Potential confounding factors in currently used antibiotic susceptibility assays for the honey bee pathogen Melissococcus plutonius

<b>Introduction.</b> <i>Melissococcus plutonius</i> is the causative agent of European foulbrood (EFB), a disease of honey bees that is endemic in many areas of the USA. Only one antibiotic, oxytetracycline (OTC), is approved for EFB management, and there have been reports of recalcitrance.<b>Gap Statement.</b> Resistant strains of <i>M. plutonius</i> have been identified in Canada and Japan, but methodology differs between studies, making reliable comparisons difficult. Additionally, no <i>M. plutonius</i> isolates from the USA have yet been tested for susceptibility to OTC, despite decades of use.<b>Aim.</b> Here, we determine the impact of media, time and persistence on the results of commonly used growth and antibiotic resistance assays using regionally representative <i>M. plutonius</i> isolates.<b>Methodology.</b> Twelve genetically diverse isolates of <i>M. plutonius</i> were tested for susceptibility to OTC using previously published assays, but with variations in media and time to determine factors that may be impacting results.<b>Results.</b> Media composition and incubation time dramatically impact antibiotic susceptibility assays for <i>M. plutonius</i>, differing widely between strains, likely due to differences in OTC stability. Assays that ended when growth appeared on antibiotic-free agar showed that all strains remained susceptible to OTC with an MIC of 2-4 µg ml^-1. However, <i>M. plutonius</i> remains viable after OTC efficacy wanes, with some strains able to persist at room temperature for at least 3.5 years.<b>Conclusion.</b> To standardize antibiotic susceptibility testing for <i>M. plutonius,</i> we recommend the use of M110 media due to stability and speed of growth. However, all strains of <i>M. plutonius</i> persist on M110 beyond the window of OTC efficacy, complicating assay results and interpretation, and additional research is needed to determine the clinical implications of these findings.