Antibacterial Activity of Some Aromatic Plant Essential Oils Against Fish Pathogenic Bacteria

Bazı Aromatik Bitki Esansiyel Yağlarının Patojenik Balık Bakterilerine Karşı Antibakteriyel Aktivitesi Öz: 24 adet bitki türünün uçucu yağları hidrodistilasyon yoluyla elde edildi ve 7 çeşit balık patojenine (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum, Yersinia ruckeri, Enterococcus faecalis, Lactococcus garvieae ve Streptococcus agalactiae) karşı antibakteriyel etkileri araştırıldı. Uçucu yağların, disk difüzyon yöntemiyle elde edilen antibakteriyel aktivite sonuçları, tüm patojenlere karşı kuvvetli aktiviteleri olduğunu göstermektedir. Genel olarak, Artemisia absinthium dışında tüm uçucu yağlar, balık patojenlerinin çoğunluğuna karşı güçlü antibakteriyel etkiler göstermiştir. Bununla birlikte, A. absinthium uçucu yağı, sadece A. hydrophila’ ya karşı zayıf antibakteriyel etki göstermiştir. Yirmi dört uçucu yağ arasından çoğunlukla yedi uçucu yağ (T. spicata, T. vulgaris, L. nobilis, C. verum, H. plicatum and A. citriodora Paláu) tüm balık patojenlerine karşı iyi antibakteriyel aktivite sergilemişlerdir. Test edilen antibiyotikler (furazolidon, oksitetrasiklin, sefalotin ve trimetoprim/sulfametoksazol) ile karşılaştırıldığında, uçucu yağların antibakteriyal etkileri çoğunlukla eşit veya güçlü olarak bulunmuştur. Uçucu yağların antibakteriyel aktivite sonuçları göz önünde bulundurarak, su ürünleri yetiştiriciliğinde bakteriyel balık hastalıklarına karşı antimikrobiyal ajanların yerine alternatif olarak kullanımları uygun olabilir. Anahtar kelimeler: Antibakteriyel aktivite, disk difüzyon, balık patojenleri, esansiyel yağlar Alıntılama Birinci Yıldırım A, Türker H. 2018. Antibacterial Activity of Some Aromatic Plant Essential Oils Against Fish Pathogenic Bacteria. LimnoFish. 4(2): 67-74. doi: 10.17216/LimnoFish.379784 Introduction The extracts of medicinal plants had been extensively used over human beings and animals for a large number of purposes for a long time. Today, the medicinal and aromatic plants came into use in the modern medicine in contrast to synthetic ones that are regarded as unsafe to human and the environment. Besides, herbal products and plant-derived compounds present potential sources of new antibiotics, anticancer agents, and anti-HIV agents (Gurib-Fakim et al. 2005). In addition to their medicinal use in human, the medicinal plants were 68 Birinci Yıldırım and Türker 2018 LimnoFish 4(2): 67-74 also used as chemotherapeutics and food additives in aquaculture due to their ability of enhancing the fish immune system (Van Hai 2015). Aquaculture is one of the main food supply among animal food products for balanced nutrition and good health, and aquaculture fish production is the fastest growing food source sector in comparison to all other animal food sources. However, the factors such as intensification of aquaculture, periodic handling, extreme temperature changes, poor water quality and poor nutritional status contribute to adverse effects on fish health. In other word, high fish density and poor physiological environment may cause an increase in spread of pathogens in aquaculture and also an increase in the susceptibility of fish to the microbial agents (bacteria, fungi, virus etc.). So, this causes high mortality and also leads to serious economic losses (Harikrishnan et al. 2011; Reverter et al. 2014). In order to avoid and control of these pathogens, the antibiotics have been frequently used in aquaculture (Romero et al. 2012). Some antibiotics such as amoxicillin, erythromycin, enrofloxacin, oxytetracycline and furazolidone, have been used successfully to control the most of fish diseases (Harikrishnan et al. 2011). However, conscious or unconscious overdose application of antibiotics might improve the resistance of these antibiotics to the bacteria and thereby a reduced efficacy of the drugs. In addition, antibiotics possess a potential risk to consumers and the environment due to their accumulation within the environment and fishes (Harikrishnan et al. 2011; Ontas et al. 2016). This situation prompted the scientists to search new and eco-friendly alternatives to antimicrobial agents. The most promising method to prevent fish diseases was the enhancement of the immune system by using immunostimulants derived from plants stimulating humoral and cellular defence mechanisms. Plant-derived immunostimulants are eco-friendly and easily prepared, and effective with fewer side effects during treatment of diseases and without any environmental and hazardous problems (Mousavi et al. 2011; Reverter et al. 2014) and also they do not lead to any drug resistance (Soltani et al. 2010). Recently, this interest in natural medicine has also been increasing in fish culture (Soltani et al. 2010). Lately, the essential oils are very popular as natural antimicrobial agents due to their rich mixture of highly functional molecules (Park et al. 2011). Numerous studies have been reported about the antibacterial properties of essential oils isolated from aromatic plants for their potential bioactive principles (Romano et al. 2005). Nowadays, some studies have been reported on the antimicrobial activities of essential oils on aquatic animal diseases (Rattanachaikunsopon and Phumkhachorn 2007; Mousavi et al. 2011) and also provided a promising managemet tool for the controlling or treating aquatic fish diseases (Olusola et al. 2013). Hence, in the present study, essential oils obtained from twentyfour different Turkish plants were studied for screening their in vitro antibacterial activities on a fish pathogenic bacteria from aquaculture industry. Material and Methods Some plants were purchased from herbalists in Bolu, Turkey and some others were grown in pots to produce their essential oils. The plants used in this study were given in Table 1. Purchased plants were grounded into fine powder and some others were cut into small pieces without drying. Briefly, 100 g of each plant material (selected organ) were seperately steam-distilled by using a Clevenger type apparatus for 4 hour (Randrianarivelo et al. 2010). The obtained essential oils were collected in sealed-brown vials seperately and covered with aluminum foil and kept in a refrigerator until use. The yield of each essential oil (ml/weight) was calculated from the weight of used plant parts (Table 1). Antimicrobial assay Fish Pathogens A. hydrophila, A. salmonicida, V. anguillarum, Y. ruckeri, E. faecalis, L. garvieae and S. agalactiae were used for antibacterial assay. A. hydrophila (ATCC 19570) and S. agalactiae (Pasteur Institute 55118) bacterial strains were obtained from Refik Saydam National Type Culture Collection (Ankara, Turkey). V. anguillarum, Y. ruckeri and L. garvieae bacterial strains were provided by Dr. İlhan Altınok, Faculty of Marine Science, Karadeniz Technical University, Surmene, Trabzon, Turkey. E. faecalis bacterial strain were provided by Dr. Cafer Erkin Koyuncu, Faculty of Fisheries, Mersin University, Mersin, Turkey. A. salmonicida bacterial strain by Dr. Şükrü Kırkan, Faculty of Veterinary Medicine, Adnan Menderes University, Aydın, Turkey. Antibacterial assay The antibacterial activity of twenty-four essential oil extracts was determined by using disc diffusion assay (Kirby-Bauer Method) (Andrews 2009). Agar culture plates were prepared as described before (Türker and Yıldırım 2015). Briefly, each bacterial strain was grown on Tryptic Soy Agar (TSA) (Acumedia) plates and incubated for 2 days at 28 oC for A. salmonicida and Y. ruckeri; at 37 oC for the other bacterial strains. The turbidity of each bacteria broth culture was adjusted to equal that of the 0.5 McFarland standard and then the broth cultures adjusted was separately inoculated on Mueller Hinton Agar plates by using cotton swabs. 10 μl of Birinci Yıldırım and Türker 2018 LimnoFish 4(2): 67-74 69 each oil was applied to sterile filter paper discs(6 mm in diameter, Glass Microfibre filters, Whatman). Standard antibiotic discs (furazolidone (100 μg), oxytetracycline (30 μg), cephalothin (30 μg) and trimethoprim/sulfamethoxazole (1.25 / 23.75 μg)) (Bioanalyse) were used for positive control placed on the inoculated Muller Hinton agar plates. Hexane were used as a negative control because essential oils collected in tiny amounts in clevenger apparatus were taken with hexane. Inoculated plates with discs were incubated at 37 oC with the exception of A. salmonicida and Y. ruckeri (at 28 oC) for 24 hours. After incubation, inhibition zone diameter (mm) was measured. Three independent experiments were done in different times. Statistical analysis The Shapiro-Wilk test (Shapiro and Wilk 1965; Royston 1995) and an inspection of the skewness and kurtosis measures showed that the sample data were not approximately normally distributed (P<0,05). A Kruskal-Wallis H test, is a rank-based nonparametric test, showed that there was a statistically significant difference among the extract treatments (P<0,05) and performed a pairwise Conover test of multiple comparisons using rank sums as post-hoc test (Conover 1999). All data were analyzed by using MedCalc Statistical Software (version 15.8). Table 1. List of the studied plant species, plant parts used and essential oil yields. Family and plant species Common name Part used Yield (ml)* Lamiaceae Lavandula angustifolia Lavender Flower 3.3 Lavandula stoechas French lavender Flower 0.7 Menthax piperita Pepper mint Leaves 0.6 Ocimum basilicum Sweet basil Leaves 0.5 Origanum majorana Wild marjoram Leaves 0.05 Thymus vulgaris Thyme Leaves 0.7 Rosmarinus officinalis Rosemary Leaves 2.0 Thymbra spicata Spiked thyme Leaves 0.6 Salvia officinalis Sage Leaves 2.2 Lauraceae Laurus nobilis Bay laurel Leaves 1.1 Cinnamomum verum Cinnamon Bark 3.0 Geraniaceae Pelargonium graveolens Rose geranium Leaves 0.2 Piperaceae Piper nigrum Black pepper Seed 4.6 Verbenaceae Aloysia citriodora Paláu Lemon verbena Leaves 0.4 Zingiberaceae Zingiber officinale Ginger Root 0.7 Apiaceae Coriandrum sativum Chinese parsley Seed 0.5 Foeniculum vulgare 


Introduction
The extracts of medicinal plants had been extensively used over human beings and animals for a large number of purposes for a long time.Today, the medicinal and aromatic plants came into use in the modern medicine in contrast to synthetic ones that are regarded as unsafe to human and the environment.Besides, herbal products and plant-derived compounds present potential sources of new antibiotics, anticancer agents, and anti-HIV agents (Gurib-Fakim et al. 2005).In addition to their medicinal use in human, the medicinal plants were also used as chemotherapeutics and food additives in aquaculture due to their ability of enhancing the fish immune system ( Van Hai 2015).Aquaculture is one of the main food supply among animal food products for balanced nutrition and good health, and aquaculture fish production is the fastest growing food source sector in comparison to all other animal food sources.However, the factors such as intensification of aquaculture, periodic handling, extreme temperature changes, poor water quality and poor nutritional status contribute to adverse effects on fish health.In other word, high fish density and poor physiological environment may cause an increase in spread of pathogens in aquaculture and also an increase in the susceptibility of fish to the microbial agents (bacteria, fungi, virus etc.).So, this causes high mortality and also leads to serious economic losses (Harikrishnan et al. 2011;Reverter et al. 2014).In order to avoid and control of these pathogens, the antibiotics have been frequently used in aquaculture (Romero et al. 2012).Some antibiotics such as amoxicillin, erythromycin, enrofloxacin, oxytetracycline and furazolidone, have been used successfully to control the most of fish diseases (Harikrishnan et al. 2011).However, conscious or unconscious overdose application of antibiotics might improve the resistance of these antibiotics to the bacteria and thereby a reduced efficacy of the drugs.In addition, antibiotics possess a potential risk to consumers and the environment due to their accumulation within the environment and fishes (Harikrishnan et al. 2011;Ontas et al. 2016).
This situation prompted the scientists to search new and eco-friendly alternatives to antimicrobial agents.The most promising method to prevent fish diseases was the enhancement of the immune system by using immunostimulants derived from plants stimulating humoral and cellular defence mechanisms.Plant-derived immunostimulants are eco-friendly and easily prepared, and effective with fewer side effects during treatment of diseases and without any environmental and hazardous problems (Mousavi et al. 2011;Reverter et al. 2014) and also they do not lead to any drug resistance (Soltani et al. 2010).
Recently, this interest in natural medicine has also been increasing in fish culture (Soltani et al. 2010).Lately, the essential oils are very popular as natural antimicrobial agents due to their rich mixture of highly functional molecules (Park et al. 2011).Numerous studies have been reported about the antibacterial properties of essential oils isolated from aromatic plants for their potential bioactive principles (Romano et al. 2005).Nowadays, some studies have been reported on the antimicrobial activities of essential oils on aquatic animal diseases (Rattanachaikunsopon and Phumkhachorn 2007;Mousavi et al. 2011) and also provided a promising managemet tool for the controlling or treating aquatic fish diseases (Olusola et al. 2013).Hence, in the present study, essential oils obtained from twentyfour different Turkish plants were studied for screening their in vitro antibacterial activities on a fish pathogenic bacteria from aquaculture industry.

Material and Methods
Some plants were purchased from herbalists in Bolu, Turkey and some others were grown in pots to produce their essential oils.The plants used in this study were given in Table 1.Purchased plants were grounded into fine powder and some others were cut into small pieces without drying.Briefly, 100 g of each plant material (selected organ) were seperately steam-distilled by using a Clevenger type apparatus for 4 hour (Randrianarivelo et al. 2010).The obtained essential oils were collected in sealed-brown vials seperately and covered with aluminum foil and kept in a refrigerator until use.The yield of each essential oil (ml/weight) was calculated from the weight of used plant parts (Table 1).

Antibacterial assay
The antibacterial activity of twenty-four essential oil extracts was determined by using disc diffusion assay (Kirby-Bauer Method) (Andrews 2009).Agar culture plates were prepared as described before (Türker and Yıldırım 2015).Briefly, each bacterial strain was grown on Tryptic Soy Agar (TSA) (Acumedia) plates and incubated for 2 days at 28 ºC for A. salmonicida and Y. ruckeri; at 37 ºC for the other bacterial strains.The turbidity of each bacteria broth culture was adjusted to equal that of the 0.5 McFarland standard and then the broth cultures adjusted was separately inoculated on Mueller Hinton Agar plates by using cotton swabs.10 µl of each oil was applied to sterile filter paper discs(6 mm in diameter, Glass Microfibre filters, Whatman ® ).Standard antibiotic discs (furazolidone (100 µg), oxytetracycline (30 µg), cephalothin (30 µg) and trimethoprim/sulfamethoxazole (1.25 / 23.75 µg)) (Bioanalyse ® ) were used for positive control placed on the inoculated Muller Hinton agar plates.Hexane were used as a negative control because essential oils collected in tiny amounts in clevenger apparatus were taken with hexane.Inoculated plates with discs were incubated at 37 ºC with the exception of A. salmonicida and Y. ruckeri (at 28 ºC) for 24 hours.After incubation, inhibition zone diameter (mm) was measured.Three independent experiments were done in different times.

Statistical analysis
The Shapiro-Wilk test (Shapiro and Wilk 1965;Royston 1995) and an inspection of the skewness and kurtosis measures showed that the sample data were not approximately normally distributed (P<0,05).A Kruskal-Wallis H test, is a rank-based nonparametric test, showed that there was a statistically significant difference among the extract treatments (P<0,05) and performed a pairwise Conover test of multiple comparisons using rank sums as post-hoc test (Conover 1999).All data were analyzed by using MedCalc Statistical Software (version 15.8).

Results
Antibacterial screening of 24 essential oils against 7 fish pathogens was shown in Table 1.As a result of our work, essential oils generally showed strong antibacterial effects against all bacteria.However, among bacteria, A. hydrophila, E. faecalis, L. garviceae and S. agalactiae were found as the most sensitive bacterial strains to the essential oils.
Against E. faecalis and L. garvieae bacterial strains, the best antibacterial effect was obtained with essential oils of L. nobilis and S. officinalis.This effect was followed by the effects of H. plicatum essential oil only on L. garvieae bacterial strain, and the effects of P. nigrum, A. citriodora Paláu essential oils against both E. faecalis and L. garvieae bacterial strains.Essential oils of these plants exhibited higher inhibitory effects than all used antibiotics.
Although all used bacterial strains were mainly sensitive against tested essential oils, mostly seven essential oils of the plants (T.spicata, T. vulgaris, L. nobilis, C. verum, H. plicatum and A. citriodora Paláu) among twenty-four essential oils exhibited good antibacterial activity against all fish pathogens in present study.Nonetheless, A. absinthium essential oil was not effective against used bacteria except A. hydrophila. A. absinthium essential oil produced the smallest inhibition zone of 8.3 mm.In addition, P. sativum showed weaker antibacterial activities against all bacteria than those of other used essential oils.Moreover, P. sativum showed similar inhibition zones as antibiotic furazolidone against L. garvieaea and also similar inhibiton zones as antibiotic cephalothin against Y. ruckeri (Table 2).
In addition to plant essential oils exhibiting the best antibacterial effects, the rest of the plant essential oils exhibited good inhibitory effects against most of the tested fish pathogens and they also exhibited more stronger antibacterial effects than antibiotics used as standard drugs in the present study.
Positive controls (antibiotic discs) showed antibacterial activity to used fish pathogens.Hexane was used as a negative control and no inhibition was observed with hexane.

Discussion
Antibacterial effect of C. lemon and A. spinosa essential oils against Y. ruckeri, A. hydrophila and L. garvieae bacterial strains have been studied by Ontas et al. (2016).Their results indicated that both essential oils possessed strong antibacterial effects against Y. ruckeri and A. hydrophila whereas weak antibacterial activity was obtained against L. garviea.However, in our study, the essential oils of many plants showed the strong antibacterial effects against Y. ruckeri, A. hydrophila and L. garviea bacterial strains.Likewise, Cermelli et al. (2008) studied the antibacterial activity of Eucalyptus globulus oil and they reported that eucalyptus oil did not exhibit any antibacterial effects against S. agalactiae.However, the essential oil of E. camaldulensis possessed strong antimicrobial effect against same fish pathogens in our study.
In another study, essential oils of two Rosmarinus officinalis L. varieties exhibited weak to moderate antimicrobial effects against K. pneumoniae, S. aureus, E.coli, B.subtilis and B.cereus (Zaouali et al. 2010).However, Roomiani et al. (2013) reported that the essential oil of R. officinalis possessed very strong antibacterial effect against Streptococcus iniae.
Mean diameter of inhibitory zones (mm ± SE) presented as zone of inhibition of bacterial growth in mm.Means with the same letter within columns are not

Table 1 .
List of the studied plant species, plant parts used and essential oil yields.