Skip to content
1887

Journal of Medical Microbiology logo Journal of Medical Microbiology

Volume 74, Issue 12

Potential confounding factors in currently used antibiotic susceptibility assays for the honey bee pathogen Open Access

Abstract

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.

Resistant strains of have been identified in Canada and Japan, but methodology differs between studies, making reliable comparisons difficult. Additionally, no isolates from the USA have yet been tested for susceptibility to OTC, despite decades of use.

Here, we determine the impact of media, time and persistence on the results of commonly used growth and antibiotic resistance assays using regionally representative isolates.

Twelve genetically diverse isolates of 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.

Media composition and incubation time dramatically impact antibiotic susceptibility assays for , 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. However, remains viable after OTC efficacy wanes, with some strains able to persist at room temperature for at least 3.5 years.

To standardize antibiotic susceptibility testing for we recommend the use of M110 media due to stability and speed of growth. However, all strains of 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.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antibiotic resistance , European foulbrood , honey bee , oxytetracycline and USA
Funding
This study was supported by the:
  • College of Agriculture and Natural Resources, Michigan State University (Award The Rackham Research Endowment)
    • Principal Award Recipient: MeghanO. Milbrath
  • North Central SARE (Award #GNC21-325)
    • Principal Award Recipient: MeghanO. Milbrath
  • College of Agriculture and Natural Resources, Michigan State University (Award Project GREEEN Proposal #GR21‐033)
    • Principal Award Recipient: MeghanO. Milbrath
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.002109
2025-12-12
2026-01-16

Metrics

Loading full text...

Full text loading...

/deliver/fulltext/jmm/74/12/jmm002109.html?itemId=/content/journal/jmm/10.1099/jmm.0.002109&mimeType=html&fmt=ahah

References

  1. Calderone NW. Insect pollinated crops, insect pollinators and US agriculture: trend analysis of aggregate data for the period 1992-2009. PLoS One 2012; 7:e37235 [View Article] [PubMed]
     [Google Scholar]
  2. Steinhauer N, Wilson M, Aurell D, Bruckner S, Williams G. United States Honey Bee Colony Losses 2022-2023: Preliminary Results from the Bee Informed Partnership. Bee Informed Partnership. n.d https://beeinformed.org/wp-content/uploads/2023/06/BIP-2022-23-Loss-Abstract.pdf accessed 22 June 2023
  3. Alburaki M, Abban SK, Evans JD, Chen YP. Occurrence and distribution of two bacterial brood diseases (American and European foulbrood) in US honey bee colonies and resistance to antibiotics from 2015 to 2022. J Apic Res 2024; 63:701–710 [View Article]
     [Google Scholar]
  4. Fowler PD, Dhakal U, Chang JH, Milbrath MO. Everything, everywhere, all at once - Surveillance and molecular epidemiology reveal Melissococcus plutonius is endemic among Michigan, US beekeeping operations. PLoS One 2025; 20:e0331903 [View Article] [PubMed]
     [Google Scholar]
  5. Fowler PD, Schroeder DC, Kevill JL, Milbrath MOG. No impact of hygienic behavior and viral coinfection on the development of European foulbrood in honey bee (Apis mellifera) colonies during blueberry pollination in Michigan. J Insect Sci 2023; 23:21 [View Article] [PubMed]
     [Google Scholar]
  6. Laate EA, Emunu JP, Duering A, Ovinge L. Potential Economic Impact of European and American Foulbrood on Alberta’s Beekeeping Industry. Agriculture and Forestry, Government of Alberta; 2020 https://open.alberta.ca/publications/potential-economic-impact-european-american-foulbrood-on-albertas-beekeeping-industry
  7. Arai R, Tominaga K, Wu M, Okura M, Ito K et al. Diversity of Melissococcus plutonius from honeybee larvae in japan and experimental reproduction of european foulbrood with cultured atypical isolates. PLoS One 2012; 7:e33708 [View Article]
     [Google Scholar]
  8. Takamatsu D, Okura M, Yoshiyama M, Wu M, Arai R et al. Detection of atypical Melissococcus plutonius in honeybees. Vet Rec 2012; 171:630 [View Article] [PubMed]
     [Google Scholar]
  9. Budge GE, Shirley MDF, Jones B, Quill E, Tomkies V et al. Molecular epidemiology and population structure of the honey bee brood pathogen Melissococcus plutonius. ISME J 2014; 8:1588–1597 [View Article]
     [Google Scholar]
  10. Takamatsu D, Morinishi K, Arai R, Sakamoto A, Okura M et al. Typing of Melissococcus plutonius isolated from European and Japanese honeybees suggests spread of sequence types across borders and between different Apis species. Vet Microbiol 2014; 171:221–226 [View Article]
     [Google Scholar]
  11. Grossar D, Haynes E, Budge GE, Parejo M, Gauthier L et al. Population genetic diversity and dynamics of the honey bee brood pathogen Melissococcus plutonius in a region with high prevalence. J Invertebr Pathol 2023; 196:107867 [View Article] [PubMed]
     [Google Scholar]
  12. de León-Door AP, Romo-Chacón A, Rios-Velasco C, Zamudio-Flores PB, Ornelas-Paz J de J et al. Prevalence, typing and phylogenetic analysis of Melissococcus plutonius strains from bee colonies of the State of Chihuahua, Mexico. J Invertebr Pathol 2018; 159:71–77 [View Article] [PubMed]
     [Google Scholar]
  13. Haynes E, Helgason T, Young JPW, Thwaites R, Budge GE. A typing scheme for the honeybee pathogen Melissococcus plutonius allows detection of disease transmission events and a study of the distribution of variants. Environ Microbiol Rep 2013; 5:525–529 [View Article] [PubMed]
     [Google Scholar]
  14. Grossar D, Kilchenmann V, Forsgren E, Charrière J-D, Gauthier L et al. Putative determinants of virulence in Melissococcus plutonius, the bacterial agent causing European foulbrood in honey bees. Virulence 2020; 11:554–567 [View Article] [PubMed]
     [Google Scholar]
  15. Moffett JO. The effect of various therapeutic agents on European foulbrood1. J Econ Entomol 1953; 46:879–881 [View Article]
     [Google Scholar]
  16. Wilson WT, Moffett JO. The effect of erythromycin and other antibiotics on the control of european foulbrood of honeybees1. J Econ Entomol 1957; 50:194–196 [View Article]
     [Google Scholar]
  17. Forsgren E. European foulbrood in honey bees. J Invertebr Pathol 2010; 103 Suppl 1:S5–9 [View Article]
     [Google Scholar]
  18. Miyagi T, Peng CY, Chuang RY, Mussen EC, Spivak MS et al. Verification of oxytetracycline-resistant american foulbrood pathogen paenibacillus larvae in the United States. J Invertebr Pathol 2000; 75:95–96 [View Article]
     [Google Scholar]
  19. Hornitzky MAZ, Smith LA. Sensitivity of Australian Melissococcus pluton isolates to oxytetracycline hydrochloride. Aust J Exp Agric 1999; 39:881 [View Article]
     [Google Scholar]
  20. Waite R, Jackson S, Thompson H. Preliminary investigations into possible resistance to oxytetracycline in Melissococcus plutonius, a pathogen of honeybee larvae. Lett Appl Microbiol 2003; 36:20–24 [View Article]
     [Google Scholar]
  21. Masood F, Thebeau JM, Cloet A, Kozii IV, Zabrodski MW et al. Evaluating approved and alternative treatments against an oxytetracycline-resistant bacterium responsible for European foulbrood disease in honey bees. Sci Rep 2022; 12:5906 [View Article] [PubMed]
     [Google Scholar]
  22. Takamatsu D, Yoshida E, Watando E, Ueno Y. Oxytetracycline resistance of Melissococcus plutonius strains in Japan. J Apic Res 2024; 63:306–309 [View Article]
     [Google Scholar]
  23. Sporns P, Kwan S, Roth LA. HPLC analysis of oxytetracycline residues in honey. J Food Prot 1986; 49:383–388 [View Article] [PubMed]
     [Google Scholar]
  24. Xuan R, Arisi L, Wang Q, Yates SR, Biswas KC. Hydrolysis and photolysis of oxytetracycline in aqueous solution. J Environ Sci Health Part B 2009; 45:73–81 [View Article]
     [Google Scholar]
  25. Budge GE, Burns N, Takamatsu D, Erler S, Forsgren E et al. Standard methods for European foulbrood research 2.0. J Apicult Res 2025; 64:403–442 [View Article]
     [Google Scholar]
  26. Budge GE, Barrett B, Jones B, Pietravalle S, Marris G et al. The occurrence of Melissococcus plutonius in healthy colonies of Apis mellifera and the efficacy of European foulbrood control measures. J Invertebr Pathol 2010; 105:164–170 [View Article] [PubMed]
     [Google Scholar]
  27. Milbrath MO, Fowler PD, Abban SK, Lopez D, Evans JD. Validation of diagnostic methods for European foulbrood on commercial honey bee colonies in the United States. J Insect Sci 2021; 21:6 [View Article] [PubMed]
     [Google Scholar]
  28. Humphries RM, Ambler J, Mitchell SL, Castanheira M, Dingle T et al. CLSI methods development and standardization working group best practices for evaluation of antimicrobial susceptibility tests. J Clin Microbiol 2018; 56:e01934-17 [View Article] [PubMed]
     [Google Scholar]
  29. Takamatsu D, Osawa A, Nakamura K, Yoshiyama M, Okura M. High-level resistance of Melissococcus plutonius clonal complex 3 strains to antimicrobial activity of royal jelly. Environ Microbiol Rep 2017; 9:562–570 [View Article] [PubMed]
     [Google Scholar]
  30. Arai R, Miyoshi-Akiyama T, Okumura K, Morinaga Y, Wu M et al. Development of duplex PCR assay for detection and differentiation of typical and atypical Melissococcus plutonius strains. J Vet Med Sci 2014; 76:491–498 [View Article] [PubMed]
     [Google Scholar]
  31. Hornitzky MAZ, Wilson SC. A system for the diagnosis of the major bacterial brood diseases of honeybees. J Apicul Res 1989; 28:191–195 [View Article]
     [Google Scholar]
  32. Olson RD, Assaf R, Brettin T, Conrad N, Cucinell C et al. Introducing the bacterial and viral bioinformatics resource center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res 2023; 51:D678–D689 [View Article] [PubMed]
     [Google Scholar]
  33. Krueger F. Trim Galore!: A wrapper around Cutadapt and FastQC to consistently apply adapter and quality trimming to FastQ files, with extra functionality for RRBS data. Babraham Institute; 2015 https://cir.nii.ac.jp/crid/1370294643762929691 accessed 27 January 2025
  34. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 2017; 13:e1005595 [View Article] [PubMed]
     [Google Scholar]
  35. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012; 19:455–477 [View Article] [PubMed]
     [Google Scholar]
  36. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014; 30:1312–1313 [View Article]
     [Google Scholar]
  37. Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML Web servers. Syst Biol 2008; 57:758–771 [View Article] [PubMed]
     [Google Scholar]
  38. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 2021; 49:W293–W296 [View Article]
     [Google Scholar]
  39. R Core Team R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing; 2025 https://www.R-project.org/
  40. Wickham H. ggplot2: Create Elegant Data Visualisations Using the Grammar of Graphics; 2016 https://ggplot2.tidyverse.org/
  41. Kitamura Y, Kusajima Y, Nakayama M, Ueda D, Kawaguchi R et al. Impact of different culture media and culture conditions on the growth and survival of Melissococcus plutonius, the causative agent of European foulbrood. J Apicul Res 20251–14 [View Article]
     [Google Scholar]
  42. Barry AL, Badal RE. Stability of minocycline, doxycycline, and tetracycline stored in agar plates and microdilution trays. Curr Microbiol 1978; 1:33–36 [View Article]
     [Google Scholar]
  43. Mitscher LA. The Chemistry of the Tetracycline Antibiotics New York: Marcel Dekker; 1978
     [Google Scholar]
  44. McKee BA, Goodman RD, Saywell C, Hepworth G, McKee BA. Oxytetracycline hydrochloride activity in honey bee larvae (Apis mellifera) following medication with various doses. Apidologie 2003; 34:269–279 [View Article]
     [Google Scholar]
  45. Wardell G. European foulbrood association with Michigan blueberry pollination, and control. PhD Diss Michigan State University; 1982
     [Google Scholar]
  46. Miles AA, Maskell JP. The neutralization of antibiotic action by metallic cations and iron chelators. J Antimicrob Chemother 1986; 17:481–487 [View Article] [PubMed]
     [Google Scholar]
  47. Scholar EM, Pratt WB. The Antimicrobial Drugs Oxford University Press; 2000 [View Article]
     [Google Scholar]
  48. Bailey L. The epizootiology of European foulbrood of the larval honey bee, Apis mellifera linnaeus. J Insect Pathol 1960; 2:67–83
     [Google Scholar]
  49. Sebastian Jose M, Bezerra da Silva MC, Obshta O, Masood F, Thebeau JM et al. Antimicrobial control and temporal dynamics of M. plutonius colonization in adult worker honey bees (Apis mellifera). PLoS One 2025; 20:e0322770 [View Article] [PubMed]
     [Google Scholar]
  50. Kramer A, Assadian O. Survival of microorganisms on inanimate surfaces. In Use of Biocidal Surfaces for Reduction of Healthcare Acquired Infections 2014 pp 7–26 [View Article]
     [Google Scholar]
  51. Walsh RL, Camilli A. Streptococcus pneumoniae is desiccation tolerant and infectious upon rehydration. mBio 2011; 2:e00092-11 [View Article] [PubMed]
     [Google Scholar]
/content/journal/jmm/10.1099/jmm.0.002109
Loading
Potential confounding factors in currently used antibiotic susceptibility assays for the honey bee pathogen Melissococcus plutonius
J. Med. Microbiol. 74, 002109 (2025); https://doi.org/10.1099/jmm.0.002109
/content/journal/jmm/10.1099/jmm.0.002109
/content/journal/jmm/10.1099/jmm.0.002109
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An error occurred
Approval was partially successful, following selected items could not be processed due to error