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Potential confounding factors in currently used antibiotic susceptibility assays for the honey bee pathogen
Peter Fowler1, Robyn Hawley2, Meghan O Milbrath1,3
1Comparative Medicine and Integrative Biology, College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA.
Abstract:
Introduction. Melissococcus plutonius 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.Gap Statement. Resistant strains of M. plutonius have been identified in Canada and Japan, but methodology differs between studies, making reliable comparisons difficult. Additionally, no M. plutonius isolates from the USA have yet been tested for susceptibility to OTC, despite decades of use.Aim. Here, we determine the impact of media, time and persistence on the results of commonly used growth and antibiotic resistance assays using regionally representative M. plutonius isolates.Methodology. Twelve genetically diverse isolates of M. plutonius 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.Results. Media composition and incubation time dramatically impact antibiotic susceptibility assays for M. plutonius, 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, M. plutonius remains viable after OTC efficacy wanes, with some strains able to persist at room temperature for at least 3.5 years.Conclusion. To standardize antibiotic susceptibility testing for M. plutonius, we recommend the use of M110 media due to stability and speed of growth. However, all strains of M. plutonius 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.
Insights
Oxytetracycline (OTC) susceptibility testing for European foulbrood (EFB) in honey bees is complicated by media and time. All tested *Melissococcus plutonius* strains remained susceptible to OTC but persisted long after antibiotic efficacy waned.
Area of Science:
- Veterinary Microbiology
- Apiculture Science
- Antimicrobial Resistance
Background:
- *Melissococcus plutonius* causes European foulbrood (EFB), a significant honey bee disease.
- Oxytetracycline (OTC) is the sole approved antibiotic for EFB management in the USA.
- Concerns exist regarding OTC recalcitrance and lack of susceptibility data for US *M. plutonius* isolates.
Purpose of the Study:
- To investigate the influence of media composition and incubation time on OTC susceptibility assays for *M. plutonius*.
- To assess the persistence of *M. plutonius* viability after OTC efficacy diminishes.
- To establish standardized methods for antibiotic susceptibility testing of *M. plutonius*.
Main Methods:
- Testing twelve diverse *M. plutonius* isolates for OTC susceptibility using established assays with modified media and incubation times.
- Evaluating Minimum Inhibitory Concentration (MIC) of OTC against *M. plutonius* strains.
- Assessing bacterial persistence under varying conditions post-antibiotic exposure.
Main Results:
- Media and incubation time significantly alter OTC susceptibility assay outcomes for *M. plutonius*.
- All tested *M. plutonius* strains demonstrated susceptibility to OTC (MIC: 2-4 µg/mL) when assays concluded upon initial growth.
- *M. plutonius* isolates exhibited prolonged viability, persisting for at least 3.5 years at room temperature, even after OTC efficacy declined.
Conclusions:
- Standardized antibiotic susceptibility testing for *M. plutonius* is recommended using M110 media for improved stability and faster growth.
- The extended persistence of *M. plutonius* beyond the window of OTC efficacy complicates assay interpretation and necessitates further research into clinical implications.
