Citation/Atıf: Sevim E, Bozdeveci A, Pinarbaş M, Kekeçoğlu M, Akpinar R, Keskin M, Kolayli S, Karaoğlu SA. 2021. Antibacterial Effects of Anatolian Propolis on Paenibacillus larvae (Anadolu Propolisinin Paenibacillus larvae Üzerine Antibakteriyel Etkisi). Arı D./U. Bee J. 21(2):177-186, DOI: 10.31467/uluaricilik.976536 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE ANTIBACTERIAL EFFECTS OF ANATOLIAN PROPOLIS ON PAENIBACILLUS LARVAE Anadolu Propolisinin Paenibacillus larvae Üzerine Antibakteriyel Etkisi Elif SEVİM1, Arif BOZDEVECİ2, Müberra PINARBAŞ2, Meral KEKEÇOĞLU3, Rahşan AKPINAR4, Merve KESKİN5, Sevgi KOLAYLI6, Şengul ALPAY KARAOĞLU2 1Kırşehir Ahi Evran University, Faculty of Art and Science, Department of Molecular Biology and Genetic, Kırşehir, TURKEY, ORCID No: 0000-0002-6455-1333. 2Recep Tayyip Erdogan University, Department of Biology, Rize, TURKEY, ORCID No: 0000-0002-0729-9143, ORCID No: 0000-0001-6064-0673, ORCID No: 0000-0003-1047-8350, Corresponding author/Yazışma Yazarı E-mail: sengul.karaoglu@erdogan.edu.tr. 3Düzce University, Faculty of Art and Science Department of Biology, Düzce, TURKEY, ORCID No: 0000-0002-2564-8343 4Samsun Veterinary Control and Research Institute, Samsun, TURKEY, ORCID No: 0000-0003-0075-9247. 5Bilecik Seyh Edebali University, Vocational School of Health Services, Bilecik, TURKEY, ORCID No: 0000-0001-9365- 334X. 6Karadeniz Technical University, Department of Chemistry, Trabzon, TURKEY, ORCID No: 0000-0003-0437-6139. Geliş Tarihi / Received: 30.07.2021 Kabul Tarihi / Accepted:20.09.2021 DOI: 10.31467/uluaricilik.976536 ABSTRACT 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 ÖZ 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) 177 U.Arı D. – U.Bee J. 2021, 21 (2): 177-186 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE 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 Çürüklüğü, Anadolu propolisi, MBC, MIC, Paenibacillus larvae GENIŞLETILMIŞ ÖZET propolis ekstraktının toplam flavonoid madde miktarı, antioksidan kapasitesi ve fenolik profili Amaç: Dünyanın birçok yerinde koloni kaybına, belirlendi. P. larvae PB35 ve SV35 suşları morfolojik, toplu arı ölümlerine ve bal veriminin düşmesine biyokimyasal ve moleküler özelliklerine göre neden olan çeşitli arı hastalıkları bulunmaktadır. tanımlandı. Daha sonra etanolik Anadolu propolis Hastalıklar arasında en dikkat çekici olanlardan bir ekstraktının antibakteriyel aktiviteleri, agar-well tanesi de Amerikan Yavru Çürüklüğü Hastalığıdır difüzyon yöntemi kullanılarak P. larvae PB35 ve (AYÇ). Hastalık larva aşamasında arının orta SV35 suşlarına karşı test edildi. Minimal inhibisyon bağırsak lümenini etkiler ve yetişkin arı hastalığına derişimi ve minimal bakteriyosidal derişimi tespit neden olmaz. Paenibacillus sp., gram pozitif, edildi. Etanolik Anadolu propolis ekstraktının P. fakültatif anaerobik, katalaz negatif. endospor larvae PB35 ve SV35 popülasyonları, gecikme oluşturan bakterilerdir. Paenibacillus larvae fazları ve üstel fazlar üzerindeki etkileri Bioscreen kolonileri küçük, düzenli, çoğunlukla kaba, düz veya kullanılarak incelendi. kabarık ve beyazımsı ile bej renklidir. Etkilenen larvada bir milyonun üzerinde spor üretebilir ve AYÇ Bulgular: Yapılan analizler neticesinde etanolik oluşumuna neden olur. Oksitetrasiklin hidroklorür Anadolu propolis ekstraktının total fenolik madde (OTC) gibi antibiyotiklerin kullanılması, etkilenen miktarının 181,73±5,20 mg GAE/g, total flavonoid kolonilerin kaybının önlenmesi ve tedavisi için madde miktarının 42,33±1,40 mg QE/g olduğu ve yaygın bir stratejidir. Ancak, antibiyotiklerin uzun propolis ekstraktının p-kumarik asit, ferulik asit, süreli kullanımında çeşitli sorunlar oluşabilmektedir. krisin ve pinocembrin bileşenleri açısından zengin Son zamanlarda bazı ülkelerde oksitetrasikline olduğu tespit edildi. Etanolik Anadolu propolisinin P. dirençli P. larvae izolatlarının olduğu birçok larvae’ye ait iki farklı suş üzerine de etkili olduğu çalışmada gösterilmiştir. Bu nedenle, kovanlarda belirlendi patojenik mikroorganizmaları kontrol etmek için Sonuç: Bu çalışmanın sonuçları, etanolik Anadolu propolis gibi doğal ürünlerin kullanımına artan bir ilgi propolis ekstraktının AYÇ'yi ortadan kaldırmak için vardır. Bu çalışmanın amacı, doğal bir kovan ürünü iyi bir potansiyele sahip olduğunu ve P. larvae olan etanolik Anadolu propolis ekstraktının (EAP) P. tehdidine karşı doğal bir ilaç olabileceğini larvae suşları (PB35 ve SV35) üzerindeki etkisini in göstermektedir. Ayrıca spor oluşturan bir bakteri vitro belirlemek ve AFB'nin kontrolü ve önlenmesi olan P. larvae'nin MBC değerlerinin elde edilmesi, için alternatif bir ürün olarak etanolik Anadolu AYÇ hastalık kontrolünde kullanım potansiyeline propolis ekstraktının kullanılabilirliğini araştırmaktır. sahip olduğunu gösteren önemli bir sonuç olarak Gereç ve Yöntem: Yapılan bu çalışmada ham değerlendirilmektedir. propolis Düzce Üniversitesi Arıcılık Uygulama ve Araştırma Merkezi’nden (DAGEM) 2016 yılında temin edildi. Ham propolis donduruldu, öğütüldü ve INTRODUCTION %70’lik etanol ile ekstrakte edildi. Hazırlanan In many parts of the world, there are various bee ekstraktın toplam fenolik madde miktarı Folin- diseases which cause loss of colonies, collective Ciocalteu metoduna göre belirlendi. Ayrıca etanolik bee deaths, and reduced honey yield. One of the Uludağ Arıcılık Dergisi – Uludag Bee Journal 2021, 21 (2): 177-186 178 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE most remarkable of among the diseases is the MATERIAL AND METHODS American Foulbrood Disease (AFB). This disease in Bacteria Strains and Propolis Sample many countries "forced notifiable" disease is on the list (Genersch 2010). The disease affects the middle Paenibacillus larvae (P. larvae) PB35 and SV35 gut lumen at the larval stage and does not cause strains were obtained from Bee Diseases disease of adult bee. Paenibacillus sp. in the Laboratory, Samsun Veterinary Control Institute. Firmicutes Phylum and Bacillales Ordo are gram- The tested Anatolian Propolis sample was obtained positive, facultative anaerobic, catalase negative from Düzce University Beekeeping Research, endospore-forming bacteria. Paenibacillus larvae Development and Application Center (DAGEM) in colonies were small, regular, mostly rough, flat or Yığılca areas in Turkey, 2016. Raw propolis sample raised, and whitish to beige (Ash et al. 1993). It is a was obtained from directly plastic traps put under bacterium that can produce over one million spores hive cover. The propolis sample was kept in a deep in effected larva thus AFB occurs. A complete freezer (-20°C). solution for the control and protection of the disease Identification of P. larvae strains is not offered because bacterial spores can survive for a long time against physical conditions (Hrabák P. larvae PB35 and SV35 strains were identified and Martínek 2007, Genersch 2010). Using according to their morphological, biochemical, and antibiotics like oxytetracycline hydrochloride (OTC) molecular characteristics. Properties of colony is a common strategy for the prevention and morphologies, Gram staining, endospore staining treatment of affected colonies (Hansen and were used for morphological characterization of Brødsgaard 1999). However, there may be various bacterial strains (CLSI 2015). For molecular problems with the extended use of these antibiotics. analyses, 16S rRNA gene sequence was performed Recently many studies showed to be oxytetracycline previously described by Sevim et al. (2017). The 16S resistant P. larvae isolates in some countries (USA, rRNA sequences for P. larvae isolates were Canada, and Argentina) (Alippi 2000, Evans 2003). deposited in GenBank (NCBI, Bethesda, MD, USA) under accession numbers MW227606 (PB35) and Propolis is a natural bee product containing essential MW227607 (SV35). oils, waxes, phenolic, and flavonoids. Propolis is collected by honeybees from tree buds, leaf, and Extraction of Anatolian Propolis other botanical resinous sources for protection of hives from a number of threats. Propolis composition Frozen raw propolis sample was grinded, and 5 g of depends on mainly the botanical origin (Kuropatnicki powdered raw sample was dissolved in 50 mL 70% et al. 2013, Baltas et al. 2016). There has been an ethanol in a glass flask (500 mL), stirred on a shaker increasing interest in the usage of natural products (Heidolph Promax 2020, Schwabach, Germany) at like propolis for controlling the pathogenic room temperature for 48 hours and after filtration, the microorganisms in hives. extract was evaporated with a Rotary evaporator at 400C (HEIDOLPH Hei-VAP Value Digital G3) and The aim of the study is to determine the effect of stored at -20°C (Aliyazıcıoglu et al., 2013, Keskin et Ethanol Extract of Anatolian Propolis (EAP) on P. al. 2020). larvae strains and is to investigate the availability of EAP as an alternative product for the control and Determination of Total Phenolic and Flavonoid prevention of AFB. Until now, no one has tested Content Anatolian propolis extract against P. larvae using Total phenolic content of the Anatolian propolis was Bioscreen C techniques. All microorganisms carried out according to Folin-Ciocalteu method (bacteria, mold, yeast, etc.) increase the turbidity of (Singleton et al. 1999). The results were expressed the liquid growth medium when growing and as mg of gallic acid equivalents per g sample. Total multiplying in it. Bioscreen C monitors this growth by flavonoid was measured by spectrometric assay measuring the turbidity of the medium in the wells of (Fukumoto and Mazza 2000). The result was a microplate. These measurements are done expressed as mg of quercetin equivalents (QE) per kinetically and recorded as optical density (OD) g sample. measurements (Anonymous 2016). In our study, the values of propolis extract stopping the development of P. larvae were determined by monitoring them with Bioscreen C at different concentrations. 179 U.Arı D. – U.Bee J. 2021, 21 (2): 177-186 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE Determination of Ferric Reducing Power Approximately 10 microliters of culture were added Antioxidant Activity to each well containing serially diluted propolis extracts and incubated at 37°C for 48 h in order to The FRAP method is the most commonly used determined MIC values. The MIC values were method for determining the antioxidant capacity of defined as the lowest concentration that showed no natural products, and it is a method based on the growth. Ampicillin (10 μg/ mL) was used as a reduction of iron(III) ion in the Fe(III)-TPTZ complex standard antibacterial agent. MYGB medium was by antioxidants (Benzie and Strain, 1996). Fe(III), used as negative control. which is reduced by the antioxidant substances in the solution, gives absorbtion at 593 nm. Results for Determination of Minimal Bacterisidal FRAP activity were expressed as µmoL Concentration (MBC) Fe2SO4.7H2O/g. The minimum bactericidal concentration (MBC) is Analyses of Phenolic Compounds in HPLC-UV the least concentration of antimicrobial agent required to kill microorganisms (Andrews 2001). The Phenolic compounds of the propolis were analyzed MBC was determined for ethanol extracts of according to method described by Can et al. (2015). Anatolian propolis. After MIC and Bioscreen C C18 analytical column C (150 mm x 4.6 mm, 5 μm; determination of the EAP tested, an aliquot of 10 μl Fortis) was used by gradient elution (Can et al. from all wells in which showed no bacterial growth 2015). 2 % acetic acid in water (A) and acetonitrile: was plated onto MYPG Agar plates without EAP. water (70:30) (B) were utilized as mobile phase for The plates were then incubated for 2 days at 37°C. elution. 25 L samples were injected at 30°C and The MBC value was determined as the lowest EAP flow rate at 0.75 ml/min. Caffeic acid phenethyl ester concentration without growth on MYPG Agar plates (CAPE) compound was carried out the same colon (Andrews 2001). with different analyses conditions (Can et al. 2015). A 0.1% formic acid in water and B 0.1% formic acid Bioscreen C techniques in acetonitrile and was monitored at 270 nm at 30°C. The effects of EAP on the P. larvae PB35 and SV35 Determination of Antibacterial Activity populations, lag phases and exponential phases were studied by using Bioscreen C (Labsystems, Antibacterial activities of EAP were tested against P. Helsinki, Finland) incubator. Triplicates of 360 µL larvae PB35 and SV35 strains using the agar-well MYPG broth including serial dilution (1/2) diffusion method. PB35 and SV35 strains were concentration of EAP and 40 µL 106 cfu/ ml bacterial suspended in 3 mL of MYPG broth. These bacterial concentrations were added in the wells of the suspensions were diluted to 106 cfu/ ml and then Bioscreen plate. Plates were then placed on were plated on the surface of MYPG agars. Wells of Bioscreen C incubator and then incubated at 37°C. 5 mm in diameter were cut from the dried agar using The optical density of the cell suspensions at 600 nm a sterile cork-borer. Each well was filled with 50 μL was monitored automatically in regular intervals of of EAP and the plates were incubated at 37°C for 48 30 min for 20 hours. Plates were shaken for 20 h. Antibacterial activity was evaluated by calculating seconds before each measurement. The control the clear zone of inhibition. Ampicillin (10 μg) was wells contained the tested culture medium without used as a control antibacterial agent. As a solvent EAP. The data were analyzed using the Excel control, 1/2 dilution of analytical grade ethanol was software in Office 365 and by calculating the used (Sevim et al. 2017). averages of three copies for each culture media Determination of Minimal Inhibition type. Concentrations Value (MICS) MICs values were determined by microdilution RESULTS technique (CLSI, 2015). The microdilution technique was performed in microtiter plates in MYPG broth. The PB35 and SV35 were isolated from the flower Ethanolic propolis extract was serially diluted in plate honey in the hives showing disease and wells with MYPG broth. In fresh cultures of honeycombs showing illness symptom, respectively. Paenibacillus larvae PB35 and SV35 strains, 0.5 The strains were identified as P. larvae according to McFarland turbidity suspensions were prepared. properties of morphological, biochemical, and Uludağ Arıcılık Dergisi – Uludag Bee Journal 2021, 21 (2): 177-186 180 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE molecular. The PB35 strain has a cream was resistant to tetracycline and norfloxacin. The appearance, rough colony morphology, and terminal partial sequences of the 16S rRNA gene were used spore. The PB35 strain was negative with respect to for further characterization of the bacterial isolates. catalase production, Voges-Proskauer, citrate, and The obtained sequences were used for a Blast starch hydrolysis test. The PB35 strain was able to search in the NCBI database and phylogenetic metabolize maltose. The SV35 strain has analysis. Using 16S rRNA gene sequences, transparent appearance, rough colony morphology dendrogram was constructed and 2 isolates were and terminal spore. All biochemical test of the SV35 identified as P. larvae (Figure. 1). Results obtained strain that was only able to metabolize glucose was for the amount of total phenolic content and phenolic negative. Two strains PB35 and SV35 have penicillin composition of propolis sample were summarized in and ampicillin resistance. While the strains PB35 Table 1. Effect of EAP on vegetative growth of PB35 was resistant to chloramphenicol, the strains SV35 and SV35 were given in Figure. 2 and Figure. 3. Table 1. Characteristic properties of the Anatolian propolis. Tablo 1. Anadolu propolisinin karakteristik özellikleri Total phenolic content (mg GAE/ g) 181.73±5.20 Total flavanoid (mgQE/ g) 42.33±1.40 Total antioxidant capacity (FRAP) µmoL Fe2SO4.7H2O/ g 680.70±6.80 Phenolic Composition (HPLC-UV analyses) (mg/ 100g) Gallic acid - Protocatechuic acid 0.55 p-OH benzoic acid 6.0 Caffeic Acid 210 Syringic Acid - p-coumaric acid 124.0 Ferulic acid 42.5 t-cinnamic acid 1.68 Catechin - Rutin 58 Epicatechin 6.40 Resveratrol - Daidzein 1.02 Luteolin 7.40 Myricetin - Hesperetin 4.20 Chrysin 46.80 Pinocembrin 50.04 Caffeic acid phenethyl ester (CAPE) 8.12 181 U.Arı D. – U.Bee J. 2021, 21 (2): 177-186 Flavonoids Phenolic acids ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE Figure 1. Phylogenetic analysis of the P. larvae PB35 and SV35 based on partial sequencing of the 16S rRNA gene. Neighborjoining analysis with p-distance method was used to construct the dendrogram. Bootstrap values shown next to nodes are based on 1000 replicates. P. larvae isolates are indicated with black squares. The scale on the bottom of the dendrogram shows the degree of dissimilarity. Bacillus subtilis RSA-5 was used as out group. Şekil 1. 16S rRNA geninin kısmi dizilimine dayalı olarak P. larva PB35 ve SV35'in filogenetik analizi. Dendrogramı oluşturmak için p-mesafe yöntemiyle komşuluk analizi kullanıldı. Düğümlerin yanında gösterilen önyükleme değerleri, 1000 kopyayı temel alır. P. larva izolatları siyah karelerle gösterilmiştir. Dendrogramın altındaki ölçek, farklılığın derecesini gösterir. Dış grup olarak Bacillus subtilis RSA-5 kullanıldı. Figure 2. Effect of EAP on vegetative growth of SV35 Şekil 2. EAP'nin SV35'in vejetatif büyümesi üzerindeki etkisi Uludağ Arıcılık Dergisi – Uludag Bee Journal 2021, 21 (2): 177-186 182 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE Figure 3. Effect of EAP on vegetative growth of PB35 Şekil 3. EAP'nin PB35'in vejetatif büyümesi üzerindeki etkisi DISCUSSION control AFB disease is crucial (Pellegrini et al. 2017, Sevim et al. 2017). It is known that propolis is a P. larvae are a major threat to hives. The infection of multifunctional honeybee product. Propolis extract P. larvae cause a collapse in honeybee colonies. To has been identified as a natural alternative for protect the hives, it has been required effective controlling AFB (Antunez et al. 2008, Bastos et al. antibacterial agents that do not leave residues in the 2008). The inhibitory effect of propolis extract bee products. For this reason, we tried to use against P. larvae was attributed to the synergy Anatolian propolis as an antibacterial and between flavonoids and phenolic acids of propolis prophylactic agent against P. larvae strains in this (Mihai et al. 2012). The higher phenolic contents study. have the higher biological active properties, as well P. larvae, caused AFB, is a serious problem in as antimicrobial activity (Can et al. 2015, El Adaouia beekeeping worldwide and highly infectious. The et al., 2020). When compared our result with spores of the pathogen are resistant to different regions on propolis, Yığılca propolis environmental conditions thus controlling the contained a higher amount of total phenolic disease is more difficult. Adult worker bees, compounds than many samples (Bankova et al. beekeeping equipment and products that are 1995, Cabral et al. 2012, Rebiai 2015). Flavanoids infected with P. larvae spores cause the spread of have high pharmaceutical properties such as the disease within and among colonies. As a result, antioxidant, anti-inflammatory, and antimicrobial beekeepers often burn infected colonies to eliminate effects (Bankova et al. 1995, Salomão et al. 2004). the source of infection (Bastos et al. 2008, Sevim et In a study it has been reported there is a relationship al. 2017). For beekeeping, the use of antibiotics is an between antimicrobial, bacteriostatic activities and alternative way to protect from AFB. Many antibiotics phenolics of propolis (Baltas et al. 2016). The last such as tetracycline derivatives (oxytetracycline and studies have demonstrated that the inhibitory effect chlortetracycline), streptomycin, sulfonamides, of propolis on bacteria depends on the synergism of tylosin, erythromycin, chloramphenicol, and many compounds in propolis. Mirzoeva et al. (1997) lincomycin have been used to date. However, using investigated the antibacterial activity of propolis them insensibly has led to both the accumulation of ethanol extract. They concluded that this activity is antibiotic residues in honey and in other hive highly related to the composition and concentration products and to antibiotic resistant in strains of P. of propolis active compounds. Phenolic and larvae. Therefore, the development of different flavonoid components of propolis were found to methods such as the use of natural products to inhibit bacterial motility. It has been claimed that the 183 U.Arı D. – U.Bee J. 2021, 21 (2): 177-186 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE antibacterial activity of propolis extracts is related to incorporation into social nest architecture as propolis the chemical composition of flavonoids (Sforcin is reported to be an effective but relatively 2007). It has been reported that the interaction undiscovered colony-level defense against between the two major groups (flavones/flavonols pathogens (Banskota et al. 2001). and flavanones/dihydroflavonols) was particularly Conclusion important in inhibiting growth of honeybee pathogens such as P. larvae by Mihai et al. (2012). In conclusion, the results of this study showed that The antimicrobial effect of propolis extracts with EAP is rich in ferulic, caffeic, coumaric acids, rutin, different floral origins was tested against P. larvae and caffeic acid phenethyl ester (CAPE). It has a strains isolated in Brazil. It has been reported that good potential to eliminate AFB by its compounds the antimicrobial activity of propolis has a minimum and could be a natural drug against the threat of P. zone diameter of 20-12 mm and minimal inhibitory larvae. In addition, obtaining MBC values of P. concentration of 1.7 and 0.12 mg/mL (Bastos et al. larvae, a spore-forming bacterium, was evaluated as 2008). In another study performed by Isidorov et al. an important result showing that it has a potential for (2017), the antimicrobial activity of propolis samples use for AFB disease control. (poplar, birch, and wire poplar) collected in the Author contributions: Conceptualization; Şengül European climate zone was tested against the P. Alpay Karaoğlu, Elif Sevim, Arif Bozdeveci, larvae and their antimicrobial activity was compared. Methodology; Şengül Alpay Karaoğlu, Rahsan The researchers declared that not only phenolic Akpınar, Arif Bozdeveci, Müberra Pınarbaş Çetin, compounds but also some other compound classes Merve Keskin, Elif Sevim, Meral Kekeçoğlu, Sevgi like phenylpropanoids, hydroxycinnamyl Kolaylı Writing – Original Draft; Şengül Alpay sesquiterpenols, glycerides, and benzoates had an Karaoğlu, Arif Bozdeveci, Elif Sevim, Merve Keskin, effect against the AFB. Writing – Final Version; Şengül Alpay Karaoğlu, Elif Due to the evolution of resistance against antibiotics Sevim, Merve Keskin, Funding Acquisition; Şengül used in the treatment of AFB, this research is a Alpay Karaoğlu. significant first step in the identification of possible Funding: This work was supported by the Recep new active compounds within Anatolian propolis in Tayyip Erdoğan University research fund (RTEU- the treatment of AFB in honey bee colonies. In case BAP 2015.53001.102.03.04. 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