Antibacterial and Antibiolm Effects Extracts From Elaeagnus Angustifolia,satureja Montana, Quercus Brantii, Tragopogon Dubius, Sonchus Asper, on the P.aeruginosa and S.aureus

Background: Due to the increase in microbial resistance worldwide and the adverse effects of chemical drugs, the study of antimicrobial compounds in plant extracts as a new source of medicine can be effective in reducing or solving this problem. The purpose of this study antibacterial and antibiolm effects extracts from Elaeagnus angustifolia,satureja montana, Quercus brantii,Tragopogon dubius,Sonchus asper, on the P.aeruginosa and S.aureus Methods: In this cross-sectional descriptive-analytical study, the study population is plants collected in Isfahan and Shahrekord and Pseudomonas and S.aureus bacteria isolated from clinical samples and standard strains. The sampling method was improbably simple and 120g was collected from each of the 9 plants and extracted by massage method. For the qualitative evaluation of antimicrobial properties, you used the agar well diffusion method and for quantitative evaluation of antimicrobial properties, you used the plate microtiter method. To evaluate the anti-biolm properties, plate microtiter method, and staining by violet crystal were used. Results: Based on the ndings of this study and the study of MBC and MIC of the mentioned plant extracts, it can be said that the best antibacterial effect on all studied strains belongs to the plant extracts of Quercus brantii, Elaeagnus angustifolia, satureja montana, Tragopogon dubius, and Sonchus asper, respectively. Among them, Quercus brantii extract has the best effect on all 4 strains. Also, in the study of anti-biolm properties, it was observed that the Quercus brantii, satureja montana, and Elaeagnus angustifolia plant extracts had an anti-biolm effect. Among them, respectively, the most belonging to the Quercus brantii, and the two extracts of Tragopogon dubius, and Sonchus asper do not have good anti-biolm properties. Conclusions: According to the results of this study on the antimicrobial and antifouling properties of these extracts, it is possible to use this plant in the future with more studies and more work on these plants. et al (2018), the antibacterial, antifungal, and antioxidant abilities of T.dubius extract were investigated.Their results showed that this extract has antibacterial effects due to its phenolic compounds [23], which is in line with the present study and the results of this study also showed that T.dubius extract has antibacterial properties against Pseudomonas aeruginosa, Staphylococcus aureus.


Background
According to the latest WHO reports in 2018 from the data of the Organization for the monitoring of Antibiotic Resistance, shows a high level of resistance to some serious bacterial infections in low-income and low-income countries. The Global Antimicrobial Surveillance System (GLASS) shows that antibiotic resistance is widespread among 500,000 people with recurrent bacterial infections in 22 countries [1]. This global threat is increasing and our ability to treat nosocomial or community-acquired infections is declining. Excessive increase in antibiotic use can cause infectious diseases caused by multidrug-resistant microorganisms Therefore, to solve this challenge, identi cation and study of antimicrobial compounds in medicinal plant extracts as a new source of medicine and an alternative method can be effective [2][3][4]. The experience of the last few decades has shown that chemical drugs, with all their effectiveness, have many side effects and there is less pure substance that does not have adverse effects. In contrast, the active ingredients in medicinal plants have a state of biological equilibrium due to their association with other substances. Therefore, they do not accumulate in the body and do not cause side effects, and therefore have a signi cant advantage over chemical drugs [5]. On the other hand, we are facing an increase in treatment-resistant microbial strains, which necessitates the need for new antibiotics. Side effects and drug resistance to chemical drugs, as well as the discovery of new substances such as vitamins, hormones, antimicrobials, antivirals, and antitumors among newly discovered plants, have led to a resurgence in the use of drugs and products. Natural plants should be created and medicinal plants should be a good source for nding different drugs with fewer side effects [6]. Pseudomonas aeruginosa is an important and opportunistic gram-negative pathogen that can cause a variety of clinical infections, including wound and urinary tract infections and circulatory infections, especially in hospitalized and immunocompromised patients. It is the second leading cause of nosocomial pneumonia in the United States and the fth leading cause of nosocomial urinary tract infections in Europe [7]. Pseudomonas aeruginosa is resistant to many disinfectants and antibiotics, including anti-Pseudomonas penicillins, ceftazidime, carbapenems, and cipro oxacin [8]. Pseudomonas aeruginosa has many pathogens that are involved in acute infection. Alginate bio lm formation is one of the most signi cant mechanisms contributing to antibiotic resistance in Pseudomonas aeruginosa [9]. Staphylococcus aureus is gram-positive and optional anaerobic cocci. Staphylococcus aureus can cause a wide range of infections, from simple skin infections (such as boils, boils, scabs, eyelashes, and abscesses) to life-threatening diseases (such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, and sepsis). Staphylococcus aureus is one of the ve most common causes of nosocomial infections, especially post-surgical wound infections. Each year, 500,000 people in US hospitals become infected with Staphylococcus aureus [10][11][12]. One of the pathogenic factors of this bacterium is the formation of bio lm, the ability of Staphylococcus aureus to bind and form a bio lm is a determining step in the chronicity of the disease [13]. Methicillin-resistant Staphylococcus aureus (MRSA) is a speci c strain of the bacterium that is resistant to most antibiotics. MRSA is more common in hospitals but also acquired in the community [14] this study aimed to investigate the methanolic extract of several plants to evaluate the antimicrobial and antibio lm properties against two drugs resistant to Pseudomonas aeruginosa and Staphylococcus aureus. The studied plants include (Elaeagnus angustifolia), (satureja montana), (Quercus.brantii), (Tragopogon dubius), (Sonchus asper), the last two of which are weeds.

Methods
In this cross-sectional descriptive-analytical study, the studied population is plants collected in Isfahan and Shahrekord and Pseudomonas and Staphylococcus aureus bacteria isolated from clinical samples and standard strains. Plants collected by botanists were approved and other similar plants were excluded from the study. The sampling method was improbably simple and 120 g was collected and extracted from each of the 9 plants. Microbial strains did not require sampling and were prepared as lyophilized ampoules from Hajar Teaching Hospital in

Determination of antibacterial properties
The desired clinical bacterial strains were prepared from two bacteria P.aeruginosa, S. aureus from each of the two microbially resistant strains by microbial collection from the laboratory of Hajar Teaching Hospital in Shahrekord. And again antibiotic pattern tests were performed on it and con rmed And two standard strains of each bacterium, with a speci c antibiogram pattern according to CLSI2018 standard, were prepared, cultured, and activated in blood agar medium and again for microbial identi cation tests [15]. To evaluate the antibacterial properties of the extracts, qualitative concentrations of 100,70,50,30,10 (mg / ml) were prepared from each of the extracts using the formula M1V1 = M2V2. It was performed by the Nathan Agar Well Diffusion method and all plates were incubated at 37 ° C for 24 hours and the next day the diameter of the growth inhibition zone was measured with a ruler [16]. The Microdilution broth method was used to determine the MIC in plate 96 of sterile cells [17]. After 24 hours of incubation at 37 ° C, the bottom of the plate was examined under light and the presence or absence of turbidity indicated the growth or non-growth of bacteria was noted in the special table. Due to the color of the extracts to ensure before and after incubation, the light absorption plate of the wells was read at OD = 260 nm. By comparing the light absorption before and after incubation, the concentration that had changed signi cantly was selected as MIC to ensure 100 Landa of the contents of all wells in Müller Hinton agar and the concentration that prevented the growth of 99.9% of bacteria. The title MBC and the well before that were chosen as MIC. The initial stock of the extract was selected according to the results of agar well diffusion and made with the help of DMSO solvent and ltered with a 0.45 micron needle lter. The initial stock of the extract was selected according to the results of agar well diffusion and made with the help of DMSO solvent and ltered with a 0.45 micron needle lter. And was selected for the microtiter test It should be noted that the initial stock is a concentration higher than the concentration of the rst well of this method.

Determination of anti-bio lm properties
To dilute the extracts of each plant in the formation of bacterial bio lm, the dilution method was used in the well. To do this, two dilutions before and after MIC were selected for this purpose. After the procedure and incubation for 24 hours, remove the contents of the wells and add sterile physiology serum Shake the microplate gently until the cells are separated by weak or free binding. Remove the contents of the wells again and add 300 microliters of 96% alcohol to the wells for 15 minutes to x the cells After removing the alcohol, the wells were stained with 2% crystal violet dye for 5 minutes and after this time, the wells were washed and 33% acetic acid was added to them and set at 630 nm in ELISA reader. Contracted and then its OD is measured [18]. Then the extract bio lm reduction was investigated using the following formula. The initial stock of the extract was selected based on the results of agar well diffusion It was made with the help of DMSO solvent and ltered with a needle lter of 0.45 microns. And for plate microtiter test was selected. It should be noted that the initial stock is a concentration higher than the concentration of the rst well of this method.

Results
Qualitative method of determining the antibacterial property or the same as diffusion to qualitatively con rm the antibacterial properties of the extracts and nd the approximate range of antibacterial properties of the extracts. The results showed that each plant had antibacterial properties to each extract and the appropriate concentration range for MIC was determined. It was found that all extracts in the tested concentrations of 100, 70, 50, 30, and 10 (mg/ml) have antimicrobial properties. And some extracts in the concentration range less than 10 also had this property. (as shown in Tables 1, 2) In this study, MIC and MBC and anti-bio lm properties of Elaeagnus angustifolia,satureja montana, Quercus brantii,Tragopogon dubius,Sonchus asper plants for standard and resistant strains of P.aeruginosa, standard and resistant strains of S. aureus are as follows, Based on the ndings of this study and the study of MBC and MIC of the mentioned plant extracts, it can be said that the best antibacterial effect on all studied strains belongs to the plant extracts of Quercus brantii, Elaeagnus angustifolia, satureja montana, Tragopogon dubius, and Sonchus asper, respectively. Among them, Quercus brantii extract has the best effect on all 4 strains. Also, in the study of anti-bio lm properties, it was observed that the Quercus brantii, satureja montana, and Elaeagnus angustifolia plant extracts had an anti-bio lm effect. Among them, respectively, the most belonging to the Quercus brantii, and the two extracts of Tragopogon dubius, and Sonchus asper do not have good anti-bio lm properties. The results of the study of Chusri et al (2012) showed that the extract of infectoria. Quercus is effective on methicillin-resistant Staphylococcus aureus bio lm formation [19] In a study by Hobby et al (2012), In relation to the effect of Quercus extract on the formation of Staphylococcus aureus bio lm, it was found that the Staphylococcus aureus was inhibited by Quercus extract [20]. Mohammadi et al (2015) In a study showed that Quercus extract strongly inhibited the formation of Streptococcus mutans bio lm at concentrations higher than 19.5 µg / ml [21]. This is in line with the results of the present study on the effect of Quercus extract on the formation of Staphylococcus aureus bio lm up to a concentration of 0.03 mg/ml. Sabir et al (2007) found that ether extract of Elaneagnu umbellate is effective against Escherichia coli bacteria Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. And its aqueous extract can inhibit the growth of Escherichia coli and Staphylococcus aureus [22]. This study is in line with the present study and this study also showed that the methanolic extract of Elaneagnu against Pseudomonas aeruginosa, Staphylococcus aureus has anti-bacterial properties. In the study of Uysal et al (2018), the antibacterial, antifungal, and antioxidant abilities of T.dubius extract were investigated.Their results showed that this extract has antibacterial effects due to its phenolic compounds [23], which is in line with the present study and the results of this study also showed that T.dubius extract has antibacterial properties against Pseudomonas aeruginosa, Staphylococcus aureus.
In the study of Shahbazi et al (2017), the antimicrobial activity of several plants was investigated, among which Tragopogon extract of the Kermanshah region was proven to have antimicrobial properties [24]. This is in line with the present study and the results of this study also showed that Tragopogon extract has antibacterial properties against Pseudomonas aeruginosa, Staphylococcus aureus.
In the study of Talei et al. (2008), antimicrobial properties were not found for the native Tragopogon of Khorramabad studied by them [25]. Which is contrary to the results of the present study.
In the study of Borjian et al. (2016), the antibacterial effects of hydroalcoholic extract of Q. brantii fruit on Listeria monocytogenes and Enterococcus faecalis were studied in the laboratory. These two bacteria have a growth inhibitory effect [26]. The results of this study also showed that the extract of the Quercus has antibacterial properties against Pseudomonas aeruginosa, Staphylococcus aureus.
In the study of Omidi et al. (2016), the results showed that the methanolic extract of Quercus brantii ,Elaeagnus had antibacterial and antibio lm effects against Pseudomonas aeruginosa [27].
The results of this study also showed that the extract of Quercus has antibacterial properties against Pseudomonas aeruginosa, Staphylococcus aureus.
In the study of Khan et al. (2016), the antibacterial and antifungal effects of Quercus against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus were investigated. [28] In the study of Jamehdor et al. (2014) the results showed that the aqueous extract of Hippophae.rhamnoides has the greatest effect on Pseudomonas aeruginosa [29]. This study contradicts the results of this study and this study showed that the highest antibacterial effect on the standard strain of Pseudomonas aeruginosa is Quercus extract and in the second degree is Elaeagnus extract.
In the study of Ateufack et al (2014), the antibacterial effect of H.asper against bacteria of the Enterobacteriaceae family was investigated and the results showed that H.asper leaves have antibacterial and antidiarrheal effects [30]. This is in line with the present study and the results of this study also showed that H.asper extract has antibacterial properties against Pseudomonas aeruginosa, Staphylococcus aureus. Azaz et al (2002), investigated the antibacterial activity of 3 species of satureja in S. Coeralen, S.pilosa, and S. Boissieri against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium. The results of this study showed that satureja has antibacterial properties [31].
In 2011, Mahboubi et al(2011) studied the essential oil of Satureja hortensis. In this study, the activity of this species against the bacteria of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli Salmonella typhimurium was proved [32].
In Özkalp et al (2009)studies, the results showed that Satureja has an inhibitory effect on the bacteria of Streptococcus pneumoniae, Salmonella enteridis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and S.mutans [34]. The results of these studies are in line with the results of the present study. In general, it can be said that the results of these studies are in line with the results of our study and con rm the inhibitory and lethal effect of extracts on Pseudomonas aeruginosa.
In general, it can be said that the results of these studies are in line with the results of our study and con rm the inhibitory and lethal effect of extracts of Elaeagnus angustifolia,satureja montana, Quercus brantii,Tragopogon dubius,Sonchus asper, on the P.aeruginosa and S.aureus.
Slight differences in MIC and MBC values obtained from this study with other studies are probably due to the use of different parts of the plant such as leaves, fruits, owers, gums, differences in plant species, harvest time, plant type, and strain under investigation.

Conclusions
According to the results of this study, the methanolic extract of the medicinal plants of, Elaeagnus angustifolia,satureja montana, Quercus brantii,Tragopogon dubius,Sonchus asper in vitro, has the anti-bacterial on the P.aeruginosa and S.aureus. And three extracts of Elaeagnus angustifolia,satureja montana, Quercus brantii leaves reduce the formation of bacterial bio lm. And due to the fact that antibiotic resistance is increasing day by day and the formation of bio lm by Pseudomonas aeruginosa causes an increase in resistance to antibiotics. Probably with more studies and more work on these plants in the future can use these plants as a therapeutic supplement along with antibiotics. Declarations