Available online: December 10, 2019 Commun.Fac.Sci.Univ.Ank.Series C Volume 28, Number 2, Pages 211-224 (2019) ISSN 1303-6025 E-ISSN 2651-3749 https://dergipark.org.tr/en/pub/communc/issue/49312/632636 INVESTIGATION OF THE ANTIMICROBIAL ACTIVITY OF Vitis vinifera L. BOĞAZKERE SEDA ÇİÇEK, TALİP ÇETER ABSTRACT. In this study, extracts from Boğazkere cultivar of Vitis vinifera L. at 10 µL, 50 µL and 100 µL concentrations were tested against 18 different bacterial and fungi strains using disc diffusion (DD) method along with minimum inhibitory concentration (MIC) and minimum bactericidal/fungisidal concentration (MBC/MFC) tests to reveal possible antimicrobial properties. Then, the obtained results were compared with 18 known antibiotics. The results revealed that 7.33-19.66 mm inhibition zones were obtained for 15-different microorganisms at 100 µL concentrations while those obtained 7.33-12.33 mm inhibition zones for 12 microorganims at 50 µL volume, where no inhibition zone was observed at 10 µL volume addition. The extracts of Boğazkere for the tested concentrations showed no antimicrobial capability against Salmonella kentucky, Enterococcus durans, Salmonella typhimurium and Candida ablicans. MIC tests showed that the extract at 0.039-20 mg/100 mL concentration range was bacteriostatic for the entire tested microorganism. Bactericidal effects of the extract were obtained for Listeria innocua at 10 mg /100 mL while that was 20 mg /100 mL for Pseudomonas fluorescens, Pseudomonas aeruginosa, Enterococcus faecium and Staphylococcus aureus. The findings show that Boğazkere grape species has antibiotic character, what makes them possible preservatives for food products. 1. INTRODUCTION Humankind has accumulated the knowledge of healing potentials of the plants from the early times of humanity, and has revealed it to the next generations. Phenolic compounds are the basis of curing potentials of the plants. Plants inherently use the phenolic compounds for defense, and those give smell, flavor and color. Antimicrobial properties of the plants derived from the chemicals that have been in use of curing microorganisms-mediated diseases for ages [1]. Received by the editors: October 13, 2019; Accepted: December 09, 2019. Key word and phrases: Antimicrobial activity, Disc diffusion method, Vitaceae, Vitis vinifera, Boğazkere. © 2019 Ankara University Communications Faculty of Sciences University of Ankara Series C: Biology 212 INVESTIGATION OF THE ANTIMICROBIAL ACTIVITY OF Vitis vinifera L. BOĞ AZKERE There are two characteristic mechanisms that microorganism use to develop resistance for antibiotics, which are natural and acquired resistance. In natural resistance, the microorganisms do not possess compound or system targeted by the antibiotics while the acquired resistance refers to that the microorganism develop a mechanism (e.g. discarding the antibiotic via newly developed proteins) to eliminate antimicrobial agent’s effect that inherently kills the microorganism [1, 2]. Antimicrobial agents can trigger a variety of reactions in living organisms. They can either act on cellular membrane or target elements within the cytosol; for example inhibition of nucleic acid synthesis, and cause organelles malfunction and cell wall metabolism problems. Besides, they can cause troubles at organ levels including cardiovascular and urinary system defects [3]. Plants within Vitaceae family are among the oldest plants cultivated worldwide owing to their strong adaptation for different climate and soil types. There are about 1200 cultivated grape types belong to Vitis vinifera species. They are used to produce wine, juice, molasses, dried fruit roll-up and as ingredient of cosmetic stuffs, in addition to that they are consumed as dried and fresh fruits. Besides high sugar and vitamin contents, grape carries high amount of antioxidants. Boğazkere grape is one of the most valuable grapes of Anatolia, which is mostly cultivated in Diyarbakır. They are mostly produced in red soils possess gravel, clay and calcareous character. Beads of Boğazkere are dark red colored and mid-sized [4-8]. Baydar et al. (2006) tested the extracts of seeds from Hasandede, Emir and Kalecik Karası grapes (extraction was performed in water/acetone/acetic acid solvent system) on 15 different microorganisms using agar-disc diffusion methods for the concentrations of 1%, 2.5%, 5% and 10%. Among the grape types, Hasandede revealed suppression capacity for all the bacteria at the tested concentrations [4]. Baydar et al. (2004) performed ethyl acetate/methanol/water mediated extraction of grape seeds, whose lipid content was removed beforehand. The extracts were tested for 15 microorganisms using disc diffusion method, where the highest antibacterial activity was observed for Listeria monocytogenes while no activity was obtained for Enterobacter aerogenes [9]. Anastasiadi et al. (2009) analysed chemical contents belong to pomace of four Vitis vinifera types and tested anti-L. monocytogenes capability using MIC test approach. The findings revealed that the extracts from plant stem and seeds could be introduced to food samples as preservatives [10]. Ege (2015) carried out acetone/water/acetic acid/methanol mediated extraction from seeds of Müşküle (white), Kara dimrit (blue-black) and Öküzgözü grape types, whose lipid content was removed beforehand. Antimicrobial activity of the extracts was tested for 4 fungi and 6 SEDA ÇİÇEK, TALİP ÇETER 213 bacteria strains using MIC test. The findings revealed that all the extracts did not show antimicrobial capability against 4 fungi and 4 bacteria strains [11]. Abtahi et al. (2011) extracted dried white, red and black grape samples in 70% alcohol, then which were tested on Escherichia coli PTCC1330, Staphylococcus aureus PTCC 1431, Salmonella typhimurium PTCC1639 and Pseudomonas aeruginosa PTCC1310 strains using MIC tests. The findings showed that the extracts gave positive results for all the tested bacteria with showing its highest activity for S. aureus strain [12]. Waqar et al. (2014) tested the antimicrobial activity of the extracts from leaves of V. vinifera on E. coli, P. aeruginosa, S. aureus and Enterococcus faecalis strains using disc diffusion method. The inhibition zones belong to S. aureus as 30 mm, E. faecalis as 28.9 mm, E. coli as 28 mm and P. aeruginosa as 23.7 mm were obtained [13]. Yadav et al. (2015) tested the antibacterial capability of water-, ethanol-, acetone-, and methanol- mediated extracts of grape-peel at three different concentrations for the antibiotic resistant S. aureus, E. faecalis, Enterobacter aerogenes, Salmonella typhimurium and E. coli using disc diffusion method. The findings revealed the highest antibacterial capacity for methanol mediated extracts. S. typhimurium and E. coli showed resistance for the tested concentrations. The extract gave the inhibition zones for S. aureus as 22 mm, E. faecalis 18 mm and E. aerogenes as 21 mm [14]. In the present work, antimicrobial activity of Boğazkere belong to Vitis vinifera species performed along with evaluating its possible preservative role for food samples. 2. MATERIAL AND METHODS 2.1 Plant Samples and Extraction Samples of Boğazkere grapes, collected in Kırşehir Toklumen vineyards of Kavaklıdere Company in September of 2017. The collected samples, protected under proper conditions until they reached the laboratory. The samples rinsed thoroughly, followed by the beads were detached from the stems using clean blade. The beads were then grinded in mortar, followed by liquid part, removed with clean cheesecloth. The obtained pomace was mixed with liquid nitrogen, and then grinded into fine particles in mortar. The particles were added to the liquid part. The mixture, was mixed with 96% ethanol solution at 1:1 ratio, which then underwent mixing on orbital shaker at 100 rpm for three days. Followed by the extraction, filtration was performed using whatman paper, where alcohol and water content was eliminated using Rotary evaporator (run in water bath and under vacuum). Samples used for disc diffusion 214 INVESTIGATION OF THE ANTIMICROBIAL ACTIVITY OF Vitis vinifera L. BOĞ AZKERE method was prepared by dissolving 2 gr of dried sample in 8 mL etanol: 2 mL pure water solvent system. In MIC test, 2 gr of dried sample was dissolved in 10 mLof water, followed by filtration through 0.2 µm sterile filter. The prepared stocks were kept under proper conditions until further usage. 2.2 Test Microorganisms The prepared extracts of Boğazkere were tested for the following standart strains or isolated microorganisms: Enterobacter aerogenes (ATCC 13048), Salmonella infantis, Listeria monocytogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa (DSMZ 50071), Pseudomonas fluorescens, Salmonella kentucky, Enterococcus faecalis (ATCC 29212), Listeria innocua, Salmonella enteritidis (ATCC 13075), Enterococcus durans, Salmonella typhimurium, Candida ablicans (DSMZ 1386), Enterococcus faecium, Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (DSMZ20044), Bacillus subtilis (DSMZ 1971), and Escherichia coli (ATCC 25922), Saratia marrescens (ATCC 13048). 2.3. Preparation of Inoculation All the tested microorganisms were grown in Nutrient agar, where the identical colonies were collected using a sterile disposable swabing tool and placed into 10 mL of 0.9 % steril NaCl solution. Based on 0.5 McFarland turbiditiy standard, bacterial colonies were prepared at 108 cfu.mL-1 while that was 107 cfu.mL-1 for C. albicans [15-17]. 2.4. Loading Extract to Empty Disks In disc diffusion method, sterile empty antibiogram discs were used to evaluate antimicrobial activity of the extracts. The stock Boğazkere extracts at 10 µL, 50 µL and 100 µL volumes, placed on the empty discs, followed by incubated at 30 ℃ for overnight under sterile condition. The dried samples, kept at +4 ℃ until further usage. SEDA ÇİÇEK, TALİP ÇETER 215 2.5 Disc Diffusion Method (DD) 0.1 mL of inoculum was evenly spread over the Mueller Hinton Agar (MHA) using swab stick. Antimicrobial vulnerability test was performed in accordance with Bauer-Kirby method [18]. For each MHA, empty, 10 µL, 50 µL and 100 µL extract impregnated discs were added onto the surface of MHA. Bacterial samples and fungus sample were incubated at 37 °C and 27 °C for 24 h. Right after 24 h incubation period, the inhibition zones were measured in millimeter (mm). The tests were performed in three parallel. 2.6. Determination of minimum inhibitory concentration (MIC) All the microorganisms showed vulnerability for the Boğazkere extracts from the disc diffusion tests were included into MIC test. MIC test was performed in sterile 96-well plate. MIC value was accepted as the concentration at which bacterial growth was not visually observed [19-20]. 100 µL of sterile Muelller Hinton Broth was added to each well of 12-well microplate. 100 µL from the stock Boğazkere extract was added to the first well of the 12-well microplate (Number 1 well). 2-times serial dilution was then applied to dilute the extract from number well 1 to well 10 (Number 10 well). 100 µL solution was then discarded from number 10 well. 10 µL from each of the selected microorganisms were added from Number 1-Number 11 wells. Wells from 1-10 were used to evaluate MIC of Boğazkere while Number 11 well was used for microbial positive control and Number 12 well was used as system control. Bacterial samples and fungi sample were incubated at 37°C and 27°C for 24 h to explore MIC values for each microbial strain. The tests were performed in three parallel. 2.7. Determination of Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) Minimum bactericidal/bacteriostatic concentration (MBC) test is to find out whether any bacterial development takes place in the wells where no bacterial growth was observed in MIC test. The findings of this test clarifies whether the MIC concentrations are bactericidal or bacteriostatic. Followed by the MIC test, samples from the wells (where no growth was visually observed) were transferred onto a fresh Nutrient Agar, followed by incubated at 37°C for overnight. In 216 INVESTIGATION OF THE ANTIMICROBIAL ACTIVITY OF Vitis vinifera L. BOĞ AZKERE the case of no growth on the fresh agar, the MIC concentration was accepted as minimum bactericidal concentration while that was accepted, as minimum bacteriostatic concentration in the case growth was clear. The tests were performed in three parallel. 2.8. Controls In disc diffusion method, sterile empty discs were used as negative control while 18-standard antibiotic discs were used for 19 microorganisms as positive control. 3. RESULTS AND DISCUSSIONS Antimicrobial activity of the extract from Vitis vinifera cv. Boğazkere was tested for 18 bacterial strains and one fungus species using disc diffusion method. The findings based on triple parallel examination with inhibition zone diameters are given in Table 1. The findings revealed that Boğazkere extract possessed antimicrobial activity for 15 microorganisms by giving inhibition zones between 7.33 and 19.66 mm. Treatment with 10 µL of the preprared stock extract did not show any antimicrobial activity for the tested microorganisms while at 50 µL treatment antibacterial activity was observed for S. infantis, L. monocytogenes, K. pneumoniae, P. fluorescens, P. aeruginosa, E. faecalis, L. innocua, E. faecium, S. aeureus, S. epidermidis, B. subtilis and E. coli with causing inhibition zones in the range of 7.33-12.33 mm. Treatment with 100 µL of the stock antibacterial activity for E. aerogenes, S. infantis, L. monocytogenes, K. pneumonia, P. fluorescens, P. aeruginosa, E. faecalis, L. innocua, S. enteritidis, E. faecium, S. aeureus, S. epidermidis, B. subtilis, E. coli and S. marcescens was observed with causing inhibition zones between 7.33 and 19.66 mm diameters. However, S. kentucky, E. durans, S. typhimurium and C. ablicans did not show any vulnerability towards the extract at the tested concentrations. Followed by the disc diffusion method, MIC tests were performed for all the microbial strains that showed vulnerability for the extract. Microbial resistance gradually increased upon decreases in the applied extract concentration. The extract between 0.039 and 20 mg/100 mL concentrations gave MIC values for the selected bacterial strains, which was given in Table 2. Further studies on MIC tests revealed that most of the MIC values were bacteriostatic while such high concentrations were more of bactericidal doses (Table 2). 217 SEDA ÇİÇEK, TALİP ÇETER TABLE 1. Antimicrobial acitivity of Boğazkere extract based on disc diffusion method (Negative test result equal to empty disk diameter = 6 mm). Zone Diameter (mm) 10 µL Mikroorganisms 50 µL 100 µL A B C Mean A B C Mean A B C Mean B. subtilis - - - - 10 9 10 9.66 12 12 12 12 C. albicans - - - - - - - - - - - - E. aerogenes - - - - - - - - 7 7 8 7.33 E. coli - - - - 9 7 10 8.66 10 9 10 9.66 E. durans - - - - - - - - - - - - E. faecalis - - - - 12 12 14 12.66 12 17 17 15.33 E. faecium - - - - 12 12 13 12.33 20 19 20 19.66 K. pneumoniae - - - - 7 8 7 7.33 10 10 10 10 L. innocua - - - - 12 10 13 11.66 16 15 16 15.66 L. monocytogenes - - - - 9 10 11 10 12 14 16 14 P. aeruginosa - - - - 7 7 8 7.33 9 8 9 8.66 P. fluorescens - - - - 10 14 14 11.33 14 15 17 15.33 S. aeureus - - - - 11 7 10 9.33 13 10 12 11.66 S. enteritidis - - - - - - - - 8 8 10 8.66 S. epidermidis - - - - 7 7 8 7.33 8 8 7 7.66 S. infantis - - - - 9 7 7 7.66 10 12 12 11.33 S. kentucky - - - - - - - - - - - - S. marcescens - - - - - - - - 9 9 10 9.33 S. typhimurium - - - - - - - - - - - - 218 INVESTIGATION OF THE ANTIMICROBIAL ACTIVITY OF Vitis vinifera L. BOĞ AZKERE TABLE 2. Results for Minimum inhibition concentration (MIC) test and Minimun Bactericidal Concentration (MBC). (Bcd: Bactericidal effect, Bst: Bacteriostatic effect). (Initial concertation of the extract was 10 mg/100 µL). BACTERIA E. aerogenes S. infantis L. monocytogenes MIC 1.25 5 10 MMC (Bcd) - MBC (Bst) 1.25 5 10 K. pneumoniae P. fluorescens 20 10 20 20 10 P. aeruginosa S. kentucky E. faecalis 0.625 20 20 - 0.625 20 L. innocua S. enteritidis 0.625 0.039 10 - 0.625 0.039 E. durans S. typhimurium C. albicans - - - E. faecium S. aeureus S. epidermidis B. subtilis 10 0.625 2.5 5 20 20 - 10 0.625 2.5 5 E. coli S. marrescens 10 5 - 10 5 Antibiogram tests revealed that Lincomycin (L2) posed antibacterial activity only for P. aeruginosa, S. aereus, B. subtilis among the tested 18 different bacterial strains with 9 mm, 25 mm and 16 mm diameter zone inhibitions. Meropenem (MEM 10), Gentamicin (CN10), Neomycin (N30) and Ciprofloxacin (CIP5) showed antibacterial activity nearly all of the tested bacteria. The highest activities were observed for B. subtilis (46 mm zone diameter) and S. marcescens (43 mm zone diameter) with Ampicillin 10 mcg and Ciprofloxacin 5mcg treatment. Similarly Meropenem at 10 mcg. Caused 40 mm zone diameter formation for B. subtilis (Table 3). 219 SEDA ÇİÇEK, TALİP ÇETER Yadav et al. (2015) tested the antibacterial capability of water-, ethanol-, acetone-, and methanol- mediated extracts of grape-peel at three different concentrations (i.e. 260 mg/TAE/ml, 540 mg/TAE/ml and 1080 mg/TAE/ml) for the antibiotic resistant S. aureus, E. faecalis, E. aerogenes, S. typhimurium and E. coli using disc diffusion method. The findings revealed the highest antibacterial capacity was from the methanol-mediated extracts. S. typhimurium and E. coli showed resistance for the tested concentrations while the rest gave vulnerability for all the extracts performed in different solvents (P <0.05). The extract gave the inhibition zones for S. aureus as 22 mm, E. faecalis 18 mm and E. aerogenes as 21 mm [15], for which zone inhibitions were obtained as 11.66 mm, 15.33 mm and 7.33 respectively upon treatment with Boğazkere extract in the present work. The difference in the findings might be related to the difference of the plant species and chemistry of the extraction solvent. TABLE 3. Disc diffusion test results of positive control antibiotics BACTERIA E. aerogenes S. infantis L. monocytogenes K .pneumoniae P. fluorescens P. aeruginosa S. kentucky E. faecalis L. innocua S. enteritidis E. durans S. typhimurium E. faecium S. aereus S. epidermidis B. subtilis E. coli S. marcescens L2 OFX5 ME M10 27 28 24 37 19 25 30 30 23 25 9 19 14 32 33 20 21 17 26 32 32 17 27 32 32 25 28 35 30 32 16 28 40 36 38 38 TE30 18 9 17 18 20 15 10 22 20 21 15 25 15 33 18 CZ30 13 15 18 19 12 15 10 9 24 VA 30 21 22 20 21 24 - AM 10 10 20 22 30 25 30 28 23 25 40 46 - K 30 24 25 12 23 18 25 22 25 26 24 25 28 20 30 CN 10 25 20 20 24 20 15 14 15 25 21 20 27 25 24 30 25 27 S 10 S 300 23 10 11 20 25 13 16 11 20 26 35 20 15 22 22 20 17 18 24 20 35 21 23 25 25 NA 30 24 24 23 17 25 24 25 28 23 39 SH 100 30 12 12 18 17 20 19 21 24 24 33 21 30 30 30 33 SXT 25 28 25 25 26 28 26 25 26 23 28 30 35 16 30 N30 20 10 10 20 12 12 21 17 12 18 18 22 22 21 34 22 22 IP 5 32 30 25 35 33 24 32 23 22 30 21 35 30 37 35 43 AM C30 10 21 25 11 30 26 30 30 25 10 30 37 10 10 18 10 C30 30 30 27 30 23 30 25 24 28 30 33 25 33 33 25 32 ( - ) No effect, Lincomycin: L2, Ofloxacin: OFX 5, Meropenem: MEM 10, Tetracycline: TE 30, Ceftazidime: CAZ 30, Vancomycin: VA 30, Ampıcillin: AM10 Kanomycin: K 30, Gentamicin: CN 10, Streptomycin: S10, Compound Sulphonamides: S 3 300, Nalidixic acid: NA 30, Spectonomycine: SH 100 Sulphamethoxazole trimethaprim: SXT 25, Chloramphenicol: C 30, Neomycin: N 30, Cıprofloxacin: CIP 5, Amoxycillın clavulanic acıd: AMC30 Baydar et al. (2006) tested the extracts of seeds from Hasandede, Emir and Kalecik Karası grapes (extraction was performed in water:acetone:acetic acid solvent system, 90:9.5:0.5) on Aeromonas hydrophila ATCC 7965, Bacillus cereus FMC 19, Enterobacter aerogenes CCM 2531, Enterococcus faecalis ATCC 15753, Escherichia coli DM, E. coli O157:H7 KUEN 1461, Klebsiella pneumoniae FMC 5, Mycobacterium smegmatis RUT, Proteus vulgaris 220 INVESTIGATION OF THE ANTIMICROBIAL ACTIVITY OF Vitis vinifera L. BOĞ AZKERE FMC 1, Pseudomonas aeruginosa ATCC 27853, Pseudomonas fluorescens EU, Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus Cowan 1 and Yersinia enterocolitica EU using agar-disc diffusion methods for the concentrations of 1%, 2.5%, 5% and 10%. Among the tested grape types, Hasandede revealed suppression capacity on all the bacteria at the tested concentrations [4]. Hasandede extract at 10% concentration gave the highest antibacterial activity for Aeromonas hydrophila ATCC 7965 with 30.67 mm inhibition zone. Extracts from all the tested grape types at 0.5% and 1% concentrations gave bacteriostatic effect for Escherichia coli DM and E. coli O157:H7 KUEN 1461 strains while all the grape types showed bacteriostatic effect for S. aureus Cowan 1 strain. In our study, extracts of Boğazkere caused 9.33-11.66 mm inhibition zone formations for S.aureus while it was between 12.66 and 15.33 mm for E. faecalis. However, the extract did not show any inhibitory effect on E. aerogenes for 10 µL and 50 µL treatment while only for 100 µL treatment 7.33 mm zone inhibition was observed. Similar to the Baydar et al. study, our MIC findings were more of bacteriostatic. Baydar et al. (2004) performed ethyl acetate/methanol/water mediated extraction of grape seeds, whose lipid content was removed beforehand. The extracts were tested for Aeromonas hydrophila, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Enterobacter aerogenes, Enterococcus faecalis Escherichia coli, Klebsiella pneumoniae Listeria monocytogenes, Mycobacterium smegmatis, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus using disc diffusion method [9]. The findings revealed that the highest antibacterial activity of methanol: water: asetic acid mediated extracts was for L. monocytogenes with 33.5 mm zone inhibition diameter at 20 % concentration. Similarly for acetone: acetic acid: water mediated extract gave the highest antibacterial capability for L. monocytogenes strain at 4 % concentration. Ethyl acetate: methanol: water mediated extract did not show any activity towards E. aerogenes at 4% concentration. However, certain extracts showed antibacterial activity for such bacterial strains even at 4% concentrations. In our study, the Boğazkere extract caused 9.33-11.66 mm zone inhibition for S. aureus strain at 50 µL and 100 µL volume application while at 10 µL concentration no activity was observed. Similarly, the extract caused 12.66-15.33 mm zone inhibition for E. faecalis strain at 50 µL and 100 µL volume application while at 10 µL concentration no activity was obtained. However, the extract gave anti-E. aerogenes activity at 100 µL treatment with 7.33 mm zone diameter. Anastasiadi et al. (2009) analysed chemical contents belong to fruit, seed, stem and pomace of four Vitis vinifera types (i.e. Mandilaria, Voidomato, Asyrtiko and Aidani) and tested antiL. monocytogenes capability using MIC test approach. The findings revealed that extracts from plant stem and seeds were very effective, and they seem a possible preservative could be introduced to food samples [10]. In our study, the Boğazkere extract gave anti- L. SEDA ÇİÇEK, TALİP ÇETER 221 monocytogenes activity at 50 µL and 100 µL volume treatment with causing 10.00 and 14.00 mm inhibition zone formation while at 10 µL no activity was observed. Ege (2015) carried out extraction from seeds of Müşküle (white), Kara dimrit (blue-black) and Öküzgözü grape types, whose lipid content was removed beforehand. The extractions, performed in dedicated solvents systems of acetone, water, acetic acid and methanol. The extract stocks were prepared at 65.536 mg/mL concentration. Antimicrobial activity of the extracts was tested Alternaria alternata, Aspergillus niger, Botrytis cinerea, Penicillium expansum, Escherichia coli 35218, Pseudomonas aeruginosa 27853, Klebsiella pneumonia 700603, Enterococcus faecalis 51299, Streptococccus pneumonia 49616 and Staphylococcus aureus 44300 using MIC test. The findings revealed that all the extracts did not show antimicrobial capability against Alternaria alternata, Aspergillus niger, Botrytis cinerea, Penicillium expansum, Escherichia coli 35218, Pseudomonas aeruginosa 27853, Klebsiella pneumoniae 700603 and Enterococcus faecalis 51299. In contrasto to this, Müşküle exract at 32.768 mg/mL and Öküzgözü at 65.536 mg/mL concentraions showed anti S. aureus activity. Similarly, Kara dimrit, Müşküle and Öküzgözü posssed anti- S. pneumonia activity at 2.048 mg/mL, 4.096 mg/mL and 32.768 mg/mL concentrations [11]. In our study, the Boğazkere extract did not show antimicrobial activity against K. pneumonia, E. aerogenes, Candida ablicans, L. monocytogenes, P. fluorescens ve S. marcescens. However, the extract at the range of 0.039 and 20 mg/100 µL gave antibacterial activity against S. infentis, P. aeruginosa, S. Kentucky, E. faecalis, E. coli, L. innocua, S. enteritidis, S. typhimurium, E. faecium, S. aeureus, S. epidermidis and B. subtilis strains. The difference between our study and the mentioned literature could be related to the different extraction solvent usage. Abtahi et al. (2011) extracted dried white, red and black grape samples in 70% alcohol, which were then tested on E. coli PTCC1330, S. aureus PTCC 1431, S. typhimurium PTCC1639 and P. aeruginosa PTCC1310 strains using MIC tests. The findings showed that the extracts gave positive results for all the tested bacteria with showing the highest activity for S. aureus strain [12]. The obtained MIC values for E. coli, S. aureus, S. typhimurium and P. aeruginosa strains were 125, 32, 125 and 250 µg/mL, respectively. In our study, the Boğazkere extract gave 10 mg/100 µL and 0,625 µg/mL MIC values for E. coli and S. aureus, respectively. In contrast to this, it did not give any effect on S. typhimurium. In the present work, Boğazkere extract showed antimicrobial effect against other microorganisms with MIC values range between 0.039 and 20 mg/µL. As it was seen from the results, different grape types have different effect on the same microorganisms. Waqar et al. (2014) tested the antimicrobial activity of the extracts from leaves of V. vinifera on E. coli, P. aeruginosa, S. aureus and E. faecalis strains using disc diffusion method. In the study, 5 mg of leaf extract was dissolved in 70% ethanol, followed by 3 discs were treated with 3 mg/0.1 mL of the dissolved extract. The inhibition zones belong to S. aureus as 30 mm, E. faecalis as 28.9 mm, E. coli as 28 mm and P. aeruginosa as 23.7 mm were obtained [13]. In our study, the Boğazkere extract gave antibacterial capability against 222 INVESTIGATION OF THE ANTIMICROBIAL ACTIVITY OF Vitis vinifera L. BOĞ AZKERE S. aureus, E. faecalis, E. coli and P. aeruginosa with 9.33-11.66 mm, 12.66-15.33 mm, 8.669.66 mm and 7.33-8.66 mm zone inhibitions, respectively. However, the extract did not show any effect on these bacteria at 10 µL concentration. The obtained difference between the two studies came from utilization of different grapes and different parts of the plant. The findings of this study along with the literature provide strong insight into that leaves, fruits and seeds of Vitis vinifera L. can provide antibacterial activity depending on the extraction method. Even at low concentrations of Vitis vinifera L. Boğazkere extracts can pose antimicrobial effect for the tested bacterial strains. Our findings are endorsing the literature revealing that the plant extracts can be alternative and viable tools to fight against microbial development, which has been under investigation for a long time. REFERENCES [1] [2] [3] [4] [5] [6] Aslankoç R, Demirci D, İnan Ü, Yıldız M, Öztürk A, Çetin M, Savran EŞ and Yılmaz B (2019). The Role Of Antioxidant Enzymes İn Oxidative Stress - Superoxide Dismutase (Sod), Catalase (Cat) And Glutathione Peroxidase (Gpx). Süleyman Demirel Üniversitesi Tıp Fakültesi Dergisi 26(3): 362-369. Cheeseman KH and Slater TF (1993). An introduction to free radical biochemistry. British Medical Bulletin 49: 481-493. Kohanski MA, Dwyer DJ and Collins JJ (2010). How antibiotics kill bacteria: from targets to networks. Nature Reviews Microbiology 8(6): 423–435. Baydar NG, Sağdıç O, Özkan G and Çetin ES (2006). Determination of antibacterial effects and total phenolic contents of grape (Vitis vinifera L.) seed extracts. International Journal of Food Science and Technology 41: 799-804. GTB (2015). 2014 Yılı Çekirdeksiz Kuru Üzüm Raporu, T.C. Gümrük ve Ticaret Bakanlığı Kooperatifçilik Genel Müdürlüğü, Şubat 2015. http://koop.gtb.gov.tr/data/53319ea4487c8eb1e43d72a0/2014%20Kuru%20%C3%9 Cz%C3%BCm%20Raporu.pdf/. Access date: 03 .11.2015 Ateş F and Karabat S. (2016). Manisa Bağcılık Araştırma Enstitüsü Müdürlüğü, Sofralık Üzüm Yetiştirmeye Yönelik Kültürel Uygulamalar. [online]http://arastirma.tarim.gov.tr/manisabagcilik/Belgeler/genelbagcilik/Kalıtelı% 20sofralık%20uzum%20yetıstırıcılıgı%20fadıme%20ates.pdf/. Access date: 11.10. 2016 SEDA ÇİÇEK, TALİP ÇETER [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 223 Monagas M, Bartolome B and Gomez-Cordoves C (2005). Updated knowledge about the presence of phenolic compounds in wine. Critical Reviews in Food Science and Nutrition 45(2): 85-118. Jayaprakasha G, Selvi T and Sakariah K (2003). Antibacterial and antioxidant activities of grape Vitis vinifera seed extracts. Food Research International 36(2): 117-122. Baydar NG, Özkan G and Sağdiç O (2004). Total phenolic contents and antibacterial activities of grape (Vitis vinifera L.) extracts. Food Control 15(5): 335-339 Anastasiadi M, Chorianopoulos NG, Nychas GJE and Haroutounian S (2009). Antilisterial activities of polyphenol-rich extracts of grapes and vinification by products. Journal of Agricultural and Food Chemistry 57(2): 457-463. Ege D (2015). Bazı Zararlı Mikroorganizmaların Kültür Ortamindaki Gelişimine Üzüm (Vitis Vinifera L.) Çekirdeği Yağinin Etkileri. Yüksek lisans tezi, Selçuk Üniversitesi Fen Bilimleri Enstitüsü, Konya. Abtahi H, Ghazavi A and Karimi M (2011). Antimicrobial activities of ethanol extract of black grape. African Journal of Microbiology Research 5(25): 4446-4448 Waqar A, Han Aİ, Waqar M, Han MA, Han A, Ramazan R, Wali S, Ahmad F, Han N, Yousaf S, Zeb M, Han EU, Rahman MU and Faysal Ş (2014). In vitro Antibacterial Activity of Vitis vinifera Leaf Extracts Against Some Pathogenic Bacterial Strains. Advances in Biological Research 8 (2): 62-67. Yadav D, Kumar A, Kumar P and Mishra D (2015). Antimicrobial properties of black grape (Vitis vinifera L.) peel extracts against antibiotic-resistant pathogenic bakteri and toxin producing molds. Indian Journal of Pharmacology 47(6): 663-667. Altuner EM, Çeter T and Işlek C (2010). Cilt enfeksiyonlarının geleneksel tedavisinde kullanılan Ononis spinosa L. külünün antifungal etkisinin araştırılması. Mikrobiyoloji Bülteni 44(4): 633-639 Altuner EM, Çeter T, Bayar E, Aydın S, Arıcı F, Süleymanoğlu G, Edis A (2011). Investigation on Antimicrobial Effects of Some Moss Species Collected From Kastamonu Region, Commun. Fac. Sci. Univ. Ank. Series C 23(1-2): 33-43 Altuner EM, Çeter T, Demirkapı D, Özkay K, Hayal U, Eser G (2011) Investigation on Antimicrobial Effects of Some Lichen Species Collected From Kastamonu Region, Commun. Fac. Sci. Univ. Ank. Series C 23(1-2): 21-31 Bauer AW, Kirby WMM, Sherris JC and Turck M (1966). Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clininical Pathology 36: 493-496. 224 INVESTIGATION OF THE ANTIMICROBIAL ACTIVITY OF Vitis vinifera L. BOĞ AZKERE Bader BMA, Çeter T and Bani B (2018). Investigation of Antimicrobial Activity of Gagea dubia. Commun. Fac. Sci. Univ. Ank. Series C; Biology 27(2): 224-231 [20] Altuner EM, Çeter T, Gür M, Güney K, Kıran B, Akwieten HE, Soulman SI (2018). Chemical Composition and Antimicrobial Activities of Cold-Pressed Oils Obtained From Nettle, Radish and Pomegranate Seeds. Kastamonu Üniversitesi Orman Fakültesi Dergisi 18(3): 236 - 247 [19] Current Address: SEDA ÇİÇEK: Kastamonu University, Arts and Sciences Faculty, Department of Biology, Kastamonu, Turkey E-mail: seda_biolog@hotmail.com https://orcid.org/0000-0001-8965-119X Current Address: TALİP ÇETER: Kastamonu University, Arts and Sciences Faculty, Department of Biology, Kastamonu, Turkey E-mail: talipceter@gmail.com https://orcid.org/ 0000-0003-3626-1758