ANTIBACTERIAL EFFECTS OF SEVEN ESSENTIAL PLANT OILS ON FISH PATHOGENS

The use of natural products which have the least harmful effects on the environment has recently been taken as a novel approach against fish diseases. References on in vitro studies have demonstrated antibacterial activity of essential oils (EOs) against certain fish pathogens. The aim of this study was to evaluate the antibacterial effect of some plant essential oils against fish pathogenic bacteria in vitro conditions. Seven plant EOs: lavender (Lavandula angustifolia), clove (Eugenia caryophyllus), peppermint (Mentha piperitae), basil (Ocimum sanctum), rosemary (Rosmarinus officinalis), cinnamon (Cinnamomum zeylanicum) and black cumin (Nigella sativa) were used to identify their antibacterial properties against Yersinia ruckeri, Aeromonas hydrophila, Vibrio anguillarum, Vibrio alginolyticus, Lactococcus garvieae and Vagococcus salmoninarum at five concentrations using disc diffusion method. Especially the EOs of clove, cinnamon and rosemary showed the strongest antibacterial activities than other oils against the three most susceptible bacterial strains (Y. ruckeri, A. hydrophila and V. salmoninarum). Besides, the EOs of clove, rosemary, cinnamon and black cumin showed similar inhibition zones with OTC against A. hydrophila. The minimum inhibitory concentrations of the used EOs found between 500 and 62.5 μl mL-1. As a result, three of the EOs used in this study were effective on both Gr (-) and Gr (+) bacteria.


INTRODUCTION
The economic losses caused by infectious diseases has been a major issue in aquaculture worldwide (Flores-Kossack et al., 2020;Hwang et al., 2020;Wang et al., 2020). Antibiotics have been utilized as a successful method to control bacterial diseases but bacteria gain resistance against many antibiotics therefore utilization of numerous antibiotic agents that have been banned or using levels decreased in several countries. The use of antibiotics 2/9 in aquaculture has been implicated as a potential hazard for the development and selection of resistant bacteria and a source of these pathogens to other animals and humans (Hatha et al., 2005;Serrano, 2005;Acar et al., 2009). There is a need for new natural solutions as an alternative to antibiotics in the control and treatment of fish diseases. Essencial oils (EOs) can be used as an alternative to antibiotics in aquaculture (Romero et al., 2012;Cunha et al., 2018). EOs have been exhibited to possess antimicrobial and antioxidant effects (Cunha et al., 2018). They can also serve as significant tools to reduce bacterial resistance (Stefanakis et al., 2013). Aromatic oil liquids called essential oils are obtained from plant materials (flowers, leaves, wood, roots, seeds, herbs and fruits). The highest levels of antibacterial activity were obtained with the use of EOs with substantial phenol or aldehyde content, such as cinnamaldehyde, citral, carvacrol, eugenol, or thymol (Cunha et al., 2018). This is followed by EOs that contain terpene alcohols. On the other hand, the activity levels of EOs containing ketones or esters, including β myrcene, α-thujone, or geranyl acetate, had been determined at significantly lower levels (Swamy et al., 2016). Zheng et al. (2005) established that volatile oils containing terpene hydrocarbons were usually inactive. We also know that EOs with high levels of phenolic compounds, including but not limited to carvacrol, eugenol, and thymol, hold significant antibacterial activities (Benzeria, 2006). Rosemary, cinnamon, lavender, black cumin and clove oils have shown antibacterial and antifungal effect (Ouattara et al., 1997;Cavanagh and Wilkinson, 2002;Soltani et al., 2014). Basil oil possess anti-inflammatory property (Singh and Majumdar, 1999). Peppermint oil has shown anticancer activity (Kumar et al., 2004). The in vitro antibacterial effect of some EOs have been determined against a different of Gram-negative and Gram-positive strains, including prominent pathogens of aquaculture such as Vagococcus salmoninarum (Metin and Biçer, 2020), Streptococcus iniae, Photobacterium damselae subspecies damselae, Aeromonas hydrophila (Gholipourkanani et al., 2019); Edwardsiella tarda, P. damselae, Vibrio harveyi, V. ichthyoenteri, L. garvieae, Streptococcus iniae and S. parauberis ; Yersinia ruckeri, Lactococcus garvieae, A. hydrophila, L. anguillarum (Ontas et al., 2016;Metin et al., 2017); Edwardsiella tarda, S. iniae, S. parauberis, L. garvieae, V. harveyi, V. ichthyoenteri and P. damselae (Park et al., 2016); S. agalactiae (Zhang et al., 2013).
Therefore, the aim of this study was to investigate the antibacterial effect of EOs (lavender, clove, peppermint, basil, rosemary, cinnamon and black cumin) on six fish pathogenic bacteria (Y. ruckeri, A. hydrophila, V. anguillarum, V. alginolyticus, L. garvieae and V. salmoninarum,) that are often the reason of bacterial fish infections in aquaculture.

Bacterial strains
EOs were evaluated against some bacterial strains. The bacterial strains obtained from the microorganism collection of Fisheries Faculty, Isparta University of Applied Sciences in Turkey. In this study, the bacterial strains used were already identified before (Y. ruckeri and A. hydrophila (Metin et al., 2017), V. anguillarum (Avsever et al., 2015), V. alginolyticus (Metin et al., 2017), L. garvieae (Altun et al., 2004) and V. salmoninarum  by using molecular methods. Also, the antibacterial effect of EOs were tested against Y. ruckeri, A. hydrophila, V. anguillarum, V. alginolyticus L. garvieae and V. salmoninarum (Table 1). Before using, each bacteria was identified again by using classical biochemical tests, API 20E, API 20 Strep and API 50 CH tests (Austin and Austin, 2007).

Antibacterial assay
Antibacterial activity was carried out by disc diffusion method (Andrews, 2004;CLSI, 2015). Briefly, bacteria suspensions were prepared as a fresh culture (24 h) and adjusted to 0.5 McFarland, which equals to 1.5x10 8 CFU mL -1 . Then inoculum (100 µL) of each test organism was spread with a sterile spreader on Mueller Hinton Agar (Merck, Darmstadt, Germany) plates. EOs were dissolved in methanol and sterilized by filtration through a 0.45 µm membrane filter. Under sterile conditions, blank steril discs (Merck, Darmstadt, Germany) were impregnated with 25 µL of different concentrations (1:1, 1:2, 1:4, 1:8, 1:16) of the EOs prepared and placed on the agar surface. In this test, as positive control oxytetracycline (20 µg) and negative control methanol were utilized. The incubation of the inoculated plates took place at 22-24°C for 24 hours then occurred inhibition zone was measured as millimeter (mm). Inhibition zones evaluated as >15 mm were declared as strong, from 8 to 15 mm as moderate, and from 1 to 8 mm as weak antibacterial activities (Bansemir et al., 2006).

Statistical analysis
The results were recorded as means ± standard error (SEM) and analyzed with SPSS version 14.0 (Tukey's comparison test using). Different letters in tables represent a significant difference at p<0.05.

Biochemical test results
To identify the bacterial isolates, this study used conventional biochemical tests and API  20E system (BioMerieux, France) and API 20 Strep test (Biomerieux, France). The results of the API tests was given in Table 2 and Table 3.
The results revealed that the plant EOs used exhibited antibacterial activity with varying values except for basil. The basil essential oil did not show any inhibition zone against bacterial strains. Therefore, it was not included in the tables. The EOs of lavender displayed zones of inhibition against pathogens respectively; V. salmoninarum (19.00 ± 0.57 mm), Y. ruckeri (15.66 ± 0.33 mm), A. hydrophila (15.33 ± 0.33 mm), V. alginolyticus (13.66 ± 0.88 mm), V. anguillarum (12.00 ± 0.57 mm). The minimum were 9.00 ± 0.57 mm inhibition zone produced against L. garvieae. It was determined that the lavender EO formed significantly high inhibition zone (Table 4) among the concentrations against V. salmoninarum (p˂0.05). In general, it was obtained that lavender EO had moderate effects on all bacterial strains.
In general clove oil EO showed the highest inhibitory effect against Y. ruckeri (21.00 ± 0.57 mm), A. hydrophila (22.00 ± 0.57 mm) and V. salmoninarum (20.66 ± 0.88 mm). Statistically, the best inhibition zone (Table 5) results of 1/1 dilution of clove EO were obtained against Y.ruckeri, A. hydrophila, and V. salmoninarum (p˂0.05). Especially it was determined that clove EO formed an  inhibition zone similar with OTC against A. hyprophila strain. Besides, it has been shown that the oil has strong and moderate effects against all bacterial strains. Peppermint EO was determined statistically different from other groups against V. salmoninarum (15.00 ± 0.57 mm) and was evaluated as a moderate effect (p˂0.05). Antibacterial activity of EO was found (Table 6) to have weak and moderate effects on the other isolates evaluated. Table 7 presents the inhibition zone formed by the rosemary EO against the fish pathogenic bacteria. The strongest activities statistically were determined by rosemary EO with inhibition zones of 22 mm against Y.ruckeri and A. hydrophila (p˂0.05) in 1/1 dilution. The minimum effects were at 11.66 mm zone inhibition observed against L. garvieae (Table 7). The rosemary EO showed strong activity against Y. ruckeri, A. hydrophila and V. salmoninarum while the rest of the strains were evaluated as moderate activity (p˂0.05). Also, similar inhibition zone was determined for both A. hydrophila strain and the positive control OTC.
The EO of cinnamon statistically exhibited maximum zone of inhibition against A. hydrophila (21.33 ± 0.88 mm) (p˂0.05). It showed strong activity (Table 8) on Y. ruckeri and A. hyrophila strains while other strains displayed moderate activity.
As presented in Table 9, it was determined that black cumin EO constitutes the highest inhibition zone against A. hydrophila strain (p˂0.05). It follows as strong activity on V. salmoninarum, Y. ruckeri and moderate effect on other strains.

Minimum Inhibition Concentration -MIC
Minimum Inhibition Concentration (MIC) of EOs (lavender, clove, peppermint, basil, rosemary, cinnamon and black cumin) which showed antibacterial activity were measured. MIC results for six selected oils ranged from 500 to 0.01 µL mL -1 . The results of minimum inhibitory concentration (MIC) of EOS were shown in Table 10.

L. garvieae V. salmoninarum
--Growth at 10°C + + Growth at 45°C + -Growth at 6.5% NaCl + -Vogues-Proskauer + ND Hippurate hydrolysis     of clove, rosemary, cinnamon and black cumin showed similar inhibition zones with OTC against A. hydrophila. All EOs except basil showed a wide range of antibacterial activity on used fish bacterial pathogens. Ontas et al. (2016) determined the antibacterial effects EOs of lemon peel (Citrus limon) and argan (Argania spinosa). Both EOs possessed significant antibacterial activity against Y. ruckeri, A. hydrophila, L. anguillarum, Edwarsiella tarda, Citrobacter freundii and L. garvieae. In that study, four pathogens (Y. ruckeri, A. hydrophila, L. anguillarum) inhibited by EOs of lemon, and argan were evaluated as strong activity (17-19 mm) except L. garvieae which showed moderate activity (10.33-11.33 mm). In another study antibacterial effect of different nano-encapsulated herbal EOs against fish pathogenic bacterial strains (A. hydrophila, S. iniae and P. damselae subspecies damselae) have been studied by Gholipourkanani et al. (2019). Their results on A. hydrophila showed that EOs possessed strong antibacterial effects against fish pathogenic bacteria. Hossain et al. (2018) reported that EO of Zingiber officinale showed antibacterial effect on Gram positive bacteria (L. garvieae) 13 mm inhibition zone. In this study, 12 mm of inhibition zone was determined against L. garvieae caused by cinnamon EO. It gave moderate antibacterial activity between all used EOs like given studies. Wimalasena et al. (2018) documented that L. garvieae survival rate inhibited by lavender EO (19 mm), in this study, it was found that lavander EO had moderate activity as 9 mm inhibition zone on the same pathogen. Park et al. (2016) obtained that L. garvieae growth was inhibited by Eucalyptus globulus EO at 15-24 mm. It seems that Eucalyptus EO has a strong effect on L. garvieae. These results showed that the use EOs on the bacteria causes different level of antibacterial activity. Yıldırım and Turker (2018) examined the antibacterial effects of 24 EOs against A. hydrophila, V. anguillarum, Y. ruckeri, Table 8. Antibacterial activity (mean ± standard error) of cinnamon EO against different bacterial fish pathogens (the diameter of the zone of inhibition, mm).

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L. garvieae determined that EOs showed high antibacterial activity against these bacteria. They reported that peppermint, rosemary, and lavander EOs possess strong antibacterial activity both Gram negative and Gram positive bacteria. A parallel result was found by Metin et al. (2017) in their study on the antibacterial activity of essential oil (Eugenia caryophyllata, M. piperita and Lavandula hybrida) determined that EOs incite effective strong antibacterial activity against A. salmonicida subsp. achromogenes, A. hydrophila, V. anguillarum, Y. ruckeri and L. garvieae. Adel et al. (2016) reported that five EOs (Eryngium campestre, Pimpinella affinis, Mentha piperita, Achillea wilhelmsii and Cuminum cyminum) showed an antibacterial activity on Y. ruckeri pathogen. Peppermint EO exhibited 21.6 mm inhibition zone compared to the present study the EO rosemary (22 mm) caused similar results on Y. ruckeri pathogen. In another research, Ozogul et al. (2015) presented that rosemary EOs antimicrobial effect on A. hydrophila obtained as 11.5 mm inhibition zone. Whereas in this work, the same EO is measured as 22 mm on A. hydrophila. It showed strong antibacterial activity in this study. The reason of that is possible to use different concentrations on obtaining of antibacterial activity or using different EOs.
Antibacterial activity of rosemary, clove, peppermint, lavender and black cumin on V. salmoninarum have been searched by Metin and Bicer (2020) in which the diameters of inhibition zone were determined as 18.66 mm for clove, and peppermint as 7.3 mm. However, other EOs did not show any effect on the bacteria. In this paper, the antibacterial effects of EOs on V. salmoninarum were obtained for clove (20.66 mm), rosemary (20.33 mm), lavender (19 mm), black cumin (18.66 mm) and peppermint (15 mm). As it can be seen, five out of seven EOs showed strong activity on V. salmoninarum pathogen.
Minimum Inhibition Concentration (MIC) values obtained in this study were between 500 and 62.5 µL mL -1 on both Gram negative and Gram positive pathogen bacteria. MIC was measured minumum as 62.5 µL mL -1 on Y. ruckeri (lavender, clove, and rosemary EOs), A. hydrophila (rosemary and cinnamon EOs) and V. salmoninarum (clove and rosemary EOs). The V. salmoninarum pathogen exhibited 1000-250 µL mL -1 MIC values by using EOs of clove, peppermint, lavender and rosemary. In other papers, MIC values changing between 31.25-1000 µL mL -1 Ontas et al., 2016). The MIC values of EOs in present study on fish pathogens appear to be between maximum and minimum range among the published studies.
In general, results of antibacterial activity of essential oils presented in this study showed different values in the existing literature. They might be affected by factors such as the concentration of EOs, method differences, and the origins of bacteria. When these EOs were evaluated, it was detected that lavender, clove, rosemary and cinnamon EOs showed strong antibacterial activity among pathogens against Gram negative (Y.ruckeri and A. hydrophila) and Gram positive (V. salmoninarum) bacteria. Although they have not been completely investigated, the present study results reveal the great potential of plants for therapeutic in line with the documents on EOs from the literature.

CONCLUSIONS
In conclusion, the antibacterial activities of commercially available EOs were investigated. The results obtained from this study showed that the highest antimicrobial activity was determined in clove, cinnamon and rosemary essential oils and they inhibited the growth of both Gram negative and Gram positive bacteria. In addition, the EOs of clove, rosemary, cinnamon and black cumin showed similar inhibition zones with OTC against A. hydrophila. In the next study, the plan will be continued on in vivo studies of preventative properties of these essential oils on fish.