2019-043682) for fluoroquinolones, macrolides, lincosamides, pleuromutilins and tetracyclines had a new core genome multilocus sequencing (cgMLST) type (ST10-like) and 91% sequence similarity to the published genome of M. bovis PG45. Closely related to PG45, a 1982 isolate (1982-M6152) shared the same cgMLST type (ST17), 97.2% sequence similarity and low MIC results. Known and potential AMR- associated genetic events were identified through multiple sequence alignment of the three genomes. Isolate 2019-043682 had 507 genes with non-synonymous mutations (NSMs) and 67 genes disrupted. Isolate 1982-M6152 had 81 NSMs and 20 disruptions. Using functional roles and known mechanisms of antimicrobials, a 55 gene subset was assessed for AMR potential. Seventeen were previously identified from other bacteria as sites of AMR mutation, 38 shared similar functions to them, and 11 contained gene-disrupting mutations. This study indicated that M. bovis may obtain high AMR characteristics by mutating or disrupting other functional genes, in addition to topoisomerases and ribosomal genes.

Highlights

  • Mycoplasma bovis is a member of the Mollicutes; membrane-bound bacteria which lack a cell wall, precluding the use of many common antimicrobial agents such as the β-lactams [1]

  • Cultures of two isolates of M. bovis (1982-M6152 and 2019-043682) and M. bovis strain PG45 were tested in triplicate for minimum inhibitory concentration of 16 antimicrobials (Table 1), with the results of MIC testing identical within each triplicate

  • Of the M. bovis genes previously linked by Sulyok et al with Antimicrobial resistance (AMR) for various classes of antimicrobial, two sites linked with fluoroquinolone resistance display multiple non-synonymous mutations (NSMs) for the high-MIC isolate 2019-043682 and no NSMs for the low-MIC

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Summary

Introduction

Mycoplasma bovis is a member of the Mollicutes; membrane-bound bacteria which lack a cell wall, precluding the use of many common antimicrobial agents such as the β-lactams [1]. M. bovis is a causative agent of pneumonia, arthritis, otitis media, and reproductive disease and is a contributor to the bovine respiratory disease (BRD) complex, known as ‘shipping fever’, which is a major source of morbidity, mortality and financial loss in calf and feedlot operations. M. bovis is capable of persisting for the life of a colonized animal, which may remain asymptomatic while acting as a source of infection for herdmates or offspring [1,2]. Given the asymptomatic nature of many M. bovis infections, and the high rates of colonization when animals are co-mingled (potentially over 90%) [4,5], conditions are favourable for the development of multi-drug resistant strains. With global rates of antimicrobial resistance increasing, understanding the molecular mechanisms underlying antimicrobial resistance, for multi-drug resistant

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