Effect of ethanol plant extracts on Staphylococcus Epidermidis, Staphylococcus Aureus

The emergence of multiresistant strains of Staphylococcus epidermidis, Staphylococcus aureus that are difficult to antibiotics and cause severe lesions of soft tissues, sepsis, and complicated surgical pathology are recognized as the one of problems of current infectious diseases of animals and humans. One of challenges in pharmacognosy is the search for alternative sources of antibacterial substances with an exhaustive resource of antibiotics of fungal origin. The use of raw medicinal plants is quite promising in this regard. The tendency of scientific research of recent decade reveals a promising range of plants of a number of families, which typically contents certain active substances (phytoncides, saponins, alkaloids, glycosides, tannins, essential oils etc.). 
The goal of the work was to establish the antibacterial effect of plant infusions on reference cryogenic strains of Staphylococcus epidermidis, Staphylococcus aureus in vitro. 
Herbal material of 50 species (seeds, grass, shoots, leaves, compound fruit, peel) obtained at different periods of the growing season was used for investigation. The material was classified, dried, and grounded. Samples of 1 g were poured with 5 cm3 of 96 % ethanol and were kept it over three weeks in a dry cold place. The obtained alcohol infusion was filtered with sterile multi-layer gauze disc filters. Before the discs were put on the surface of agar with inoculation of the corresponding culture, they were dried in a sterile laminar box under ultraviolet rays. Antibacterial activity of various tinctures was determined by the disk diffusion method in agar with the measurement of the diameter of the growth suppression zone of the culture using a template ruler. 
Concerning the above mentioned point, herein, we report the results of the use of tinctures Staphylococcus epidermidis, Staphylococcus aureus in vitro. Obtained data has been systematized, summarized and evaluated. 
The paper presents the results of the effectiveness of phytopreparations on Staphylococcus epidermidis, Staphylococcus aureus in vitro. The antibacterial effect of plant tinctures of Cephalotaxus harringtonia, Hedera helix, Geranium sanguineum on cryogenic strains Staphylococcus epidermidis, Staphylococcus aureus. We consider it possible to recommend the investigated extracts of Cephalotaxus harringtonia, Hedera helix, Geranium sanguineum for further research in the fight against polyresistant strains of the above-mentioned microorganisms. 
The obtained results give grounds to recommend herbal tinctures to combat multi-resistant strains of Staphylococcus epidermidis, Staphylococcus aureus.

According to Dancer et al. (2014), Staphylococcus aureus, including methicillin-resistant strains, is a major cause of nosocomial infections. The rise of specific strains in hospitalized patients and even in the community calls for a better understanding of prevention and control measures.
Research results Karam et al. (2017), suggest that 80% of S. aureus and 80% of S. epidrmidis isolates developed Methicillin resistance. The findings of the current work have shown that most of the methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE) formed a weak biofilm. Results showed that the number of biofilm cells strongly reduced to undetectable limits in the presence of gentamicin. Vandecandelaere et al. (2017) found that S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. This indicates that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required to clarify the exact mechanism (s) involved. Kiranasari et al. (2018) have established that extract of Syzigium aromaticum, Piper betle and Aleurites moluccana were show anti bacterial activity against MRSA (Methicillin-resistant Staphylococcus aureus), Staphylococcus aureus and Staphylococcus epidermidis. Kırmusaoglu (2017) found the rate of methicillin resistance of biofilm producer Staphylococcus strains are higher than non-biofilm producer Staphylococcus strains, and MRSE strains were more related with biofilm production (80%, 56/70) than MSSE strains (64%, 138/217) significantly.
Іn studies McFadden (2016) explored the role Staphylococcus aureus autolysins play in biofilm formation, pathogenesis and resistance to both cell wall targeting and protein synthesisinhibiting antibiotics. Using a variety of mutant strains in the USA300 background lacking genes encoding autolysins, sortases, histidine-kinase signaling systems, as well as regulatory proteins, the role of these genes in MRSA could be elucidated. The results suggest a variety of negative phenotypes that correlate with the loss of these key autolysins and regulatory genes. Decreases in biofilm formation, antibiotic resistance, and pathogenesis were seen in many of the mutants. This indicates a possible relationship between autolysins and many of the characteristics of pathogenesis in Staphylococcus aureus. Anitua et al. (2012) got the potential antimicrobial effects of a product (plasma rich in growth factors; PRGF® Endoret®) against both methicillin sensitive and methicillin resistant Staphylococcus aureus and Staphylococcus epidermidis. The microbiological activity of PRGF Endoret against both staphylococcal strains was performed by counting the number of the surviving bacterial colonies after incubation at 0, 4 and 8 h with the different formulations.
Antibacterial potency with change of volume was increased in propotion to increase volume and increased on 6 days, but bacteria was increased. Antibacterial potency of Tangpo-san on S. epidermidis wasn't appeared continuousсделав вывод, что Antibacterial potency of Tangpo-san on cultivation of S. aureus showed continuous, but on cultivation of S. epidermidis was not showed continuous (Seo, 2007).
The purpose of this articleis to establish the antibacterial effect of in vitro herbal infusions on reference strains Staphylococcus epidermidis, Staphylococcus aureus.
Material and methods. 50 species of plant raw materials (seeds, grass, shoots, leaves, breeding, flax, fruit bodies, skin) of different vegetation periods were harvested in the Dnipropetrovsk botanical garden and the recreational zone of the city of Dnipro The collected raw materials were sorted and dried in a drying cabinet ML-309 (Poland) at a temperature of 60 ºС for 5-6 days. Subsequently, the raw material was placed in a grain mill grain laboratory LSMK and crushed to a particle size of 0.5-1.0 mm. The resulting vegetable raw material was packed in disposable polyethylene bags with locks and marketed with stickers. 1 g of appropriate crumbled raw material was weighed using laboratory electron analytical grade ESJ-200-4 (USA) and placed in sterile vials of 10 cm 3 and poured into 5 cm 3 of 96 % ethanol with the appropriate labeling of the vials. Alcoholic tinctures in a ratio of 1: 5 were kept for three weeks by infusion in a dark cool place. After holding, the tincture was filtered through sterile multi-layer gauze filters in sterile vials, which were placed in 50 sterile disks of filter paper 6 mm in diameter, which were kept in appropriate versions of tinctures for 10 days. Before placing the disks on the agar surface with the sowing of the corresponding culture, they were dried in a sterile laminar box (BMB-II-Laminar-C-1,2 CYTOS (Germany) under ultraviolet rays for 30 minutes.
The antibacterial activity of various plant infusions was determined by the method of disk diffusion in agar. From the daily culture of reference cryogenic strains of Staphylococcus epidermidis, Staphylococcus aureus was prepared according to the standard of turbidity of a bacterial suspension of 0.5 unit density McFarland 1.5 × 108 CFUs (colony forming units), which was determined using densitometry Densimeter II. The obtained charge was transplanted onto a Himedia agar with subsequent cultivation in a thermostat TSO-80/1 (Russia) for 24 hours at 37 ° C. On top of the seedings, discs impregnated with appropriate plant infusions were placed on the six-wheel drive in time, as a positive control, placed disks with antibiotics (1 disk contains 30 μg tetracycline, 5 μg ciprofloxacin, 15 μg azithromycin).
A day later, the diameter of the growth inhibition zone (GIZ) of the culture was measured using a template ruler to measure the size of the microorganism growth retardation zones (Antibiotic Zone Scale-C, model PW297, India).
Results and discussion. The results of the influence of ethanol extracts on the growth of Staphylococcus epidermidis are given in Table 1.
Analyzing the effectiveness of the effect of the experimental drugs on Staphylococcus aureus (Table 2), we determined the fluctuations of the growth inhibition zone of more than 10 mm with the use of Juníperus sabína, Styphnolobium japonicum, Cotinus coggygria, Ginkgo biloba, Quercus castaneifolia, Ptelea trifoliata, Toxicodendron orientale (GIZ 10-13 mm), which in 2 and more times below control (tetracycline, ciprofloxacin, azithromycin). Intermediate sensitivity is defined for Clematis flammula, Celastrus scandens, Rhus trilobata (triloboida), which is 4-10 mm insignificantly below control.
We detected a highly sensitive antibacterial effect on the Staphylococcus aureus strain under the action of Cephalotaxus harringtonia, Hedera helix, Geranium sanguineum, and GIZ ranged from 21 to 28 mm. Moreover, if the effect of Cephalotaxus harringtonia at the control level (ciprofloxacin and azithromycin), then Hedera helix, Geranium sanguineum exceeded the control: by 2.1; 1.0 mm and 4.2; 1.0 mm respectively. The highest antibacterial effect is obtained from tetracycline (GIZ within 26-29 mm).

C O N C L U S I O N
In vitro experiment revealed a positive antibacterial effect from the use of extracts of Cephalotaxus harringtonia, Hedera helix, Geranium sanguineum on cryogenic strains Staphylococcus epidermidis, Staphylococcus aureus. We consider it possible to recommend the investigated extracts of Cephalotaxus harringtonia, Hedera helix, Geranium sanguineum for further research in the fight against polyresistant strains of the above-mentioned microorganisms.