In vitro antimicrobial screening of the ethanolic extract obtained from leaves of ficus platypoda (miq.) A. Cunn. Ex miq. (moraceae) against fish bacterial pathogens

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Institute of Biology and Environmental Protection, Pomeranian University in Slupsk

In vitro antimicrobial screening of the ethanolic extract obtained from leaves of ficus platypoda (miq.) A. Cunn. Ex miq. (moraceae) against fish bacterial pathogens

Aquaculture has expanded rapidly to become a major economic and food- producing sector in the world, and is expected to grow further to meet the future demand [5, 14]. In the medium term, increased output is likely to require expansion in new environments, further intensification and efficiency gains for more sustainable and cost-effective production. The trend towards enhanced intensive systems with key monocultures remains strong and, at least for the foreseeable future, will be a significant contributor to future supplies. Dependence on external feeds (including fish), water and energy are key issues [5]. Due to the intensification of rearing methods and systems, the industry has been overwhelmed with a number of trans-boundary aquatic animal diseases caused by viruses, bacteria, fungi and parasites, with newer pathogens being identified every year [14]. Aquacultural practices frequently result in high population densities and other stresses (such as intercurrent disease). These practices increase the risk of infection establishment and spread. As aquaculture expands and new species are farmed, diseases will continue to emerge and affect both wild and farmed fish adversely. The rate and extent of emergence can be reduced by the application of biosecurity programmes designed to mitigate the risk factors for disease emergence [16].

To control disease outbreaks, several chemotherapeutics and antibiotics have been used indiscriminately, which in turn have led to residual problems affecting the environment and human health. Medicinal plants used as natural and innocuous compounds are of high value and have potential in aquaculture [10, 35]. A growing interest has emerged in the effects of various medicinal plants, including Chinese herbs, used as dietary supplements, on the growth, disease resistance, and immune response in different aquatic animals [1]. More than 60 different medicinal plant species have been studied for the improvement of fish health and disease management in aquaculture [35].

Genus Ficus L. (Moraceae) are an important medicinal plant product and can play a major role in preventing or controlling infectious microbes in fish culture. The family Moraceae traditionally treated as a member of the order Urticales comprises 37 genera and 1050-1100 species worldwide, most of which are distributed in the tropics and subtropics. The Moraceae are largely woody and highly diverse in the morphology of both vegetative and reproductive structures. They include terrestrial trees, shrubs, climbers, hemi-epiphytes, and rarely subshrubs and herbs. The pantropical genus Ficus L. is by far the largest in the family and includes approximately 750 species with the hotspot of species richness in SE Asia. The genus is represented by evergreen and (semi)deciduous trees, shrubs, and lianas, often with aerial adventitious roots. More than a half of the species are hemi-epiphytes that start life as an epiphyte in the crown of another tree and then send roots down to the ground enveloping the trunk of the host tree. In the most extreme case, the adventitious roots growing down from the branches form additional woody trunks, enabling the tree to spread out laterally and cover a wide area (the banyan-type tree) [4, 6, 7].

Ficusplatypoda (Miq.) A. Cunn. ex Miq. is a monoecious evergreen lithophytic tree reaching up to 10 m in height, with hyaline and ferruginous hairs covering leaves and leafy twigs (Photo 1). The species is naturally distributed in northwestern Australia where it normally restricted to sandstone outcrops, but is occasionally found on limestone. Its leaves are 5-17 cm long and 3-13 cm wide, alternate, ovate to widely elliptic, with acute to rounded apex and cordate to truncate or obtuse base. Figs are 1 -3 cm in diameter, pedunculate and pilose, spheroid to broadly ellipsoid, at maturity pale yellow to orange, pink, red, or purple [8].

Some biological studies indicate that leaves of Ficus species exhibit anti-ulcer, hypotensive, hypoglycemic, hypolipidemic, anti-inflammatory, anxiolytic, anticonvulsant, antinociceptive, antipyretic, anti-microbial, anti-candidal, insecticidal, pesticidal activities, antiarthritic and antioxidant activity [21]. Moreover, in our previous studies, therapeutic potential for the use of various plants of Ficus genus in the control of bacterial diseases were evaluated against fish pathogens (Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri) in in vitro study with promising results [25-33].

Therefore, the aim of this study was to assess the antibacterial efficacy of etha- nolic extract obtained from leaves of Ficus platypoda against fish pathogens, Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri and to evaluate the possible use of this plant in preventing infections caused by these bacteria in aquaculture.

Photo 1. A specimen of Ficus platypoda (Miq.) A. Cunn. ex Miq. grown at exhibition glasshouse at Botanical Garden of Ivan Franko National University in Lviv (Ukraine) (A) and leaf morphology of F. platypoda (adaxial leaf surface) (B).

Materials and methods. The leaves of F. platypoda were sampled in at Botanical Garden of Ivan Franko National University in Lviv, Ukraine. The sampled leaves of F. platypoda were brought into the laboratory for antimicrobial studies. Freshly crushed leaves were washed, weighted, and homogenized in 96 % ethanol (in proportion 1:10) at room temperature, and centrifuged at 3,000 g for 5 minutes. Supernatants were stored at -20 °C in bottles protected with laminated paper until required.

Aeromonas hydrophila (strain E 2/7/15) and Pseudomonas fluorescens (strain E 1/7/15) isolated locally from internal organs of rainbow trout (Oncorhynchus mykiss Walbaum) with clinical features of furunculosis (kidneys were gray, liver was pale and fragile, enlarged spleen with exudate in the body cavity), as well as Citrobacter freundii isolated locally from gill of eel (Anguilla anguilla L.) with clinical features of disease were used as test organisms. Samples of internal organs (kidneys, spleen, liver) weighting 2 g were taken and homogenized before preincubation in TSB broth (Tripti- case Soy Broth, Oxoid) for 24 hrs. After preincubation, bacterial culture was transferred to two different cultivation media: TSA (Tripticase Soy Agar, Oxoid) and BHIA (Brain Heart Infusion Agar, Oxoid) supplemented with 5 % of sheep blood (OIE Fish Diseases Commission, 2000). After 48 hrs of incubation at 27 °C, characteristic pink colonies were selected for further examination.

The isolates of Y. ruckeri were collected from clinically healthy fish and fish with clinical symptoms of yersiniosis. Internal tissues (predominantly pronephros and gills) as well as intestinal swabs were sampled. Tissue samples were homogenized and inoculated on nutritional agar with 5% blood (Columbia Blood Agar, Oxoid). Following a 24 h incubation period at 25 °C ± 2 °C, distinctive colonies were transferred onto TSA. Round, elevated, shining and whitish colonies without haemolytic properties were considered typical of Y. ruckeri. After 24h-incubation at 25 °C ± 2 °C, an oxidase test and Gram-staining were performed. Gram-negative and oxidase-negative isolates were cultured on TSA medium and incubated for 24 h at 25 °C ± 2 °C.

Bacterial species were identified with the use of the oxidase test and API E test kit (BioMerieux, France). The results of the test were interpreted in accordance with the manufacturer's protocol, after 24 hrs of incubation at 27 °C. Codes ++V-V--+V+++-- +-VV+ in API E test were identified as A. hydrophila. The strain was obtained from Diagnostics Laboratory of Fish and Crayfish Diseases, Department of Veterinary Hygiene, Provincial Veterinary Inspectorate in Olsztyn (Poland). For characterization of Y. ruckeri isolates, bacteria were Gram-stained and then morphologically evaluated. 24h bacterial culture was wet-mounted and a microscopic smear on the slide was prepared. Following fixation over the flame, the slide was Gram- stained with a Gram colour set (Merck) according to the manufacturer's instructions. The shape of the bacteria was determined by observing the microorganisms under a light microscope at 1000x with immersion oil [11, 36]. Motility was examined on a wet mount. A drop of distilled water was put on a cover slip and bacteria were mounted on it with drops of distilled water put on the corners of a slip. The slip was then covered with a special microscopic slide with an indentation and the whole set was vigorously turned. The motility of the bacteria was evaluated under a light microscope at 400x [11, 36].

Oxidase test was performed according to manufacturer's instruction (Merck). Biochemical properties of individual Y. ruckeri isolates were investigated with the API 20E system (BioMerieux, France). Tests were performed according to the manufacturer's instructions. The results, namely, the presence or a lack of reaction, were read based on the key featured in the operating procedure provided by the manufacturer of the assay. The results were analysed with the Apiweb software (BioMerieux, France) to identify the investigated bacterium.

Strains tested were plated on TSA medium (Tryptone Soya Agar) and incubated for 24 hrs at 25 °C. Then the suspension of microorganisms was suspended in sterile PBS and the turbidity adjusted equivalent to that of a 0.5 McFarland standard. The disc diffusion assay (Kirby-Bauer Method) was used to screen for antibacterial activity [3]. Muller-Hinton agar plates were inoculated with 200 and 400 pL of standardized inoculum (108 CFU/mL) of bacterium and spread with sterile swabs.

Sterile filter paper discs impregnated by extract were applied over each of the culture plates, 15 min after bacteria suspension was placed. The antimicrobial susceptibility testing was done on Muller-Hinton agar by disc diffusion method (Kirby-Bauer disk diffusion susceptibility test protocol). A negative control disc impregnated by sterile ethanol was used in each experiment. The sensitivity of strain was also studied to the commercial preparation with extracts of garlic (in dilution 1:10, 1:100 and 1:1000). After culturing bacteria on Mueller-Hinton agar, the disks were placed on the same plates and incubated for 24 hrs at 25°C. The diameters of the inhibition zones were measured in millimeters, and compared with those of the control and standard susceptibility disks. Activity was evidenced by the presence of a zone of inhibition surrounding the well.

Fig. 1. Antimicrobial activity of ethanolic extracts obtained from F. platypoda against Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yer¬sinia ruckeri. Muller-Hinton agar plates inoculated with 200 and 400 ^L of standard¬ized inoculum (10 CFU/mL) of bacterium.

Each test was repeated six times and the average values of antimicrobial activity were calculated. All statistical calculation was performed on separate data from each species with STATISTICA 8.0 software (StatSoft, Poland) [37]. The following zone diameter criteria were used to assign susceptibility or resistance of bacteria to the phytochemicals tested: Susceptible (S) > 15 mm, Intermediate (I) = 11-14 mm, and Resistant (R) < 10 mm [17].

Research result. Antimicrobial activity of ethanolic extracts obtained from F. platypoda against Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluo- rescens, Yersinia ruckeri expressed as mean of diameters of inhibition zone is presented in Figs 1-5.

In our study, the A. hydrophila strain (200 and 400 pl of standardized inoculum) revealed intermediate susceptibility to ethanolic extract obtained from leaves of F. platypoda (inhibition zone diameters ranged from 8.5 to 11.5 mm) (Figs 1, 2).

Fig. 2. Antimicrobial activity of ethanolic extracts obtained from F. platypoda (54) against Aeromonas hydrophila. Muller-Hinton agar plates inoculated with 200 (A) and 400 ^L of standardized inoculum (10 CFU/mL) of bacterium (B).

Our results also demonstrated that the C. freundii (200 and 400 pl of standardized inoculum) was resistant to ethanolic extract obtained from leaves of F. platypoda (inhibition zone diameters were ranged between 6 and 9.5 mm) (Fig. 3).

Fig. 3. Antimicrobial activity of ethanolic extracts obtained from F. platypoda (54) against Citrobacter freundii. Muller-Hinton agar plates inoculated with 200 (A) and 400 ^L of standardized inoculum (10 CFU/mL) of bacterium (B).

Ethanolic extract derived from F. platypoda leaves exhibited intermediate antibacterial activity against Pseudomonas fluorescens causing a zone of inhibition, comprising at least 10.5-11.4 mm for 200 pL and 7-8.4 mm for 400 pL of standardized inoculum (108 CFU/mL) of bacterium strain (Figs 1, 4).

Fig. 4. Antimicrobial activity of ethanolic extracts obtained from F. platypoda (54) against Pseudomonas fluorescens. Muller-Hinton agar plates inoculated with 200 (A) and 400 pL of standardized inoculum (10 CFU/mL) of bacterium (B).

Y. ruckeri (200 and 400 pl of standardized inoculum) revealed intermediate susceptibility to ethanolic extract obtained from leaves of F. platypoda (inhibition zone diameters were ranged between 8.1 and 12.2 mm) (Fig. 5).

Fig. 5. Antimicrobial activity of ethanolic extracts obtained from F. platypoda (54) against Yersinia ruckeri. Muller-Hinton agar plates inoculated with 200 (A) and 400 pL of standardized inoculum (10 CFU/mL) of bacterium (B).

The antimicrobial activity profile of ethanolic extract obtained from leaves of F. platypoda against the tested pathogen strains indicated that P. fluorescens and Y. ruckeri was the most susceptible bacteria (200 pl of standardized inoculum) among all the bacterial test strains (Figs 1, 4, 5). A. hydrophila was found less sensitive strain (inhibition zone diameters ranged from 8.5 to 11.5 mm) and C. freundii was found to be least susceptible to ethanolic extract obtained from leaves of F. platypoda (Figs 1-4).

The comprehensive review of usefulness of some medicinal plants (herbal drugs) against fish diseases has been presented by many researchers [19, 20, 22, 23]. In our previous studies, therapeutic potential for the use of various plants of Ficus genus in the control of bacterial diseases were evaluated against fish pathogens in in vitro study with promising results [25-33]. Antibacterial properties of plant extracts have been by far the most studied bioactivity with potential application in aquaculture systems [19].

This investigation is in line with our previous works which have revealed a great potential of Ficus species as plants with potent antimicrobial properties. In our previous study, the in vitro antimicrobial activity of the ethanolic leaf extracts of various Ficus species against Citrobacter freundii was evaluated. The results proved that the extracts from F. drupacea, F. septica, F. deltoidea as well as F. hispida, F. mucuso, F. pumila, F. craterostoma exhibit a favorable antibacterial activity against C. freundii (200 pL of standardized inoculum) [28]. Our results also proved that the ethanolic extracts obtained from F. pumila, F. binnendijkii `Amstel Gold', F. carica, F. erecta, F. hispida, F. mu- cuso, F. palmeri, F. religiosa possess considerably sufficient antibacterial potential against C. freundii. Among various species of Ficus screened ethanolic extracts of the leaves of ten Ficus species: F. hispida, F. binnendijkii, F. pumila, F. rubiginosa, F. erecta, F. erecta var. sieboldii, F. sur, F. benjamina, F. craterostoma, F. lyrata, F. palmeri (the species are listed in the order of effectiveness against pathogen tested) were the most effective against P. fluorescens (200 pL of standardized inoculum) [27]. Moreover, previous investigation has shown that the most effective against P. fluorescens (400 pL of standardized inoculum) were the ethanolic extracts obtained from leaves of ten Ficus species: F. craterostoma, F. cyathistipula, F. drupacea `Black Velvet', F. hispida, F. macrophylla, F. mucuso, F. pumila, F. villosa. In our study, most ethanolic extracts obtained from Ficus spp. proved effective against the bacterial strain of Gram-negative A. hydrophila tested, with 10-12 mm zones of inhibition being observed. A. hydrophila demonstrated the highest susceptibility to F. pumila. The highest antibacterial activity against A. hydrophila (200 pL of standardized inoculum) was displayed by F. benghalensis, F. benjamina, F. deltoidea, F. hispida, F. lyrata leaf extracts. Among various species of Ficus genus exhibiting moderate activity against

A. hydrophila (400 pL of standardized inoculum), the highest antibacterial activity was displayed by F. benghalensis, F. benjamina, F. deltoidea, F. hispida, F. lyrata leaf extracts [29, 32].

The preliminary screening assay indicated that F. hispida leaves extract possess great potential for the therapy of bacterial infections and may be used as a natural antiseptic and antimicrobial agent [30]. Our results from the disc diffusion assay indicated that the A. hydrophila revealed intermediate susceptibility concerning to ethanolic extract obtained from leaves of F. hispida (inhibition zone diameters were ranged from 8 to 12 mm). The most effective at least causing a zone of inhibition 14-16 mm was ethanolic extract from F. hispida against P. fluorescens both in 200 pL of standardized inoculum of bacterium (inhibition zone diameters were ranged from 15 to 16 mm) and 400 pL (14-15 mm). Our results demonstrated that the C. freundii revealed intermediate susceptibility F. hispida (inhibition zone diameters were ranged between 11 and 15 mm) [30].

In line with these general findings, there are copious evidences that various species of genus Ficus exhibit antimicrobial properties against broad spectrum of microorganisms. The scientific research on Ficus spp. indicated that these plants have received increasing interest in recent years. It is well documented that various Ficus spp. have been used against Gram-positive and Gram-negative bacteria [21]. For instance, Valsa- raj and co-workers (1997) evaluated activity of ethanolic extracts from F. benghalensis aerial roots and F. religiosa leaves, among a large number of plants, against four bacterial strains (Bacillus subtilis ATCC 6633, Escherichia coli ATCC 11229, Pseudomonas aeruginosa ATCC 9027, and Staphylococcus aureus ATCC 6538) and two fungi (Aspergillus niger IMI 076837 and Candida albicans IMI 349010), using the agar dilution method for the former and agar-well diffusion method for the latter. Although both Ficus extracts showed inhibitory activity against bacteria, none of them affected the fungal species tested [34].

Farrukh and Ahmad (2003) investigated antimicrobial activity of ethanolic extracts of 22 Indian medicinal plant species against seven bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, S. paratyphi, S. typhi, Shigella dys- enteriae, and Staphylococcus aureus) and five filamentous fungi species (Alternaria alternata, Aspergillus niger, Fusarium chlamydosporum, Rhizoctonia bataticola, and Trichoderma viride). Of these, extracts from F. carica fruits and F. religiosa leaves showed generally weak inhibition with higher expression of antibacterial action. With respect to C. albicans both extracts were active and produced inhibition zone of 1015 mm in diameter. No inhibition of any organism tested caused the extracts from F. rumphii and F. benghalensis leaves [9].

Rajiv and Rajeshwari (2012) screened antimicrobial activity of F. religiosa bark, leaf, stem, and fruit aqueous extracts against a number of major pathogens (A. hydrophi- la, Enterobacter aerogenes, E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Aspergillus niger, and Candida albicans) and conducted their phytochemical analysis. All tested extracts appeared active against the pathogens at concentrations 25-100 mg/ml, the widest inhibition zone (15-16 mm) resulting from the highest concentration. Fruit extract showed generally the weakest activity and only the leaf extract affected the whole set of tested organisms at maximal concentration. Antifungal properties of the extracts were generally weaker pronounced than antibacterial ones. All extracts at all concentrations tested affected C. albicans, although the strongest inhibition showed the maximal concentration extracts from stems and fruits (inhibition zone diameter 15 mm) and slighter effect was produced by leaf (12 mm) and bark (11 mm) extracts. Qualitative phytochemical analysis showed the bark extract to have the richest chemical composition (sugar, alkaloids, phenols, and tannins present), being poorer in fruits (phenols and flavonoids), stem (sugar and tannins), and leaves (only tannins). Glycosides and terpenoids faeatured all extracts tested. Hence the most specific chemicals appeared to be alkaloids (found only in bark) and flavonoids (only in fruits), while tannins were common for the plant parts with the highest antimicrobial activity in general (i.e., bark, leaves, and stem). Although the authors present the results of phytochemical analysis, they do not make any inferences concerning possible contribution of particular chemical classes to the antimicrobial activity of plant extracts [18].

Atindehou and co-workers (2002) tested crude ethanol extracts from 115 plant species against Gram-negative bacteria (E. coli and Pseudomonas aeruginosa), Grampositive bacteria (Enterococcus faecalis and Staphylococcus aureus), and fungi (Candida albicans and Cladosporium cucumerinum). Among the examined plants, there were three Ficus species, namely F. exasperata, F. mucuso, and F. sur. The Gramnegative bacteria appeared unaffected by any plant extract tested, whereas the Grampositive bacteria and fungi were inhibited by at least several plant species. Among Ficus species tested, F. exasperata and F. mucuso had no significant effect on any microorganism, while F. sur appeared among the most active plant species against Grampositive bacteria. No effect was shown for F. sur against C. albicans [2].

Kubmarawa and co-workers (2007) carried out an antimicrobial and phytochemical screening of 50 Nigerian plant species ethanolic extracts, among which were five species of Ficus (i.e., F. abutifolia (Miq.) Miq., F. platyphylla Del., F. polita Vahl, F. sycomorus L., and F. thonningii Blume). Microbial strains used in the study were Bacillus subtilis NCTC 8236, E. coli ATCC 9637, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 13709, and Candida albicans ATCC 10231. Ficus stem bark extracts demonstrated comparatively low antimicrobial activity, with the broadest activity spectrum being of F. thonningii extract (active against all microorganisms except P. aeruginosa and S. aureus). Extracts from F. polita and F. sycomorus showed no activity at all. C. albicans showed generally low susceptibility compared to other organisms tested. Among Ficus species, only F. thonningii extract affected C. albicans (with MIC value of 1.0 mg/ml). Phytochemical analysis revealed the presence of only saponins and volatile oil in F. thonningii extract and saponins and flavonoids in F. polita extract, while richer chemical content was found in F. abutifolia (tannins, alkaloids, and volatile oil), F. platyphylla (saponins, flavonoids, alkaloids, and volatile oil), and F. sycomorus (glycosides, tannins, flavonoids, and volatile oil) extracts. However, the authors do not make any speculations regarding the contribution of particular chemical classes to antimicrobial activity of plant extracts tested. Authors also suggest the presence of some compound classes (such as alkaloids) in plants to be affected by climatic and environmental factors [12].

Maregesi and co-workers (2008) screened n-hexane, methanol, and water extracts from 39 plant species of 22 families against a number of bacteria (Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 15442, Klebsiella pneumoniae ATCC 13883, and Salmonella typhimurium ATCC 13311), fungi (Aspergillus niger ATCC 16404 and Candida albicans ATCC 10231), and viruses (Herpes simplex Virus type 1, Vesicular Stomatitis Virus T2, Semliki forest A7, and Coxsackie B2). Gram-positive bacteria generally appeared more sensitive to the extracts than Gram-neative ones. Stem bark extracts of F. sycomorus, the only Ficus species examined, were active against Gram-positive bacteria and fungi with MIC 1000 pg/ml and higher, while they showed comparatively strong antiviral activity. C. albicans was inhibited by F. sycomorus n-hexane extract with MIC 1000 pg/ml, whereas by the methanol and water extracts with higher MIC values [15].

Consequently, the antimicrobial property of F. benghalensis leaf extract may be manifested due to its constituents. Antibacterial flavonoids might be having multiple cellular targets, rather than one specific site of action [13]. One of their molecular actions is to form complex with proteins through nonspecific forces such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation. Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhe- sins, enzymes, cell envelope transport proteins, and so forth. Lipophilic flavonoids may also disrupt microbial membranes [13]. It is reported that flavones have been used as Efflux pump inhibitors (EPIs). Flavones also exhibit bactericidal activity by interference with iDNA synthesis. A series of flavones was studied for their DNA-gyrase inhibitory activities. It was proposed that the ring-B of flavones is involved in intercalation or hydrogen bonding with the stacking of nucleic acid bases, thus imparting inhibitory action on DNA and RNA synthesis [24]. antimicrobial bacteria fish infection

Conclusions

1. In summary, ethanolic extract obtained from leaves of F. platypoda investigated possessed antimicrobial activity against fish pathogens. A. hydrophila strain (200 and 400 pl of standardized inoculum) revealed intermediate susceptibility (inhibition zone diameters ranged from 8.5 to 11.5 mm). C. freundii (200 and 400 pl of standardized inoculum) was resistant to ethanolic extract (inhibition zone diameters were ranged between 6 and 9.5 mm).

2. Ethanolic extract derived from F. platypoda leaves exhibited intermediate antibacterial activity against Pseudomonas fluorescens causing a zone of inhibition, comprising at least 10.5-11.4 mm for 200 pL and 7-8.4 mm for 400 pL of standardized inoculum of bacterium strain. Y. ruckeri (200 and 400 pl of standardized inoculum) revealed intermediate susceptibility to ethanolic extract (inhibition zone diameters were ranged between 8.1 and 12.2 mm).

3. The results also indicate that scientific studies carried out on medicinal plants having traditional claims of effectiveness might warrant fruitful results in aquaculture.

4. Further studies aimed at the isolation and identification of active substances from the ethanolic fractions of various Ficus species could also disclose compounds with better antibacterial value. It is believed that screening of all the investigated plants for other biological activities including immunostimulating and antioxidant activities is essential in aquaculture practice.

Acknowledgements. This study was carried out during Olha Kasiyan's Scholarship Program supported by The International Visegrad Fund in the Department of Zoology and Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University in Slupsk (Poland). We thank to The International Visegrad Fundfor the supporting our study.

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Abstract

Ficus platypoda (Miq.) A. Cunn. ex Miq. were screened for antimicrobial activity against different strains of bacteria which are known to cause different types of fish infections, i.e. Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri. The tests were carried out using agar well diffusion method with the use of crude ethanolic extract. Muller-Hinton agar plates were inoculated with 200 and 400 pL of standardized inoculum (108 CFU/mL) of bacterium and spread with sterile swabs. Aeromonas hydrophila ( strain E 2/7/15) isolated locally from gills of rainbow trout (Oncorhynchus mykiss Walbaum) and Pseudomonas fluorescens (strain E 1/7/15) isolated locally from internal organs of rainbow trout with clinical features offurunculosis, Citrobacter freundii isolated locally from gills of eel (Anguilla anguilla L.) with clinical features of disease, as well as Yersinia ruckeri collected from clinically healthy fish andfish with clinical symptoms of yersiniosis were used as test organisms. Our results showed that ethanolic extract obtained from leaves of F. platypoda investigated possessed antimicrobial activity against fish pathogens. A. hydrophila strain (200 and 400 pl of standardized inoculum) revealed intermediate susceptibility (inhibition zone diameters ranged from 8.5 to 11.5 mm). C. freundii (200 and 400 pl of standardized inoculum) was resistant to ethanolic extract (inhibition zone diameters were ranged between 6 and 9.5 mm).

Ethanolic extract derived from F. platypoda leaves exhibited intermediate antibacterial activity against P. fluorescens causing a zone of inhibition, comprising at least 10.5-11.4 mm for 200 pL and 7-8.4 mm for 400 pL of standardized inoculum of bacterium strain. Y. ruckeri (200 and 400 pl of standardized inoculum) revealed intermediate susceptibility to ethanolic extract (inhibition zone diameters were ranged between 8.1 and 12.2 mm). The results also indicate that scientific studies carried out on medicinal plants having traditional claims of effectiveness might warrant fruitful results in aquaculture. Further studies aimed at the isolation and identification of active substances from the ethanolic fractions of various Ficus species could also disclose compounds with better antibacterial value. It is believed that screening of all the investigated plants for other biological activities including immunostimulating and antioxidant activities is essential in aquaculture practice.

Keywords: Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluo- rescens, Yersinia ruckeri, antimicrobial activity, disc diffusion technique, ethanolic extract.

Целью этого исследования была оценка антимикробной эффективности этанольного экстракта, полученного из листьев Ficusplatypoda (Miq.) A. Cunn. ex Miq. относительно бактериальных патогенов рыб (Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri) и возможность применения этого растения для предотвращения инфекций, вызванных этими бактериями в аквакультуре. Листья F. platypoda были собраны в Ботаническом саду Львовского национального университета им. Ивана Франко (Львов, Украина). Свежие листья промывали, измельчали, взвешивали и гомогенизировали в 96 %-ном этаноле для получения 10 % экстрактов. Aeromonas hydrophila (штамм E 2/7/15), Pseudomonas fluorescens (штамм E 1/7/15), а также Yersinia ruckeri выделенные из внутренних органов радужной форели (Oncorhynchus mykiss Walbaum) с клиническими признаками фурункулеза и иерсиниоза. Citrobacter freundii выделен из внутренних органов угря (Anguilla anguilla L.) с клиническими признаками заболевания.

Эти штаммы мы использовали в этом исследовании как тестовые в диско-диффузионном методе Кирби-Бауэра (1966). В чашки с агаром Muller-Hinton инокулировали 200 и 400 мкл посевного материала (108 КОЕ/мл) бактерий. Наши результаты показали, что этанольный экстракт, полученный из листьев F. platypoda, обладает средней антимикробной активностью относительно патогенных рыб. У штамма A. hydrophila (200 и 400 мкл посевного материала) выявлена промежуточная восприимчивость (диаметры зон ингибирования колеблются от 8,5 до 11,5 мм). Штамм C. freundii (200 и 400 мкл посевного материала) был устойчив к этанольному экстракту (диаметры зоны ингибирования варьировались от 6 до 9,5 мм). Этанольный экстракт, полученный из листьев F. platypoda, проявлял промежуточную антибактериальную активность относительно P. fluorescens (диаметр зоны ингибирования роста штамма составлял 10,5-11,4 мм для 200 мкл посевного материала и 7-8,4 мм для 400 мкл посевного материала). Изолят Y. ruckeri (200 и 400 мкл посевного материала) проявил промежуточную восприимчивость к этанольному экстракту, полученному из листьев F. platypoda (диаметры зон ингибирования варьировались от 8,1 до мм).

Результаты также показывают, что научные исследования лекарственных растений могут обеспечить плодотворные результаты в аквакультуре в дальнейших перспективах. Будущие исследования, направленные на выделение и идентификацию активных веществ из этанольных фракций различных видов Ficus, помогут также изучить соединения с лучшей антибактериальной ценностью. Скрининг всех исследуемых растений относительно других видов биологической активности, включая иммуностимулирующую и антиоксидантную, будет иметь важное значение в практической аквакультуре.

Ключевые слова: Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri, антимикробная активность, диско-диффузионный метод Байера-Кирби, этанольный экстракт

Метою цього дослідження була оцінка антимікробної ефективності ета- нольного екстракту, отриманого з листя Ficus platypoda (Miq.) A. Cunn. ex Miq. щодо бактеріальних патогенів риб (Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri) і можливість застосування цієї рослини для запобігання інфекцій, викликаних цими бактеріями в аквакультурі. Листя F. platypoda були зібрані в Ботанічному саду Львівського національного університету ім. Івана Франка (Львів, Україна). Свіже листя промивали, подрібнювали, зважували і гомогенізували в 96 %-ному етанолі для отримання 10 % екстрактів. Aeromonas hydrophila (штам E 2/7/15), Pseudomonas fluorescens (штам E 1/7/15), а також Yersinia ruckeri були виділені з внутрішніх органів райдужної форелі (Oncorhynchus mykiss Walbaum) з клінічними ознаками фурункульозу і ієрсиніозу. Citrobacter freundii був ізольований з внутрішніх органів вугра (Anguilla anguilla L.) з клінічними ознаками захворювання. Ці штами ми використовували в цьому дослідженні як тестові в диско-дифузійному методі Кірбі-Бауера (1966). У чашки з агаром Muller-Hinton наносили 200 і 400 мкл посівного матеріалу (108 КУО/мл).

Наші результати показали, що етанольний екстракт, отриманий з листя F. platypoda, проявив середню антимікробну активність щодо патогенів риб. У штаму A. hydrophila (200 і 400 мкл посівного матеріалу) виявлена проміжна антимікробна чутливість (діаметри зон інгібування росту коливалися від 8,5 до 11,5 мм). Штам C. freundii (200 і 400 мкл посівного матеріалу) був стійкий до етанольного екстракту (діаметри зон інгібування варіювалися від 6 до 9,5 мм). Етанольний екстракт, отриманий з листя F. platypoda, проявив середню антибактеріальну активність щодо P. fluorescens (діаметр зон інгібування росту штаму становив 10,5-11,4 мм для 200 мкл інокулята і 7-8,4 мм для 400 мкл інокулята бактеріального штаму). Ізолят Y. ruckeri (200 і 400 мкл посівного матеріалу) виявив середню чутливість щодо екстракту, отриманого з листя F. platypoda (діаметри зон варіювалися від 8,1 до 12,2 мм).

Результати також показують, що наукові дослідження лікарських рослин можуть забезпечити плідні результати в аквакультурі в подальших перспективах. Майбутні дослідження, спрямовані на виділення і ідентифікацію активних речовин з етаноль- них фракцій різних видів Ficus, допоможуть також дослідити сполуки з кращою антибактеріальною цінністю. Скринінг всіх досліджуваних рослин щодо інших видів біологічної активності, включаючи імуностимулюючу і антиоксидантну, матиме важливе значення в практичній аквакультурі.

Ключові слова: Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri, антимікробна активність, диско-дифузійний метод Байєра-Кірбі, етанольний екстракт

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