Conducting antimicrobial susceptibility testing (AST) in a comparable manner requires the availability of a standardized method. Organizations, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), provide standardized protocols for a range of fastidious bacteria but not for Mycoplasma hyorhinis. We developed a broth microdilution method for testing M. hyorhinis in a standardized and harmonized way using a modified Friis broth devoid of antimicrobial or otherwise bacterial growth-inhibiting agents. The type strain M. hyorhinis DSM 25591 was chosen to establish the methodology. The antimicrobial agents of interest were doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin, tested by using commercial SensititreTM microtiter plates. In addition, the suitability of the methodology was evaluated via variation of the individual ingredients of the modified Friis broth by either using different batches or choosing other distributors. Despite these alterations, the method provided reliable results. We obtained repeatable minimal inhibitory concentrations for all six tested field isolates and the M. hyorhinis type strain. With this newly proposed method, we aim to provide an improved AST method for diagnostic laboratories and monitoring purposes with better comparability between times and countries. In addition, this new method will allow for an improvement of targeted treatments using antimicrobial agents and thereby reduce the options for resistance development.

Antimicrobial susceptibility testing (AST) should be conducted in a standardized manner prior to the start of an antimicrobial treatment. For fastidious bacteria, such as porcine Mycoplasma (‘Mesomycoplasma’) spp., specifically M. hyorhinis, neither guidelines or standards for the performance of AST, nor quality control strains for the validation of AST results are approved by organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The CLSI- and EUCAST-approved quality control strains Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were chosen to validate AST by broth microdilution using modified Friis broth, developed as growth medium for porcine Mycoplasma (‘Mesomycoplasma’) spp. The antimicrobial agents doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin were examined using customized SensititreTM microtiter plates. Minimal inhibitory concentrations, determined after 24, 48, and 72 h, were mostly within the CLSI-approved quality control ranges for defined antimicrobial agents. We propose the use of the combination of E. faecalis ATCC 29212 and S. aureus ATCC 29213 as surrogate quality control strains for the validation of future AST results obtained for M. hyorhinis by broth microdilution using modified Friis broth.

The whole-genome sequence of the type strain Mycoplasma (Mesomycoplasma) hyorhinis DSM 25591 is reported and compared to the available sequences of the corresponding type strains from other strain collections to ascertain conformity. Knowledge of the identity of type strains is of importance for their application in standardized test systems.