STUDY THE ANTIBACTERIAL ACTIVITY OF AQUEOUS EXTRACTION OF ONION ( Allium cepaL) AGAINST Staphylococcus aureus ISOLATED FROM OTITIS MEDIA.

This study was aimed to determine the antibacterial activity of onion ( Allium cepaL .) aqueous extract against S. aureus bacteria isolated from patients with otitis media. Twenty swabs samples were taken from patients with otitis infection; these samples were cultured on different agar media .Results revealed that half of these isolates were related to S. aureus and the five of these isolates were subject to sensitivity test against several antibiotic groups. Most of isolates 100% were resistance to amoxicillin, chloramphenicol, cefoxitin and pencillin, and 60 % of were showed resistance to cefotaxtime and tetracycline, where as 20 % were resistance to Ciprofloxacin. Depending on the results of antibiotic sensitivity, the two isolates(S2,S5) were chosen for determining the minimum inhibitory concentration (MIC) test, S2 isolate showed high-level of resistance at (16 and 128 µg/ml) to chloraemphenicol, amoxicillin, ampicillin and Cefotaxime respectively, while S5 at concentration (32, 64 and 128 µg\ml) were resistance to amoxicillin, ampicillin, chloraemphenicol and cefotaxtime. The MIC of onion extract against S. aureus was determined at two incubation periods The results were indicated that after 24 hrs, MIC of extract at 64 µg/ml was affected in inhibit the growth of S2 isolate, While 32 µg/ml was inhibited the growth of S5 isolate.However, after 72 hrs, It was found that the MIC at 32, 64 and 128 µg/ml were more effective in inhibited the growth of S2 and S5 isolates.

.Onion (Allium cepa, L.) considrd as essential dietary intake and have been of critical for medical approach (34).Allium considered as the largest and important representative genus of the Liliaceae family comprises 450 species.Onion (Allium cepa) is a bulbous plant broadly cultivated in almost every nation of the world (18).They are easily propagated, transported and stored and it has diverse biological importance like treatment of cold, heart disease, diabetes, coughs and sore throat (7).Onions has different phytochemical constituents inrichness which clarified is rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids and flavonoids, which have been found to have antibacterial activities (12).Onion also contained proteins, carbohydrates ,sodium, potassium and phosphorus (26).Onion was expended all through Europe amide and was later thought to guard against evil spirits and the plague, all that due to strong odor (20).In folkloric medicine generally utilized onion to prevent infections and is the oldest cultivated plants utilized both as nourishment and for therapeutic applications (27).The beneficial uses of onion in reduce the risk of certain disease was also investigated for example gastric ulcers by scavenging free radicals and by preventing development of the ulcerforming microorganism, Heliobacter pylori.Also, In vitro onion appered to have antibacterial, antiparasitic and antifungal potential (14).Anciently, Otitis media is inflammation of the middle ear , or middle ear infection.Which occurs in the area between the ear drum (the end of the outer ear) and the inner ear, including a duct known as the Eustachian tube (17).In children and adults, these disease considered as a major problem since ancient time which require specific treatment because it is leading to hearing loss in children (9) (15).

MATERIALS AND METHOD Collection of plant materials
The plants were collected from the confined markets during September (2017), and identified previously by National Herbarium of Iraq.

Preparation the aqueous onion extract
The onion bulb was cleaned from dust by using sterile distilled water, then removing the outer cover of the bulb.(21).Peeled out manually and soaked with distill water, 200g of the onion bulb were cut into small pieces and grinded, then soak with 100 ml of distilled water for 24hrs (13).The extract was filtered with Whitman no. 1 filter paper and evaporate at 45ºC for drying.Then stored in sterile condition at 4ºC until use to prepare the required concentrations (31).

Preparation of different concentrations of onion
Five concentrations (128, 64, 32, 16 and 8 μg/ml) of onion extract were prepared according to method of Mohamed Eltaweel.2013.(28) by resuspending the onion extract in sterile distilled water.

Bacterial isolation
Twenty ear samples were collected from patients suffering from middle ear infection (otitis) from Al-Yarmook Teaching Hospital in Baghdad from the periods (1/9/2017-1/11/2017) using sterile cotton swab these were processed for direct examination and cultivation on blood agar media, macConkey agar and brain heart agar media (Bangalore, India) and were identified initially as Staphylococcus species according to their morphological, physiological and biochemical properties as indicated by Baird-Parker AC. 1963 (24)

Minimum inhibitory concentration
In this experiment Broth micro dilution method was performed, in two fold dilutions of antibiotics were done in broth media and broth was inoculated with 10 6 CFU/ml of the tested organisms (2).After incubation for (18-24) hrs, the MIC was determined as concentration in which no visible growth was observed (4).

Antibacterial potential the plant
The method of Karam, I. F.A. 2016 (22) was followed in assessment of antibacterial activity of plant crude extract in accordance agar-well diffusion method.After growing bacteria in a nutrient broth for 18 hour and standardized to 0.5 McFarland standards (10 6 cfu/ml).200 μl of the standardized cell suspensions were spread on a Mueller-Hinton agar, following that wells bored into agar with 6mm.Then 50 μl of the aqueous crude extract suspensions were added at different concentrations (8,16,32, 64 and 128 μg/ml) and allowed to stand at room temperature for about 2 h and then incubated at 37°C.The same procedure was followed for control set by using the solvent and distilled water.Then the zones of inhibition after (24 h) was measured.

RESULTS AND DISCUSSION
The results in Table 1 indicated that resistance to antibiotics was widely distributed among isolates, however, they varies according to types of the isolates and kinds of antibiotics, All isolates (S1, S2, S3, S4 and S5) were resistance for (amoxicillin, chloramphenicol, cefoxitin, penicillin) respectively, Where as three of isolates showed resistance to cefotaxime (S2, S3 and S5),While(S1and S4) were sensitive to cefotaxime and three of them resistance to tetracyclin (S1, S2, S3) ,Also (S4,S5) were sensitive to tetracyclin, while all isolates showed sensitivity to ciprofloxacin except (S5) which was resistance to this antibiotic.The results of the antibacterial activities of the onion extracts against S. aureus organisms were shown in Table 3, after 24hr using Minimal inhibitory concentration was determined via macro broth dilution method.It was found that at 8µg\ml of onion extract a slightly inhibitory effect on S5 while have no effect on S2.However at 16 and 32µg\ml showed good inhibitory were observed against S2 and S5.While at 64 and 128µg\ml completely inhibit the growth of S2 and S5 The pH of each of the onion solution were (6.8, 7.0, 7.2, 7.4, 7.6 and 7.8).Result demonstrated that no pH effect on the onion activity has been observed.The onion extract have antibacterial activity due to the presence of flavonoids and polyphenols which has been reported to have broad spectrum of antibacterial activity (19).Also these activity belongs to they do not act directly on bacteria but create an adverse environment for them, thus causing threate their survival and they have also been found to decrease the resistant strains of microorganisms (3) .This property belongs to the onion constituent sulphur compounds, thromboxanes and by inhibiting the action of platelet-activating factor (PAF) exhibits its antimicrobial activity mainly by immediate and total inhibition of RNA synthesis, although DNA and protein syntheses are also partially inhibited (16).The greater sensitivity with largest zone of inhibition was observed with ciprofloxacin.This result was in agreement with another by Reese, R., R. Betts, and B. Gumustop.2000 (32) who found that ciprofloxacin was effective in the treatment of the virulent gram positive bacteria including S. aureus.From this result of Table (1) one could conclude that ciprofloxacin remain the first choice when most isolates were sensitive to it.Because of resistance for ciprofloxacin included effective suction pump of the antibiotic from inside to outside to escape its effect and prevent the accumulation of antibiotic inside bacterial cell (29) .Ciprofloxacin inhibit bacterial DNA gyrease, so preventing the super coiling of DNA, a process that is necessary for compacting chromosomes into the bacterial cell (10) and this was concluded that plasmid mediated β-lactamase conferred high level resistance to penicillin and cephalosporin antibiotics and the level of resistance depend on the amount of β-lactamase produced so the mechanism (35) (36) that account for increase production of plasmid-determined β-lactamase include mutations or insertion elements that alter the promoter strength, microscopy plasmid and gene duplications (33) .The number of multiple antibiotic resistance strains has been increasing since resistance is mainly mediated by R-plasmids, which determined βlactamase in Gram positive rod (25) .All the Rplasmids carried the markers of resistance to chloraemphenicol, tetracycline, ampicillin, gentamycin and streptomycin (5).Results of MIC showed that S. aureus infections are more difficult to treat because the organism's high intrinsic resistance antimicrobial agents; this resistance is partly due to its relatively low outer membrane permeability (6).However there are other mechanisms that included decreasing the passage into or increasing the efflux of drug from bacterial cell and modification of the target site (6).Our results its matches with (11) who founded that both lower and higher concentration of petroleum ether, ethyl acetate and chloroform extracts of onion which inhibited the Staphylococcus aureus growth.In contrast, the Butanol, ethanol and water extracted of onion at any concentration were ineffective to control the growth of Staphylococcus aureus.Our results its matches with Mohamed Eltaweel.2013 (28) who founded that the onion extract has antibacterial activity against Staphylococcus aureus.It has shown that dilute solutions of onion can completely inhibit the growth of S. aureus at the concentration of more than16 µg/ml.From our study it was concluded that S. aureus were highly prevalence among teenager age of otitis patients and high prevalence of antibiotic resistance was observed among S. aureus isolates especially to penicillin cefotaxin and tetracycline and it was found that onion extract has considerable inhibitory effects against the tested S. aureus isolates.

Fig 1 .
Fig 1. Percentage resistant of S. aureus isolated from otitis media against several antibiotics Percentage of resistance

Table 2 . MIC value of some antibiotics for S. aureus (S2, S5) isolates
Tow isolates were selected due to multiple antibiotic resistance therapy, in order to evaluate bacteria susceptibility to antibiotic that revealed earlier by disk diffusion test when examined against antibiotics.The results of MIC were shows in Table2, In this Table

Indicates growth of S. aureus and -: no growth Indicates growth of S. aureus and -: no growth
Table 4 reveals the results of MIC of onion extract on S. aureus after 72 hrs.It was found that the concentrations (32, 64 and 128 µg/ml) were more effective in inhibition growth of (S2, S5) while (8 µg/ml) showeed heavy growth of S. aureus.