Elif SEVİM, Arif BOZDEVECİ, Müberra PINARBAŞ ÇETİN, Meral KEKEÇOĞLU, Rahşan AKPINAR, Merve KESKİN, Sevgi KOLAYLI, Şengül ALPAY KARAOĞLU

Anadolu Propolisinin Paenibacillus larvae Üzerine Antibakteriyel Etkisi

Paenibacillus larvae (P. larvae), bal arısı larvalarında Amerikan Yavru Çürüklüğü hastalığına (AYÇ) neden olan patojenik bir bakteridir. P. Larvae tedavisinde kullanılan antibiyotikler bal arısı kolonilerinin ciddi kaybına neden olmakta ve bu nedenle antibiyotik kullanımına bağlı olarak bu hastalığın kontrolü için alternatif stratejiler geliştirmek gerekmektedir. Son çalışmalar, P. larvae’ya karşı doğal mücadele ajanlarını araştırmaya yöneliktir. Çalışmamızda Anadolu propolisi etanolik ekstresinin (EAP) Türkiye'de izole edilen P. larvae suşları PB35 ve SV35'e karşı potansiyel antimikrobiyal etkinliğinin ortaya konması amaçlanmıştır. EAP, toplam fenolik madde (TPC), flavonoid madde miktarları (TFC) ve bazı fenolik bileşikler açısından karakterize edilmiştir. Analizlere göre, EAP'nin total fenolik madde miktarı ve flavonoid madde miktarları sırasıyla 181,73±5,20 mg Gallik Asit Eşdeğeri (GAE)/g TFC olarak 42,33±1,40 mg Kuersetin Eşdeğeri (QE)/g ve farklı miktarlarda ferulik, kafeik, kumarik asitler, rutin ve kafeik asit fenetil ester (CAPE) içerdiği bulundu. EAP'nin antimikrobiyal aktivitesi, agar kuyu difüzyon, mikrodilüsyon ve Bioscreen C teknikleri kullanılarak belirlendi. EAP'nin Minimal İnhibisyon Konsantrasyon (MIC) değerleri, hem mikrodilüsyon hem de Bioscreen C tekniği kullanılarak PB35 ve SV35 suşuna karşı 74,87 μg/ml olarak belirlendi. Her iki teknikte de PB35 ve SV35 suşlarına karşı EAP'nin minimal bakterisidal konsantrasyon (MBC) değerleri sırasıyla 149 ve 598,4 μg/ml olarak belirlendi.

Anahtar Kelimeler:

Amerikan yavru çürüğü, Anadolu propolisi, MBC, MIC, Paenibacillus larvae

ANTIBACTERIAL EFFECTS OF ANATOLIAN PROPOLIS ON PAENIBACILLUS LARVAE

Paenibacillus larvae (P. larvae) is a pathogenic bacterium causing American Foulbrood Disease (AFB) in honeybee larvae. It is necessary to develop alternative strategies for the control of this disease due to the serious honeybee colonies loses and the use of antibiotics. Recent studies are aimed at the investigating natural fighting agents against P. larvae. In our study, it was aimed to demonstrate potential antibacterial efficacy of ethanol extract of Anatolian Propolis (EAP) against P. larvae strains PB35 and SV35 which were isolated in Turkey. The total phenolic content (TPC) and flavonoid content (TFC) of EEAP were determined as 181.73±5.20 mg Gallic Acid Equivalents (GAE)/g, and 42.33±1.40 mg Quercetin Equivalents (QE)/g, respectively. It was found that EAP contains different amounts of ferulic, caffeic, coumaric acids, rutin, and caffeic acid phenethyl ester (CAPE). The antibacterial activity of the EAP was determined by using agar-well diffusion, microdilution, and Bioscreen C techniques. The Minimal Inhibition Concentration (MIC) values of the EAP were determined as 74.87 μg/ml against strain PB35 and SV35 using both microdilution and Bioscreen C teqnique. In both techniques, Minimal Bactericidal Concentration (MBC) values of the EAP were evaluated as 149 and 598.4 μg/ml against strain PB35 and SV35, respectively. The fact that EAP shows low concentrations of bacteriostatic (MIC) and bactericide (MBC) activity values against P. larvae strains, spore-forming bacilli, which are agents of AFB disease, suggests that it may be a potential source in AFB disease control.

Keywords:

American Foulbrood, Anatolian propolis, MBC, MIC, Paenibacillus larvae,

PDF

___

Alippi, A.M. (2000). Is Terramycin losing its effectiveness against AFB? The Argentinian experience. Bee Biz, 11, 27–29. doi.org/10.1080/00218839.2004.11101124.
Aliyazıcıoglu, R., Sahin, H., Erturk, O., Ulusoy, E., Kolayli, S. (2013). Properties of phenolic composition and biological activity of propolis from Turkey. International Journal of Food Properties, 16(2), 277–287.
Andrews, J.M. (2001). Determination of minimum inhibitory concentrations. Antimicrob Chemother, 48: 5–16. doi.org/10.1093/jac/48.suppl_1.5.
Anonymous (2016). User’s manuel Bioscreen C. Oy Growth Curves Ab Ltd., Available at http://www.bioscreen.fi/images/Bioscreen%20User%20Manual%202015.pdf Accessed date: 10. 05. 2019.
Antúnez, K., Harriet, J., Gende, L., Maggi, M., Eguaras, M., Zunino, P. (2008). Efficacy of natural propolis extract in the control of American Foulbrood. Veterinary Microbiology, 131(3–4), 324–331. doi.org/10.1016/j.vetmic.2008.04.011.
Ash, C., Priest, F.G., Collins, M.D. (1993). Molecular identification of rRNA group 3 Bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus. Antonie Van Leeuwenhoek, 64, 253–260. doi.org/10.1007/BF00873085.
Baltas, N., Karaoglu, S.A., Tarakci, C., Kolayli, S. (2016). Effect of propolis in gastric disorders, inhibition studies on the growth of Helicobacter pylori and production of its urease. Journal of Enzyme Inhibition and Medicinal Chemistry, 31(sup2), 46–50. doi.org/10.1080/14756366.2016.1186023.
Bankova, V., Christov, R., Kujumgiev, A., Marcucci, MC., Popov, S. (1995). Chemical composition and antibacterial activity of Brazilian propolis Zeitschrift für Naturforschung C, 50(3–4), 167–172. doi.org/10.1515/znc-1995-3-402.
Banskota, A.H., Tezuka Y., Kadota S. (2001). Recent progress in pharmacological research of propolis. Phytotherapy Research, 15, 561–571. doi.org/10.1002/ptr.1029.
Bastos, E.M.A., Simone, M., Jorge, D.M., Soares, A.E.E., Spivak, M. (2008). In vitro study of the antimicrobial activity of Brazilian propolis against Paenibacillus larvae. Journal of Invertebrate Pathology, 97(3), 273–281. doi.org/10.1016/j.jip.2007.10.007.
Benzie I.F., Strain, J.J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Analytical Biochemistry, 239(1), 70–76. doi: 10.1006/ abio.1996.0292.
Bogdanov, S. (2006). Contaminants of bee products. Apidologie, 37(1), 1–18. doi.org/10.1051/apido:2005043.
Cabral, I.S.R., Oldoni, T.L.C, Alencar, S.M.D., Rosalen, P.L., Ikegaki, M. (2012). The correlation between the phenolic composition and biological activities of two varieties of Brazilian propolis (G6 and G12) Brazilian. Journal of Pharmaceutical Sciences, 48(3), 557–564. doi.org/10.1590/S1984-82502012000300023.
Can, Z., Yildiz, O., Şahin, H., Asadov, A., Kolayli, S. (2015). Phenolic profile and antioxidant potential of propolis from Azerbaijan. Mellifera, 15(1), 16–28.
CLSI (2015). Methodes for dilution antimicrobial susceptibility test for Bacteria that grow aerobically; Approvard Tenth Edition. Available at https,//clsi.org/media/1632/m07a10_sample.pdf Accessed date: 18. 11. 2020.
El Adaouia Taleb, R., Djebli, N., Chenini, H., Sahin, H., Kolayli, S. (2020). In vivo and in vitro anti‐diabetic activity of ethanolic propolis extract. Journal of Food Biochemistry, 44(7), e13267.
Evans, J.D. (2003). Diverse origins of tetracycline resistance in the honey bee bacterial pathogen Paenibacillus larvae. Journal of Invertebrate Pathology, 83, 46–50. doi.org/10.1016/S0022-2011(03)00039-9.
Fukumoto, L.R., Mazza G. (2000). Assessing Antioxidant and Prooxidant Activities of Phenolic Compounds. J Agric Food Chem, 48, 3597–3604. doi.org/10.1021/jf000220w.
Genersch, E. (2010). American Foulbrood in honeybees and its causative agent, Paenibacillus larvae. Journal of Invertebrate Pathology, 103, 10–19. doi.org/10.1016/j.jip.2009.06.015.
Hansen, H., Brødsgaard, C.J. (1999). American Foulbrood, A review of its biology, diagnosis and control. Bee World, 80, 5–23. doi.org/10.1080/0005772X.1999.11099415.
Hrabák, J., Martínek, K. (2007). Screening of secreted proteases of Paenibacillus larvae by using substrate-SDS-polyacrylamide gel electrophoresis. Journal of Apicultural Research, 46, 160–164. doi.org/10.1080/00218839.2007.11101388.
Isidorov, VA., Buczek, K., Zambrowski, G., Miastkowski, K., Swiecicka, I. (2017). In vitro study of the antimicrobial activity of European propolis against Paenibacillus larvae. Apidologie, 48(3), 411–422. doi.org/10.1007/s13592-016-0485-z.
Keskin, Ş., Yatanaslan, L., Karlıdağ, S. (2020). Anadolu’nun farklı illerinden toplanan propolis örneklerinin kimyasal karakterizasyonu (Chemical characterization of propolis samples collected from different provinces of Anatolia). Uludağ Arı Dergisi – Uludağ Bee Journal, 20(1), 81–88. doi.org/10.31467/uluaricilik.714317.
Kuropatnicki, A.K., Szliszka, E., Krol, W. (2013). Historical aspects of propolis research in modern times. Evidence-Baset Complementary and Alternative Medicine, 2013, 964149. doi.org/10.1155/2013/964149.
Lopez, M.I., Feldlaufer, M.F., Williams, A.D., Chu, P.S. (2007). Determination and confirmation of nitrofuran residues in honey using LC-MS/MS. Journal of Agricultural and Food Chemistry, 55(4), 1103-1108. doi.org/10.1021/jf0625712.
Mihai, C.M., Mărghitaş, L.A., Dezmirean, D.S., Chirilă, F., Moritz, R.F., Schlüns, H. (2012). Interactions among flavonoids of propolis affect antibacterial activity against the honeybee pathogen Paenibacillus larvae. Journal of Invertebrate Pathology, 110(1), 68–72. Doi.org/10.1016/j.jip.2012.02.009.
Mirzoeva, O.K., Grishanin, R.N., Calder, P.C. (1997). Antimicrobial action of propolis and some of its components: The effects on growth, membrane potential and motility of bacteria. Microbiological Research, 152, 239–246. doi.org/10.1016/S0944-5013(97)80034-1.
Pellegrini, M.C., Alonso‐Salces, R.M., Umpierrez, M.L., Rossini, C., Fuselli, S.R. (2017). Chemical composition, antimicrobial activity, and mode of action of essential oils against Paenibacillus larvae, etiological agent of American foulbrood on Apis mellifera. Chemistry & Biodiversity, 14(4), e1600382. doi.org/10.1002/cbdv.201600382.
Rebiai, A. (2015). Evaluation of antioxidant capacity of propolis collected in various areas of Algeria using electrochemical techniques. Int J Electrochem Sci, 10, 9641–9651.
Salomão, K., Dantas, A.P., Borba, C.M., Campos, L.C., Machado, D.G., Aquino Neto, F.R., de Castro, S.L. (2004). Chemical composition and microbicidal activity of extracts from Brazilian and Bulgarian propolis. Letters in Applied Microbiology, 38(2), 87–92. doi.org/10.1111/j.1472-765X.2003.01458.x.
Sevim, E., Baş, Y., Celik, G., Pinarbaş, M., Bozdeveci, A., Özdemir, T., Karaoğlu, Ş.A. (2017). Antibacterial activity of bryophyte species against Paenibacillus larvae isolates. Turkish Journal of Veterinary and Animal Sciences, 41(4), 521–531. doi.org/10.3906/vet-1611-70.
Sforcin, J.M. (2007). Propolis and the immune system: a review. Journal of Ethnopharmacology, 113, 1–14. doi.org/10.1016/j.jep.2007.05.012.
Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. In Methods in Enzymology, 299, 152–178. doi.org/10.1016/S0076-6879(99)99017-1.