臺灣博碩士論文加值系統

本研究分為兩個部分，實驗一以新鮮草藥：黑小茴香籽 (Nigella sativa) 使用乙醇及蒸餾水萃取後，以紙錠擴散法 (Paper disk diffusion method)，針對五種常見魚類致病病菌進行抑菌環實驗：親水性產氣單胞菌 (Aeromonas hydrophila)、愛德華氏菌 (Edwardsiella tarda)、創傷弧菌 (Vibrio vulnificus)、溶藻弧菌 (Vibrio alginolyticus) 和腸炎弧菌 (Vibrio parahaemolyticus)。結果顯示僅有黑小茴香籽萃取物產生抑菌環，其餘三種草藥之萃取物均無抑菌效果。黑小茴香籽乙醇萃取物有較好的表現，乙醇萃取物的抑制圈顯示創傷弧菌的抑菌能力最好 (14.6±0.5 mm)，其次是溶藻弧菌 (10.0±0.0 mm)、腸炎弧菌 (8.3±0.5 mm)和愛德華氏菌 (8.3±0.5 mm)，對親水性產氣單胞菌效果最差 (8±0.0 mm)；黑小茴香蒸餾水萃取物的抑菌力較弱，僅創傷弧菌出現抑菌環 (9±1.0 mm)。
實驗二以肉汁培養基巨量稀釋法 (Broth macro-dilution assay)，比較黑小茴香籽乙醇萃取物、蒸餾水萃取物和現有之六種動物用藥抗生素：安默西林 (Amoxicillin)、紅黴素 (Erythromycin)、氟甲磺氯黴素 (Florfenicol)、氟滅菌 (Flumequine)、歐索林酸 (Oxolinic acid)和土黴素 (Oxytetracycline) 對同前五種致病菌的最低抑菌濃度 (Minimum inhibitory concentration, MIC) 以及最低殺菌濃度 (Minimum bactericidal concentration, MBC)。每組皆分別以低菌量 (5x102 CFU/ml)和高菌量 (5x105 CFU/ml) 進行實驗。黑小茴香籽乙醇萃取物之MIC和MBC皆在480 - 7,800 mg/L，黑小茴香籽蒸餾水萃取物的MIC和MBC分別為15,620 - 125,000 mg/L和15,620 - 250,000 mg/L。黑小茴香籽萃取物對創傷弧菌之抑菌效果相較其他四種致病菌更佳。六種動物用藥對所有致病菌皆有效：安默西林的MIC和MBC在高菌量和低菌量組皆為1-1,024 mg/L、紅黴素的MIC和MBC在高菌量和低菌量組分別為1-64 mg/L和8-512 mg/L、氟甲磺氯黴素的MIC和MBC在高菌量和低菌量組分別為0.5-4 mg/L和1-512 mg/L、氟滅菌的MIC和MBC在高菌量和低菌量組分別為0.0625-1 mg/L和0.0625-64 mg/L、歐索林酸的MIC和MBC在高菌量和低菌量組分別為0.25-8 mg/L 和0.5 – 1,024mg/L、土黴素的MIC和MBC在高菌落和低菌落組別分別為0.125-256 mg/L和0.25-512 mg/L。實驗結果顯示，黑小茴香籽的乙醇萃取物與水萃取物具備抑菌效果，但與現有動物用藥相比，效果不佳。

This study was divided into two parts, black cumin seed (Nigella sativa) extracted with ethanol and distilled water, subsequently the disc diffusion assay performed to inhibit the bacterial pathogens growth (Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio vulnificus). The inhibition zone of ethanol extract of black cumin seed showed that V. vulnificus (14.6 ± 0.5 mm) was sensitive, followed by V. alginolyticus (10.0 ± 0.0 mm), V. parahaemolyticus (8.3 ± 0.5 mm) and E. tarda (8.3 ± 0.5 mm) while A. hydrophila (8 ± 0.0 mm) resistant to this extract. The distilled water extract of black cumin seed showed weak activity against the tested bacteria, only V. vulnificus was inhibited about 9 ± 1.0 mm.
In the second experiment, broth macro-dilution assay was used to observe the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of black cumin seed extracts and animal drugs (amoxicillin, erythromycin, florfenicol, flumequine, oxolinic acid and oxytetracycline) against fish bacterial pathogens. The MIC and MBC were done with two levels of pathogens population, low (5x102 CFU/ml) and high (5x105 CFU/ml). The values of MIC and MBC for ethanol extract of black cumin seed at both levels pathogens population for all pathogens were varied from 480 to 7,800 mg/L whereas for distilled water extract of black cumin seed ranged about 15,620-125,000 mg/L and 15,620- 250,000 mg/L, respectively. The inhibitory effect of black cumin seed extracts against V. vulnificus was better compared to the other pathogens. Six kinds of animal drugs are effective against all pathogens. Both MIC and MBC of amoxicillin were distributed from 1 to 1,024 mg/L at both levels pathogens population. The MIC and MBC ranges for erythromycin, florfenicol, flumequine, oxolinic acid and oxytetracycline at high- and low-level pathogens population were 1 to 64 mg/L and 8 to 512 mg/L, 0.5-4 mg/L and 1-512 mg/L, 0.0625 to 1 mg/L and 0.0625-64 mg/L, 0.25 to 8 mg/L and 0.5-1,024 mg/L and 0.125 to 256 mg/L and 0.25 to 512 mg/L, respectively. Experimental results show that the ethanol and distilled water extract of black cumin seed have inhibitory effects against all pathogens, but ineffective compared with the existing animal drugs.

Acknowledgement i
Chinese Abstract ii
Abstract iv
Table of Contents v
List of Tables vii
List of Appendix viii
Chapter
I. Introduction 1
1.1. Background 1
1.2. Statement of Problem 2
1.3. Objective 3
II. Literature Review 4
2.1. Plant Extracts as Antibacterial 4
2.1.1. Black cumin seed (Nigella sativa) 4
2.1.2. Betel Leaves (Piper betle) 5
2.1.3. Papaya Leaves (Carica papaya) 6
2.1.4. Sousop Leaves (Annona muricata) 7
2.2. Aquatic Pathogens in Aquaculture 7
2.2.1. Aeromonas hydrophila 8
2.2.2. Edwardsiella tarda 8
2.2.3. Vibrio alginolyticus 9
2.2.4. Vibrio parahaemolyticus 9
2.2.5. Vibrio vulnificus 9
2.3. Animal Drugs 10
2.3.1. Amoxicillin 11
2.3.2. Erythromycin 11
2.3.3. Florfenicol 11
2.3.4. Flumequine 12
2.3.5. Oxolinic acid 12
2.3.6. Oxytetrecyline 12
2.4. Inhibitory Methods 13
2.4.1. Disc Diffusion Methods 13
2.4.2. Broth Macro-dilution 14
III. Materials and Methods 15
3.1. Preparation of Plant Extract 15
3.2. Test Organisms 15
3.3. Antibacterial Assay 16
3.3.1. Preparation of Animal Drugs 16
3.3.2. Standardization of Bacterial Inoculum 16
3.3.3. Disc Diffusion Assay 16
3.3.4. Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC) Assay 17
IV. Results 18
4.1. Disc Diffusion Assay 18
4.2. Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC) Assay 18
V. Discussion 22
Conclusion 28
References 29

Ahmad, I., & Beg, A. Z. (2001). Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multidrug resistant human pathogens. Journal of Ethnopharmacology, 74(2), 113-123.

Ahmed, M. A. E. A., & Kamel, G. M. (2005). Efficacy and tissue residue of flumequine in treatment of Clarias gariepinus experimentally infected with Aeromonas hydrophila. Egyptian Journal of Experimental Biology (Zoology), 1, 15 – 22.

Ahmed, A., Howlader, M. S., Dey, S. K., Hira, A., Hossain, M. H., & Uddin, M. M. N. (2013). Phytochemical screening and antibacterial activity of different fractions of Operculina turpethum root and leaf. American Journal of Scientific and Industrial Research, 4(2), 167-172.

Akinbowale, O. L., Peng, H., & Barton, M. D. (2006). Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia. Journal of Applied Microbiology, 100(5), 1103-1113.

Alabi, O. A., Haruna, M. T., Anokwuru, C. P., Jegede, T., Abia, H., Okegbe, V. U., & Esan, B. E. (2012). Comparative studies on antimicrobial properties of extracts of fresh and dried leaves of Carica papaya (L) on clinical bacterial and fungal isolates. Applied Scientific Research, 3(5).

Alderman, D. J., & Hastings, T. S. (1998). Antibiotic use in aquaculture: development of antibiotic resistance–potential for consumer health risks. International Journal of Food Science and Technology, 33(2), 139-155.

Ali, B. H., & Blunden, G. (2003). Pharmacological and toxicological properties of Nigella sativa. Phytotherapy Research, 17, 299–305.

Ali, I., Khan, F. G., Suri, K. A., Gupta, B. D., Satti, N. K., Dutt, P., Afrin, F., Qazi, G.N. & Khan, I. A. (2010). In vitro antifungal activity of hydroxychavicol isolated from Piper betle L. Annals of Clinical Microbiology and Antimicrobial, 9(7), 1-9.

Ali, M. H., Chowdhury, F. S., Ashrafuzzaman, M., & Al Nayem, M. (2014). Identification, pathogenecity, antibiotic and herbal sensitivity of Edwardsiella tarda causing fish disease in Bangladesh. Current Research in Microbiology and Biotechnology, 2(1), 292-297.

Al-Naggar, T. B., Gómez-Serranillos, M. P., Carretero, M. E., & Villar, A. M. (2003). Neuropharmacological activity of Nigella sativa L. extracts. Journal of Ethnopharmacology, 88(1), 63-68.
Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy, 48(supplement 1), 5-16.

Arambewela, L. S. R., Arawwawala, L. D. A. M., & Ratnasooriya, W. D. (2005). Antidiabetic activities of aqueous and ethanolic extracts of Piper betle leaves in rats. Journal of Ethnopharmacology, 102(2), 239-245.

Arici, M., Sagdic, O., &Gecgel, U. (2005). Antibacterial effect of Turkish black cumin (Nigella sativa L.) oils. Grasas y Aceites, 56(4), 259-262.

Ayandiran, T. A., Ayandele, A. A., Dahunsi, S. O., & Ajala, O. O. (2014). Microbial assessment and prevalence of antibiotic resistance in polluted Oluwa River, Nigeria. The Egyptian Journal of Aquatic Research, 40(3), 291-299.

Banala, R. R., Nagati, V. B., & Karnati, P. R. (2015). Green synthesis and characterization of Carica papaya leaf extract coated silver nanoparticles through X-ray diffraction, electron microscopy and evaluation of bactericidal properties. Saudi Journal of Biological Sciences, 22(5), 637-644.

Barnes, A. C., Lewin, C. S., Hastings, T. S., & Amyes, S. G. (1991). In vitro susceptibility of the fish pathogen Aeromonas salmonicida to flumequine. Antimicrobial Agents and Chemotherapy, 35(12), 2634-2635.

Basile, A., Senatore, F., Gargano, R., Sorbo, S., Del Pezzo, M., Lavitola, A., Ritieni, A., Bruno, M., Spatuzzi, D., Rigano, D. & Vuotto, M. L. (2006). Antibacterial and antioxidant activities in Sideritis italica (Miller) Greuter et Burdet essential oils. Journal of Ethnopharmacology, 107(2), 240-248.

Bray, W. A., Williams, R. R., Lightner, D. V., & Lawrence, A. L. (2006). Growth, survival and histological responses of the marine shrimp, Litopenaeus vannamei, to three dosage levels of oxytetracycline. Aquaculture, 258(1), 97-108.

Bruun, M. S., Madsen, L., & Dalsgaard, I. (2003). Efficiency of oxytetracycline treatment in rainbow trout experimentally infected with Flavobacterium psychrophilum strains having different in vitro antibiotic susceptibilities. Aquaculture, 215(1), 11-20.

Buller, N. B. (2014). Bacteria from fish and other aquatic animals: a practical identification manual. Cabi.

Calvo, M. A., Arosemena, E. L., Shiva, C., & Adelantado, C. (2012). Antimicrobial activity of plant natural extracts and essential oils. Science against Microbial Pathogens: Communicating Current Research and Technological Advances, Mendez-Vilas, A.(Ed.). Formatex Research Center, Spain, 1179-1185.

Cerf, O., Carpentier, B., & Sanders, P. (2010). Tests for determining in-use concentrations of antibiotics and disinfectants are based on entirely different concepts:“resistance” has different meanings. International Journal of Food Microbiology, 136(3), 247-254.

Chaieb, K., Kouidhi, B., Jrah, H., Mahdouani, K., & Bakhrouf, A. (2011). Antibacterial activity of Thymoquinone, an active principle of Nigella sativa and its potency to prevent bacterial biofilm formation. BMC Complementary and Alternative Medicine, 11(1), 29.

Cheikh-Rouhou, S., Besbes, S., Hentati, B., Blecker, C., Deroanne, C., & Attia, H. (2007). Nigella sativa L.: Chemical composition and physicochemical characteristics of lipid fraction. Food Chemistry, 101(2), 673-681.

Chen, H., Liu, S., Xu, X. R., Liu, S. S., Zhou, G. J., Sun, K. F., ...& Ying, G. G. (2015). Antibiotics in typical marine aquaculture farms surrounding Hailing Island, South China: Occurrence, bioaccumulation and human dietary exposure. Marine Pollution Bulletin, 90(1), 181-187.

Chitmanat, C., Tongdonmuan, K., Khanom, P., Pachontis, P., & Nunsong, W. (2003, February). Antiparasitic, antibacterial, and antifungal activities derived from a Terminalia catappa solution against some tilapia (Oreochromis niloticus) pathogens. In III WOCMAP Congress on Medicinal and Aromatic Plants-Volume 4: Targeted Screening of Medicinal and Aromatic Plants, Economics 678 (pp. 179-182).

Christofilogiannis, P. (2001). Current inoculation methods in MIC determination. Aquaculture, 196(3), 297-302.

Citarasu, T. (2010). Herbal biomedicines: a new opportunity for aquaculture industry. Aquaculture International, 18(3), 403-414.

Conti, G. O., Copat, C., Wang, Z., D'Agati, P., Cristaldi, A., & Ferrante, M. (2015). Determination of illegal antimicrobials in aquaculture feed and fish: An ELISA study. Food Control, 50, 937-941.

CLSI. (2012). Methods for Dilution Antimicrobial Susceptibility Test for Bacteria That Grow Aerobically; Approved Standard-Ninth Edition. CLSI document M07-A9. Wayne, PA: Clinical and Laboratory Standards Institute.

Daniel, P. (2009). Drugs and chemicals in aquafeeds: The problems and solutions. Options Méditerranéennes, 86, 85-94.

Declercq, A. M., Boyen, F., den Broeck, W., Bossier, P., Karsi, A., Haesebrouck, F., & Decostere, A. (2013). Antimicrobial susceptibility pattern of Flavobacterium columnare isolates collected worldwide from 17 fish species. Journal of Fish Diseases, 36(1), 45-55.

Della Rocca, G., Zaghini, A., Zanoni, R., Sanguinetti, V., Zanchetta, S., Di Salvo, A., & Malvisi, J. (2004). Seabream (Sparus aurata L.): disposition of amoxicillin after single intravenous or oral administration and multiple dose depletion studies. Aquaculture, 232(1), 1-10.

de-la-Re-Vega, E., García-Galaz, A., Díaz-Cinco, M. E., & Sotelo-Mundo, R. R. (2006). White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae. Fish and Shellfish Immunology, 20(3), 405-408.

Direkbusarakom, S. (2004). Application of medicinal herbs to aquaculture in Asia. Walailak Journal of Science and Technology, 1(1), 7-14.

El-Nagerabi, S. A., Al-Bahry, S. N., Elshafie, A. E., & AlHilali, S. (2012). Effect of Hibiscus sabdariffa extract and Nigella sativa oil on the growth and aflatoxin B 1 production of Aspergillus flavus and Aspergillus parasiticus strains. Food Control, 25(1), 59-63.

Essawi, T., & Srour, M. (2000).Screening of some Palestinian medicinal plants for antibacterial activity. Journal of Ethnopharmacology, 70(3), 343-349.

Fagutao, F. F., Yasuike, M., Santos, M. D., Ruangpan, L., Sangrunggruang, K., Tassanakajon, A., ...& Aoki, T. (2009). Differential gene expression in black tiger shrimp, Penaeus monodon, following administration of oxytetracycline and oxolinic acid. Developmental & Comparative Immunology, 33(10), 1088-1092.

Ferreira, L. E., Castro, P. M. N., Chagas, A. C. S., França, S. C., & Beleboni, R. O. (2013). In vitro anthelmintic activity of aqueous leaf extract of Annona muricata L. (Annonaceae) against Haemonchus contortus from sheep. Experimental Parasitology, 134(3), 327-332.

Florence, N. T., Benoit, M. Z., Jonas, K., Alexandra, T., Désiré, D. D. P., Pierre, K., & Théophile, D. (2014). Antidiabetic and antioxidant effects of Annona muricata (Annonaceae), aqueous extract on streptozotocin-induced diabetic rats. Journal of Ethnopharmacology, 151(2), 784-790.

Forouzanfar, F., Bazzaz, B. S. F., & Hosseinzadeh, H. (2014). Black cumin (Nigella sativa) and its constituent (thymoquinone): a review on antimicrobial effects. Iranian Journal of Basic Medical Sciences, 17(12), 929.

Galina, J., Yin, G., Ardo, L., & Jeney, Z. (2009). The use of immunostimulating herbs in fish.An overview of research. Fish Physiology and Biochemistry, 35(4), 669-676.

Gavamukulya, Y., Abou-Elella, F., Wamunyokoli, F., & AEl-Shemy, H. (2014). Phytochemical screening, anti-oxidant activity and in vitro anticancer potential of ethanolic and water leaves extracts of Annona muricata (Graviola). Asian Pacific Journal of Tropical Medicine, 7, S355-S363.

Gomes, J., Vilela, C. L., Bexiga, R., Nunes, G. D., Pereira, N., & Cavaco, L. M. (2007). Fish antibiotherapy: bioencapsulation of flumequine using adult brine shrimp (Artemia salina). Aquaculture Research, 38(6), 613-617.

Guardiola, F. A., Cerezuela, R., Meseguer, J., & Esteban, M. A. (2012). Modulation of the immune parameters and expression of genes of gilthead seabream (Sparus aurata L.) by dietary administration of oxytetracycline. Aquaculture, 334, 51-57.

Guz, L. E. S. Z. E. K., & Kozinska, A. (2004). Antibiotic susceptibility of Aeromonas hydrophila and A. sobria isolated from farmed carp (Cyprinus carpio L.). Bull Vet Inst Pulawy, 48, 391-395.

Hajhashemi, V., Ghannadi, A., & Jafarabadi, H. (2004). Black cumin seed essential oil, as a potent analgesic and antiinflammatory drug. Phytotherapy Research, 18(3), 195-199.

Halawani, E. (2009). Antibacterial activity of thymoquinone and thymohydroquinone of Nigella sativa L. and their interaction with some antibiotics. Advances in Biological Research, 3(5-6), 148-52.

Hamizah, S., Roslida, A. H., Fezah, O., Tan, K. L., Tor, Y. S., & Tan, C. I. (2012). Chemopreventive potential of Annona muricata L leaves on chemically-induced skin papillomagenesis in mice. Asian Pacific Journal of Cancer Prevention, 13(6), 2533-2539.

Hao, Y. Y., Brackett, R. E., & Doyle, M. P. (1998). Efficacy of plant extracts in inhibiting Aeromonas hydrophila and Listeria monocytogenes in refrigerated, cooked poultry. Food Microbiology, 15(4), 367-378.

Harzallah, H. J., Kouidhi, B., Flamini, G., Bakhrouf, A., & Mahjoub, T. (2011). Chemical composition, antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone. Food Chemistry, 129(4), 1469-1474.

Hatha, M., Vivekanandhan, A. A., & Joice, G. J. (2005). Antibiotic resistance pattern of motile aeromonads from farm raised fresh water fish. International Journal of Food Microbiology, 98(2), 131-134.

Ho, S. P., Hsu, T. Y., Chen, M. H., & WANG, W. S. (2000). Antibacterial effect of chloramphenicol, thiamphenicol and florfenicol against aquatic animal bacteria. Journal of Veterinary Medical Science, 62(5), 479-485.

Hosseinzadeh, H., Tafaghodi, M., Mosavi, M. J., & Taghiabadi, E. (2013). Effect of aqueous and ethanolic extracts of Nigella sativa seeds on milk production in rats. Journal of Acupuncture and Meridian Studies, 6(1), 18-23.

Hung, S. W., Kong, P. H., Shih, C. W., Tu, C. Y., Ho, S. P., & Wang, W. S. (2007). The antibiotic susceptibility and expression of amoxicillin-resistant genes in Vibrio strains isolated from hard clams, Meretrix in Taiwan. 臺灣獸醫學雜誌, 33(1), 20-29.
Inglis, V., & Richards, R. H. (1991). The in vitro susceptibility of Aeromonas salmonicida and other fish‐pathogenic bacteria to 29 antimicrobial agents. Journal of Fish Diseases, 14(6), 641-650.

Ishiwu, C. N., Umenwanne, C. P., Obiegbuna, J. E., & Uchegbu, N. N. (2014). In vitro Assessment of Antibacterial Effect of Extracts of Ocimum Gratissimum and Carica Papaya Leaves. International Journal of Applied, 4(1).

Julianti, T., De Mieri, M., Zimmermann, S., Ebrahimi, S. N., Kaiser, M., Neuburger, M., Raith, M., Brun, R.& Hamburger, M. (2014). HPLC-based activity profiling for antiplasmodial compounds in the traditional Indonesian medicinal plant Carica papaya L. Journal of Ethnopharmacology, 155(1), 426-434.

Kamruzzaman, M., Bari, S. N., & Faruque, S. M. (2013). In vitro and in vivo bactericidal activity of Vitex negundo leaf extract against diverse multidrug resistant enteric bacterial pathogens. Asian Pacific Journal of Tropical Medicine, 6(5), 352-359.

Keyes, K., Hudson, C., Maurer, J. J., Thayer, S., White, D. G., & Lee, M. D. (2000). Detection of Florfenicol Resistance Genes inEscherichia coli Isolated from Sick Chickens. Antimicrobial Agents and Chemotherapy, 44(2), 421-424.

Kim, J., Marshall, M. R., & Wei, C. I. (1995). Antibacterial activity of some essential oil components against five foodborne pathogens. Journal of Agricultural and Food Chemistry, 43(11), 2839-2845.

Kim, C. M., Park, R. Y., Choi, M. H., Sun, H. Y., & Shin, S. H. (2007). Ferrophilic characteristics of Vibrio vulnificus and potential usefulness of iron chelation therapy. Journal of Infectious Diseases, 195(1), 90-98.

Kim, J. S., Lee, G. G., Kim, J., Kwon, J. Y., & Kwon, S. T. (2008). The development of rapid real‐time PCR detection system for Vibrio parahaemolyticus in raw oyster. Letters in Applied Microbiology, 46(6), 649-654.

Labella, A., Gennari, M., Ghidini, V., Trento, I., Manfrin, A., Borrego, J. J., & Lleo, M. M. (2013). High incidence of antibiotic multi-resistant bacteria in coastal areas dedicated to fish farming. Marine Pollution Bulletin, 70(1), 197-203.

Lancini, G., Parenti, F., & Gallo, G. G. (1995). Antibiotics: a multidisciplinary approach. Plenum Press.

Maisarah, A. M., Amira, B. N., Asmah, R., & Fauziah, O. (2013). Antioxidant analysis of different parts of Carica papaya. International Food Research Journal, 20(3), 1043-1048.

Mascaretti, O. A. (2003). Bacteria versus Antibacterial Agents. American Society of Microbiology.

Matasyoh, J. C., Maiyo, Z. C., Ngure, R. M., & Chepkorir, R. (2009). Chemical composition and antimicrobial activity of the essential oil of Coriandrum sativum. Food Chemistry, 113(2), 526-529.

Mazzola, P. G., Jozala, A. F., Novaes, L. C. D. L., Moriel, P., & Penna, T. C. V. (2009). Minimal inhibitory concentration (MIC) determination of disinfectant and/or sterilizing agents. Brazilian Journal of Pharmaceutical Sciences, 45(2), 241-248.

Michel, C., Kerouault, B., & Martin, C. (2003). Chloramphenicol and florfenicol susceptibility of fish‐pathogenic bacteria isolated in France: comparison of minimum inhibitory concentration, using recommended provisory standards for fish bacteria. Journal of Applied Microbiology, 95(5), 1008-1015.

Miranda, C. D., & Rojas, R. (2007). Occurrence of florfenicol resistance in bacteria associated with two Chilean salmon farms with different history of antibacterial usage. Aquaculture, 266(1), 39-46.

Moghadamtousi, S. Z., Karimian, H., Rouhollahi, E., Paydar, M., Fadaeinasab, M., & Kadir, H. A. (2014). Annona muricata leaves induce G 1 cell cycle arrest and apoptosis through mitochondria-mediated pathway in Human HCT-116 and HT-29 colon Cancer cells. Journal of Ethnopharmacology, 156, 277-289.

Mojica, E. R. E., & Aga, D. S. (2011). Antibiotics Pollution in Soil and Water: Potential Ecological and Human Health Issues. Encyclopedia of Environmental Health. Elsevier, Burlington, 97-110.

Mohanty, B. R., & Sahoo, P. K. (2007). Edwardsiellosis in fish: a brief review. Journal of Biosciences, 32(3), 1331-1344.

Muroga, K. (2001). Viral and bacterial diseases of marine fish and shellfish in Japanese hatcheries. Aquaculture, 202(1), 23-44.

Nair, M. K. M., Vasudevan, P., & Venkitanarayanan, K. (2005). Antibacterial effect of black seed oil on Listeria monocytogenes. Food Control, 16(5), 395-398.

Novotny, L., Dvorska, L., Lorencova, A., Beran, V., & Pavlik, I. (2004). Fish: a potential source of bacterial pathogens for human beings. A review. Veterinarni Medicina-UZPI (Czech Republic).

O'grady, P., Moloney, M., & Smith, P. R. (1988). Bath administration of the quinoline antibiotic flumequine to brown trout Salmo trutta and Atlantic salmon S. salar. Diseases of Aquatic Organisms, 4(1), 27-33.

Pianotti, R. S., Mohan, R. R., & Schwartz, B. S. (1968). Biochemical effects of oxolinic acid on Proteus vulgaris. Journal of Bacteriology, 95(5), 1622-1626.

Pridgeon, J. W., & Klesius, P. H. (2013). Major bacterial diseases in aquaculture and their vaccine development. Animal Science Reviews.

Punitha, S. M. J., Babu, M. M., Sivaram, V., Shankar, V. S., Dhas, S. A., Mahesh, T. C., Immanuel, G.& Citarasu, T. (2008). Immunostimulating influence of herbal biomedicines on nonspecific immunity in Grouper Epinephelus tauvina juvenile against Vibrio harveyi infection. Aquaculture International, 16(6), 511-523.

Rahman, T., Akanda, M. M. R., Rahman, M. M., & Chowdhury, M. B. R. (2009). Evaluation of the efficacies of selected antibiotics and medicinal plants on common bacterial fish pathogens. Journal of the Bangladesh Agricultural University, 7(1), 163-168.

Rakholiya, K., & Chanda, S. (2012). In vitro interaction of certain antimicrobial agents in combination with plant extracts against some pathogenic bacterial strains. Asian Pacific Journal of Tropical Biomedicine, 2(2), S876-S880.

Rattanachaikunsopon, P., & Phumkhachorn, P. (2010). Potential of cinnamon (Cinnamomum verum) oil to control Streptococcus iniae infection in tilapia (Oreochromis niloticus). Fisheries Science, 76(2), 287-293.

Reverter, M., Bontemps, N., Lecchini, D., Banaigs, B., & Sasal, P. (2014). Use of plant extracts in fish aquaculture as an alternative to chemotherapy: current status and future perspectives. Aquaculture, 433, 50-61.

Robert-Pillot, A., Copin, S., Gay, M., Malle, P., & Quilici, M. L. (2010). Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR. International Journal of Food Microbiology, 143(3), 190-197.

Rodgers, C. J. (2001). Resistance of Yersinia ruckeri to antimicrobial agents in vitro. Aquaculture, 196(3), 325-345.

Saeidi, S., Hassanpour, K., Ghamgosha, M., Heiat, M., Taheri, R. A., Mirhosseini, A., & Farnoosh, G. (2014). Antibacterial activity of ethyl acetate and aqueous extracts of Mentha longifolia L. and hydroalcoholic extract of Zataria multiflora Boiss. plants against important human pathogens. Asian Pacific Journal of Tropical Medicine, 7, S186-S189.

Salih, B., Sipahi, T., & Dönmez, E. O. (2009). Ancient Nigella seeds from Boyalı Höyük in north-central Turkey. Journal of Ethnopharmacology, 124(3), 416-420.

Shah, S. Q., Colquhoun, D. J., Nikuli, H. L., & Sørum, H. (2012). Prevalence of antibiotic resistance genes in the bacterial flora of integrated fish farming environments of Pakistan and Tanzania. Environmental Science & Technology, 46(16), 8672-8679.

Shiau, Yu-Chen. (2009). Studies on the protection of divalent vaccine of Edwardsiella tarda and Aeromonas hydrophila in Japanese eel (Anguilla japonica). Thesis (Unpublished). National Taiwan Ocean University, Taiwan.

Sokmen, A., Jones, B. M., & Erturk, M. (1999). The in vitro antibacterial activity of Turkish medicinal plants. Journal of Ethnopharmacology, 67(1), 79-86.

Smith, P. (2012). Antibiotics in aquaculture; reducing the use and maintaining the efficacy. Infectious Diseases in Aquaculture, pp. 161-189.

Su, M. S., Liao, I. C., & Guo, J. J. (2000). The use of chemicals in aquaculture in Taiwan, Province of China. In Use of Chemicals in Aquaculture in Asia: Proceedings of the Meeting on the Use of Chemicals in Aquaculture in Asia 20-22 May 1996, Tigbauan, Iloilo, Philippines (pp. 193-205). SEAFDEC Aquaculture Department.

Starliper, C. E., Ketola, H. G., Noyes, A. D., Schill, W. B., Henson, F. G., Chalupnicki, M. A., & Dittman, D. E. (2015). An investigation of the bactericidal activity of selected essential oils to Aeromonas spp. Journal of Advanced Research, 6(1), 89-97.

Tafalla, C., Bøgwald, J., & Dalmo, R. A. (2013). Adjuvants and immunostimulants in fish vaccines: current knowledge and future perspectives. Fish & Shellfish Immunology, 35(6), 1740-1750.

Tan, D. (2015). Exploration of phage-host interactions in the fish pathogen Vibrio anguillarum and antiphage defense strategies. Disertation.

Tan, J. B. L., & Lim, Y. Y. (2015). Critical analysis of current methods for assessing the in vitro antioxidant and antibacterial activity of plant extracts. Food Chemistry, 172, 814-822.

Tekwu, E. M., Pieme, A. C., & Beng, V. P. (2012). Investigations of antimicrobial activity of some Cameroonian medicinal plant extracts against bacteria and yeast with gastrointestinal relevance. Journal of Ethnopharmacology, 142(1), 265-273.

Tendencia, E. A., & de la Peña, L. D. (2001). Antibiotic resistance of bacteria from shrimp ponds. Aquaculture, 195(3), 193-204.

Thirumalai, T., Tamilselvan, N., & David, E. (2014). Hypolipodemic activity of Piper betel in high fat induced hyperlipidemic rat. Journal of Acute Disease, 3(2), 131-135.

Vegara, S., Funes, L., Martí, N., Saura, D., Micol, V., & Valero, M. (2011). Bactericidal activities against pathogenic bacteria by selected constituents of plant extracts in carrot broth. Food Chemistry, 128(4), 872-877.

Vital, P. G., & Rivera, W. L. (2011). Antimicrobial activity, cytotoxicity, and phytochemical screening of Voacanga globosa (Blanco) Merr.leaf extract (Apocynaceae). Asian Pacific Journal of Tropical Medicine, 4(10), 824-828.

Viuda-Martos, M., Mohamady, M. A., Fernández-López, J., ElRazik, K. A., Omer, E. A., Pérez-Alvarez, J. A., & Sendra, E. (2011). In vitro antioxidant and antibacterial activities of essentials oils obtained from Egyptian aromatic plants. Food Control, 22(11), 1715-1722.

Whitman, K. A. (2004). Finfish and shellfish bacteriology manual: techniques and procedures (pp. i-xv+). Iowa state press. Pp 161-178.

Yano, Y., Satomi, M., & Oikawa, H. (2006). Antimicrobial effect of spices and herbs on Vibrio parahaemolyticus. International Journal of Food Microbiology, 111(1), 6-11.

Yeh, R. Y., Shiu, Y. L., Shei, S. C., Cheng, S. C., Huang, S. Y., Lin, J. C., & Liu, C. H. (2009). Evaluation of the antibacterial activity of leaf and twig extracts of stout camphor tree, Cinnamomum kanehirae, and the effects on immunity and disease resistance of white shrimp, Litopenaeus vannamei. Fish & Shellfish Immunology, 27(1), 26-32.

Zhou, S., Hou, Z., Li, N., & Qin, Q. (2007). Development of a SYBR Green I real‐time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood. Journal of Applied Microbiology, 103(5), 1897-1906.