CC BY
75
UDK 639.3:615.32:615.38
Halyna Tkachenko1, Lyudmyla Buyun2, Olha Kasiyan3, Elzbieta Terech-Majewska4,
Zbigniew Osadowski1
department of Zoology and Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University in Slupsk, Poland; Arciszewski Str. 22b, 76-200 Slupsk, Poland; 2M.M. Gryshko National Botanical Garden, National Academy of Science of Ukraine, Kyiv, Ukraine; 3Danylo Halytsky Lviv National Medical University, Lviv, Ukraine;
^Department of Epizootiology, University of Warmia and Mazury in Olsztyn, Poland
THE ANTIBACTERIAL ACTIVITY OF THE ETHANOLIC LEAF EXTRACT OF
FICUS PUMILA L. (MORACEAE) AGAINST FISH BACTERIAL PATHOGENS
The aim of this study was to test the antibacterial properties of ethanolic extract prepared from Ficus pumila L. leaves against fish pathogens, Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluo-rescens, Yersinia ruckeri to evaluate the possible use of this plant in preventing infections caused by these bacteria in aquaculture. The antimicrobial susceptibility testing was done on Muller-Hinton agar by disc diffusion method (Kirby-Bauer disk diffusion susceptibility test protocol). Muller-Hinton agar plates were inoculated with 200 and 400 fL 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 (On-corhynchus mykiss Walbaum) and Pseudomonas fluorescens (strain E 1/7/15) isolated locally from internal organs of rainbow trout with clinical features of furunculosis, 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 and fish with clinical symptoms of yersiniosis were used as test organisms. In our study, the A. hydrophila and C. freundii strain (200 and 400 fl of standardized inoculum) revealed intermediate susceptibility to ethanolic extract obtained from leaves of F. pumila (inhibition zone diameters ranged from 10 to 11 mm). Ethanolic extract derived from F. pumila leaves exhibited the highest antibacterial activity against Pseudomonas fluorescens causing a zone of inhibition, comprising at least 13-14 mm for 200 fL and 9-10 mm for 400 fL of standardized inoculum (108 CFU/mL) of bacterium strain. Y. ruckeri isolate (200 and 400 fl of standardized inoculum) revealed high susceptibility to ethanolic extract obtained from leaves of F. pumila (inhibition zone diameters ranged between 12 and 14 mm). Further studies aimed at the isolation and identification of active substances from the ethanolic extracts obtained from leaves of F. pumila could also disclose compounds with better therapeutic value. It is believed that screening of plants from Ficus genus for other biological activities including antimicrobial activity is essential. Finally, it seems important not only to determine the substances involved in the activity against pathogens but also to establish the induced response in the fish physiology.
Key words: Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri, antimicrobial activity, disc diffusion technique, ethanolic extrac.t
Г.М. Ткаченко, Л.И. Буюн, О.П. Касиян, Э. Терех-Маевская, З. Осадовский
АНТИМИКРОБНАЯ ЭФФЕКТИВНОСТЬ ЭТАНОЛЬНОГО ЭКСТРАКТА, ПОЛУЧЕННОГО ИЗ ЛИСТЬЕВ FICUS PUMILA L. (MORACEAE) ОТНОСИТЕЛЬНО БАКТЕРИАЛЬНЫХ ПАТОГЕНОВ РЫБ
Целью этого исследования была оценка антимикробной эффективности этанольного экстракта, полученного из листьев Ficus pumila L. относительно бактериальных патогенов рыб (Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri), и возможность применения этого растения для предотвращения инфекций, вызванных этими бактериями в аква-культуре. Листья F. pumila были собраны в Национальном ботаническом саду им. Н.Н. Гришко НАН Украины (Киев, Украина). Свежие листья промывали, измельчали, взвешивали и гомогенизировали
в 96%-м 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, а также Citrobacter freundii, изолированный из внутренних органов угря (Anguilla anguilla L.) с клиническими признаками заболевания, мы использовали в этом исследовании как тестовые штаммы в дискодиффузионном методе Кирби-Бауэра (1966). В чашки с агаром Muller-Hinton инокулировали 200 и 400 мкл стандартизованного инокулята (108 КОЕ/мл) бактерий. В нашем исследовании штамм A. hydrophila и C. freundii (200 и 400 мкл стандартизированного инокулята) продемонстрировал среднюю восприимчивость к этанольному экстракту, полученному из листьев F. pumila (диаметры зон ингибирования роста штамма варьировались от 10 до 11 мм). Самая высокая антибактериальная активность экстракта продемонстрирована относительно штамма Pseudomonas fluorescens (диаметр зоны ингибирования роста штамма составлял 13-14 мм для 200 мкл стандартизированного инокулята и 9-10 мм - для 400 мкл инокулята бактериального штамма). Изолят Y. ruckeri (200 и 400 мкл посевного материала) проявил высокую восприимчивость к этанольному экстракту, полученному из листьев F. pumila (диаметры зон ингибирования составляли от 12 до 14 мм). Дальнейшие исследования, направленные на выделение и идентификацию активных веществ из этанольных экстрактов, полученных из листьев F. pumila, помогут целенаправленно улучшить терапевтическую ценность экстрактов. Важно не только определить вещества, участвующие в активности относительно патогенов, но и установить индуцированный физиологический ответ рыб в исследованиях in vivo.
Ключевые слова: Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri, антимикробная активность, дискодиффузионный метод Байера-Кирби, этанольный экстракт.
Introduction
Infectious diseases in aquaculture are of major concern to the industry and are typically controlled by eradication of the pathogen, treatment with antibiotic or chemotherapeutics [6]. Many plants were shown to have potential for being effective herbal drugs against the fish and other aquaculture pathogens [17]. Therefore, use of natural products has been considered as an alternative to antibiotics in fish health management to control bacterial infections in aquaculture. Additionally, it is an attractive method for increasing the protective capabilities of fish [15]. One of the potential plants that can be used as antimicrobial to enhance survival and immune competence is Ficus genus plants.
Among 37 genera of Moraceae comprising 1050-1100 species in total, Ficus L. is the largest one with ca 750 species of tropical and subtropical distribution worldwide [4]. Ficus trees have widely been used by humans over their history in a variety of industries and fields of activity. Virtually all parts of their body are utilized by local people in various medicinal practices to cure wounds, sores, stomach and eye problems, headaches and toothaches, and even tumours and cancer, etc. A number of species are known helpful in healing disorders of digestive and respiratory systems, parasitic infections, and also as painkillers, tonics, and ecbolics [12].
Ficus L. is one of the largest genera of angiosperms, with about 750 species of terrestrial trees, shrubs, hemi-epiphytes, climbers and creepers occurring in the tropics and subtropics of the world [4]. Ficus trees have widely been used by humans over their history in a variety of industries and fields of activity. Virtually all parts of their body are utilized by local people in various medicinal practices to cure wounds, sores, stomach and eye problems, headaches and toothaches, and even tumours and cancer, etc. A number of species are known helpful in healing disorders of digestive and respiratory systems, parasitic infections, and also as painkillers, tonics, and ecbolics [12].
Ficus pumila L. is a (gyno)dioecious evergreen root-climber naturally occurring in continental southern and eastern Asia. Leaves are distichous, 1-10 cm long and 0.7-6 cm wide, elliptic to
oblong or ovate. The species represents dimorphy of leaves and stems: sterile climbing stems possess short adventitious roots and small asymmetric leaves, while the fertile non-climbing stems without roots bear large symmetric coriaceous leaves. A separate plant may possess either two or one stem (and leaf) type, with the fertile branches tenting to develop in mature plants reaching the forest canopy. Figs are axillary or below the leaves, solitary, 3.5-6.5 cm in diameter, pedunculate and obovoid, pubescent, purple to blackish at maturity [4].
Ficus (Moraceae) species are reported to have antimicrobial activity against several pathogenic bacteria and have been used as traditional medicines for the treatment of human diseases [2, 19, 20]. Moreover, in line with the growing interest in the antibacterial properties of different plants, in our previous researches, we have used ethanolic extracts derived from leaves of various Ficus species to assess antibacterial activity against harmful fish pathogens, Aeromonas hydro-phila, Citrobacterfreundii, Pseudomonas fluorescens [21-26].
Therefore, the aim of this study was to test the efficacy of ethanolic extract prepared from F. pumila leaves against fish pathogens, Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, and Yersinia ruckeri to evaluate the possible use of this plant in preventing infections caused by these bacteria in aquaculture.
Materials and methods
Collection of Plant Material and Preparing of Plant Extract. The leaves of F. mucuso were sampled in M.M. Gryshko National Botanical Garden (Kyiv, Ukraine). The whole collection of tropical and subtropical plants at M.M. Gryshko National Botanical Garden (Kyiv, Ukraine) (including Ficus spp. plants) has the status of a National Heritage Collection of Ukraine. The sampled leaves of Ficus spp. 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.
Method of Culturing Pathological Sample and identification Method of the Bacteria. Aeromonas hydrophila (strain E 2/7/15) isolated locally from gill of rainbow trout (Oncorhyn-chus mykiss Walbaum) 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 (Tripticase Soya Broth, Oxoid) for 24 hrs. After preincubation, bacterial culture was transferred to two different cultivation media: TSA (Tripticase Soya 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 organs (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.
Preliminary characterization of isolates. 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 [9, 28]. 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 [9].
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). 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) to identify the investigated bacterium.
Bacterial Growth Inhibition Test of Plant Extracts by the Disk Diffusion Method. 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 |iL 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.
Statistical analysis. 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) [29]. 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 [21-26].
Results
Antimicrobial activity of ethanolic extracts obtained from F. pumila against Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri expressed as mean of diameters of inhibition zone is presented in Figs 1-5.
Fig. 1. Antimicrobial activity of ethanolic extracts obtained from F. pumila against Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Yersinia ruckeri. Muller-Hinton agar plates inoculated with 200 and 400 ^L of standardized inoculum (108 CFU/mL) of bacterium
In our study, the A. hydrophila and C. freundii strain (200 and 400 ^l of standardized inoculum) revealed intermediate susceptibility to ethanolic extract obtained from leaves of F. pumila (inhibition zone diameters ranged from 10 to 11 mm) (Figs 1-3).
A
B
Fig. 2. Antimicrobial activity of ethanolic extracts obtained from F. pumila (53) against Aeromonas hydrophila. Muller-Hinton agar plates inoculated with 200 (A) and 400 ^L of standardized inoculum
(108 CFU/mL) of bacterium (B)
Our results demonstrated that the C. freundii (200 and 400 ^l of standardized inoculum) revealed intermediate susceptibility to ethanolic extract obtained from leaves of F. pumila (inhibi tion zone diameters were ranged between 10 and 12 mm) (Fig. 3).
Fig. 3. Antimicrobial activity of ethanolic extracts obtained from F. pumila (53) against Citrobacter freundii. Muller-Hinton agar plates inoculated with 200 (A) and 400 |iL of standardized inoculum (108 CFU/mL)
of bacterium (B)
Ethanolic extract derived from F. pumila leaves exhibited the highest antibacterial activity against Pseudomonas fluorescens causing a zone of inhibition, comprising at least 13-14 mm for 200 цЬ and 9-10 mm for 400 цЬ of standardized inoculum (10 CFU/mL) of bacterium strain. Y. ruckeri isolate (200 and 400 ц1 of standardized inoculum) revealed high susceptibility to ethanolic extract obtained from leaves of F. pumila (inhibition zone diameters ranged between 12 and 14 mm) (Figs 1, 4, 5).
A B
Fig. 4. Antimicrobial activity of ethanolic extracts obtained from F. pumila (53) against Pseudomonas fluorescens. Muller-Hinton agar plates inoculated with 200 (A) and 400 |iL of standardized inoculum
(108 CFU/mL) of bacterium (B)
Y. ruckeri (200 and 400 of standardized inoculum) revealed intermediate susceptibility to ethanolic extract obtained from leaves of F. pumila (inhibition zone diameters were ranged between 12 and 14 mm) (Fig. 5).
Fig. 5. Antimicrobial activity of ethanolic extracts obtained from F. pumila (53) against Yersinia ruckeri. Muller-Hinton agar plates inoculated with 200 (A) and 400 (iL of standardized inoculum (108 CFU/mL)
of bacterium (B)
Discussion
The antimicrobial activity profile of ethanolic extract obtained from leaves of F. pumila against the tested pathogen strains indicated that Yersinia ruckeri was the most susceptible bacterium (200 and 400 ^l of standardized inoculum) among all the bacterial test strains (Figs 1, 5). Similarly, P. fluorescens was found to be sensitive strain (13-14 mm for 200 ^L and 9-10 mm for 400 ^L of standardized inoculum of bacterium strain) although A. hydrophila and C. freundii was found to be least susceptible to ethanolic extract obtained from leaves of F. pumila (Figs 1-4). Of all the bacterial strains included in the test, Y. ruckeri (200 and 400 ^l of standardized inoculum dilution) and P. fluorescens (200 ^l) were found to be the most susceptible and C. freundii, which is an isolate from gills of eel, was found to be the least inhibited bacterium (Figs 1, 3-5).
The comprehensive review of usefulness of some medicinal plants (herbal drugs) against fish diseases has been presented by many researchers [5, 7, 8, 13, 19, 20]. 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 [21-26]. Antibacterial properties of plant extracts have been by far the most studied bioactivity with potential application in aquaculture systems [18]. Castro and co-workers (2008) have revealed by agar diffusion assay that 31 methanolic extracts of Brazilian plants presented antibacterial activity against the fish pathogenic bacteria, i.e. Streptococcus agalactiae, Flavobacterium columnare and A. hy-drophila. F. columnare being the most susceptible microorganism to most of the tested extracts [5]. Wei and Musa (2008) also studied the susceptibility by assay of minimum inhibitory concentration (MIC) of two Gram positive bacteria (Staphylococcus aureus and Streptococcus agalactiae), four Gram negative bacteria (C. freundii, Escherichia coli, Vibrio parahaemolyticus and Vibrio vulnificus) and 18 isolates of Edwardsiella tarda to aqueous extract of garlic (500, 250, 125, 62.5 mg/mL), and found that all garlic extracts were effective against the tested pathogenic bacteria [27].
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. del-toidea as well as F. hispida, F. mucuso, F. pumila, F. craterostoma exhibit a favorable antibacterial activity against C. freundii (200 |iL of standardized inoculum) [21]. Our results also proved that the ethanolic extracts obtained from F. pumila, F. binnendijkii 'Amstel Gold', F. carica, F. erecta, F. hispida, F. mucuso, 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 |iL of standardized inoculum) [22]. Moreover, previous investigation has shown that the most effective against P. fluorescens (400 |iL 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 |iL 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 |iL of standardized inoculum), the highest antibacterial activity was displayed by F. benghalensis, F. benjamina, F. deltoidea, F. hispida, F. lyrata leaf extracts [23, 26].
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 Gramnegative bacteria [19]. For instance, Rajiv and Rajeshwari (2012) screened antimicrobial activity of F. religiosa bark, leaf, stem, and fruit aqueous extracts against a number of major pathogens (Aeromonas hydrophila, 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. Antibacterial properties of the extracts were generally better pronounced than untifungal ones. All extracts at all concentrations tested affected P. aeruginosa, although the strongest inhibition showed the maximal concentration extracts from leaves and stems (inhibition zone diameter 14 mm) and slighter effect was produced by bark (13 mm) and fruit (12 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 featured 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) [16].
Al Askari et al. (2013) screened aqueous and ethanolic leaf extracts of F. carica from different regions of Morocco against 16 bacterial strains (Acinetobacter baumannii, Escherichia coli, Hafnia alveie, Klebsiella pneumonia, Proteus mirabilis, Pseudomonas aeruginosa, P. fluorescens, Salmonella arizonae, S. entericatyphimurium, S. enteritidis, Staphylococcus aureus, S. aureus meticillin-resistant, S. epidermidis, Streptococcus pyogenes, S. sanguins, and Yersinia
enterocolitica) and 8 yeasts (Candida famata, C. glabrata, C. parapsilosis, C. tropicalis, two strains for each species). In general, aqueous extract was found more active against Grampositive bateria than Gram-negative ones and it was not active against yeasts. Ethanolic extract demonstrated stronger inhibitory activity compared to aqueous extract and inhibited growth of both bacteria and fungi. E. coli showed comparatively high susceptibility among all bacteria tested. Aqueous extracts from plants collected in different regions affected E. coli with the inhibition zone diameter values of 0-11 mm (MIC 100 pg/ml), while ethanolic extracts showed zone diameters of 13.3-18.7 mm (MICs 25-50 pg/ml) [1].
Nair and Chanda (2006) screened aqueous and ethanol extracts from 20 plant species, among which were four species of Ficus (F. benghalensis, F. racemosa, F. religiosa, and F. tisela), against seven Gram-negative (Pseudomonas aeruginosa ATCC27853, Pseudomonas testosteroni NCIM5098, Proteus mirabilis NCIM2241, Proteus vulgaris NCTC8313, Enterobacter aerogenes ATCC10240, Escherichia coli ATCC25922, and Citrobacter freundii ATCC10787) and five Gram-positive (Staphylococcus epidermidis ATCC12228, Bacillus cereus ATCC11778, Streptococcus fecalis ATCC29212, Streptococcus cremoris NCIM2179, and Streptococcus agalactiae NCIM2401) bacterial strains. Aqueous extracts generally showed less activity than ethanol extracts and Gram-positive bacteria were generally more affected than Gram-negative ones. The examined Ficus species, of which bark extracts were used, showed low inhibition activity in general. Among bacteria tested, E. coli appeared the most resistant. Neither Ficus extract acted against E. coli [14].
Further studies focused on antimicrobial evaluation of methanolic extracts, hexane-ethyl acetate and ethyl acetate-methanol extract fractions, and isolated compounds from stem bark of F. ovata Vahl., testing a range of microbe clinical isolates, including Gram-positive bacteria (methi-cillin-resistant Staphylococcus aureus LMP805, Streptococcus faecalis LMP806, and Bacillus icereus LMP716), Gram-negative bacteria (P-lactamase positive Escherichia coli LMP701, am-picillin-resistant Klebsiella pneumoniae LMP803, carbenicillin-resistant Pseudomonas aerugi-nosa LMP804, chloramphenicol-resistant Salmonella typhi LMP706, and chloramphenicol-resistant Citrobacter freundii LMP802), and fungi (Candida albicans LMP709U and Micro-sporum audouinii LMP725D) [11]. The crude extracts and certain fractions and compounds were found active against all organisms tested. E. coli was most strongly inhibited (MIC 78 pg/ml) by Hex-EtOAc 25% fraction and two compounds, namely taraxeryl acetate (terpenoid) and proto-catechuic (phenolic) acid. Other compounds showed MIC values of 156 pg/ml, fractions 156 to 312 pg/ml, and crude extract 312 pg/ml against E. coli. Among all organisms tested, E. coli demonstrated relatively high susceptibility to the evaluated antimicrobial agents. In general, the most inhibitory-active compounds appeared to be 2-hydroxyisoprunetin (isoflavonoid) and protocate-chuic acid, affecting (nearly) all pathogens tested with relatively low MIC values. Antimicrobial activity of flavonoids and isoflavonoids was suggested to come from their ability to complex with bacterial cell wall, while that of terpenoids to cause membrane disruption, both resulting in the microbial growth inhibition [10, 11].
Conclusions
In conclusion, ethanolic extract obtained from leaves of F. benghalensis investigated possessed activity against at least two strains of bacteria (Y. ruckeri and P. fluorescens). The data on susceptibility patterns of pathogens responsible for fish diseases are very important for aquaculture. The results also indicate that scientific studies carried out on medicinal plants having traditional claims of effectiveness might warrant fruitful results. Therefore, it seems a promising strategy to apply plant-derived products to gain control of infections in fish used for aquaculture. Further studies aimed at the isolation and identification of active substances from the ethanolic extract obtained from leaves of F. benghalensis could also disclose compounds with better therapeutic value.
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 Fund for the supporting our study.
References
1. Al Askari G., Kahouadji A., Khedid K., Ouaffak L., Mousaddak M., Charof R., Mennane Z. In vitro antimicrobial activity of aqueous and ethanolic extracts of leaves of Ficus carica collected from five different regions of Morocco // Journal of Materials and Environmental Science. 2013. 4(1). P. 33-38.
2. Ali M., Chaudhary N. Ficus hispida Linn.: A review of its pharmacognostic and eth-nomedicinal properties // Pharmacogn. Rev. 2011. 5(9). P. 96-102.
3. Bauer A.W., Kirby W.M., Sherris J.C., Turck M. Antibiotic susceptibility testing by a standardized single disk method // Am. J. Clin. Pathol. 1966. 45(4). P. 493-496.
4. Berg C.C., Corner E.J.H. Moraceae. Ficus. Flora Malesiana // National Herbarium Neder-land. The Netherlands. 2005. Ser. I, 17(2). 1-730.
5. Castro S.B.R., Leal C.A.G., Freire F.R., Carvalho D.A., Oliveira D.F., Figueiredo H.C.P. Antibacterial activity of plant extracts from Brazil against fish pathogenic bacteria // Braz. J. Microbiol. 2008. 39. P. 756-760.
6. Chinabut S., Puttinaowarat S. The choice of disease control strategies to secure international market access for aquaculture products // Dev. Biol. (Basel). 2005. 121. P. 255-261.
7. Farrukh A., Ahmad I. Broad-spectrum antibacterial and antifungal properties of certain traditionally used Indian medicinal plants // World Journal of Microbiology and Biotechnology. 2003. 19. P. 653-657.
8. Ji S.-C., Jeong G.-S., Gwang-Soon I., Lee S.-W., Yoo J.-H., Takii K. Dietary medicinal herbs improve growth performance, fatty acid utilization, and stress recovery of Japanese flounder // Fish. Sci. 2007. 73. P. 70-76.
9. Kocwowa E. Cwiczenia z mikrobiologii ogolnej. Panstwowe Wydawnictwo Naukowe. Warszawa, 1981. P. 78-85.
10. Kuete V., Nana F., Ngameni B., Mbaveng A.T., Keumedjio F., Ngadjui B.T. Antimicrobial activity of the crude extract, fractions and compounds from stem bark of Ficus ovata (Moraceae) // Journal of Ethnopharmacology. 2009. 124. P. 556-561.
11. Kuete V., Ngameni B., Simo C.C.F., Tankeu R.K., Ngadjui B.T., Meyer J.J.M., Lall N., Kuiate J.R. Antimicrobial activity of the crude extracts and compounds from Ficus shlamydo-carpa and Ficus cordata (Moraceae) // Journal of Ethnopharmacology 120: 17-24.
12. Lansky E.P., Paavilainen, H.M. Figs: the genus Ficus. In: Hardman R. (ed.) Traditional herbal medicines for modern times. CRC Press, Boca Raton, 2011. Vol. 9. P. 1-357.
13. Lin D.J., Hua Y.N., Zhang Q.Z., Xu D.H., Fu Y.W., Liu Y.M., Zhou S.Y. Evaluation of medicated feeds with antiparasitical and immune-enhanced Chinese herbal medicines against Ich-thyophthirius multifiliis in grass carp (Ctenopharyngodon idellus) // Parasitol. Res. 2016. 115(6). P.2473-2483.
14. Nair R., Chanda S. Activity of some medicinal plants against certain pathogenic bacterial strains // Indian Journal of Pharmacology. 2006. 38(2). P. 142-144.
15. Pachanawan A., Phumkhachorn P., Rattanachaikunsopon P. Potential of Psidium gua-java supplemented fish diets in controlling Aeromonas hydrophila infection in tilapia (Oreo-chromis niloticus) // J. Biosci. Bioeng. 2008. 106(5). P. 419-424.
16. Rajiv P., Rajeshwari S. Screening for phytochemicals and antimicrobial activity of aqueous extract of Ficus religiosa Linn. // International Journal of Pharmacy and Pharmaceutical Sciences. 2012. 4. P. 207-209.
17. Ramudu K.R., Dash G. A review on herbal drugs against harmful pathogens in aquaculture // Am. J. Drug Discov. Dev. 2013. 3(4). P. 209-219.
18. Reverter M., Bontemps N., Lecchini D., Banaigs B., Sasal P. Use of plant extracts in fish aquaculture as an alternative to chemotherapy: Current status and future perspectives // Aquaculture. 2014. 433. P. 50-61.
19. Salem M.Z.M., Salem A.Z.M., Camacho L.M., Ali H.M. Antimicrobial activities and phytochemical composition of extracts of Ficus species: An over view // Afr. J. Microbiol. Res. 2013. 7(33). P. 4207-4219.
20. Sirisha N., Sreenivasulu M., Sangeeta K., Chetty C.M. Antioxidant properties of Ficus species, a review // Int. J. Pharma Techn. Res. 2010. 4. P. 2174-2182.
21. Tkachenko H., Buyun L., Terech-Majewska E., Osadowski Z. Antibacterial activity of ethanolic leaf extracts obtained from various Ficus species (Moraceae) against the fish pathogen, Citrobacter freundii // Baltic Coastal Zone - Journal of Ecology and Protection of the Coastline. 2016. 20. P. 117-136.
22. Tkachenko H., Buyun L., Terech-Majewska E., Osadowski Z., Sosnovskyi Y., Hon-charenko V., Prokopiv A. In vitro antibacterial efficacy of various ethanolic extracts obtained from Ficus spp. leaves against fish pathogen, Pseudomonas fluorescens // Globalisation and regional environment protection. Technique, technology, ecology. Scientific editors Tadeusz Noch, Wioleta Mikolajczewska, Alicja Wesolowska. Gdansk: Gdansk High School Publ., 2016. P. 265-286.
23. Tkachenko H., Buyun L., Terech-Majewska E., Osadowski Z. In vitro antimicrobial activity of ethanolic extracts obtained from Ficus spp. leaves against the fish pathogen Aeromonas hydrophila // Arch. Pol. Fish. 2016. 24. P. 219-230.
24. Tkachenko H., Buyun L., Terech-Majewska E., Osadowski Z.Screening for antimicrobial activities of the ethanolic extract derived from Ficus hispida L.f. leaves (Moraceae) against fish pathogens // Науч. тр. Дальрыбвтуза (Scientific Journal of DALRYBVTUZ). 2017. Т. 41. P. 56-64.
25. Tkachenko H., Buyun L., Terech-Majewska E., Osadowski Z.Screening for antimicrobial activities of the ethanolic extract derived from Ficus mucuso Welw. ex Ficalho leaves (Moraceae) against bacterial fish pathogens // Науч. тр. Дальрыбвтуза (Scientific Journal of DALRYBVTUZ). 2017. 42. P. 25-36.
26. Tkachenko H., Buyun L., Terech-Majewska E., Osadowski Z., Sosnovskyi Y., Hon-charenko V., Prokopiv A. The antimicrobial activity of some ethanolic extracts obtained from Ficus spp. leaves against Aeromonas hydrophila // Тр. ВНИРО (Trudy VNIRO). 2016. 162. P. 172-183.
27. Wei L., Musa N. Inhibition of Edwardsiella tarda and other fish pathogens by Allium sativum L. (Alliaceae) extract // Am.-Eur. J. Agric. Environ. Sci. 2008. 3. P. 692-696.
28. Whitman K.A., MacNair N.G. Finfish and shellfish bacteriology manual: techniques and procedures. Blackwell Publishing Company, Iowa, USA, 2004.
29. Zar J.H. Biostatistical Analysis. 4th ed., Prentice-Hall Inc., Englewood Cliffs, New Jersey, 1999.
Сведения об авторах: Ткаченко Галина Михайловна, e-mail: кандидат биологических наук, e-mail: [email protected];
Буюн Людмила Ивановна, доктор биологических наук, e-mail: [email protected];
Касиян О.П.; Терех-Маевская Э.;
Осадовский Збигнев, доктор биологических наук, профессор, e-mail: zbigniew. [email protected].
