Abstract

According to increased bacterial resistance to common antibiotics, tendency toward using herbal drugs is increasing. Many researches have been executed about antibacterial and preservative effects of herbal extract like extracts of lamiacceae family (like Shiraz oregano herb). In this study, anti microbial effects of Zataria multiflora extract on Urinary Tract E. coli Infections in comparison with eleven antibiotic disks in antibiogram pattern is evaluated. After providing microbe suspension and comparing it to Mc Farland 0.5 to obtain 1.5x10⁸ bacterium per milliliter the susceptibility of the mentioned bacteria was determined in Muller-Hinton agar by embedding the disks of Betadine, Zataria multiflora and antibiotic disks. Fallowing incubation of media for 24 h at 37°C the inhibition zone of Zataria, Betadine and antibiotic disks were measured. Surprisingly, according to antibiogram results, the most sensitivity of E. coli was to Z. multiflora extract and inhibitory zone produced by eleven antibiotic disks and betadine are significantly less than Zataria induced. According to obtained results and limitations of increasingly usage of chemical antimicrobial substances there is a need to replacement of these substances with natural and herbal extracts.

---

INTRODUCTION

Lamianceae family is one of the biggest herbals’ families that have global distribution and has 200 genus and 2000-5000 species of aroma. Shiraz oregano (Zataria multiflora) is a member of this family that is basically originated in Iran, Afghanistan and Pakistan with multiple, thin, hard and forked stalks. The extract contains thymol, carvacrol (Mohagheghzadeh et al., 2004; Saleem et al., 2004), zatrinal, oleanolic acid, betulic acid, rosmarinic acid (Javidnia et al., 1999) and monoterpenoids, sesquiter-penoids, p-cymene, y-terpinene (Mohagheghzadeh et al., 1999, 2004). Avishan (Persian name of Oregano) is widely used in medicine, alimentary, hygienic and cosmetic industries. Aqueous and alcoholic extracts of Z. multiflora have been therapeutically used for relieving nociceptive pain (Hosseinzadeh et al., 2000; Ramezani et al., 2004), recurrent aphthous stomatitis (Jafari et al., 2003) and prevent growth of oral streptococci (Owlia et al., 2004), Plasmodiumm falciparum (Ziegler et al., 2004) and Trichomonas vaginalis (Abdollahy et al., 2004) as well as used as an insect repellent (Saleem et al., 2004). Also, Mahmoudabadi et al. (2006), showed that methanolic extract of the aerial parts of Z. multiflora has anti-Candida effect. According to the recent report (Ettehad and Arab, 2007), Salmonella typhi is another susceptible agent to Z. multiflora. Its anti-fungal properties have been also investigated in vitro (Fataneh, 1991). The antimicrobial effect of Zataria on gastrointestinal disease is reported such as its beneficial effect on inflammatory bowel disease among mouse (Khalili and Vahidi, 2006; Abdollahy et al., 2004).

Clinically Urinary Tract Infection (UTI) is confirmed, while pathogenic microorganisms are Paraclinically detected in the urine, urethra, bladder, kidney, or prostate. In most cases, growth of >10⁵ organisms per milliliter from a properly collected midstream clean-catch urine sample indicates infection.

In the vast majority of UTIs, bacteria gain access to the bladder via the urethra. Many different microor-ganisms can infect the urinary tract but the most common agent is the gram negative bacilli. Escherichia coli cause about 80% of acute infections. Other gram negative rods, especially Proteos and Klebsiella and occasionally Entrobacter, account for a smaller proportion of uncomplicated infections. UTIs are subdivided into catheter associated (or nosocomial) infections and non-catheter-associated (or community-acquired) infections. Catheter associated UTIs can sometimes be prevented in patients catheterized for <2 weeks by use of a sterile closed collecting system, by attention to aseptic technique during insertion and care of the catheter and by measures to minimize cross-infection. Despite precautions, the majority of patients catheterized for >2 weeks eventually develop bacteriuria. Colonization of the urine of catheterized or diabetic patients by Candida and other fungal species is common and sometimes progresses to symptomatic invasive infection. Except in acute uncomplicated cystitis in women, a quantitative urine culture or a comparable alternative diagnostic test should be preformed to confirm infection before empirical treatment is begun. When, culture results become available, antimicrobial sensitivity testing should be used to further direct therapy. Above concerns encouraged us to conduct a research study on the effect of zataria on urinary tract E. coli infections. Urine E. coli positive patients with different cause of UTI selected to test the antibacterial effect of Z. multiflora pure extract and and we compared its antimicrobial effects against E. coli with betadine and eleven antibiotic disks in Antibiogram pattern.

MATERIALS AND METHODS

E. coli isolated from the urine of the patients, was cultured in Muller-Hinton agar for 24 h. About 3-4 colonies of species of E. coli were suspended in a tube containing 0.5 mL of saline. In order to standardize the number of bacteria, Mc Farland 0.5 standard was prepared by adding 99.5 mL of 1% sulfuric acid to 0.5 mL of 1.175% barium sulfate solution. Then, the suspension of bacterium was compared to Mc Farland 0.5 to obtain 1.5x10⁸ bacterium per milliliter (CFUmL^-1).

To determine the susceptibility of the mentioned bacteria, Muller-Hinton agar (Merk) was prepared and the bacterial strains were transferred on to the surface of the medium using sterile cotton swabs. Blank disks were loaded with one drop (2.3 mg Thymole and 12.7 mg carvacrol manufactured by Barig Essance Co. Iran) and also one drop of Betadine 10% solution (manufactured by Behsadin Co. Iran) were put on the surface of the plates left 1 h at 4°C to allow better diffusion of the extract in to the media prior then simultaneously, these disks and 11 antibiotic disks such as Nitrofurantoin, Ceftriaxone, Ciprofloxacin, Cephalexin, Nalidixic acid, Co-trimoxazole, Cefixime, Cefotaxime, Gentamycin, Cefotizoxime, Amikacin (manufactured by pad tan Co. Iran) were embedded in two separate plates of Muller-Hinton agar. Following incubation of media for 24 h at 37°C, the inhibition zone of Zataria and antibiotic disks and also Betadine were measured and the average of results were recorded. Note that the measurements of the antibiotics were compared to NCCLS Standard Tables and the results were recorded as Sensitive, Intermediate and Resistance for each antibiotic disk.

RESULTS

Surprisingly, according to antibiogram results (Table 1), the most inhibition zone and sensitivity obtained for E. coli was produced by Z. multiflora extract and the inhibitory zone produced by the most sensitive antibiotics Ciprofloxacin (Cp) and Ceftizoxime (Ct) in antibiogram pattern are significantly less than Zataria induced and by using ANOVA-Tukey test Zataria was more significant when compared to betadine and eleven antibiotics that were used in antibiogram pattern (p = 0.00)

As Table 2 shows, the most sensitivity percentage obtained for E. coli was to Z. multiflora (100%), while Cefotizoxime (Ct) (77.77%), Ciprofloxacin (Cp) (75.92%), Cefotaxime (Ctx) (62.96%) and Ceftriaxone (Cro) (57.4%), ranked next, whereas it was resistance to Cephalexin (Cn) (79.62%) and Co-trimoxazole (Sxt) (70.62%) followed by Nalidixic acid Na (37.03%) Amikacin (An) (61.11%) Gentamycin (Gm) (55.55%) Nitrofurantoin (Fm) (53.7%) Cefixime (Cfm) (46.29%) were the most antibiotics that placed in the intermediate part. The results of this experiment shows that the antimicrobial effects of Zataria multiflora pure extract on UTI derived E. coli was more than Betadine and antibiotics and zataria was sensitive in all experiments.

Table 1:	Average of inhibition zone and comparison the significancy for E. coli
The mean difference is significant at the 0.05 level

Table 2:	Comparison the sensitivity percentage of Z. multiflora with eleven antibiotics against E. coli
S: Sensitive; I: Intermediate; R: Resistance; Fm: Nitrofurantoin; Cro: Ceftriaxone; Cp: Ciprofloxacin; Cn: Cephalexin; Na: Nalidixic acid; Sxt: Co-trimoxazole; Cfm: Cefixime; Ctx: Cefotaxime; Gm: Gentamycin; Ct: Cefotizoxime; An: Amikacin

DISCUSSION

Comparison between results reported about antibacterial effects of different essences is very difficult. Differences in various methods for evaluating of antibacterial effect of different plant essences, resources of essences and different genus of tested bacteria are from its reasons (Ettehad and Arab, 2007). Iranian scientists, Avicenna and Razi have published several books on herbal medicine a few centuries ago that are still in use in many libraries in Europe.

In Iranian traditional herbal medicine Z. multiflora is used for antiseptic, analgesic and carminative properties (Mohagheghzadeh et al., 2004; Hosseinzadeh et al., 2000; Zargari, 1990). It is also, used for treatment of 'Women disease' (Candidiasis vagina) in Iranian folklore (Rojhan, 2000). Also, the leaf powder of Z. multiflora is used as nutritional flavoring. Various studies has been performed about antibacterial effects of essence of herbs belong to Lamiaceae family and some important compounds in essence of this family like Carvacrol and Thymol. Karaman et al. (2001) showed powerful bacteriostatic effect of Thymus Revolatus essence on gram positive bacteria among Staphylococcus aerous. They illustrated high level of Carvacrol in essence as possible reason of this effect. Similar study by Rasooli and Mirmostafa (2004) about bactericidal effects of Thymus pubescus essence (with high amount of Carvacrol) on geram positive and geram negative and E. coli was executed and like Karaman et al. (2001), high amount of Carvacrol in essence was mentioned as reason of powerful antibacterial effect of Z. multiflora. The anti-Candida activity of methanolic extract of Z. multiflora indicated against isolates of Candida recently (Mahmoudabadi et al., 2006). They also mentioned that this anti-candida activity is due to both rosmarinic acid and thymol that extracted only into methanol (Mahmoudabadi et al., 2006; Mohagheghzadeh et al., 2004). Fataneh (1991) have shown that Z. multiflora has anti-fungal activity. They tested several isolates of dermatophytes and saprophytic fungi against Z. multiflora extract. Amanlou et al. (2006) have shown that Z. multiflora has antierythma in denture stomatitis compared to miconazol gel. Ramezani et al. (2004) reported six fractions of the extracts of aerial parts of Z. multiflora that have anti-nociceptive activity. Abdollahy et al. (2004) showed anti oxidative stress potential of Z. multiflora in rats, Present study shows that the antimicrobial effects of Zataria multiflora pure extract on UTI derived E. coli was more than Nitrofurantoin, Ceftriaxone, Ciprofloxacin, Cephalexin, Nalidixic acid, Co-trimoxazole, Cefixime, Cefotaxime, Gentamycin, Cefotizoxime and Amikacin that were used in antibiogram pattern. Surprisingly, according to antibiogram results, the most sensitivity of E. coli was to Z. multiflora extract (100%) and the inhibitory zone produced by Betadine that is normally used as an antiseptic solution before catheterization is significantly less than Zataria induced.

Zataria was sensitive in all experiments and it played a major roll in an experiment that the most sensitive antibiotics were Amikacin (16 mm) and Co-trimoxazole (13 mm) and according to NCCLS tables, they placed in intermediate part and the inhibitory zone produced by Z. multiflora for E. coli was (42 mm). In addition, high potency of E. coli in mutation against drugs may be another reason, which supports the study. According to obtained results and limitations of increasingly usage of chemical antimicrobial substances there is a need to replacement of these substances with natural and herbal extracts.

CONCLUSION

According to obtained results from this research, previous publications and increasing limitations of chemical and synthetic antibacterial agents usage due to drug resistance and some side effects, we recommend Zataria multiflora as a potent bactericide and it can have a potent anti urinary tract E. coli infections activity, especially in the cases that there is no significant antibiotic against E. coli.

ACKNOWLEDGEMENTS

Researchers wish to thank Dr. Mahzad Akbarpour from Embryology Department of Royan Institute to review the manuscript and personnel of Fazel Medical Laboratory for their helpful assistance.

How to cite this article:

Neda Shams Alizadeh, Effat Merghati Khoei, Mehrdad Fazelimanesh and Ahmad Astaneh. Antibacterial Effects of Zataria multiflora Boiss (Shirazi Avishan Extract) on Urinary Tract Escherichia coli Infections.
DOI: https://doi.org/10.36478/rjbsci.2009.891.894
URL: https://www.makhillpublications.co/view-article/1815-8846/rjbsci.2009.891.894