Antimicrobial activity of Loranthus europaeus L. and Lawsonia inermis L. extracts against clinical Methicillin-resistant Staphylococcus aureus isolated from boil infections

This study included to collect 106 samples boil swab from patients, males and females, at Baqubah Teaching Hospital whose ages ranged between (1-80) years old. At the period of 1 st July 2016 to the end of January 2017. Aimed to identification of bacterial isolates and determine the antibiotic resistance of isolates. In addition to determine the activity of aqueous and alcohol of Loranthus europaeus and Lawsonia inermis extracts on bacterial isolates. The results showed a significant difference (p <0.05) in the percentage of infected females (58.49%) compared to the infected males (41.51%). The study involved isolation and diagnosis of (41) (38.67%) of Staphylococcus aureus, of which, 5 (12.20%) were methicillin-resistant isolates. Bacterial isolates were identified initially depending on cultural and microscopic features and biochemical reactions test. Antibiotics sensitivity of Staph. aureus conducted for 14 β-lactam antibiotic groups. All of bacterial isolates (100%) were resistant to ceftriaxone, cefotaxime and cefepime, and (92.68%) to cefoxitin and penicillin G, while (100%) of isolates were sensitive to Imipenem and Meropenem. Regarding plant extracts, various concentrations of hot aqueous extracts were prepared for each one of them (12.5, 25, 50, 100 and 200) mg/ml against methicillin-resistant Staph. aureus . When Loranthus europaeus’s extract was used, the diameters of inhibition zone were (14.57, 15.28,16.00, 23.71 and 25.00 mm) while (0.00, 11.85, 18.00, 21.28 and 24.14 mm) when Lawsonia inermis’s extract was used. Like aqueous extracts, same range of concentrations were prepared for alcoholic extracts. The diameters of inhibition zone were (7.57, 8.42, 10.57,13.28, 17.28), (11.14, 17.00, 20.85, 25.42, 28.42) when alcoholic extracts of Loranthus europaeus and Lawsonia inermis, respectively were used. Comparing the activity of hot and alcoholic aqueous extracts of the tested plants, alcoholic extracts of Lawsonia inermis had a higher inhibitory effect against S. aureus isolates.


Introduction
Boils are deep inflammation of hair follicles, leading to accumulation of pus and dead tissues.Boils appear as red swollen nodules in many parts of the body caused by Staph.Aureus [1] and especially Methicillin -resistant Staph.aureus (MRSA), the most serious pathogen of the skin and soft tissue.This disease is often recurrent and may spread among family members, where some healthy people are considered as carriers of Staph.aureus [2].Various tissue diseases caused by Staph.aureus are nosocomial infections spreading through USA, and Europe [3].However, Staph.aureus now appears in the general public as well as the healthy, with 10% infection in some hospitals and nearly 65% in others [4].Spreading of these infections and antibiotic resistance are due to the abuse and overuse of antibiotics as well as horizontal gene transfer among microbial community [5].Resistance of Staph.aureus to antibiotics such as Oxacillin, Methicillin and βlactam group antagonists is due to the production of β-lactamase enzymes.These enzymes are reducing the amount of Penicillin Binding Protein (PBP2), and enhancing bacterial resistance to antibiotics.This binding protein, which is encoded by mecA gene, has a low affinity for the β-lactam antagonists, lead to resistance for all β -lactam antagonists [6].These bacteria cause killing and rapid diseases such as necrotizing pneumonia and necrotizing fasciitis [7].Biologists refereed to the activity of medical herbs as effective antimicrobial agents and as alternative treatment for antibiotics.The side effects of these antibiotics on human health also encouraged many people to use alternative medical herbs as effective therapeutic substances [8].The interest of companies to manufacture pharmaceuticals and medical drugs from raw plants has increased because of the importance of plant extracts as safe substance compared to antibiotics, in addition to their availability, accessibility at cheapest price, stability, and bioactivity [9].Lowsonia inermis plant extracts were used as traditional treatment among Arabs for different skin infections [10] The leaves, flowers, seeds, roots and bark were used as ancient traditional medicine for various diseases, such as arthritis, headache, ulcer, leprosy, heart disease, and diabetes [11].It is also used as an antifungal, antiviral and antiparasital agent [12].Loranthus europaeus is used as treatment for eczema, foot ulcer, granulomatous tumor, and tuberculosis [13].It has also been used since ancient times to treat various dermatologic infections including genital warts, psoriasis, basal cell carcinoma, Baghdad boil or sore, as well as boils and abscesses [14].This study aimed to evaluate of the antimicrobial effect of Loranthus europaeus and Lawsonia inermis extracts against clinical isolates of Methicillin resistant Staph.aureus isolated from boil infection.

Materials and Methods
A total of (106) swabs were collected from boil infection patients who visited the consultative clinic of Baqubah teaching hospital in Diyala governorate for the period from the first of July 2016 to the end of January 2017.The ages of patients ranged from (1-80) years old for both sexes.The samples were cultured on the blood agar and incubated at 37 °C for 24 hrs.The pure single colonies were then transferred and cultured on the Mannitol Salt Agar and incubated at 37 ° C for 24-48 hrs.The appearance of yellow golden medium is an indication of Mannitol fermentation by these bacteria [15].Initially, Staph.aureus were identified depending on the cultural, microscopic, and biochemical tests [16].using API Staph System.[17], and Vitek 2 device, which consists of 64 biochemical tests.

Antibiotic susceptibility testing
The disk diffusion method was done using Mueller Hinton agar medium.colonies (3-5) with the same morphologic features growing on the blood agar were transferred by sterile-inoculating loop to a tube containing 5 ml of physiological saline solution.Turbidity of bacterial growth was compared with standard McFarland No 0.5, which gives an approximate number of cells (1.5×10 8 cell/ml).Aliquots, 100µl of bacterial suspension was transferred by the micropipette, and spread by swab on overall the surface of the Muller-Hinton Agar.Then, the inoculated agar plates were left to dry at room temperature for (10)(11)(12)(13)(14)(15) min.The antibiotic discs listed in table (1) were then applied into the Muller-Hinton Agar using sterile forceps.The agar plates incubated at 37 °C for 24 hours.The diameter of the inhibition zones around the antibiotic discs was measured and compared with the standard chart to decide wither bacteria are sensitive or resistant.The isolates of MRSA were identified by measuring the sensitivity of the bacteria to the Oxacillin [18].

Preparation of Hot Aqueous Extract
According to Chanda and Parekh method [19], 50 g of dry plant was weighed and dissolved in 500 ml of boiled distilled water.After cooling to 60°C and placed in a 1000 ml flask, the solution was placed on the hot plate-Magnetic stirrer for 2 hrs.Following that, the mixture was filtered using medical gauze, then filtrated by Whatman No.1 filter papers.The supernatant was transferred into centrifuge tubes at a speed of 3000 cycles/min for 10 min.After that, the supernatant was evaporated by rotary evaporator under low pressure at (40-50°) C. The remainder extract was dried using dishes with a large surface area in the oven at of 40°C until the water was evaporated completely.The powder of aqueous extract was placed in tightly sealed glass tubes.The tubes were labeled and kept in the refrigerator at 4°C until use.

Preparation of alcoholic extract
According to Jameela method [20], 50 g of dry plant was weighed and dissolved in 500 ml of ethanol 70% and put in a 1000 ml tightly-closed flask and kept in opaque place for 7 days.The mixture was filtered using gauze, and complete the extraction according the previous aqueous extract.

Effect of Loranthus europaeus and Lawsonia inermis on bacterial growth
Well diffusion assay was used according to Al-Okaili method [21]. .Bacterial suspension was prepared from (3-5) colonies with the same morphologic features growing on the blood agar.These colonies transferred by standard wire loop into a tube containing 5 ml of physiological saline solution.Turbidity of bacterial growth was compared to the standard McFarland No 0.5, which gives an approximate number of cells (1.5 × 10 8 cell/ml).Aliquots 100 µl of bacterial suspension was transferred by the micropipette, and spread by swab overall agar plate.The inoculated agar plates were left to dry at room temperature for 5 min.Wells with 6 mm in diameter were made on agar.After that, 20 µl of the extract were added to each well at pre-determined concentrations (12.5, 25, 50, 100, and 200 mg/ml) and left for 1 h to ensure proper distribution.Distilled water and phosphate buffer saline were added as control for boiled aqueous and alcoholic extract, respectively.Following that, agar plates were incubated at 37°C for 24 hrs.The activity of each plant extract was determined by measuring the diameter of inhibition zone in millimeters by a standard scale.

Result and Discussion
The data of current study showed that the number of females infected with boils 62(58.49%)was more than male 44(41.51%)as shown in (table 2).These results were in agreement with Shallcross [22]..The variation in infection number and percentage between the sexes may be due to the quantity and quality of the normal flora of skin in different sexes [23].Furthermore, different methods of sample collection in addition to the physiological condition of humans especially depression, secretion of female hormones may play a role in decreasing the number of males infection [24].[25], in which methicillin-resistant S. aureus was (11.5%) of 343 samples.A mass of pus is accumulated in hair follicles after S. aureus infection which usually occurs after puberty.This mass develops and opens either spontaneously or as a result of external pressure [26].
The reason for the dominance of Staph.aureus infection is due to its ability to develop antibioticresistant and production of β-lactamase enzymes.Moreover, S. aureus rapidly multiply and possess many virulence factors such as production of enzymes: catalase, coagulase, deoxyribonuclease, lipase, hemolysin, leucocidin, PVL, enterotoxin and hyaluronidase.Capsule and surface proteins play an important role in the invasion of host tissues and the spread of bacteria.The β-Lactamase-produced enzymes contribute to bacterial resistance to many antibiotics [27].In the current study, the sensitivity of (41) isolates of S. aureus were examined to 14 commonly used βlactum antibiotics.All S. aureus isolates were 100% resistance to ceftriaxone, cefotaxime and cefepime, and 92.68% of isolates were resistant cefoxitin and penicillin G. Our findings are in agreement with the results of Al-Timeemi [28].The percentage of bacterial resistance was moderate toward some antibiotics, such as Ampicillin/Sulbactam and Augmentin (consists of two antibiotics Amoxicillin and Clavulanic acid).Amoxicillin inhibit cell wall synthesis, while clavulanic acid encounter with penicillinase production.On the other hand, the isolates showed low resistance to Augmentin/Sulbactam, cefachlor, cefazolin, and oxacillin (85.54%, 36.58%,26.83%, and 12.19%), respectively.All Staph.aureus were sensitive to Carbapenems, which include Meropenem and Imipenem.The broad spectrum of these antibiotics associated with their effectivity against many skin and soft tissue pathogens [29].the Grampositive and Gram-negative bacteria [30].table (4), β-lactam antibiotics inhibit bacterial cell wall synthesis by interfering with peptidoglycan synthesis.Resistance to β-lactam antibiotics is possibly due to production of β-lactamase which break the β-lactam ring or reduce the affinity of antibiotic binding with the bacterial penicillin-binding proteins.Moreover, the binding site could be modified by adenylation, phosphorylation and acetylation enzymes, or a chromosomal mutation.all of these reasons lead to loss of the antibiotic affinity to bind with the target protein and reduction of bacterial cell permeability to antibiotics [31].

Number of isolates
Sensitivity % In this study, the inhibitory effect of hot and alcoholic extracts of Loranthus europaeus and Lawsonia inermis was determined against Staph.aureus.These extracts showed variable inhibitory effect against the tested bacterial isolates.Table (4) showed that the hot aqueous extract of the Loranthus europaeus was significantly more effective at the highest concentration (200 mg/ml) followed by the concentrations (100, 50, and 25 mg/ml) with which, the inhibition zones were(123.7,16.00 and 15.28 mm), respectively.While, the lowest effect was at the concentration 12.5 mg/ml with which, the diameter of inhibition zone was 14.57 mm.There was no significant difference in bacterial growth inhibition between the concentrations 12.5 and 25 mg/ml at (p<0.05).In addition, the results showed that the alcoholic extract of Loranthus europaeus had an inhibitory effect on the methicillin-resistant S. aureus bacteria, which was 17.28 mm at the concentration of 200 mg/ml, followed by the concentrations (100, 50, 25 mg/ml) with which, the diameter of inhibition zones were (13.28 , 10.57, 8.42 mm), as shown in table (5).Also, there was no significant difference in Staph.aureus inhibition between the concentrations (12.5, 25.50 mg/ml), while the differences were significant (p <0.05) among other concentrations.There was a significant difference between the hot aqueous and alcoholic extracts at (P <0.05).Our study was on contrary to the findings of Salem (2017) [32].who indicated that the inhibitory concentration of hot Loranthus europaeus's extract against Staph.aureus was at a concentration of 40 mg with diameter of inhibition zone of (14 mm), while it was (17 mm) were produced when alcoholic extract of the same plant used at the same concentration.Antimicrobial activity could be attributed to the active substances of boiled extract of Loranthus europaeus such as tannins, terpenoids, flavonoids and alkaloids phyto-constituents, which are considered as antibacterial compounds.However, Uzochukwu Osadebe [33]., and EL-Sharquie [34], reported that the alcoholic extract contains polysaccharides and aldehydes, which accelerate wound healing and have antimicrobial activity, preventing secondary bacterial infections.The study of Al-Rubaee [35].also showed that the wounds of laboratory rabbits infected with S. aureus were healed when treated with Loranthus europaeus extract.Tannins and Flavonoides, existed in Loranthus europaeus, deposit in the bacterial cell membrane, and inhibit the action of metabolic enzymes leading to bacterial death [36].This work showed that the hot aqueous extract of Lawsonia inermis effectively inhibited the growth of methicillin-resistant S. aureus producing inhibition zone (24.14mm) at 200 mg /mL followed by (21.28, 18.00, 11.85 mm) when (100, 50, 25 mg/ml) respectively, were used.while no effect at the concentration of 12.5 mg/ml was noticed, table (6).In addition, the alcoholic extract of Lawsonia inermis had an inhibitory effect on Staph.aureus with inhibition zone of 28.42 mm at 200 mg/ml.With the following concentrations (100, 50, 25 mg/ml), the inhibition zones were (25.42, 20.85, 17.00 mm), respectively.In addition, the diameter of inhibition zone was 11.14 mm at the concentration of 12.5 mg/ml (table 7).Comparing the results of inhibitory effect of aqueous and alcoholic extract of Lawsonia inermis, it was found that there was a significant difference (P<0.05).This may be due to the effect of alcohol in the extraction of active substances [37] Al-Hamooshi at Mosul University reported that the alcoholic extract of Lawsonia inermis had a higher inhibition activity than its aqueous extract .Alcohol is a good solvent of active substances such as phenols, an effective substances which form a complex with extracellular substances of the cell wall disrupt the cell membrane permeability of the bacteria.In addition, alcohol acts on protein denaturation agent in the bacterial cell and suppress the action of enzymes that responsible for metabolic reactions [38].The antimicrobial effect of alcoholic extract of Lawsonia inermis's leaves against Staph.aureus was due to its compositions: comarins, toxic oxidizing compounds, which are highly pass through the cell wall of bacteria [39].In addition, it contains lowsone, mannite, flavonoid and tannin compounds which inhibit the action of transporting enzymes and proteins of the cell membrane [40].Jahan [47] reported that Lawsonia inermis extract, prepared from various parts of the plant and with different solutions, showed antimicrobial activity.Sudharameshwari et al [41] mentioned that the activity of Lawsonia inermis is due to the presence of active substances such as alkaloids, phenols, anthocyanin, xanthoprotein, carboxylic acid, cumarins and sterols.In the present study, we found that the boiled aqueous extract has higher inhibitory effect than the alcoholic extract of Loranthus europaeus.However, the alcoholic extract of Lawsonia inermis has higher inhibition ability than its aqueous extract against MRSA.When comparing between the most effective inhibitor among the two extracts, there was a preference in affectivity of the alcoholic extract of Lawsonia inermis.It is also observed that the active ingredients of medical plants are affected by their quantity and quality [42].When using ethanol in Lawsonia inermis extract, its anti-microbial activity is increased.Ethanol may increase the concentration of the active substance compared to its aqueous extract in water.In addition, the chemical components of the culture medium may oppose or reduce the effectiveness of the extract against bacteria [43].

Table ( 2) Distribution of patients with boils by sex
S. aureus were the dominant bacterial isolates.This findings were in line with the study of Ibler and Kromann[2].In our study, 5 (12.20%) isolates were methicillinresistant Staph.aureus and 36 (87.80%) identified as methicillin-sensitive isolates (table 3).The results of our study are in agreement with study performed in Egypt by Abdel-Maksoud et al.,