Evaluation of inhibitory and plasmid curing activity of Citrus aurantium L fruit extracts against some bacteria isolated from urinary tract infections

In this study, the sensitivity and resistance of six types of bacterial isolates, Escherichia coli, pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, and Klebsiella pneumoniae, against 10 antibiotics were tested for 10 antibiotics, Ciprofloxicine (CF) 25 g / ml Amoxicillin (AX) 50 g / ml, Ampicillin (Am) 50 μg / ml, Erythromycin (Ery) 15 μg / ml, Gentamycin ( GN) 25 g / ml, Rifampicin (RA) 50 μg / ml, Streptomycin (Sm) 25 μg / ml, Tetracyline (TE) 15 μg / ml Trimethprim (Tri) 10 μg / ml Nalidixic acid (Nal) 30 μg / ml. The bacterial isolates showed different resistance to most antibiotics. One isolate of each bacterium was selected for subsequent experiments. The aqueous and ethanol extracts of Citrus aurantium L fruit were prepared. The inhibitory effect of the prepared plant extracts on the selected bacteria was investigated by using the Agar diffiuision method and the Turbidity method. The prepared plant extracts showed a clear growth inhibitory activity, which was estimated by measuring the inhibition zone with a millimeter of studied bacteria. The results obtained showed that the value of the growth inhibitory activity increased with increasing the concentration of the extracts. Sub minimum inhibition concentration of plant extracts was used as a curing to remove antibiotic resistance of the bacterial species under study. Varying values for the percentage loss of antibiotic resistance were obtained as a result of the efficacy of the extracts, but some extracts did not show curing efficacy. The results of the curing experiments were supported by the characterization of the plasmid DNA on the agarose gel of the curing isolates. Electrophoresis showed the disappearance of the plasmid DNA bundles from the agarose gel for most of the bacteria under study compared to the non-curing isolates. Keyword: Bacteria Urinary tract infections, Plasmid DNA, Citrus aurantium L extracts ايرتكبلا ضعبل جنرانلا رامث تاصلختسمل يديمزلابلا دييحتلاو ةيطيبثتلا ةيلاعفلا مييقت تاباهتلا نم ةلوزعملا ةيلوبلا يراجملا 1 * ، دبع دمحا يميعنلا ميهاربإ قازرلا ، 2 يوافصلا زيزع دماح للاط راون 1 ،* 2 مولع مسق ةايحلا قارعلا ،لصوملا ،لصوملا ةعماج ،ةفرصلا مولعلل ةيبرتلا ةيلك ، Journal of Education and Science (ISSN 1812-125X), Vol: 30, No: 3, 2021 (236-251) Special Issue for Proceeding of 3 National (1 international conference of biology) (ICBSUM 2021) 5, 6 July College of Education for Pure Science, University of Mosul, Mosul, Iraq. Downloaded from https://edusj.mosuljournals.com/ 237 ةصلاخلا : يهو ةيموثرجلا تلازعلا نم عاونا ةتس ةمواقمو ةيساسح رابتخا مت ةسا ردلا هذه يف Escherichia coli ، pseudomonas aeruginosa ، Staphylococcus aureus ، Proteus vulgaris ، Proteus mirabilis و Klebsiella pneumonia هاجت 10 يهو ةيويح تاداضم Ciprofloxicine (CF) 25 g / ml ، Amoxicillin (AX) 50 g / ml ، Ampicillin (Am) 50 μg / ml ، Erythromycin (Ery) 15 μg / ml ، Gentamycin ( GN) 25 g / ml ، Rifampicin (RA) 50 μg / ml ، Streptomycin (Sm) 25 μg / ml ، Tetracyline (TE) 15 μg / ml ، Trimethprim (Tri) 10 μg / ml ، Nalidixic acid (Nal) 30 μg / ml . ةنيابتم ةمواقم ةيموثرجلا تلازعلا ترهظا بلغلأ ميثا رجلا نم عون لك نم ةدحاو ةلزع رايتخا متو ةيويحلا تاداضملا ءا رجلأ .ةقحلالا براجتلا جنرانلا تابن رامثل ةيلوناثيلااو ةيئاملا تاصلختسملا ترضح Citrus aurantium L. fruit . نع يرحتلا مت راشتنلاا ةقيرط مادختساب كلذو ةراتخملا ميثا رجلا عاونا ىلع ةرضحملا ةيتابنلا تاصلختسملل ومنلل يطيبثتلا ريثأتلا راكلأاب Agar diffiuision method ةقيرطو سايق ةراكعلا Turbidity method ةطبثم ةيلاعف حضاو لكشب ترهظا ةرضحملا ةيتابنلا تاصلختسملا . مت ومنلل ومنلل ةطبثملا ةيلاعفلا ةميق نا اهيلع لوصحلا مت يتلا جئاتنلا تحضواو .ةسوردملا ميثا رجلل ململب طيبثتلا ةقطنم سايقب اهريدقت ( ىندلاا طبثملا تحت زيكرتلا .تاصلختسملا زيكرت ةدايزب دادزت Sub-MIC ) Sub minimum inhibition concentration ماعك مدختسا ةيتابنلا تاصلختسملل ديحم ل ةلا زلإ ةيويحلا تاداضملا ةمواقم عاونلأل ةنيابتم ميق ىلع لوصحلا مت .ةسا ردلا ديق ةيموثرجلا .ةديحم ةيلاعف تاصلخستملا ضعب رهظت مل نكل تاصلختسملا ةيلاعفل ةجيتنك ةيويحلا تاداضملل ةمواقملا نادقف ةبسنل تاملكلا ةيحاتفملا : اندلا ،ةيلوبلا يراجملا ايرتكب جنرانلا تاصلختسم ،يديمزلابلا . Introduction: Urinary tract infections (UTI) are the most common type of bacterial infection worldwide. During 2016, it is estimated that 150 million cases are recorded annually worldwide [1]. Infection occurs in both sexes and all ages, and females are more susceptible to infection than males due to the anatomical and physiological differences of the urogenital system in addition to the difference in lifestyle, and the incidence of infection increases in the elderly men. And when the infection occurs, different parts of the urinary system may have the infection [2]. Microbial infection, especially with the following species Pseudomonas aeruginosa, Proteus vulgaris, Proteus mirabilis, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae are among the main causes of UTIs [3,4]. As it leads to the emergence of various signs and symptoms, including pain when urinating, dysuria, bloody urine, heartburn, frequent urination, nausea and vomiting [2]. As a result of the emergence of multidrug-resistant bacterial strains, the incidence of urinary tract infections has increased dramatically and this disease has become a major social and economic burden worldwide [2]. Natural herbs are highly effective in removing bacterial resistance with high efficiency, and they are available and cheap in addition to the side effects are very little or no. So, it has attracted the interest of researchers interested in exploring the treatment of UTIs with medicinal plants. There is a need for more discoveries in order to reveal the plant components responsible for treating urinary tract diseases and their mechanism of action [2]. Citrus aurantium belongs to the family of Citrus aurantium (Sour orange), and its scientific name is Citrus aurantium. It also follows the genus Citrus, which contains four economically important groups, the most important of which is the orange group of Citrus aurantium. [5]. Also known as Seville orange, sour orange, or bitter orange, Citrus aurantium is a small tree about five meters high with scented white blossoms [6, 7]. Citrus fruit originated Journal of Education and Science (ISSN 1812-125X), Vol: 30, No: 3, 2021 (236-251) Special Issue for Proceeding of 3 National (1 international conference of biology) (ICBSUM 2021) 5, 6 July College of Education for Pure Science, University of Mosul, Mosul, Iraq. Downloaded from https://edusj.mosuljournals.com/ 238 in East Africa, Arabia and Syria, and is cultivated in Spain, Italy and North America [6,7]. Citrus aurantium is a very common plant that is available and cheap in Diyala Province / Iraq [8]. Medical part the following parts of Citrus aurantium are used for medicinal purposes (flowers, fruits, roots, leaves, and essential oils) [10-9].


Introduction:
Urinary tract infections (UTI) are the most common type of bacterial infection worldwide. During 2016, it is estimated that 150 million cases are recorded annually worldwide [1]. Infection occurs in both sexes and all ages, and females are more susceptible to infection than males due to the anatomical and physiological differences of the urogenital system in addition to the difference in lifestyle, and the incidence of infection increases in the elderly men. And when the infection occurs, different parts of the urinary system may have the infection [2]. Microbial infection, especially with the following species Pseudomonas aeruginosa, Proteus vulgaris, Proteus mirabilis, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae are among the main causes of UTIs [3,4]. As it leads to the emergence of various signs and symptoms, including pain when urinating, dysuria, bloody urine, heartburn, frequent urination, nausea and vomiting [2]. As a result of the emergence of multidrug-resistant bacterial strains, the incidence of urinary tract infections has increased dramatically and this disease has become a major social and economic burden worldwide [2]. Natural herbs are highly effective in removing bacterial resistance with high efficiency, and they are available and cheap in addition to the side effects are very little or no. So, it has attracted the interest of researchers interested in exploring the treatment of UTIs with medicinal plants. There is a need for more discoveries in order to reveal the plant components responsible for treating urinary tract diseases and their mechanism of action [2]. Citrus aurantium belongs to the family of Citrus aurantium (Sour orange), and its scientific name is Citrus aurantium. It also follows the genus Citrus, which contains four economically important groups, the most important of which is the orange group of Citrus aurantium. [5]. Also known as Seville orange, sour orange, or bitter orange, Citrus aurantium is a small tree about five meters high with scented white blossoms [6,7]. Citrus fruit originated in East Africa, Arabia and Syria, and is cultivated in Spain, Italy and North America [6,7]. Citrus aurantium is a very common plant that is available and cheap in Diyala Province / Iraq [8].
Medical part the following parts of Citrus aurantium are used for medicinal purposes (flowers, fruits, roots, leaves, and essential oils) .

Medicinal uses:
Citrus fruits, peel, leaves, seeds, and essential oils are used in food, perfumes, medicinal and cosmetic applications, as well as in the food, confectionery, and perfume industry (11). They are widely used in nutritional supplements to control appetite, weight, athletic performance and energy. Citrus products are also used in the form of food such as juices and bitter orange jam ( (12,11. It is believed that Citrus has the following vital activities as it is considered an antioxidant (13,14), Anti-inflammatories (15), antimicrobials (16,17), and may be responsible for preventing cancer and degenerative diseases (14). These vital activities of aurantium are due to the presence of bioactive compounds such as phenols, flavonoids, essential oils and vitamins (18,19). Citrus aurantium is also used in the treatment of many diseases such as anxiety (20), lung and prostate cancer (21), digestive disorders and obesity (22,23). Citrus aurantium is rich in natural vitamin C and antioxidants that increase the activity of the immune system (24,25). Citrus aurantium is traditionally used to treat the intestines and some disorders (such as constipation, cramps, diarrhea and abdominal cramps), respiratory tract convulsions (such as cough, cold, bronchitis and tuberculosis), obesity, menstrual disorders, cardiovascular disease (angina, high blood pressure) and Stress and depression (26).
For the purpose of determining the effectiveness of the Citrus fruits on antibiotic-resistant bacteria, the study was designed to: ❖ study the sensitivity and resistance of isolated species to antibiotics study the inhibitory and curing effect of Citrus aurantium extracts on bacteria under study and Characterization of the plasmid DNA content by using gel electrophoresis technique of bacterial species before and after treatment with Citrus fruit extracts.

Research Method
Citrus fruits were obtained from the local market in Mosul.

Antibiotic resistance and sensitivity of isolated bacteria:
The medium was prepared by adding the appropriate antibiotic to the sterilized agar nutrient medium cooled to (45-50 °C) and the final concentration (µg/ml) which prepared stock solutions for antibiotics according to the method used by [27] by dissolving a certain amount of the antibiotic in the appropriate solvent, then sterilizing all the stock solutions by filtering (0.22). micrometer) and kept until use at a temperature of -20 °C, according to [28].
An electric grinder was used to crush the Citrus fruits, as 40 gm of the ground fruit were added to 160 ml of sterile distilled water, so that the ratio became (4: 1) weight: volume. Then crushed the plant under study using the Grater3, Moleny-supplied Blender Crusher in an ice bath for one hour, after which using the electric magnetic stirrer stir the mixture for 60 minutes in order to detonate the plant cell walls for the purpose of soaking Leave the mixture in the refrigerator for 24 hours, use Several layers of gauze to filter the mixture, then filter the mixture by using a Buechner funnel and using Whatmann No.1 filter paper, thus preparing the aqueous plant extract. In airtight glass bottles and in an atmosphere free of moisture, the samples were preserved by freezing for later use [29].

Preparation of alcoholic extracts:
Prepared according to the method of the researcher [30]. And modified according to the method of the researcher [31]. Dissolve in 400 ml of ethyl alcohol at a concentration of 95% 40 g of the vegetable form in an ice bath, then shake the mixture well and leave in the refrigerator for 24 hours, after which it was filtered through a Buechner funnel and then placed in the Rotary Vaccume Evaporator which is provided by Electrothermal. The device is based on evaporation under vacuum pressure and a temperature of up to 40 ° C. The layer formed after evaporation from the crude extract was collected, and then it was frozen by freezing in sterile glass bottles with a tight lid until use.

Antimicrobial activity:
The inhibitory efficacy of alcoholic and aqueous extracts on the growth of the studied bacteria was tested depending on the [32] where the broth medium fed with the bacteria to be tested was inoculated and after incubation for 24 hours at a temperature of 37 ° C, the suspension was diluted using physiological saline and by comparison with the standard control tube of McFarland's solution, which is equivalent to 108 cells / Then, 0.1 ml of the bacterial suspension was spread on the nutrient media using a cotton swab, then the dishes were left for half an hour at a temperature of 37 ° C in order to absorb. Using the drilling method, he studied the effect of plant extracts on the bacteria to be studied, where holes were made in the dishes containing the nutrient pellet medium using a 6 mm stainless steel borer, then 0.1 ml of each concentration of aqueous and alcoholic extracts was placed in each hole, after which the dishes were incubated for 24 hours. At a temperature of 37 ° C, the damping zones around each hole are measured in mm [33].

Measurement of minimal inhibition concentration (MIC) using agar dilution technique:
The MIC of extracts was tested by the agar dilution method [34], With some modifications. Serial doubling concentrations of plant extracts were prepared, whose value ranged between (12.5-200) mg / ml, and different proportions of these extracts were added to Muller-Hinton agar medium, and after hardening of the media, the dishes were inoculated as one drop with bacterial suspensions and the dishes were incubated at a temperature of 37 ° C for 24 hours. . The lowest inhibitory concentration is then estimated after 24 hours, which represents the lowest concentration of the plant or chemical extract in which no visible bacterial growth was observed, while (Sub-MIC) represents the lowest inhibitory concentration in which the bacteria can grow, which was used in the curing experiments [35,36].
The method of [37]. was followed, a culture of isolated bacteria under study was prepared where it was grown in the presence of the plant extract and at the required sub-MIC for 24 hours at a temperature of 37 ° C, after which several decimal dilutions were brought to the bacterial culture and 0.1 ml was taken From the last three years, it was spread on the nutrient agar and incubated at 37 ° C for 24 hours. The plates were incubated at a temperature of 37°C for a period of 24 hours after which the colonies were transferred to media containing the antibiotics used under study separately, Colonies that fail to grow on medium of antibiotics are curing colonies. Calculates the percentage of bacterial colonies lost to antigen resistance. The physical loss of curing plasmids is inferred by the electrophoresis process on gel electrophoresis [38].

Isolation of plasmid DNA:
The plasmid was extracted from bacterial cells using Promega's prepackaged kit: 5 ml of the broth medium containing a suitable antibiotic are inoculated with a young colony of isolated bacteria under study and incubated for 24 hours, the bacterial growth is transferred to 1.5 ml tubes, then a centrifugation is carried out at maximum speed for one minute and the saliva is completely discarded. Added 100 µl of Solution I, and are re-suspended sediment using a mixing device Vortex. Add 200 μl of Solution II and mix by gently inverting several times without using a mixer. Add 150 μl of Solution III and mix directly several times as in the previous step. Conduct the centrifuge for 10 minutes.Transfer 400 μl of the filtrate to a new 1.5 ml tube. Add 0.8 ml of 95% ethanol and mix by stirring the tube and leave at room temperature for 5 minutes. Centrifuge at maximum speed for 10 minutes and get rid of the odorant ethanol. he precipitates was washed with 200 μl of 70% ethanol and mixed gently, then centrifuged for 5 minutes and the ethanol was removed. The tube is placed upside down on blotting paper to get rid of the ethanol residue until the precipitate is completely dry. The precipitate representing the plasmids was dissolved with 50 μl of TE solution and preserved until use.

Agarose gel preparation and electrophoresis of isolated plasmid DNA:
For DNA migration and detection, a 1% acarose gel is prepared and to obtain this concentration, 0.5 g of acarose powder is dissolved in (50) ml of X1 TBE and 3 μl of red safe dye is added, this is done using a heat source with constant stirring until boiling and left to cool To a temperature of (60-50) C. Then the gel solution is poured into the Tray basin of the relay device after the special comb is installed to form the Wells pits at the edges of the gel, taking into account that the pouring is quietly to avoid the formation of bubbles, and that they are formed by using a pipette, then leave the gel until it solidifies.
Then the Tray is placed in an electrophoresis sump containing an appropriate amount of X1 TBE solution, after which the comb is gently lifted. Transfer samples are prepared by mixing (5) μl of DNA sample with (3) μl of loading solution. After that, the relay is switched on by passing the electrical current at a voltage difference of (5) volts / cm, and the process takes (2-1.5) hours. The gel is then imaged under ultraviolet rays using a Gel Documentintation to be able to visualize the plasmid DNA bundles. From the results of Table (1) the bacteria showed a variation in their resistance to antibiotics, as most of the isolates gave multiple resistance to most of the antibiotics used, with the exception of bacteria Protus vulgaris that gave resistance to amoxicillin and ampicillin by 100%, and by 50% to trimethoprim. Bacterial resistance to antibiotics has become one of the major problems that threaten public health. In recent recent years, infections caused by intestinal bacteria that are multidrug-resistant have been recorded in almost all European countries for several reasons, including the indiscriminate and widespread use of antibiotics in the treatment of infections caused by bacteria, which exacerbated The crisis, as it led to the emergence of strains that have multiple resistance to most groups of antibiotics, this problem appears clearly in hospitalized patients, as it causes great economic losses in health care centers as it increases treatment costs [39]. . Bacteria have more than one type of resistance, the first of which is self-resistance, and internisc resistance occurs as a result of the possession of these bacteria possessing natural characteristics that distinguish them from others responsible for them by genetic factors that they possess, which makes them less susceptible to antibiotics, including the impermeability of the outer wall of these bacteria, as well as their ability to spit out the antibiotics. The cell. As for the other type of resistance to these bacteria, it is the acquired resistance. Acquired resistance appears as a result of exposure of the sensitive strains of these bacteria to antibiotics and their transformation into resistant strains by several means, including, Chromosomal mutations, Pump-flow pumps, transposon elements, Effervescent plasmids and other methods of Resist plasmid. [40]. Table (2) The inhibitory effect and MIC of the Citrus aurantium L fruit extracts with different concentrations on the bacterial species (diameter of the inhibition zone, measured in mm).

Inhibition Activity of Plant Extracts Test
(-) no effect From the results of the above table, it appears that the extracts of the Citrus fruits have a clear effect on inhibiting the growth of the bacteria under study. The diameters of the inhibition circuit varied between high and weak inhibition and non-inhibition, and the concentration was given 200 mg / cm3 the highest effect of either the aqueous or alcoholic extract on the bacteria under study, and the concentration value ranged. The minimum inhibitory level is between (0.125-1) mg / cm3. Citrus aurantium contains many primary and secondary metabolites responsible for the therapeutic effects of Citrus aurantium contains vitamins, minerals, phenolic compounds and terpenoids, Among the various components of narang are the flavonoids belonging to phenols which are important due to their pharmacological and physiological role and important health benefits [41,42]. Journal of Education and Science (ISSN 1812-125X)     From the above pictures, the results of the curing are shown, the removal of antibiotic resistance, through the absence of bacterial growth or weak growth after adding the aqueous or alcoholic extract of Citrus aurantium. Through the above results it was found that many of the bacterial species under study have remained resistant to a number of antibiotics, including (TE, Tr) after curing and this may indicate that the gene related to resistance to these antibiotics is located on the bacterial chromosome and was not affected by this process. As for the discrepancy after curing between the bacterial species under study in terms of their resistance to antibiotics, it is because the plasmid responsible for carrying antibiotic resistance is R-plasmid, which consists of two molecules and each molecule contains genes responsible for antibiotic resistance. The results of the curing showed that the bacterial species S. aureus, K. pneumonia and P. aeruginosa were the most affected by the curing test for both aqueous and alcoholic extracts of Citrus aurantium, while the bacterial species E. coli and Pr. vulgaris and Pr. Mirabils are the other affected by the curing using water and alcoholic extracts of Citrus aurantium, but at a lower level than the bacterial species S. aureus, K. pneumonia and P. aeruginosa, and it may be due to what these aqueous and alcoholic extracts contain of effective compounds, including tannins, phenols, alkaloids, flavonoids, and others [43].

MIC
Bacterial growth Weak growth No growth area

Effect of aquous and alcoholic extracts as curing agent of growth of isolated bacteria:
The method of testing the turbidity mentioned in the materials and working methods was used to study the effect of the extracts used under study on the growth of bacterial species before and after curing. The above table notes that the curing agent (aqueous and alcoholic extracts) caused a clear decrease in the optical density values of the bacterial colonies after the neutralization process. There are many factors that affect the numbers of bacteria, including temperature and pH, which affect the activity of enzymes, proteins, and cell membranes of the bacteria. Any change in these two factors will be reflected in the growth and preparation of the bacteria (44). Also, plant extracts may contribute with the materials they contain. Effective in influencing the growth and viability of bacteria (45). 1  2  3  4  5  6 The apparent contrast between the bacterial species after curing in terms of their resistance to antibiotics is due to the fact that the plasmid that carries antibiotic resistance is of the R-plasmid, as it is in the form of two molecules and each molecule carries genes responsible for resistance. From the above results of curing, we find that the rates of curing efficiency of plant extracts on the bacteria under study showed a variation in the rates of curing and loss of resistance to antibiotics. The plasmid DNA of the parent cell and the distribution of these plasmids during the process of cell division leads to the production of nuclear cells with few plasmids and at other times lacking plasmids. Consequently, the bacteria missing the plasmids are transformed from resistance to antibiotics to sensitive [46].