Afrika kedibalığı (Heterobranchus bidorsalis) için Antimikrobiyal Ajanlar olarak Tridax procumbens Yaprak Özü Potansiyelleri

Balıklara olan talebin artması, yeni balık hastalıklarının ortaya çıkışı ve ortaya çıkışı ile eski balık yetiştiriciliğinin tekrar yaşanmasına neden olan yoğun akuakültür uygulamalarına da yol açmıştır. Doğal ve çevre dostu materyalleri kullanarak farmasötik ilaçlara alternatif terapi yapılmasının savunulması bu çalışmayı gerektirdi. Tridax procumbens özütlerinin antimikrobiyal özellikleri, patojenik balık bakterilerine (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aereus, Aeromonashydrophilia ve Listeria monocytogenes) karşı test edilmiştir. Bakteriler kültürlendi, minimum inhibisyon konsantrasyonu (MIC) ve inhibisyon zonu (ZI) standart yöntemler kullanılarak gerçekleştirildi. Yüz sekiz (108) Hetrobranchus bidorsalis yavruya 54 gün süreyle tamamen randomize edilmiş olarak günde iki kez T. procumbens esaslı diyetler (% 40 ham protein) verilmiştir. Elde edilen veriler, betimsel istatistikler ve ANOVA kullanılarak α0.05'de analiz edilmiştir. Elde edilen sonuçlar, T. procumbens ekstraktının alkaloidler, flavonoidler, taninler, steroidler ve karptenoidler içerdiğini, ekstraktaki saponin ve terpoidlerin bulunmadığını göstermektedir. Etkili MİK 40 mg / ml idi ve ZI, 12.85 ± 0.39 ila 14.20 ± 0.74 mm arasında değişiyordu. Patojenler L. monocyogenes hariç T. procumbens ekstraktına duyarlıydı. TVC ve TCC, tedavi edilen grupta anlamlı olarak azaldı. Sonuçlar, T. procumbens yapraklarının, su ürünleri yetiştiriciliğinde potansiyel kullanımını göstermiş olabilecek antimikrobiyal özelliklere sahip olduğunu ortaya koymuştur. Ayrıca, T. procumbens'in test edilen organizmalara karşı antimikrobiyal etkinliğe sahip olduğunu ve balık sağlığı yönetiminde kullanılabileceğini belirtti.

The increase in the demand for fish has led to intensive aquaculture practices which has also ushered in a manifestation and emergence of new fish diseases and reoccurrence of old ones. The advocacy for alternative therapy to drugs using natural and environmental friendly materials necessitated this study. The antimicrobial properties of Tridax procumbens extracts were tested against pathogenic fish bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aereus, Aeromonashydrophilia, and Listeria monocytogenes). Bacteria were cultured, minimum inhibitory concentration (MIC) and zone of inhibition (ZI) were carried out using standard methods. One hundred and eight (108) Hetrobranchus bidorsalis juveniles were fed T. procumbens based diets (40% crude protein) two times daily in a completely randomized designed for 54 days. Data obtained were analyzed using descriptive statistics and ANOVA at α0.05. The results shows that T. procumbens extract contained alkaloids, flavonoids, tannins, steroids and carptenoids while saponin and terpnoids were not detected in the extract. Effective MIC was 40mg/ml and ZI ranged from 12.85±0.39 to 14.20±0.74 mm. Pathogens were sensitive to the T. procumbens extract except for L. monocyogenes. The TVC and TCC were significantly reduced in the treated group. The results revealed T. procumbens leaves have antimicrobial properties which may had shown its potential utilization in aquaculture. It also indicated that T. procumbens have antimicrobial activity against tested organisms and could be used fish health management.

Giriş

Following the increase in the consumption of fish which majorly comes from aquaculture [1-2] the need for the safest animal source of protein especially fish has become obvious and cannot be over-emphasized. The demand has led to intensive aquaculture practices which is characterized by high yield and economic returns which has also ushered in a manifestation and emergence of new fish diseases and reoccurrence of old ones [3] and thus could resulted in great biological and economical losses. Since fish is capable of harbouring pathogens/diseases or serves as carrier, which could be transmitted to man through contact or consumption of infected fish it has become necessary to ensure food safety [4]). Reported that import rejection of seafood and fish was approximately 15% out of total product rejection and thus bacteria pathogens became the major reason for product rejection [5].

In an attempt to combat these challenges, several methods have applied. One of the such methods is the use of chemotherapy. However, in recent times, strong recommendation against continuous and indiscriminate use of drugs have been encouraged due to residual side effects, environmental degeneration and noticeable development of antibiotic-resistant pathogens [6-7]. Therefore, the need for alternative therapy using natural and environmental friendly materials cannot be over emphasized.

African catfish (Heterobranchus bidorsalis) is widely distributed fish in Africa, especially in Nigeria. H. bidorsalis belongs to the family Clariidae. It has ability survive in wide range of water and environmental parameters such as low dissolved oxygen levels which is usually harmful or lethal to other species. It has ability to consume varieties of conventional and non-conventional feed including kitchen waste [8-9]. It has been cultured singly and/with other species in form of polyculture in Nigeria.

In Nigeria, use of medicinal plants in health management is very common locally, because traditional ways of treating diseases are well established and has contributed to the pharmaceutical industries. Tridax procumbens (family-Asteraceae) known as “Ebueigba” in Yoruba language is a perennial plant which available virtually in all seasons. It is a weak straggling herb about 12-24 cm long with few leaves 6-8 cm long and grows on available space including backyard in Nigeria. The leaves of this plant including other aerial parts except flowering tops is said to be useful in the treatment of inflammatory conditions, heal wound, anti-diabetic activity, anti-arthritic activity, preventing hair loss, diarrhoea and serve as insect repellent. Despite of the enormous advantage and benefit of T. procumbens it is majorly regarded as weed except in feeding of rabbit and therefore there is need to investigate the antimicrobial potential of T. procumbens in the control of fish diseases.

Oils from these plants were reportedly having inhibiting and bactericidal properties [10]. It contains phenolic compounds, which possess antimicrobial activity. These compounds were classified as generally recognized as safe (GRAS) as such could be used in the control and management of bacterial in food [11-12]. The oil from the plants have been reported to perform actively on many bacteria (Staphylococcus, Salmonella, Listeria monocytogenes, Shigella, and Escherichia coli) and fungi (Trichophyton rubrum, T. mentagrophytes, Penicillium islandicum and Candida albicans) respectively [13-14].

This study was therefore aimed at screening for the antimicrobial properties of T. procumbens against bacteria of fish with a possibility of determining the minimum inhibitory concentration by which the plant extract can be included in fish diets.

Gereç ve Yöntem

Plant Collection and Identification: Tridax procumbens leaves were obtained from open space adjacent to Department of Aquaculture and Fisheries Management, University of Ibadan, Nigeria. The plant was authenticated at the herbarium of the Forestry Research Institute of Nigeria (FRIN), Ibadan and a voucher specimen was placed under FHI110831.

Preparation and Extraction of Plant Materials: The plant was rinsed with distilled water and aired at ambient temperature for 2 hours. Five [5] g of T. procumbens fresh leaves was weighed on a digital ScoutPro sensitive scale in the Department of Aquaculture and Fisheries Management, University of Ibadan, covered with sterilized cotton wool and transferred into Soxhlet apparatus as described by [11,15]. Briefly, about 170 mL of ethyl-acetate were poured in a spiral tube of the equipment. The extraction last for six hours until no further drop of extract in triplicates. The filtrate were concentrated on a rotary evaporator at 45°C for chemicals elimination, stripped into a sterile bottle and kept in a refrigerator until use.

Phytochemical Analysis: The extracts of fresh leaves of T. procumbens were both qualitatively and quantitatively examined for the presence of secondary metabolites using various analytical tests and reagents. The metabolites examined were Alkaloids [16-17], Flavonoids [17], Tannins [18], Saponins (18), Terpenoids [19], Steroids [20] and Carotenoids [21].

Media Preparation: MacConkey, Nutrient, Plate Count Agars, nutrient broth and peptone water were prepared according to Manufacturers’ instruction (all agar were LAB-M products).

Microbial Analysis: The organs (liver, kidney, gill, intestine, flesh/muscle and spleen) were aseptically collected and weighed into sterile universal bottles while skin samples were collected using skin swab. The Samples were inserted into peptone water (0.1%) and allowed to release the available bacterial for a period of 2-3 hours. One mL was taken from each sample bottles and diluted in ten folds and subsequently serially diluted with dilution factor 10-4. Two ml were taken from each sample and dispensed into two petri dishes (1 mL to each). The first dish received plate count agar (PCA) for total viable count (TVC) (LAB M, LAB149) while the second petri dish received MacConkey agar (LAB M, LAB002) for total coliform count (TCC) using the pure plate count method. The media were prepared according to manufacturers’ instruction. Each dilution was overlaid with PCA and MacConkey respectively that has been cooled to 500C. At this temperature, agar is still in liquid form. The dishes were then gently swirled to mix the bacteria with the liquid agar [15]. The mixtures were allowed to harden. When the mixture is hardened, the individual cells are fixed in place and incubated (Newlife Laboratory Incubator NL-9052-1) for 24 hours at 37 ⁰C to allow a distinguish colonies to form. The colonies formed were counted using Wincom Colony Counter (16W, 220V±10%, 50 Hz). The experiments were replicated three times. The total viable count and total coliform count were expressed in Log10CFU/g [6,21].

Antimicrobial Assay: Five pure bacteria isolates were obtained from Laboratory of Department of Microbiology, University of Ibadan, Nigeria. The isolates were: Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aereus and Aeromonashydrophilia.

Detection of Antagonistic Activity: Agar well diffusion methods were adapted. From the extracts, 0.1 g were dissolved in 2 mL of Dimethyl Sulfoxide (DMSO) (22) and reconstituted to have 50, 40, 30, 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 mg/mL. A cork borer of 6 mm diameter was used to create wells on the plates. Each well was filled with the extracts at the working concentration sterilized droppers and incubated at 37 ⁰C for 24 to 48 hours. The zones of inhibition were measured and minimum inhibitory concentrations were determined.

Feeding Trial:
Feed Preparation: The best two concentrations that produced better zones of inhibition were incorporated in to feed formulated at 40% crude protein and fed to fish for 54 days. Organs were asceptically collected and examined for TCC and TVC. The feed ingredients (Table 1) were pelleted (2.0 mm) and packed in a polythene bags and store in a cool dry place until use with a well label on it.

Experimental Procedure: A total number of one hundred and eight (180) Heterobranchus bidorsalis juveniles (12.20±1.12 g) were used for the experiment. The fish were acclimatized for 2 weeks in plastic tanks before the experiment. Fish were weighed and distributed in to nine rectangular plastic tanks (60 x 38 x 27 cm) tanks in a completely randomized design. Each tank contained twenty fish and water was replaced on three days interval. The experiment had three treatments containing 0.0% (control), 0.05% (T1) and 0.04% (T2) of T. procumbens extracts in triplicates. The fish were fed at 3% body weight daily (1.5% was given in the morning by 8:00 am and 1.5% in the evening by 5:00 pm) throughout the experimentation period of 54 days. Water quality parameters were measured while the experiment lasted for fifty-four days. The protocol of this study was subjected to ethical consideration and received approval from University of Ibadan Animal Care Use and Research Committee (UI-ACUREC; U.I.ACUREC/IA/16/0005-UI-ACUREC/App/03/2017/008)

Statistical Analysis: The data obtained were subjected to descriptive statistics and one-way analysis of variance (ANOVA) at α0.05 using SPSS IBM version 20. Microbial load was expressed as log10 and reported as mean. The means were separated using Duncan multiple range test.

Bulgular

Secondary metabolites observed were alkaloids (156.67±3.02 mg/100 g), flavonoids (130.12±5.29 mg/100 g), tannins (107.21±4.17 mg/100 g), steroids (112.306±8.39 mg/100 g) and carptenoids (84.77±3.85 mg/100 g) while saponin (23.08±8.01 mg/100 g) and terpnoids (18.02±1.61 mg/100 g) were detected in minute quantities in the extract (Table 2) of the T. procumbens. It was observed that most of the parameters examined were present and significantly different (P=0.02).

Extract of T. procumbens at concentrations of 0.00 to 2.5 mg/mL did not inhibit the tested pathogens. However, concentrations from 10 mg/mL to 50 mg/mL showed inhibition of the organisms except L. monocytogenes which did not show any inhibition. No growth of E. coli and A. hydrophilia were observed at 20 mg/mL while P. aeruginosa and S. aereusdid not grow at 10 mg/mL and 5 mg/mL respectively and thus 20 mg/mL is minimum inhibitory concentration (Table 3).

The antagonistic performance of T. procumbens extract against the tested pathogens revealved that highest zone of inhibition was observed in 50 mg/mL extract tested on S. aereus (16.23±1.33 mm) while the least was recorded in control (0.00±0.00 mm) at 0.00 mg/mL concentration. More so, there were progressive increase in the zones of inhibition observed in tested organisms with increase in the concentration of the extract except in L. monocytogenes which did not show any clear zone of inhibition (Table 4). There were significant differences among the extracts against the pathogens (P<0.05).

Results of microbial load investigations indicated that both total viable bacteria count (TVC) and total coliform count (TCC) were higher in fish tissues before feed trial than after the feed trial. After the feeding trial, the highest TCC and TVC were recorded in fish treated control diet and the least in group fed fortified diet. Skin had highest TCC and TVV while the least TVC and TCC were recorded in muscle. There were significant differences in the TVV and TCC among the treatments across the organs (Table 5).

The mean dissolved oxygen was 6.80±1.10 mg/L; temperature was 25.15±2.66 and pH was 7.28±0.81 (Table 6).

Tartışma

Phytochemicals in the plants has been used as an indices in predicting the antimicrobial activities of the plants. The leaves had as alkaloids, flavonoids, tannin, steroids and carotenoids which are considered relatively abundance. The result of this study is in agreement with the work of [23-25] who reported the presence of flavonoids, tannin, alkaloids, saponin in the methanolic extract of T. procumbens. The continuous and indiscriminate use of synthetic drugs has ushered in drug resistance pathogens, residual effects among others. The trend of minimum inhibitory concentration observed in this study follows the trend reported by [26-27].

The presence of flavonoids, an agent that have counter-action against free radicals which gesticulating disorder like inflammation and hepatotoxins could be responsible for inhibition and reduction of bacteria load in fish tissues. Tannin is refers to as antioxidant and antimicrobial agents [28] and thus its presence could be associated with the effective performance of T. procumbens in the diet of fish and in the invitro study. The lower MIC observed in P. aeruginosa and S. aereus revealed that T. procumbens at lower concentrations would be adequate to control the pathogens in fish farming. Similar observations were made by [6,29] who reported 500μg/ml of onion bulb and walnut leaves against P. aeruginosa.

The widest zone of inhibition obtained against S. aereus showed that the pathogen is highly sensitive to the extract. According to the [25] acceptable zone of inhibition of effective plant extract should be 13 mm and above. Therefore, T. procumbens extract at 40 and 50 mg/mL had zones of inhibitions within the recommended values. The zones of inhibition obtained in the study for E. coli, S. aereus, P. aeruginosa and A. hydrophila corroborate with the findings [6,30] who reported 9 to 13.5 mm zone of inhibition of plants extract against pathogenic bacteria including the tested organism of this study. The factors responsible for the higher zones of inhibition in this study may be attributed to antibacterial properties in the plants such as tannin.

The result of this study shows that there were amazing reduction in the TVC and TCC in groups treated T. procumbens extract based diet. According to international Commission on the Microbiological Specification of Foods [31] acceptable level of bacteria load in fish should be between 102 – 107per gram or cm2 equivalent to about 5.70 Log10cfu/cm2 for skin and in the same vein [32] recommended 105 per gram for other part of fish as acceptable level. Therefore, the result obtained in this study before fed extract based diet and control after the trial had value higher than the recommended values. However, fish fed extract based diets had values in both TVC and TCC lower than the recommended values which is significantly different when compared to control. The result of this study is in agreement with the findings of [3,6]. Furthermore, it was noticed that skin and gill had higher bacteria load than other tissues which may be connected to the fact that they are external organs. These external organs usually have direct and frequent contact with the environment and thus lead increase bacterial load on the sites.

The results revealed T. procumben sleaf have antimicrobial properties which may had shown it potential utilization in aquaculture. It also indicated that T. procumbens is an effective plants and can be used to control many common pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aereus and Aeromonas hydrophilia with minimum inhibitory concentration is 40 mg/mL without deleterious effect on the water.

References

1. Food and Agriculture Society (FAO). The State of World Fisheries and Aquaculture: Contributing to food security and nutrition for all, World review, 2016: 24.
2. Lazo JP. Preface to current status of scallop culture in Brazil, President’s Column. Mag Wrd Aquac Soc 2016; 42: 1-2.
3. Adeshina I, Umma SD, Adesanmi O, Adewale YA. Distribution of Bacteria Load in Fish Tissues, Pond Water and Sediment in Oyo State, Nigeria. Alex J Vet Sci 2016; 51: 1-9.
4. Rapid Alert System for Food and Feed (RASFF), Food and feed importation, Tech Reprt RASFF 2013; 46:1-72.
5. Ababouch L, Gandini G, Ryder J. Causes of detentions and rejections in international fish trade. FAO Fish Tech Pap 2005; 473:1-82.
6. Bello OS, Olaifa FE, Emikpe BO, Ogunbanwo ST. Potentials of walnut (Tetracarpidiumconophorum Mull. Arg) leaf and onion (Allium cepa Linn) bulb extracts as antimicrobial agents for fish. Afri J Micro Res 2013; 7: 2027-2033.
7. Jadhav VS, Khan SI, Girkar MM, Gitte MJ. The role of immunostimulants in fish and shrimp aquaculture. Aquatic animal health. Aquaculture Asia Magazine July-September 2006: 24-27.
8. Omitoyin BO. Introduction of fish farming in Nigeria. Ibadan: University Press, 2007.
9. Sule OD. Morphometric Characterization and Embryonic Development of C. gariepinus. PhD Thesis, Bayero University Kano, 2003.
10. Cosentino S, Tuberoso CIG, Pisano B, et al. In-vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. L Appl Micro 1999; 29: 130-135.
11. Bayoub K, Baibai T, Mountassif D, Retmane A, Soukri A. Antibacterial activities of the crude ethanol extracts of medicinal plants against Listeria monocytogenesand some other pathogenic strains. Afri J Biotech 2010; 9: 4251-4258.
12. Ponce AG, Fritz R, del Valle C, Roura SI. Antimicrobial activity of essential oils on the native microflora of organic Swiss chard. Lebensm.-Wiss. u.-Techn 2003; 36: 679-684.
13. Akinyemi KO, Mendie UE, Smith ST, Oyefolu AO, Coker AO. Screening of some medicinal plants for anti salmonella activity. J Herb Pharmocot 2004; 5: 45-60.
14. Lopez P, Sanchez K, Batlle R, Nerin C. Solid and vapor phase anti-microbial activities of six essential oils susceptibility of selected food borne bacterial and fungal strains. J Agric Fd Chem 2005; 53: 6939-6946.
15. Hema R, Kumaravel S, Sivasubramanian C. GC-MS study on the potentials of Syzygiumaromaticum. Res 2010; 12: 1-4.
16. FDA, Everything added to food in the United States (EAFUS). FDA 2008. www.fda.gov/Food/FoodIngridients Packagin/ucm115326.htm.pdf http://www.fda.gov/Food/ FoodIngredientsPackaging/ucm115326.htm/15.10.2016.
17. Joshi B, Sah GP, Basnet BB, et al. Phytochemical extraction and antimicrobial properties of different medicinal plants: Ocimum sanctum (Tulsi), Eugenia caryophyllata (Clove), Achyranthes bidentata (Datiwan) and Azadirachta indica (Neem). J Micro Antimicro 2011; 3: 1-7.
18. Mayuri PN. Screening of Ailanthus (Excelsa Roxb.) for Secondary Metabolites. J Curr Pharm Res 2012; 10: 19-219.
19. Odebiyi OO, Sofowora EA. Phytochemical screening of Nigerian medicinal plants. Lyodia 1978; 3: 150-156.
20. Marcano L, Hasenawa D. Analysis of Phytochemicals in Leaves and Seeds. Agro J 1991; 83: 445-452.
21. American Public Health Association (APHA), Standard Methods for the Examination of Water and Wastewater, 16th Edition. Washington: American Public Health Association, American Water Works Association, Water Pollution Control Federation, 1985.
22. Gupta C, Garg A, Uniyal R, Gupta S. Comparison of Antimicrobial Activities of Clove Oil and Its Extract On Some Food Borne Microbes. Int J Micro 2008; 7: 1-7.
23. Harnly JM, Doherty RF, Beecher GR, et al. Flavonoid Content of U.S. Fruits, Vegetables, and Nuts. J Agric Fd Chem 2006; 54: 9966-9977.
24. Ikewuchi CC, Ikewuchi JC, Ifeanacho MO. Phytochemical composition of Tridax procumbens linn leaves: Potential as a functionalf. Fd Nutr Sci 2015; 6: 992-1004.
25. Kumar SS, John R, Lakshmi-Narayanan G. Antimicrobial activity of Tridax procumbens leaf. International. J Pharma Sci Res 2015; 6: 517-518.
26. Das BK, Pradhan J, Sahu S. The effect of Euglena virdis on immune response of rohu, Labeorohita (Ham.). Fish Shellfish Immun 2009; 26: 871-876.
27. Harikrishnan R, Kim MC, Kim JS, Balasundaram C, Heo MS. Probiotics and herbal mixtures enhance the growth, blood constitutents and non-specific immune response in Paralichthysolivaceus against Streptococcus parauberis. Fish Shellfish Immun 2011; 31: 310-317.
28. Namkung W, Thiagarajah JR, Phuan PW, Verkman AS, Inhibition of Ca2+-Activated Cl− Channels by Gallotannins as a Possible Molecular Basis for Health Benefits of Red Wine and Green Tea. FASEB J 2010; 24: 4178-4186.
29. Selvamohan T, Ramadas V, Kishore SSS. Antimicrobial activity of selected medicinal plants against some selected human pathogenic bacteria. Adv Appl Sci Res 2012; 3: 3374-3381.
30. Food and Agriculture Organization (FAO). Report of the FAO expert consultation on the trade impact of Listeria in fish product FAO Fisheries Report No. 64. FIIU/ESNS/R604, Amsherst, MA, United States, 1999; 17-20.
31. International Commission on the Microbiological Specification of Foods (ICMSF). Microorganisms in food, Vol. 2, Sampling for microbiological analysis, principles and specific applications. Univ. of Toronto Press, Toronto, Canada.1982.
32. Basri DF, Fan SH. The potential of aqueous and acetone extract of galls of QuercusInfectoria as antimicrobial agents. Ind J Pharma 2005; 37: 26-29.