HACETTEPE ÜNİVERSİTESİ ARI VE ARI ÜRÜNLERİ UYGULAMA VE ARAŞTIRMA MERKEZİ

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1 HACETTEPE ÜNİVERSİTESİ ARI VE ARI ÜRÜNLERİ UYGULAMA VE ARAŞTIRMA MERKEZİ Sayı: SCIENTIFIC REPORT Surface-Bonded Preventative Action and Antimicrobial Activity of Organosilicon Quaternary Ammonium Chloride on American Foulbrood Pathogen: Paenibacillus larvae ABSTRACT The hydrolysis product of a quaternary amine-containing organosilicon salt (Si-QAC), 3- (trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride exhibited antimicrobial activity against a broad range of microorganisms while chemically bonded to a variety of surfaces. In this study, the chemical was tested for American Foulbrood (AFB) pathogen: Paenibacillus larvae. AFB is a serious disease in honey bees (Apis Mellifera) and common among bee colonies in Turkey and an economic threat to the Turkish Beekeeping Industry. Si-QAC was examined for 28 different local strains of Paenibacillus larvae spore, vegetative and spore-vegetative forms and P. larvae strain ATCC Si-QAC (BeeGuard ) was prepared by the Nanotechnology Company, Istanbul. All strains were growth in Brain-Heart Broth Medium (Sigma, 42gr/L) and then transferred 0.1 ml bacteria (1x108 CFU/mL) MYPG Medium. The experiment was set up for spore and vegetative forms in 4 parts: 1.Inoculation of P.larvae spores/vegetative forms to Brain Heart Agar medium treated with Si-QAC before, 2.Addition of Si-QAC to the medium and after drying process inoculation of P.larvae spores/vegetative forms, 3.Inoculation of P.larvae spores/vegetative forms to Brain-Heart Agar medium sprayed Si-QAC before, 4. Spraying of Si-QAC to the medium and after drying process inoculation of the P.larvae spores/vegetative forms. The results show that Si-QAC inhibits the bacterial growth significantly by in-vitro experiments. It has also bactericide activity on the spore form of P. larvae. The extended studies were completed by cage experiments to identify the toxicity level. No toxicity was observed with honey bees exposed to direct or indirect contact with Si-QAC. This is considered the main advantage of this product.

2 INTRODUCTION Beekeeping is one of the most important agricultural activities for not only producing honey and honey bee products but also in the pollination in all agriculture areas. Honey bees are always threatened by many pathogens causing diseases. American foulbrood (AFB) is a serious disease in honey bees (Apis mellifera L.). It is very common among the colonies in Turkey, as well as many locations worldwide. AFB is a major economic threat to the Turkish Beekeeping Industry. AFB is caused by Paenibacillus larvae, a spore-forming gram-positive, microaerophilic bacterium. P. larvae is endemic to bee colonies worldwide (Ozkirim and Keskin, 2002). The organism can be readily cultured from 20% of all colonies in Turkey. P. larvae spores can survive in raw and processed honey and is distributed by the honey feed to honeybees in winter. It can also found in comb foundation. Each year, bee waxes across turkey are collected regionally and processed in the same location, furthering the spread of spores. Colonies acquire P.Larvae spores from unsterilized, newlyformed, comb foundations (Ozkirim and Keskin,2005). For years, alternatives to antibiotics for the treatment of AFB have been actively researched. For over a decade alkoxysilanes have been utilized by a number of industries as coupling agents to reinforce or impart desirable properties to a variety of materials. A number of enzymes were found to remain biologically active when bonded to inorganic surfaces by alkoxysilanes. Their activity was shown to persist after repeated washing procedures. 3-(trimethoxysilyl)- propyldimethyloctadecyl ammonium chloride (Si-QAC) is representative of a group of Alkoxysilanes. Si-QAC (BeeGuard ) was prepared by Nanotechnology Company, Istanbul. The specific site of action of this class of compounds has been attributed by various investigators to membrane phenomenon, i.e., membrane lyses, membrane enzyme inactivation, or interference with ion transport (Lawrence, 1968). The antimicrobial activity of Si-QAC is thought to be due to the disruption of membrane function with possible cell lyses, caused by the high concentration of charged chemical (quaternary ammonium chloride) on the substrate (Vetter et al., 2009). In this study, Si-QAC was tested for antimicrobial activity against P. larvae by in-vitro and cage experiments. MATERIAL-METHOD Laboratory Experiments Si-QAC was examined for 28 different local strains of Paenibacillus larvae spore, vegetative and spore-vegetative forms and P. larvae strain ATCC 9545(Table 1.). Si-QAC was prepared by Nanotechnology Company, Istanbul. All strains were growth in Brain-Heart Broth Medium (Sigma, 42gr/L) and then transferred 0.1 ml bacteria (1x108 CFU/ml) Brain Heart Agar Medium. The experiment was set up for spore and vegetative forms in 4 parts: 1.Inoculation of P.larvae spores/vegetative forms to Brain Heart Agar medium added Si-QAC before, 2.Addition of Si-QAC to the medium and after drying process inoculation of P.larvae spores/vegetative forms,

3 3. Inoculation of P.larvae spores/vegetative forms to Brain-Heart Agar medium sprayed Si-QAC before, 4. Spraying of Si-QAC to the medium and after drying process inoculation of P.larvae spores/vegetative forms. All plates were set up three replicated for each strain of P. larvae to investigate the preventative activity of Si-QAC against the three forms of P. larvae. At the second stage of laboratory experiments, the classic antibiogram test was used to measure inhibition zone for antimicrobial activity. The sterile absorbent paper prepared like antibiotic disks in 3.5 cm diameter. All strains were growth in Brain-Heart Broth Medium (Sigma, 42gr/L) and then transferred 0.1 ml bacteria (1x10 CFU/ml) Brain Heart Agar Medium (Sigma, 42gr/L). After inocula- 8 tion of 0.1 ml P. larvae bacterial solution, the paper disk were placed the middle of the plates and o 0.5 ml of Si-QAC solution was absorbed by these paper disks. All plates were incubated 37 C 24 hours. Table local strains of P. larvae from different places of Turkey P. larvae Strain Code Province 01/15 Adana 01/17 Adana 01/33 Adana 01/37 Adana 01/38 Adana 31/09 Hatay 31/28 Hatay 31/27 Hatay 31/08 Hatay 31/11 Hatay 01/41 Adana 48/38 Muğla 31/18 Hatay 48/35 Muğla

4 31/32 Hatay 01/29 Adana 48/07 Muğla 31/24 Hatay 31/02 Hatay 01/19 Adana 48/27 Muğla 01/23 Adana 31/26 Hatay 48/31 Muğla 01/40 Adana 31/21 Hatay 01/43 Adana 31/16 Hatay 9545 ATCC Cage Experiments 50 newly emerged bees were used for each cage in the cage experiment. Before using cages, Si-QAC solution was applied to all sides of wooden cage by spraying. 10 ml of the solution were sprayed 200 cm2 surfaces of the cages. After waiting ten minutes for drying process, 50 newly emerged bees were put inside the cages. Total 20 cages were used, 10 cages as a treatment group, 10 cages as control group without any chemical or disinfectant. All cages were observed for 18 days. Honey bees in cages were fed with 5 ml sugar syrup and 3 gr pollen cake per day. Dead bees were noted. The aim of the cage experiments was to determine the occurrence of any toxicity of Si-QAC on the honeybees.

5 RESULTS Results of Laboratory Experiments In laboratory experiments, the results show that Si-QAC has antimicrobial activity significantly on P. larvae growth in all forms (spore, spore-vegatative, vegatative). All results were analysed statistically by One Way ANOVA and Mann-Withney Test. In order to measure the preventative activity of Si-QAC against P. larvae, the surface of plates were washed by 1 ml of sterilled saline solution (0.9% NaCl) and cleaned by sterilled cotton stick. After collecting all cells in the saline solution from the culture into the micro tube, the cotton stick were suspended in the solution. 0.1 ml of the solution were taken out and counted cells again by heamocytometer to determine the increase or decrease of the number of the bacteria (Figure1. Heamocytometer used for counting, Figure 2. The areas used for bacterial counting, Figure 3. BHI Plates in Laboratory Experiments). Figure 1. Neubauer heamocytometer

6 Figure 2. The areas on the heamacytometer used for counting Figure 3. The inhibition zone of Si-QAC on the spore and vegetative forms of P. larvae (VK:Vegatative Control, V1, V2, V3: Three replicated experiments of vegetative form treated with Si-QAC., SK:Spore Control, S1,S2, S3: Three replicated experiments of vegetative form treated with Si-QAC)

7 Table 2. The number of cells in the Si-QAC treated Plates (CFU/ml) P. larvae Strain Code 01/15 01/17 01/33 01/37 01/38 31/09 31/28 31/27 31/08 31/11 01/41 48/38 31/18 48/35 31/32 01/29 48/07 31/24 31/02 01/19 48/27 01/23 31/26 48/31 Spore form Spore-vegatative form Vegatative form

8 01/40 31/21 01/43 31/ All Strains (Median) % Reduction % Reduction= control- sample/ control x 100 The Number of Cells AfterTreatment of Si-QAC in Three Forms of P. larvae N u m b e r o f C e l l s Strains of P. larvae Spore form Spore-vegatative form Vegatative form Results of Cage Experiments In the cage experiments, it is found that there is no difference between control and Si-QAC treated cages statistically for honey bee mortality rate (One way ANOVA, Mann-Withney Test P 0.005)

9 Figure 4. Cage experiment photos It was observed that 50 newly emerged bees in each cage consumed app. 3.5 ml of sugar syrup and 1.8 gr of polen cake per day. The comparison results between two groups demonstrated that there is a correlation between all data and there is no toxic effect of Si-QAC on honey bees (Table 2). Figure 1. The Comparison of Control and Si-QAC Treated Cages Honey Bee Mortality Check time (in a couple of days) Control Si-QAC

10 It is emphasized that Si-QAC inhibits the bacterial growth by means of biocidal and biophysical effects only in the places where it is applied. Due to the nature of the surface-bonded and waterproof properties of Si-QAC, it is advisible not to apply on larval and adult forms of honey bees directly. On the other hand, the results show that Si-QAC has an important role to inhibit the growth of P. larvae which can be survive in the hive or the soil between years. Although only one spore can grow by itself; the prevention of infection by AFB is reduced using Si-QAC in hives. The preventive action of organosilicon materials combats the disfigurement of wood as well as help to control moisture issues (Vetter et al., 2009). It suggests that Si-QAC can also prevents fungal growth in the wooden hive used generally all over the world. Anyway, the Antimicrobial effect of Si-QAC was reported by Walters et al in REFERENCES Lawrence, C.A. (1968), Quaternary ammonium surface-active disinfectants. In disinfection, Sterilization and Preservation (C.A Lawrence and S.S Block, Eds.), pp Lea and Febiger Philadelphia. Ozkirim, A., Keskin, N., (2002), Distribution of the major bacterial brood diseases diagnosed in Ankara and its surroundings, Mellifera, 2-4: Ozkirim, A., Keskin, N., (2005), The Culture of Bacillus spp. From Comb Foundation, Hacettepe Journal of Biology and Chemistry, 34: Vetter, D. L., Bulcke, J. W. D., Imke D. W., Stevens, Acker, J. V.,(2009), Preventive Action of organosilicon treatments against disfigurement of wood under laboratory and outdoo conditions, International Biodeterioration and Biodegrradation, 63 : Analysed and written by Dr. Aslı Özkırım Hacettepe University Bee Health Laboratory Beytepe-Ankara-TURKEY Phone: Fax: ozkirim@hacettepe.edu.tr Dr. Aslı Özkırım Assoc. Director of Bee and Bee Product Research and Application Center (HARUM)