https://doi.org/10.17795/iji-23036

The Effect of Withania somnifera Extract on Drug Resistant Strains of Escherichia coli

authors:

avatar Mohammad Bokaeian 1 , avatar Barat Ali Fakheri 2 , avatar Nafise Mahdi Nejad 2 , avatar Hemadollah Zarei 2 , avatar Saeide Saeidi 1 , * , avatar Taher Mohasseli 3

Infectious Disease and Tropical Medicine Research Center, Zahedan University of Medical Sciences, Zahedan, IR Iran
Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Zabol, Zabol, IR Iran
Department of Biotechnology, Faculty of Agricultureal, Shahid Bahonar University, Kerman, IR Iran
International Journal of Infection: Vol.2, issue 2; e23036
published online: April 25, 2015
article type: Research Article
received: August 24, 2014
accepted: August 30, 2014

how to cite: Bokaeian M, Fakheri B A, Mahdi Nejad N, Zarei H, Saeidi S, et al. The Effect of Withania somnifera Extract on Drug Resistant Strains of Escherichia coli. Int J Infect. 2015;2(2):e23036. https://doi.org/10.17795/iji-23036.

Abstract

Background:

Due to the increased resistance of pathogenic bacteria even towards new antibiotics, researchers have attempted to find herbal antimicrobial agents to replace chemical drugs.

Objectives:

The purpose of this study was to evaluate the effect of W. somnifera extracts on drug resistant E. coli strains isolated from clinical samples.

Materials and Methods:

We studied all drug resistant strains of E. coli collected from patients with urinary tract infections hospitalized at Zabol hospitals from January 2010 to January 2012. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of plant extracts were determined by the dilution method with various concentrations of the bacteria. Sensitivity to multiple antibiotics was defined by the Kirby-Bauer disc diffusion test.

Results:

The results showed that the isolated E. coli strains were sensitive to these antibiotics: erythromycin (52.94%), tetracycline (76.47%), ceftazidime (41.17%), cefixime (35.29%), penicillin (76.47%), ampicillin (58.82%) and nalidixic acid (41.17%). Examination of the herbal extracts showed that the highest maximum inhibitory concentration (MIC) against drug resistant E. coli was 200 ppm. The lowest MIC was 50 ppm, where three strains of E. coli were inhibited at this concentration.

Conclusions:

Our results indicated that Withania somnifera extracts had antibacterial effects on drug resistant E. coli strains isolated from clinical samples. Further studies are required to evaluate the effective compounds of this plant on microbial agents.

1. Background

Withania somnifera is an evergreen plant from the Solanaceae family. It grows widely in India, East Mediterranean regions and East Asia. In ancient times, roots and leaves of this plant were used for treatment of infectious diseases such as wound infections, painful abscesses, conjunctivitis, tuberculosis, and for sexually transmitted infections such as gonorrhea and syphilis (1-4). Previous studies have shown the antimicrobial activity of Withania somnifera extracts (1, 5). These extracts have also been used for dyspepsia and abdominal pain (6). Also, the entire plant, including its roots, leaves and fruits, due to containing polyphenols and antioxidant compounds is useful for human health (7). Previous studies have shown that in animal models, this extract increases memory and learning power (8). Escherichia coli belongs to the Enterobacteriaceae family that causes many infections such as urinary tract infection, sepsis, peritonitis, neonatal meningitis, gall bladder infections and pneumonia (9-12). Indiscriminate and careless use of antibiotics has led to the emergence of drug resistance to this bacterium.

2. Objectives

The aim of this study was to evaluate the effect of Withania somnifera extract on isolated E. coli strains from clinical samples.

3. Materials and Methods

3.1. Isolation of Escherichia coli

We evaluated all strains of E. coli isolated from urine cultures of hospitalized patients at Zabol hospitals (South-eastern Iran) with urinary tract infections from January 2011 to January 2012. The samples were examined microscopically by Gram staining. Gram-negative samples were inoculated on plates of nutrient agar, cysteine lactose electrolyte deficient (CLED) agar, MacConkey's and blood agar. Samples were then incubated at 37°C for 24 hours (13). The colonies that showed fermenting of lactose on MacConkey’s agar and CLED agar media were purified and identified according to their morphology as circular, rose-pink to red colonies on MacConkey’s agar medium and yellow colonies on CLED agar. The isolates were identified by biochemical tests such as catalase enzyme test, potassium hydroxide test, indole and methyl red test, Voges Proskauer reaction, urease and citrate test, H2S and oxidase test (14).

3.2. Agar Disc Diffusion Assay

Resistance to tetracycline, erythromycin, cefixime, ceftazidime, penicillin, ampicillin, and nalidixic acid was tested by the disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) protocols. Antibiotic discs were obtained from the Patended -Iran. Escherichia coli isolates were evaluated based on their size of zones of inhibition and classified as susceptible (S), intermediate resistant (I) or resistant (R) according to the CLSI criteria (15).

3.3. Plant Material

The leaves of W. somnifera were collected from Zabol (located South-east of Iran) and approved by the Faculty of Agriculture of Zabol University. Specimens were then dried at room temperature. Samples were crushed and transferred to glass containers and maintained until the extraction procedure.

3.4. Preparation of Extracts

Twenty grams of fresh leaf materials were macerated with 60 mL of 95% ethanol, in a grinding machine for about 10 to 15 minutes to separate the extract phases during one day (shaking occasionally with a shaker). The supernatant was filtered through Whatman No. 1 filter paper. The extracts were preserved aseptically at 5°C for further use.

3.5. Minimum Inhibitory Concentration of Plant Extracts

The broth microdilution method was used to determine the minimum inhibitory concentration (MIC). All tests were carried out in Mueller Hinton broth supplemented with Tween 80 at a final concentration of 0.5% (v/v). Next, serial doubling dilutions of the extract were made in a 96-well microtiter plate ranging from 0.3 mg/mL to 10.00 mg/mL. To each well, 10 μL of indicator solution (prepared by dissolving a 10-mg extract in 2 mL of DMSO) and 10 μL of Mueller Hinton broth were added. Finally, 10 μL of bacterial suspension (106 CFU/mL) was added to each well to obtain a concentration of 104 CFU/mL. The plates were loosely held together with a cling film to warrant that the bacteria do not become dehydrated. Next plates were prepared in triplicates, and placed in an incubator at 37°C for 18-24 hours. Color change of the samples was then visually evaluated. The lowest concentration at which color change occurred was considered as the MIC value. The average of the three values was calculated providing the MIC values for the tested extracts. The MIC is defined as the lowest concentration of the extract at which the microorganism does not demonstrate visible growth. Microorganism growth was indicated by turbidity. The MBC was defined as the lowest concentration of the extract at which the incubated microorganisms were completely killed. The results are expressed as mean and or rank in order of importance as a percentage. The data were subjected to one-way analysis of variance (ANOVA), using the SPSS-17 software. P < 0.05 was considered significant.

4. Results

The results showed that 17 strains of isolated E. coli were resistant to antibiotics with different percentages; erythromycin (52.94%), tetracycline (76.47%), ceftazidime (41.17%), cefixime (35.29%), penicillin (76.47%), ampicillin (58.82%) and nalidixic acid (41.17%). Results from W. somnifera extract showed that the highest MIC (minimum inhibitory concentration) was 200 ppm. Four strains were inhibited at this concentration, and the lowest MIC was 50 ppm and three strains of E. coli were inhibited at this MIC (Table 1).

Table 1.

Minimum Inhibitory Concentration/Minimum Bactericidal Concentration of Withania somnifera Extract on drug Resistant Bacterial Strains a

BacteriaThe least MIC/ MBC, PPM
1No growth/No growth
2100/200
3100/100
4200/200
5No growth/No growth
6No growth/No growth
7200/200
8100/100
950/100
10100/200
11100/200
12100/200
13200/400
1450/100
15200/200
16100/100
1750/50

a Abbreviations: MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration.

5. Discussion

Our results showed that the isolated E. coli strains were resistant to the following antibiotics: erythromycin, tetracycline, ceftazidime, cefixime, penicillin, ampicillin and nalidixic acid with different percentages and the extract of W. somnifera had antibacterial effect on these strains. Due to the wide and indiscriminate use of antibiotics, we have faced drug resistant bacteria especially in during the recent years, even against new drugs such a quinolones, new penicillin groups and cephalosporins. Therefore, we need new substances to help us treat infectious diseases. Among these agents, W. somnifera has been shown to have antibacterial effects on some drug resistant bacteria. A study at Imam Ali hospital (in Chaharmahal Bakhtiari) showed that isolated E. coli were resistant to gentamicin (62.50%), co-trimoxazole (51.51%), nalidixic acid (21.21%), amikacin (66.67%), ciprofloxacin (41.86%) and ceftriaxone (57.14%) (16). In a report by Mohammadi Mehr et al., E. coli strains isolated from clinical samples were resistant to antibiotics including: gentamicin (27.77%), ampicillin (80.55%), sulfamethoxazole (47.22%), nitrofurantoin (13.88%), ceftazidime (63.89%) and amikacin (80.55%) (17). In Madani’s study from Kermanshah, E. coli showed the greatest amount of resistance to ampicillin (91.4%), co-trimoxazole (61.1%), cefixime (46.8%), gentamicin (43.3%) and ceftazidime (38.8%) (18). The result of the herbal extraction experiment showed that the highest MIC was 200 ppm. Four strains were inhibited at this concentration. The lowest MIC was 50 ppm and two strains of E. coli were inhibited at this concentration. In Kaur’s research, treatment of infected mice with a combination of Asparagus racemosus and Withania somnifera not only resulted in successful reduction of parasite level, but also, generated protective Th1 type of immune responses with normalization of biochemical and hematological tests suggesting their role as important anti-leishmanial agents (19). Bensod in his study showed that extracts of Withania somnifera with 100 µg concentration in each disk, made an inhibitory zones (15 ± 0.5 mm) against Aspergillus fumigatus (20). In Kuboyama’s study, Withania somnifera and its constituents showed various activities against Alzheimer’s disease and spinal cord injury. Withania somnifera extracts also showed ameliorative effects against other neurodegenerative disease models such as Parkinson’s disease and Huntington’s disease (21). Mir Ba et al. reported that Withania somnifera is a rich source of withaferin A and other bioactive withanolides, which are used for medical purpose (22). Results by Heidari Sour Shojani et al. in Iman Ali Hospital in Chaharmahal Bakhtiari showed that isolated E. coli were resistant to gentamicin (62.50%), co-trimoxazole (51.51%), nalidixic acid (21.21%), amikacin (66.67%), ciprofloxacin (41.86%) and ceftriaxone (57.14%) (16). In the report by Mohammadi Mehr et al., E. coli strains isolated from clinical samples were resistant to antibiotics including: gentamicin (27.77%), ampicillin (80.55%), sulfamethoxazole (47.22%), nitrofurantoin (13.88%), ceftazidime (63.89%) and amikacin (80.55%) (17).

In conclusion, our results and other previous results suggest that Withania somnifera extracts have anti-bacterial effect on even drug resistant bacteria such as E. coli and also, have a wide range of efficacy on neurodegenerative diseases such as Alzheimer’s disease.

Acknowledgements

References

  • 1.

    Kurup PA. The antibacterial principle of Withania somnifera. I. Isoation and antibacterial activity. Antibiot Chemother (Northfield Ill). 1958;8(10):511-5. [PubMed ID: 24544974].

  • 2.

    Devi PU, Akagi K, Ostapenko V, Tanaka Y, Sugahara T. Withaferin A: a new radiosensitizer from the Indian medicinal plant Withania somnifera. Int J Radiat Biol. 1996;69(2):193-7. [PubMed ID: 8609455].

  • 3.

    Gupta LG, Rana AC. Withania somnifera (Ashwagandha): a review. Pharmacol Rev. 2007;1(1):129-36.

  • 4.

    Yousaf Z, Masood S, Shinwari ZK, Khan MA, Rabani A. Evaluation of taxonomic status of medicinal Species of the genus Hyoscyamous, Withania, Atropa and Datura based on poly acrylamide gel electrophoresis. Pak J Bot. 2008;40(6):229.39-2297.

  • 5.

    Bhatnagar SS, Santapau H, Desa JD, Maniar AC, Ghadially NC, Solomon MJ, et al. Biological activity of Indian medicinal plants. I. Antibacterial, antitubercular and antifungal action. Indian J Med Res. 1961;49:799-813. [PubMed ID: 13868832].

  • 6.

    Hemalatha S, Kumar R, Kumar M. Withania coagulans Dunal: A Review. Pharmacognosy Reviews. Phcog. Pharmacogn Rev. 2008;2(4):351-8.

  • 7.

    Alam N, Hossain M, Khalil MI, Moniruzzaman M, Sulaiman SA, Gan SH. High catechin concentrations detected in Withania somnifera (ashwagandha) by high performance liquid chromatography analysis. BMC Complement Altern Med. 2011;11:65. [PubMed ID: 21854608]. https://doi.org/10.1186/1472-6882-11-65.

  • 8.

    Dhuley JN. Nootropic-like effect of ashwagandha (Withania somnifera L.) in mice. Phytother Res. 2001;15(6):524-8. [PubMed ID: 11536383].

  • 9.

    Alizadeh Taheri P, Navabi B, Shariat M. Neonatal urinary tract infection: clinical response to empirical therapy versus in vitro susceptibility at Bahrami Children's Hospital- Neonatal Ward: 2001-2010. Acta Med Iran. 2012;50(5):348-52. [PubMed ID: 22837090].

  • 10.

    Khalesi N, Sharaky T, Haghighe M. Prevalence of urinar tract infection in neonates with prolonged jaundice referred to Aliasghar Hospital in Zahedan. J Qazvin Univ Med Sci. 2005;11(3):14-8.

  • 11.

    Dong F, Xu XW, Song WQ, Lu P, Yu SJ, Yang YH, et al. Characterization of multidrug-resistant and metallo-beta-lactamase-producing Pseudomonas aeruginosa isolates from a paediatric clinic in China. Chin Med J (Engl). 2008;121(17):1611-6. [PubMed ID: 19024085].

  • 12.

    Lagatolla C, Edalucci E, Dolzani L, Riccio ML, De Luca F, Medessi E, et al. Molecular evolution of metallo-beta-lactamase-producing Pseudomonas aeruginosa in a nosocomial setting of high-level endemicity. J Clin Microbiol. 2006;44(7):2348-53. [PubMed ID: 16825348]. https://doi.org/10.1128/JCM.00258-06.

  • 13.

    Betty AF, Daniel FS, Alice SW. Diagnostic microbiology. 10th ed. Mosby publishing; 1998.

  • 14.

    Holt JG, Krieg NR, Sneath PH, Staley JT, Williams T, Hensyl WR. Bergy's manual of determination bacteriological. Baltimore Meryland: Williams and Wilkins; 1994.

  • 15.

    Clinical and Laboratory Standards Institute. M100-S16 Performance standards for antimicrobial susceptibilitytesting; sixteenth informational supplement. Wayne PA USA: CLSI; 2006.

  • 16.

    Heidari Sour Shojani A, Heidari M, Dousti A. [Epidemiology of urinary tract infection and the pattern of drug resistance E.coli among referral patients to Imam Ali Hospital in Chahartmahal and Bakhtiari Province in Iran]. J Med Fac Sharkord. 2012;15(2):9-15.

  • 17.

    Mohammadi Mehr M, Faizabadi M, Bahadori A. [The pattern of drug resistant Gram negative bacilli among hospitalized patients in ICU of Golestan and Family hospitals in Tehran]. Mil J Iran. 2010;8(4):283-90.

  • 18.

    Madani H, Khazaei S, Kanani M, Shahi M. [The pattern of drug resistance E.coli among referral patients to Imam Reza Hospital in Kermanshah, Iran]. J Med Fac Kermanshah. 2009;12(3):287-95.

  • 19.

    Kaur S, Chauhan K, Sachdeva H. Protection against experimental visceral leishmaniasis by immunostimulation with herbal drugs derived from Withania somnifera and Asparagus racemosus. J Med Microbiol. 2014;63(Pt 10):1328-38. [PubMed ID: 25082945]. https://doi.org/10.1099/jmm.0.072694-0.

  • 20.

    Bansod S, Rai M. Antifungal activity of essential oils from Indian medicinal plants against human pathogenic Aspergillus fumigatus and A. niger. World J Med Sci. 2008;3(2):81-8.

  • 21.

    Kuboyama T, Tohda C, Komatsu K. Effects of Ashwagandha (roots of Withania somnifera) on neurodegenerative diseases. Biol Pharm Bull. 2014;37(6):892-7. [PubMed ID: 24882401].

  • 22.

    Mir BA, Mir SA, Koul S. In vitro propagation and withaferin A production in Withania ashwagandha, a rare medicinal plant of India. Physiol Mol Biol Plants. 2014;20(3):357-64. [PubMed ID: 25049463]. https://doi.org/10.1007/s12298-014-0243-5.