Experimental investigation of the antimicrobial activity of the preparation and its influence on the viability of honey bees (Apis mellifera)

Maksym Zastulka, Tatiana Romanyshyna, Anastasiia Lakhman, Oleksandr Galatyuk
Received: 12.06.2025 Revised: 02.09.2025 Accepted: 24.09.2025
Retrieved from Vol. 28, No. 10, 2025 Pages: 9-20
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Abstract

The epizootic spread of bacterial infections in apiculture, intensified by the phenomenon of colony collapse disorder, necessitates the development of new and safe preventive agents. Due to legislative restrictions on the use of antibiotics, priority is given to the study of antiseptics with immunomodulatory properties. The aim of this work was a comprehensive assessment of the effectiveness of the preparation Kombiyod, namely the determination of its antimicrobial activity in vitro and the establishment of optimal safe concentrations to enhance the viability of honeybees in vivo. The study included the disc diffusion method using pure strains of enterobacteria and mixed microbial cultures isolated from diseased bees in vitro, as well as a cage experiment evaluating the viability and physiological state of bees when the preparation was added to sugar syrup (0.01-2.05%) in vivo under laboratory conditions. The results demonstrated the bacteriostatic efficacy of the preparation in vitro at concentrations of 0.1-0.5%. A dose-dependent effect was observed in bees under laboratory conditions: concentrations of 0.5-2.05% caused acute toxicity with a mortality peak on the 7th day, whereas optimal concentrations of 0.05-0.1% exhibited a stimulating effect –maximum lifespan of 17-18 days, a 25% increase in viability compared to the control, and a probable immunomodulatory effect associated with iodine deficiency correction. Based on the obtained results, a 0.1% solution of the preparation in sugar syrup is recommended for oral administration and a 0.2% aqueous solution for hive sanitation in the presence of bees. The therapeutic concentration range has been established for further research. The findings may serve as a foundation for developing new prevention schemes of bee bacterioses based on iodine-containing preparations, considering the identified dose-dependent effects

Keywords

honeybees (Apis mellifera); povidone-iodine; antimicrobial activity; enterobacteria; microbiological studies; bee viability; immunomodulatory effect

  1. Alburaki, M., Abban, S.K., Evans, J.D., & Chen, Y.P. (2024). 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. Journal of Apicultural Research, 63(4), 701-710. doi: 10.1080/00218839.2024.2329854.
  2. Aziz, M. A., & Alam, S. (2024). Diseases of honeybee (Apis mellifera). IntechOpen. doi: 10.5772/intechopen.1003947.
  3. Balázs, V.L., Nagy-Radványi, L., Filep, R., Kerekes, E., Kocsis, B., Kocsis, M., & Farkas, Á. (2021). In vitro antibacterial and antibiofilm activity of Hungarian honeys against respiratory tract bacteria. Foods, 10(7), article number 1632. doi: 10.3390/foods10071632.
  4. Boisson, M., et al. (2019). Multicentre, open-label, randomised, controlled clinical trial comparing 2% chlorhexidine-70% isopropanol and 5% povidone iodine-69% ethanol for skin antisepsis in reducing surg ical-site infection after cardiac surgery: The CLEAN 2 study protocol. BMJ Open, 9(6), article number 026929. doi: 10.1136/bmjopen-2018-026929.
  5. Brhich, A., Hachimi, T., Chatoui, H., Ait Sidi Brahim, M., Hnini, R., Chatoui, R., Merzouki, H., & Merzouki, M. (2025). Evaluation of pesticide effects on honeybee health and colony collapse: Findings from a beekeeper survey in the Beni Mellal-Khenifra Region, Morocco. Journal of Environmental & Earth Sciences, 7(2), 89-100. doi: 10.30564/ jees.v7i2.7645.
  6. Chen, Z., Chen, S., Huang, Y., Qin, Z., Shi, F., & Lin, L. (2024). Effects of enrofloxacin and povidone-iodine on immunity, intestinal microbes, and transcription pathways of grass carp. Intestinal Microbes, and Transcription Pathways of Grass Carp. doi: 10.2139/ssrn.4648779.
  7. Daisley, B.A., Pitek, A.P., Mallory, E., Chernyshova, A.M., Allen-Vercoe, E., Reid, G., & Thompson, G.J. (2023). Disentangling the microbial ecological factors impacting honey bee susceptibility to Paenibacillus larvae infection. Trends in Microbiology, 31(5), 521-534. doi: 10.1016/j.tim.2022.11.012.
  8. Damico, M.E., Beasley, B., Greenstein, D., & Raymann, K. (2025). Testing the effectiveness of a commercially sold probiotic on restoring the gut microbiota of honey bees: A field study. Probiotics and Antimicrobial Proteins, 17, 991-1000. doi: 10.1007/s12602-023-10203-1.
  9. Demyanenko, D.V., Berezovsky, A.V., Vashchyk, Y.V., Fotina, T.I., & Nazarenko, S.M. (2021). Efficiency of the “Combiiod” means for disinfection of water supply system in the conditions of a poultry factory. Scientific and Technical Bulletin оf State Scientific Research Control Institute of Veterinary Medical Products and Fodder Additives аnd Institute of Animal Biology, 22(2), 118-123. doi: 10.36359/scivp.2021-22-2.13.
  10. DesJardins, N.S., Harrison, J.F., & Smith, B.H. (2023). The effects of anthropogenic toxins on honey bee learning: Research trends and significance. Apidologie, 54, article number 59. doi: 10.1007/s13592-023-01040-w.
  11. Dinata, R., et al. (2023). Pharmacological and therapeutic potential of honey bee antimicrobial peptides: Bee antimicrobial peptides. Indian Journal of Biochemistry and Biophysics (IJBB), 60(5), 365-384. doi: 10.56042/ijbb. v60i5.552.
  12. Eggers, M. (2019). Infectious disease management and control with povidone iodine. Infectious Diseases and Therapy, 8, 581-593. doi: 10.1007/s40121-019-00260-x.
  13. Galatyuk, O., Romanyshyna, T., Lakhman, A., Lysenko, O., & Shimanska, V. (2020). The pathogenic bee enterobacteria resistance to the experimental iodine-containing disinfectant “Jodis des no. 2”. Scientific Horizons, 23(1), 71-78. doi: 10.33249/2663-2144-2020-86-1-71-78.
  14. Hussain, R., Hasan, M., Iqbal, K.J., Zafar, A., Tariq, T., Saif, M.S., Gul Hassan, Sh., Shu, X., Caprioli, G., & Anjum, S.I. (2023). Nano-managing silver and zinc as bio-conservational approach against pathogens of the honey bee. Journal of Biotechnology, 365, 1-10. doi: 10.1016/j.jbiotec.2023.01.009.
  15. Kampf, G. (2024). Povidone iodine. In Antiseptic stewardship: Biocide resistance and clinical implications (pp. 915964). Cham: Springer International Publishing. doi: 10.1007/978-3-031-66074-0_18.
  16. Karbownik-Lewińska, M., Stępniak, J., Iwan, P., & Lewiński, A. (2022). Iodine as a potential endocrine disruptor – a role of oxidative stress. Endocrine, 78, 219-240. doi: 10.1007/s12020-022-03107-7.
  17. Killam, S.M., Daisley, B.A., Kleiber, M.L., Lacika, J.F., & Thompson, G.J. (2024). A case for microbial therapeutics to bolster colony health and performance of honey bees. Frontiers in Bee Science, 2, article number 1422265. doi: 10.3389/frbee.2024.1422265.
  18. Lamas, Z.S., Chen, Y., & Evans, J.D. (2024). Case report: Emerging losses of managed honey bee colonies. Biology, 13(2), article number 117. doi: 10.3390/biology13020117.
  19. Law of Ukraine No. 3447-IV “On Protection of Animals from Cruel Treatment”. (2006, February). Retrieved from https://zakon.rada.gov.ua/laws/show/3447-15#Text.
  20. Lepelletier, D., Maillard, J.Y., Pozzetto, B., & Simon, A. (2020). Povidone iodine: Properties, mechanisms of action, and role in infection control and Staphylococcus aureus decolonization. Antimicrobial Agents and Chemotherapy, 64(9), article number 10.1128/aac.00682-20. doi: 10.1128/aac.00682-20.
  21. Makhayeva, D.N., Irmukhametova, G.S., & Khutoryanskiy, V.V. (2023). Advances in antimicrobial polymeric iodophors. European Polymer Journal, 201, article number 112573. doi: 10.1016/j.eurpolymj.2023.112573.
  22. Meehan, J.P. (2025). Dilute povidone-iodine irrigation: the science of molecular iodine (I2) kinetics and its antimicrobial activity. JAAOS-Journal of the American Academy of Orthopaedic Surgeons, 33(2), 65-73. doi: 10.5435/ JAAOS-D-24-00471.
  23. Mohammadian, B., Toopchi, B., Moharrami, M., & Rahimi, A. (2024). Determination of factors influencing honeybee colony collapse disorder phenomenon using analytic hierarchy process. Honeybee Science Journal, 14(27), 60-70. doi: 10.22034/hbsj.2024.364586.1153.
  24. Morthorst, J.E., Holbech, H., De Crozé, N., Matthiessen, P., & LeBlanc, G.A. (2023). Thyroid-like hormone signaling in invertebrates and its potential role in initial screening of thyroid hormone system disrupting chemicals. Integrated Environmental Assessment and Management, 19(1), 63-82. doi: 10.1002/ieam.4632.
  25. Motta, E.V., & Moran, N.A. (2024). The honeybee microbiota and its impact on health and disease. Nature Reviews Microbiology, 22, 122-137. doi: 10.1038/s41579-023-00990-3.
  26. Nekoei, S., Rezvan, M., Khamesipour, F., Mayack, C., Molento, M.B., & Revainera, P.D. (2023). A systematic review of honey bee (Apis mellifera, Linnaeus, 1758) infections and available treatment options. Veterinary Medicine and Science, 9(4), 1848-1860. doi: 10.1002/vms3.1194.
  27. OECD. (n.d.). Guidelines for the testing of chemicals. Retrieved from https://www.oecd.org/en/topics/sub-issues/ testing-of-chemicals/test-guidelines.html.
  28. Peña-Chora, G., Toledo-Hernández, E., Sotelo-Leyva, C., Damian-Blanco, P., Villanueva-Flores, A.G., AlvarezFitz, P., Palemón-Alberto, F., & Ortega-Acosta, S.A. (2023). Presence and distribution of pests and diseases of Apis mellifera (Hymenoptera: Apidae) in Mexico: A review. The European Zoological Journal, 90(1), 224-236. doi: 10.1080/24750263.2023.2182920.
  29. Shi, F., Chen, Z., Yao, M., Huang, Y., Xiao, J., Ma, L., Li, L., & Qin, Z. (2024). Effects of glutaraldehyde and povidoneiodine on apoptosis of grass carp liver and hepatocytes. Ecotoxicology and Environmental Safety, 272, article number 116078. doi: 10.1016/j.ecoenv.2024.116078.
  30. Silva Morais, L., et al. (2023). Africanized honeybee (Apis mellifera) semen freezing using Tris-based and Collins extenders. Tropical Animal Health and Production, 55, article number 329. doi: 10.1007/s11250-023-03762-6.
  31. Singh, G., & Rana, A. (2025). Honeybees and colony collapse disorder: Understanding key drivers and economic implications. Proceedings of the Indian National Science Academy, 91, 750-766. doi: 10.1007/s43538-02500399-x.
  32. Smriti Rana, A., Singh, G., & Gupta, G. (2024). Prospects of probiotics in beekeeping: A review for sustainable approach to boost honeybee health. Archives of Microbiology, 206, article number 205. doi: 10.1007/s00203024-03926-4.
  33. Tlak Gajger, I., Tomljanović, Z., Mutinelli, F., Granato, A., & Vlainić, J. (2024). Effects of disinfectants on bacterium Paenibacillus larvae in laboratory conditions. Insects, 15(4), article number 268. doi: 10.3390/insects15040268.
  34. Truong, A.T., Kang, J.E., Yoo, M.S., Nguyen, T.T., Youn, S.Y., Yoon, S.S., & Cho, Y.S. (2023). Probiotic candidates for controlling Paenibacillus larvae, a causative agent of American foulbrood disease in honey bee. BMC Microbiology, 23, article number 150. doi: 10.1186/s12866-023-02902-0.
  35. Turganbay, S., Sabitov, A., Askarova, D., Jumagaziyeva, A., Iskakbayeva, Z., Seisembekova, A., Bukeyeva, T., & Azembayev, A. (2025). Dextrin/polyvinyl alcohol/iodine complexes: Preparation, characterization, and antibacterial, cytotoxic activity. Engineered Science, 35, article number 1595. doi: 10.30919/es1595.
  36. Verwold, C., Ortega-Hernandez, A., Murakami, J., Patterson-Fortin, L., Boutros, J., Smith, R., & Kimura, S.Y. (2021). New iodine-based electrochemical advanced oxidation system for water disinfection: Are disinfection byproducts a concern? Water Research, 201, article number 117340. doi: 10.1016/j.watres.2021.117340.
  37. Wang, D., Huang, X., Lv, W., & Zhou, J. (2022). The toxicity and antibacterial effects of povidone-iodine irrigation in fracture surgery. Orthopaedic Surgery, 14(9), 2286-2297. doi: 10.1111/os.13422.
  38. Yakan, S., Akpınar, R., Genişel, M., Çelik, S., & Bozdeveci, A. (2025). Honey bee pathogen prevalence and interactions in the ağrı province of Türkiye. Sociobiology, 72(3), article number 11603. doi: 10.13102/ sociobiology.v72i3.11603.
  39. Yu, X., Sun, N., Cheng, Y., & Yang, X. (2022). Povidone-iodine modulates the antioxidant capacity, immunity, and resistance to Aeromonas hydrophila of the Chinese mitten crab, Eriocheir sinensis. Aquaculture International, 30(6), 2953-2967. doi: 10.1007/s10499-022-00942-3.
  40. Zhyla, M., Pyatnychko, O., Kotsyumbas, H., Shkodyak, N., Stronskyi, Y., & Palonko, R. (2024). Evaluation morphofunctional state of broiler chickens’ body under the applica-tion of various doses of Combiiod. Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies. Series: Veterinary Sciences, 26(113), 156-164. doi: 10.32718/nvlvet11324.
Zastulka, M., Romanyshyna, T., Lakhman, A., & Galatyuk, O. (2025). Experimental investigation of the antimicrobial activity of the preparation and its influence on the viability of honey bees (Apis mellifera). Scientific Horizons, 28(10), 9-20. https://doi.org/10.48077/scihor10.2025.09