The Effect of Alcoholic Extracts of Zingiberofficinale Anti-E.Coli Isolates Isolated from Urinary Tract Infection

The Escherichia coli isolated from UTIs, were identified with biochemical tests and vitek test.The ethanolic extracts of Zingiberofficinale(Z.officinale) were tested against E.coli by using the good agar diffusion test, the alcoholic extracts from (25 100) mg/ml showed antimicrobial activity against tested microorganism. The diameter of inhibition zone increase at high concentrations and rang from (1820mm) , and these results compared with antibiotics sensitivity discs were used by discs diffusion method against E.coliisolates, they were resisted to all antibiotics used in this study. It could be concluded that alcoholic extract of Z.officinalehad good antimicrobial effects, and may be able to use for treatment UTIs caused by E.coli because it not make any side effect and can be used occasionally to prevent the infect by this bacteria.


Introduction
In developing countries, urinary tract infections (UTIs) are one of the most common diseases among diverse age groups [1]. Females are more affected compared to males, UTIs can be categorized as acquired or nosocomial. E.coli is the most common organism responsible for UTI in both communities acquired and nosocomial and cause other infections such as diarrhea and bacteremia. E. coli can cause these infections because it has many virulence factors like adhesion, iron uptake, toxins, capsular polysaccharides , and proteins [2]. UTI caused by other bacteria like Klebsiella, Proteus, Pseudomonas, and Enterobacter. Increasing bacterial resistance to antibiotics has become the main concern due to ISSN: 0067-2904

Mohamad
Iraqi Journal of Science, 2019, Vol. 60, No.10, pp: 2136-2140 2137 misuse of antibiotics, sensitivity pattern should be made in UTI , So, it is necessary to look for the most sensitive antibiotics for proper treatment [3]. Z.officinale is a common natural herb and widely used as medicinal and food beverage purpose. The presence of pharmacological activities such as antiinflammatory, anti-oxidatives and anti-cancer from the extraction of it mainly due to the existence of gingerols and shogaols [4].Z.officinalehasessential oils like udesmol, γ-terpinene, acurcumene,alloaromadendrene,zingiberene (, α-pinene, δ-cadinene,elemol andfarnesal [5].The aim of this study is to determine the antimicrobial effectiveness of Z.officinale alcoholic extracts on E.coli isolates isolate from UTI and compare it with the effect of different antibiotics against this pathogen.

Materials and Methods: Isolation of bacteria:
Fifty samples of urine were collected from Al-Yarmuk, and Al-Karama hospitals from patients (male amd female) suffering from UTIs. biochemical tests: The isolated bacteria were identified with morphological characters and biochemical tests.TheMacConkey agar (Himedia/India) was used for primary identification of E. coli and other gram negative bacteria; eosin methylene blue media used also in identification. Indole and Simmons citrate tests to identify E. coli from other Enterbacteriacae [6].The identification was confirmed by vitek system.

Antibiotics sensitivity test:
In vitro sensitivity test was done according to Kirby-Bauer [7]; after dilution to the standard turbidity of McFarland tube no. 0.5. With a sterile swab dip it into the broth culture of the organism and then were inoculated on Mueller Hinton agar plates. After the streaking is complete, allow the plate to dry for 5 minutes. Antibiotic discs can be placed on the top of agar by using sterilized forceps. The inoculated plates incubate over night at 37°C. In this study eleven antibiotics discs(Bioanalyse/Turkey) were used they are: Chlorotetracyclin (30µg), Sparfloxacin (10µg), Imipenem (10µg), Ticarcillin (10µg), Amoxicillin (5µg),Trimthoprim(30µg), Azithromycin (25µg), Cephradine (10 µg) and Ceftriaxon (15µg), Table-1, then measured the diameter of inhibition zone in millimeter by ruler for antibiotics used. Compare the measurement obtained with the standard table on NCCLS to determine the sensitivity zone [8]. Dried powder of Zingiberofficinale was used in current study was bought from stores of plants in Baghdad city.

Soxhlet Extraction
The initial concentrations of bioactive compounds presented in the Zingiberofficinale matrix were identified through the soxhlet extraction. 25.0g of dried plant powder was extracted with 200 ml of 80% (v/v) aqueous ethanol by Soxhlet apparatus used for 8 hrs, then the extract was dried in oven with 40° C, and the power of plant extract was kept in a refrigerator (4) C 0 for further use [9], Figure-1.

Minimum inhibitory concentrations (MIC) determination of plants extracts:
The MIC of crude extracts was determined by broth macro-dilution assay. A set of test tubes with concentrations of plant extract (25,50,75 and 100) mg/ml. Tubes were inoculated with bacteria. After incubation, tubes were examined for changes in turbidity as an indicator of growth. The first test tube that appeared clear was considered as MIC of Zingiber officinale extract, against E. coli with used positive and negative control for comparison [9].

Well method
Plant extracts concentrations determined by well method to determine the presence of inhibition zones or not forE.coliisolates by made wells in the Mullar-hinton agar by cork borer after the spread of bacteria plates and adding the suspensions to the wells then incubated for 18 hrs. at 37C o [10] Result and discussion Identification of bacteria by biochemical tests: Fifty urine samples were collected from patients with UTIs. The samples were cultured on MacConkey agar, the bacteria gram negative appeared pink colonies because of lactose ferments in  Table-3 shows E. coli which isolated from UTIs were resisted to all antibiotics used in this study, and this disagree with other studies that E. coli were resistant to commonly used antibiotics but sensitive to Nitrofurantoin, Amikacin and Cefotaxime are considered appropriate for empirical treatment of E. coli in the study area of [11]. Microorganisms causing UTI vary in their susceptibility to antimicrobials agents from region to others; while the extracts of plants were very active against these isolates. Other study reported that the most effective antibiotic on E.coliwas Trimethoprim [12]. Other study showed that lowest resistance ofE.coli isolates isolated from UTI to ciprofloxacin, ceftizoxime [13]. This result may relate to misuse and random use of antibiotics which lead to high resistance to all antibiotics under testing. The mechanisms of acquired resistance fall into one of the five categories, and these are 1)Enzymatic modification or destruction of the antibiotic, 2)Reduced antibiotic uptake into the b a c t er i u m, 3   Z.officinale had antibacterial activity to various bacteria like E.coli and S.aureus ,the zone of inhibition was 6 mm. [15].Other study showed that MIC concentrations range of Z.officlnale between 75 mg/ mL and 250 mg/ mL [16].The antimicrobial potency of plants is believed to be due to tannins, saponins, phenolic compounds, essential oils and flavonoids [17]. It is interesting to note that even crude extracts of these plants showed good activity against multidrug resistant strains where modern antibiotic therapy has limited effect.
Ginger is truly a world domestic remedy. It is also used in India and other places like the ancient Chinese where the fresh and dried roots were considered distinct medicinal products. Fresh ginger has been used for cold-induced diseases, nausea, asthma, cough, colic, heart palpitation, swelling, dyspepsia, less of appetite, and rheumatism, in short for the same purposes as in ancient china, In 2140 nineteenth century ginger serves as a popular remedy for cough and asthma when the juice of fresh ginger was mixed with a little juice of fresh garlic and honey, A paste of powdered dried ginger was applied to the temples to relieve headache and fresh ginger was mixed with a little honey, tapped off with a pinch of burnt peacock feathers to alley nausea [18].

Conclusion
From the result, E.coli isolates which isolated from UTIs showed high resistance to antibiotics , this may be related to random use of antibiotics.