HOW TO ESCAPE 'THE ESKAPE PATHOGENS' USING PLANT EXTRACTS

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DOI: 10.15587/2519-8025.2019.193155

O. Pallah, T. Meleshko, S. Tymoshchuk, L. Yusko, L. Bugyna Метою роботи було визначення вмісту біологічно активних речовин, а саме поліфенолів та антоціанів,в  екстрактах плодів аличі, чорниці, йошти, черешні, сливи, червоної та чорної смородини, та досліджен- ня впливу екстрактів плодів цих рослин на ріст та біоплівкоутворенняклінічних ізолятів "збудників  ESKAPE"in vitro. Матеріали і методи. Антибіотикорезистентність штамів клінічного походження, а саме Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,  Enterobacter cloacae визначали методом дифузії Кірбі-Бауера. Вміст біологічно активних речовин визна- чали методом тонкошарової хроматографії. Вплив ягідних екстрактів на вищезгадані ізоляти вивчали  методом їх сумісного культивування. Здатність клінічних ізолятів до утворення біоплівки вивчали за  допомогою спектрофотометричного методу з використанням кристалічного фіолетового, в якості ба- рвника.  Результати. Проаналізувавши результати чутливості до антибіотиків клінічних ізолятів, було вста- новлено, що вони стійкі до всіх антимікробних препаратів. Аналіз вмісту біологічно активних речовин  екстрактів ягід показав, що вони містять велику кількість антоціанів та поліфенолів, та володіють антибактеріальними властивостями. Було встановлено, що ізоляти клінічного походження здатні до  біоплівкоутворення, а відібрані нами екстракти ягід володіють здатністю інгібувати сформовані біоп- лівки ізолятами клінічного походження, а саме: Klebsiella pneumoniae та Pseudomonas aeruginosa.  Висновки. Досліджуючи антибактеріальні властивості антоціанів та поліфенолів, екстрагованих з ягід, можна зробити висновок, що вони здатні гальмувати ріст не лише виділених нами планктонних форм штамів, але й пригнічують утворені ними біоплівки Ключові слова: ізоляти клінічного походження, біоплівки, екстракти їстівних рослин  Copyright © 2019, O. Pallah, T. Meleshko, S. Tymoshchuk, L. Yusko, L. Bugyna.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0).

1. Introductions Acronymically termed ESKAPE pathogens are able to escape the biological impact of antibiotics and collectively represent new paradigms in pathogenesis, transmission and resistance [1]. They are primarily known as agents of nosocomial diseases, those microbes that make the most detrimental complications to major diseases and often cause death [2]. In recent years ESKAPE Pathogens appeared to  be among the most dangerous ones, with the highest fre- quency of isolations in current hospital conditions [3].  The strategy for their prevention and effective treatment  became one of the crucial challenges not only for dona- tives, but also for other segments of the population. One  of the intentions of the health care system is to solve this issue by deploying strong controlling over dissemination in the environment (including resistome) of the majority  of ESKAPE Pathogens and prohibition for using antibiot- ics for animal breeding and some other methodology.  The first step was to specify the methods for re- searching antibiotic sensitivity of clinical and non- clinical isolates of opportunistic bacteria as confirmation  of the feasibility of using and replenishment of EUCAST  database and developing a newly created unified domes- tic electronic resource [4]. The above-mentioned strains  do not show any sensitivity to most of the currently known antibiotic agents. One of the ways to overcome  the existing situation so far is the creation and develop- ment of new antibiotics, which is a promising trend,  though, in recent years, almost unknown. Another prom- ising trend is the development of new antimicrobials of  other (natural) origin, namely the use of bioactive sub- stances (extracts), of not only medicinal but also edible  plants. These promising antimicrobial compounds are al- so defined by antioxidant (anti-inflammatory) and antibi- ofilm-forming properties [5].  It is known, that polyphenolic compounds, includ- ing anthocyanins, have the antimicrobial effect against a  relatively wide range of microorganisms [6]. Anthocya- nins exhibit the antimicrobial effect through induced cell  damage by the destruction of a cell wall, membrane, and intercellular matrix [7]. 2. Literary review  Phenolic compounds are one of the most im- portant bioactive substances that give fruits a relevant  taste. Anthocyanins are pigments, responsible for the quality of fruit, and are markers of ripening, as most fruits accumulate anthocyanins only in the ripening phase  [8]. Anthocyanins, glycosides of anthocyanidins are de- rivatives of phenolic compounds and are natural plant  pigments that give a color to fruit from red to purple.  Previous studies showed that the alkaloids that are pre- sent in berries have anti-diarrheal, antimicrobial and anti-  Scientific Journal «ScienceRise:Biological Science» No5-6(20-21)2019  25  inflammatory properties [9, 10]. Today, there are many  papers, devoted to the use of anthocyanins in the treat- ment of cancer [11, 12], the study of their biological  properties [13]. The antioxidant activity of mahogany berries was studied and it was proved, that polyphenols,  especially anthocyanins, are the main compounds, re- sponsible for antioxidant properties of berries [14].  Flavonoids are powerful antioxidants, free radical  scavengers, and metal chelators; they inhibit lipid perox- idation and exhibit different physiological effects: anti- inflammatory, anti-allergic, anticarcinogenic, antihyper- tensive, anti-arthritic and antimicrobial effects [15, 16].  3. The aim and objectives of the study The aim of the work was to determine the content of biologically active substances, namely polyphenols  and anthocyanins, in the extracts of cherryplum, jostaber- ries, blueberries, sweet cherries, plums, red and black  currants, and to study the effect of these extracts on the growth and biofilm formation of clinical isolates of ESKAPE pathogens in vitro. To achieve the aim, the following tasks were set:  1. To carry out the phytochemical screening of bi- ologically active substances, namely to determine the  gross content of anthocyanins and polyphenols in the ex- tracts of berries and fruits of edible plants.  2. To investigate the ability of edible plant ex- tracts to inhibit the growth of ESKAPE Pathogens.  3. To research the ability of edible plant extracts to inhibit biofilm formation by ESKAPE Pathogens. 4. Materials and methods In our study, we used E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp. opportunistic pathogenic bacteria strains. The strains were isolated from patients of the  Transcarpathian Regional Clinical Center for Neurosur- gery and Neurology inpatient department and the Trans- carpathian Regional Cardiology Health Center inpatient  department according to methodological guidelines [17].  For strains, isolation and identification of microorgan- isms of different genera, selective and chromogenic me- dia were used as well as the following test systems: ОХІ  test, ІNDOLtest, (produced by «PLIVA Lachema Diag- nostika s.r.o.» Czech Republic), ENTERO-test 24,  STREPTOtest16, STAPHYtest16, (PLIVA-Lachema di- agnostica s.r.o, Brno city, Czech Republic).  Antibiotic resistance of isolated strains  Susceptibility determination of strains: E. faeci- um, S. aureus, K. pneumoniae, A. baumannii, P. aeru- ginosa, Enterobacter spp. to antibiotics was defined by  the Kirby-Bauer diffusion method [18]. The effect of 56 modern antibiotics, most commonly used in medicine to control pathogenic and opportunistic pathogens, was studied. Extraction of bioactive substances Methanol-free extracts of red currant (Ribes rubrum), sweet cherries (Prunus avium), plums (Prunus domestica), jostaberry (Ribes x nidigrolaria), blueberries (Vaccinium myrtillus), black currant (Ribes nigrum),  cherry plum (Prunus cerasifera) were obtained by vacu- um evaporation.  We determined the quantitative content of poly- phenols and anthocyanins in methanol extracts by thin- layer chromatography (TLCH). We prepared the extract  from 50 g of each kind of berries and used for extraction 100 ml of methanol of 80 % concentration. To determine the gross content of biologically active substances, the optical density at 765 nm was measured and compared with a standard scale of gallic acid and cyanidin-3- rutinoside [19, 20]. In order to study the antibacterial properties of berries extracts of edible plants, we used the method of  cultivation of the studied extracts with previously select- ed microorganisms: S. aureus, K. pneumoniae, P. aeru- ginosa, Enterobacter spp. The initial concentration of the  selected strains was 1.5 × 108  CFU / ml (N0). The ob- tained data were expressed as the logarithm of the num- ber of surviving bacteria (Nt) to the initial number of bac- teria (N0) ˗ lg (Nt / N0) for a certain culturing time (4 h,  14 h, and 24 h). For a positive control, we used selected strains of bacteria 1.5 × 108  CFU / ml, and for a negative  control, plant extracts were used. To define the ability of clinical isolates to form biofilms, the suspensions of the tested microorganisms’ strains were prepared according to McFarland with 0.5 of dullness, corresponding to a concentration of 1.5 x 108  CFU / ml. 5 ml of the prepared bacterial sus- pension were introduced into sterile testing tubes and  cultured at 37 ° C for 5 days. After incubation, the contents of the testing tubes were removed and 3.5 ml of distilled water and 350 μl of 1 % alcoholic solution of crystalline violet were added. The testing tubes were incubated for 45 min. at the room temperature. After incubation, the dye was removed and the  testing tubes were washed three times with distilled wa- ter. 4 ml of 96 % ethyl alcohol were added into washed  testing tubes and left for 45 minutes at the room tempera- ture. Thereafter, spectrophotometric measurements were  performed at wavelengths of 630 nm and 492 nm. The op- tical densities were measured on an SF-46 spectrophotom- eter and a KFK-3 photoelectric colorimeter in quartz cells.  Statistical processing of the results of experiments was carried out using a software OriginLab 2017 version 94E. 5. Results and discussion We have analyzed the antibiotic sensitivity of  ESKAPE Pathogens, isolated from patients of the Trans- carpathian Regional Clinical Center for Neurosurgery  and Neurology inpatient department and the Transcarpa- thian Regional Cardiology Health Center inpatient de- partment. From the obtained data, it was obvious that P.  aeruginosa isolates were the highly resistant strains. On- ly cefepime and vancomycin proved to be effective  among 56 applied antibiotics (Table 1). The moderate re- sistance was observed in relation to gatifloxacin,  levofloxacin, meropenem, ciprofloxacin, gentamicin,  ceftriaxone. K. pneumoniae strains were also character- ized by the high resistance. The strains were sensitive on- ly to gentamicin and phosphomycin out of all antibiotics  used. E. faecalis strains were susceptible to 6 out of 56 tested antibiotics, namely: nalidixic acid, ampicillin, netilmicin, rifampicin, ciprofloxacin, sparfloxacin.  Scientific Journal «ScienceRise:Biological Science» No5-6(20-21)2019  26  S. aureus strains were susceptible only to gen- tamicin, meropenem, nalidixic acid, oleandomycin,  ciprofloxacinum, streptomycin, fosfomycin, azithromy- cin, netilmicin. A. baumannii strains are also defined by  the high polyresistance to the antibiotics.  Enterobacter cloacae strains were susceptible to  levofloxacin, amikacin, gentamicin, aztreonam, ampicil- lin/sulbactam, netilmicin, rifampicin, ticarcilin, nalidixic  acid, fosfomycin (200), ciprofloxacin.

Having analyzed the results of antibiotic sensi- tivity of strains, isolated from patients of the Trans- carpathian Regional Cardiology Health Center inpa- tient department (Table 2), we found E. faecalis  strains to be resistant to almost all antibiotics, so it can be concluded, that this strain is more resistant than strains, isolated from patients of the Transcarpathian  Regional Clinical Center for Neurosurgery and Neu- rology. A. baumannii strains were not found among  the isolated strains. This can be explained by the fact  that different types of disinfectants are used to disin- fect the premises of the inpatient departments of Neu- rosurgery and Neurology Clinical Center and Cardiol- ogy Health Center. As part of BacFoodNet project, it  was revealed, that one of the effective methods of con- trolling the polyresistance of nosocomial strains is the  use of Cold Plasma Treatment [21]. Therefore, the use of antibiotic drugs is limited due to the polyresistance of isolated microorganisms

The gross content of anthocyanin and polyphenol compounds was defined in methanolic berry extracts by thin-layer chromatography. According to the results of the  study, presented in Table 3, the highest content of polyphe- nols and anthocyanins was detected in the blueberry extract.  The high content of anthocyanins and polyphenols was also found in the black currant extract, this can be explained by  the fact that these berries have a dark color, which is provid- ed by anthocyanins. Significantly less content of anthocya- nins and polyphenols was found in jostaberry, red currant,  plum, and sweet cherry extracts. The determination of an- thocyanins and polyphenols gross content in the cherry  plum extract showed no anthocyanins in this extract. It con- tains polar phenols, such as catechin and flavonoids.

Taking into account the results of gross content of anthocyanins and polyphenols in methanol extracts, we researched the ability of these extracts to inhibit the  growth of opportunistic pathogens. For this purpose, non- methanol extracts of berries were obtained by vacuum  evaporation, which were used to study their antibacterial properties.  We revealed (Table 4) that after 4 hours of incu- bation the cherry plum extract had the antimicrobial ef- fect against K. pneumoniae, E. cloacae, P. aeruginosa,  S. aureus, the number of which significantly decreased. After 14 hours of incubation, no growth was observed in all strains, tested by us, except for K. pneumoniae,  the number of which decreased. After 24 hours of ex- tracts cultivation with selected strains the bacterial  growth was absent. 4-hour incubation of the sweet cherry extract with  the tested microorganisms’ strains showed the low an- tagonistic effect, compared to the cherry plum extract.  The studied extract showed the antibacterial effect against E. cloacae, S. aureus strains, but P. aeruginosa strains were not susceptible to the impact of the sweet cherry extract.  After 14 hours this extract showed the antibacteri- al effect to all tested microorganisms. There was no  growth of E. cloacae and S. aureus microorganisms. Af- ter 24 hours of incubation no growth was observed in all  microorganisms. In the combined cultivation of the plum extract with clinical isolates for 4 hours no antagonistic effect was  observed against P. aeruginosa strain. However, the other strains were susceptible to the impact of the extract. After 14 hours of incubation, there was no growth of S. aureus,  E. cloacae, K. pneumoniae. The number of other microor- ganisms’ strains decreased significantly. After 24 hours of  incubation the growth of all strains was absent. After 4 hours of incubation, the antagonistic effect of jostaberry extract in P. aeruginosa was not observed.  Other strains revealed the antibacterial effect of the ex- tract. There was no growth of S. aureus and E. cloacae  after 14 hours of incubation. Other strains showed the an- tibacterial effect of the extract. After 24 hours, the num- ber of tested microorganisms did not significantly de- crease, compared to the data, obtained after 14 hours of  incubation. The lack of growth is observed in P. aeruginosa, S. aureus only. The low antibacterial effect was shown by the red currant extract against E. cloacae and K. pneumoniae.  After 14 hours of incubation, the number of mi- croorganisms changed significantly. No growth was ob- served in P. aeruginosa and K. pneumoniae, and no  growth of S. aureus was observed after 24 hours of incu- bation, in addition to the above-mentioned bacteria.  After 4 hours of incubation, the weak antibacterial effect was shown by blueberry extract against E. cloacae.  The number of microorganisms remained almost un- changed in the subsequent incubation periods – 14 and  24 hours, but was still less, compared to a similar one during 4-hour incubation. These extracts demonstrated the antagonistic effect on all tested strains.

The most frequently isolated strains were K. pneumoniae and P. aeruginosa, accounting for up to 70 % of selection frequency. Considering the fact that most bacteria do not exist in free-floating state but in formed associations – biofilms, we researched the ability of K. pneumoniae and P. aeruginosa strains, which did not show an antibiotic sensitivity, to form biofilms. Defining the optical density of K. pneumoniae and P. aeruginosa strains biofilms at the optical density of  630 nm showed that for the first day of cultivation the val- ues of the formed biofilm were in the range from 0.039 to  0.026 units of optical density (UN OD). The greatest abil- ity to form biofilms was observed in P. aeruginosa –  0.039±0.001. K. pneumoniae biofilms were significantly  less, but also at the high level – 0.026±0.003. Such an op- tical density indicates that biofilm has begun to form.  Starting on the second day, the optical density of sample biofilms increased, which was also observed during the third and fourth incubation days. The maximum optical density was reached on the fifth day of cultivation and made up 0.240±0.012 for P. aeruginosa and 0.135±0.005 for K. pneumoniae, respectively. Analyzing the data, we  came to the conclusion that the formation of a biofilm be- gins on the first day of incubation.

The results were expressed as the mean ± standard deviation (SD) (*P<0.05) significant differences with the  сherry plum extract on biofilm formation by P. aerugino- sa, K. pneumoniae.  6. Discussion  The literature data analysis showed that the activi- ty of polyphenols against gram-negative bacteria is high- er, compared to gram-positive strains due to the outer  membrane of gram-positive bacteria, which acts as a barri- er to permeability, which leads to a decrease in the absorp- tion of compounds into a cell. The mechanisms that are re- sponsible for the toxicity of pure phenolic compounds to  microorganisms include inhibition of enzyme by oxidized compounds, possibly through the reaction with sulfhydryl  groups or through more nonspecific interactions with pro- teins that lead to their inactivation and loss of function.  However, analyzing the obtained experimental data, we found that the cherry plum extract, in which anthocyanins were not found, but flavonoids were present, inhibited the growth of both gram-positive and gram-negative bacteria. This extract showed the best antibacterial effect. Analyz  ing the obtained experimental data on antibacterial proper- ties of jostaberry, sweet cherry, blueberry, plum, black  currant and red currant extracts against the tested strains of  microorganisms, virtually all strains proved to be suscepti- ble, both gram-positive and gram-negative. They inhibited  the growth of both gram-positive (S. aureus, E. cloacae) and gram-negative microorganisms (K. pneumoniae, P. aeruginosa). However, there is little information on the antimicrobial ability of phenols present in berries. In our studies, anthocyanin-rich extracts inhibited gram-negative and gram-positive bacteria. All the data, we obtained earlier, and the results of the studies in this paper demonstrate that the cherry plum  extract possesses the best antibacterial effect. Other ex- tracts also have a significant ability to inhibit the growth  of microorganisms belonging to ESKAPE Pathogens, which makes them promising antibacterial agents. Antibacterial agents of biological origin in aerosol form can be used in medicine. The disadvantage of edible plant extracts is their  limited shelf life due to the possibility of oxidation of bi- ologically active compounds.  7. Conclusions  Scientific Journal «ScienceRise:Biological Science» No5-6(20-21)2019  30 1. The phytochemical analysis has revealed a high content of polyphenols and anthocyanins in the methanol extracts of edible fruits (cherry plum, jostaberry, blueberry, cherry, plum, red and black currants). The highest content of aforementioned phytochemicals was registered in the blueberry, the lowest one - in the plam. 2. Methanol extracts of the investigated edible fruits exert the inhibitory effect on the growth of clinical isolates of ESKAPE pathogens. Most pronounced inhibitory effect was revealed for the extracts of cherry plum and plum, which contain the lowest amount of polyphenolics and anthocianins.  3. Extracts of investigated edible fruits also inhibit biofilm formation by clinical isolates of ESKAPE pathogens. The most powerful inhibitory effect was registered for the extracts of black currant and cherry plum. Taking into account the safety of edible fruit extracts, they could be considered as promising antimicrobial agents. Conflict of interest There is no conflict of interest 

References  1. Rice, L. B. (2008). Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE. The Journal of Infectious Diseases, 197 (8), 1079–1081. doi: http://doi.org/10.1086/533452 2. Salmanov, A. H., Mariievskyi, V. F., Doan, S. I. (2010). Antybiotykorezystentnist nozokomialnykh shtamiv Pseudomonas aeruginosa v khirurhichnykh statsionarakh Ukrainy v 2008 rotsi. Shpytalna khirurhiia, 3, 86–89. 3. Kulia, A. F., Sabo, Yu., Koval, H. M., Boiko, N. V. (2011). Porivnialnyi analiz metodiv vyznachennia antybiotykochutlyvosti umovno-patohennykh bakterii–zbudnykiv oportunistychnykh infektsii liudyny. Mikrobiolohichnyi zhurnal, 73 (5), 47–53. 4. Othman, L., Sleiman, A., Abdel-Massih, R. M. (2019). Antimicrobial Activity of Polyphenols and Alkaloids in Middle Eastern Plants. Frontiers in Microbiology, 10. doi: http://doi.org/10.3389/fmicb.2019.00911 5. Al Maqtari, Q. A. A., Al Maqtari, M. A. (2014). In vitro antibacterial activity of different Yemeni leaves extracts of Lawsonia inermis against some bacterial pathogens. International Journal of Research Studies in Biosciences, 2 (10), 52–57. 6. Jimenez-Garcia, S. N., Guevara-Gonzalez, R. G., Miranda-Lopez, R., Feregrino-Perez, A. A., Torres-Pacheco, I., Vazquez-Cruz, M. A. (2013). Functional properties and quality characteristics of bioactive compounds in berries: Biochemistry, biotechnology, and genomics. Food Research International, 54 (1), 1195–1207. doi: http://doi.org/10.1016/j.foodres.2012.11.004 7. Chen, W., He, Y., Zhou, Y., Shao, Y., Feng, Y., Li, M., Chen, F. (2015). Edible Filamentous Fungi from the SpeciesMonascus: Early Traditional Fermentations, Modern Molecular Biology, and Future Genomics. Comprehensive Reviews in Food Science and Food Safety, 14 (5), 555–567. doi: http://doi.org/10.1111/1541-4337.12145 8. Volleková, A., Košt’álová, D., Kettmann, V., Tóth, J. (2003). Antifungal activity ofMahonia aquifoliumextract and its major protoberberine alkaloids. Phytotherapy Research, 17 (7), 834–837. doi: http://doi.org/10.1002/ptr.1256 9. Kechinski, C. P., Guimarães, P. V. R., Noreña, C. P. Z., Tessaro, I. C., Marczak, L. D. F. (2010). Degradation Kinetics of Anthocyanin in Blueberry Juice during Thermal Treatment. Journal of Food Science, 75 (2), C173–C176. doi: http://doi.org/10.1111/ j.1750-3841.2009.01479.x 10. Abbas, M., Saeed, F., Anjum, F. M., Afzaal, M., Tufail, T., Bashir, M. S. Et. al. (2016). Natural polyphenols: An overview. International Journal of Food Properties, 20 (8), 1689–1699. doi: http://doi.org/10.1080/10942912.2016.1220393 11. Kong, J.-M., Chia, L.-S., Goh, N.-K., Chia, T.-F., Brouillard, R. (2003). Analysis and biological activities of anthocyanins. Phytochemistry, 64 (5), 923–933. doi: http://doi.org/10.1016/s0031-9422(03)00438-2 12. Coklar, H., Akbulut, M. (2017). Anthocyanins and phenolic compounds of Mahonia aquifolium berries and their contributions to antioxidant activity. Journal of Functional Foods, 35, 166–174. doi: http://doi.org/10.1016/j.jff.2017.05.037 13. Kandaswami, C., Middleton, E. (1994). Free radical scavenging and antioxidant activity of plant flavonoids. Free radicals in diagnostic medicine. Boston: Springer, 351–376. doi: http://doi.org/10.1007/978-1-4615-1833-4_25 14. Hertog, M. G., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F., Pekkarinen, M. (1995). Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Archives of internal medicine, 155 (4), 381–386. doi: http://doi.org/10.1001/archinte.155.4.381 15. Pro zatverdzhennya metodychnyh vkazivok shhodo vyznachennya chutlyvosti mikroorganizmiv do antybakterialnyh preparativ (2007). Nakaz MOZ Ukrainy No. 167. 05.04.2007. Available at: http://mozdocs.kiev.ua/view.php?id=6958 16. Al-Boushi, M. A., Haj Hamdo, H., Herbali, J. (2014). Extraction and study of the phenolic compounds in the leaves and sticks of the Syrian sumac plant (Rhus coriaria L). International Journal of ChemTech Research, 6, 2414–2415. 17. Wrolstad, R. E., Acree, T. E., Decker, E. A., Penner, M. H., Reid, D. S., Schwartz, S. J., Sporns, P. (Eds.) (2005). Handbook of food analytical chemistry, volume 1: Water, proteins, enzymes, lipids, and carbohydrates. John Wiley & Sons, 784. 18. Andrzejewska, J., Sadowska, K., Klóska, Ł., Rogowski, L. (2015). The effect of plant age and harvest time on the content of chosen components and antioxidative potential of black chokeberry fruit. Acta Scientiarum Polonorum Hortorum Cultus, 14, 105–114. 19. Albadawi, D. A., Mothana, R. A., Khaled, J. M., Ashour, A. E., Kumar, A., Ahmad, S. F. et. al. (2017). Antimicrobial, anticancer, and antioxidant compounds from Premna resinosa growing in Saudi Arabia. Pharmaceutical Biology, 55 (1), 1759–1766. doi: http://doi.org/10.1080/13880209.2017.1322617 20. Nazzaro, F., Fratianni, F., De Martino, L., Coppola, R., De Feo, V. (2013). Effect of Essential Oils on Pathogenic Bacteria. Pharmaceuticals, 6 (12), 1451–1474. doi: http://doi.org/10.3390/ph6121451 21. Naz, S., Siddiqi, R., Ahmad, S., Rasool, S. A., Sayeed, S. A. (2007). Antibacterial Activity Directed Isolation of Compounds from Punica granatum. Journal of Food Science, 72 (9), M341–M345. doi: http://doi.org/10.1111/j.1750- 3841.2007.00533.x  Received date 15.10.2019 Accepted date 20.11.2019 Published date 30.12.2019  Scientific Journal «ScienceRise:Biological Science» No5-6(20-21)2019  31 Oleksandra Pallah, Assistant, Junior Researcher, Department of Clinical and Laboratory Diagnosis and Pharmacology, Molecular Microbiology and Immunology Center for Molecular Microbiology and Immunology, State Higher Educational Institution «Uzhgorod National University», Narodna sq., 3, Uzhhorod, Ukraine, 88000 E-mail: ssarvash@gmail.com Tamara Meleshko, Senior Lecturer, Junior Researcher, Department of Clinical and Laboratory Diagnosis and Pharmacology, Molecular Microbiology and Immunology Center for Molecular Microbiology and Immunology, State Higher Educational Institution «Uzhgorod National University», Narodna sq., 3, Uzhhorod, Ukraine, 88000 E-mail: meleshkotv@ukr.net Svitlana Tymoshchuk, Assistant, Junior Researcher, Department of Clinical and Laboratory Diagnosis and Pharmacology, Molecular Microbiology and Immunology Center for Molecular Microbiology and Immunology, State Higher Educational Institution «Uzhgorod National University», Narodna sq., 3, Uzhhorod, Ukraine, 88000 E-mail: zub.sveta@gmail.com Lesya Yusko, PhD, Lecturel, Junior Researcher, Department of Clinical and Laboratory Diagnosis and Pharmacology, Molecular Microbiology and Immunology Center for Molecular Microbiology and Immunology, State Higher Educational Institution «Uzhgorod National University», Narodna sq., 3, Uzhhorod, Ukraine, 88000 E-mail: lesus@ukr.net Larisa Bugyna, Researcher, Molecular Microbiology and Immunology Center for Molecular Microbiology and Immunology, State Higher Educational Institution «Uzhgorod National University», Narodna sq., 3, Uzhhorod, Ukraine, 88000 E-mail: larina.bh@gmail.com