Study of Antibacterial Activity of Some Plant Extracts Against Enterohemorrhagic Escherichia coli O157:H7

 

Mona A. El-Sayed¹, Mohamed M. Kamel², Mohamed A. El-Raei², Samir M. Osman²,

Lina Gamil¹, Hisham A. Abbas¹*

¹Department of Microbiology, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt

²National Research Center, Department of Water Microbiology, Cairo, Egyt

*Corresponding Author E-mail:- h_abdelmonem@yahoo.com

This study aimed to investigate the antibacterial activity of 14 plant extracts against Escherichia coli O157:H7. Screening of antibacterial activity was made by the agar diffusion method. Among the plants tested, twelve extracts showed strong antibacterial activity with mean inhibition zones ranging from 20.17 to 14.17 mm. The extract of Allium Kurrat showed the greatest inhibition zone. The minimum inhibitory concentration (MIC) was determined by the agar dilution method. Comparing the minimum inhibitory concentrations, the extracts of Eruca sativa and Allium Kurrat showed high activity against Escherichia coli O157:H7 with MIC values of 2.5 mg/ml. These plant extracts may provide an alternative therapeutic option for Escherichia coli O157:H7 infections.

 

 

INTRODUCTION:

Enterohaemorrhagic Escherichia coli O157:H7 is considered the causative agent of important human infections such as diarrhea¹, haemorrhagic colitis², and may result in complications including haemolytic–uremic syndrome (HUS) in some people, particularly children under five years of age and the elderly^3,4.

 

Two Verocytotoxins (VT) are involved in the pathogenicity of Enterohemorrhagic Escherichia coli O157:H7 termed VT1 and VT2^5,6. Antimicrobial treatment of Escherichia coli O157:H7does not improve the course of disease, and it may precipitate kidney complications. Ciprofloxacin, co-trimoxazol, cefixime, fosfomycin and tetracycline in subinhibitory concentrations causes a significant increase in the production of VT in Escherichia coli O157:H7    strains^7-9.

 

Moreover, antidiarrhoeals, such as loperamide, should be avoided as they may prolong the duration of the infection. For these reasons, alternative therapy is needed to eradicate E. coli O157:H7 such as natural products¹⁰. Plants contain several secondary metabolites such as tannins, alkaloids and flavonoids, which can exert in vitro antimicrobial activities¹¹.

 

The objective of this study is the screening of the antibacterial effect of plant extracts as alternatives for treating E. coli O157:H7 infection by agar diffusion method and determination of minimum inhibitory concentration.

 

MATERIALS AND METHODS:

Media and chemicals:

Tryptic soya agar (Difco, France) and Tryptic soya broth (TSB) (LAB M, UK) were used in this study. Other chemicals used in the study were of pharmaceutical grade.

 

Bacterial strains:

Escherichia coli O157:H7 was kindly provided by the National Research center, Cairo, Egypt. The isolate was maintained in TSB with glycerol at -20°C.

 

Preparation of extracts:

For each plant, the dried part was crushed and percolated in ethanol 70% till exhaustion. Ethanolic extracts were filtered and the filtrates were evaporated under reduced pressure.

 

Screening for Antibacterial Activity against E. coli O157:H7:

Antibacterial Activity of plant extracts against E. coli O157:H7 was carried out according to the modified agar diffusion method¹². An overnight culture of E. coli O157:H7 was adjusted to reach a cell density of approximately 10⁷ to 10⁸ cfu/ml and was surface inculcated onto Tryptic soya agar plates, and then fixed volumes of the plant extracts (20 mg/ml) were aseptically added to cups made in these plates. Plates were incubated for 24h at       37 °C. After incubation, the diameters of inhibition zones were measured. Ciprofloxacin was used as a control at a concentration of 25µg/ml.

 

Determination of minimum inhibitory concentrations (MIC):

The MICs of the tested plant extracts were determined by agar dilution method according National Committee for Clinical Laboratory Standards (NCCLS) guidelines¹³. Specific volumes of Tryptic soya agar were added, each to a different concentration of plant extracts and mixed well before poured into Petri dishes. Agar was left to harden and then the plates were dried and stored at 4-8°C protected from light till the time of inoculation. An overnight culture in TSB was diluted with sterile saline to have a turbidity that matches that of 0.5 McFarland standard and then diluted 1:10 to achieve a cell density of approximately     10⁷ cfu/ml. Aliquots of 1-2µl of bacterial suspension were added to the surface of the agar and the plates were incubated at 37 °C for 24 h.  The lowest concentration that inhibits visible growth was defined as the MIC.

 

RESULTS:

Screening for antibacterial activity of plant extracts

A total of 14 plant extracts were screened for their antimicrobial activity against E. coli O157:H7 using the agar diffusion method. The inhibition zones ranged between 21 and 14.17 mm. Out of the 14 plant extracts tested in this study, 12 were found to be potentially active against E. coli O157:H7 (Table 1). These extracts were Allium cepa, Allium Kurrat, Eruca sativa, Petroselinum crispum, Anethum graveolens L., Apium graveolens, Raphanus sativus L., Punica granatum, Cinnamon zeylanicum Nees, Eugenia aromatica L., Brassica oleracea, and Ruta graveolens L. These extracts exhibited zones of inhibitions ranged from 21 mm to 17.17 mm. On the other hand, the extracts of Citrullus colocynthis L. and Glycyrrhiza glabra L. showed lower antibacterial activity against E. coli O157:H7 with inhibition zones of 14.83 and 14.17 mm, respectively.

E. coli O157:H7 was sensitive to ciprofloxacin with average inhibition zone diameter of 29.67 mm.

 

Table 1. Antibacterial activity of the plant extracts against E. coli O157:H7

Determination of MIC:

The MICs of the most active 12 plant extracts and ciprofloxacin against E. coli O157:H7 were determined by agar dilution method according to (NCCLS) guidelines¹³. E. coli O157:H7 was sensitive to ciprofloxacin. The extracts of Eruca sativa and Allium Kurrat exhibited the highest antibacterial activity against E. coli O157 where their MICs values were ≤ 2.5mg/ml (Table 2). Moderate antibacterial activity was exerted by the extract of Raphanus sativus L. Punica granatum, Allium cepa, Petroselinum crispum, Anethum graveolens L. and Apium graveolens with MIC values ≤ 5mg/ml.

 

On the other hand, the least antibacterial activity was shown by Ruta graveolens L., Eugenia aromatica L., Cinnamon zeylanicum Nees and Brassica oleracea extracts with MIC values of 10 mg/ml.

 

 

Table 2. MIC values of plant extracts against E. coli O157:H7

Plant	Family	MIC (mg/ml)
Eruca sativa Allium cepa Allium Kurrat Raphanus sativus L. Punica granatum Petroselinum crispum Anethum graveolens L. Apium graveolens Brassica oleracea Cinnamon zeylanicum Nees. Eugenia aromatica L. Ruta graveolens L. Ciprofloxacin	Cruciferae Liliaceae Alliaceae Brassicaceae Lythraceae Apiaceae Apiaceae Umbelliferae Brassicaceae Lauraceae Myrtaceae Rutaceae	2.5 5 2.5 5 5 5 5 5 10 10 10 10 1.56 µg/ml

 

 

DISCUSSION:

The antibacterial activity of 14 plant extracts was screened against E. coli O157:H7 by agar diffusion method and the MIC was determined by the agar dilution method. The extracts of Citrullus colocynthis and Glycyrrhiza glabra were the least active, while those of Allium kurrat and Eruca sativa showed the best antibacterial activity. The extracts of Cinnamomum zeylanicum Nees, Ruta graveolens L., Allium cepa, Eugenia aromatica L., Brassica oleracea, Raphanus sativus L., Punica granatum, Petroselinum crispum, Anethum graveolens L. and Apium graveolens exhibited significant activity against E. coli O157:H7.

 

The activity of plant extracts against pathogenic bacteria was investigated in many studies. Voravuthikunchai et al.¹⁴ found that the ethanolic extract of Punica granatum produced a significant antimicrobial activity against E. coli O157:H7. The antimicrobial activity may be attributed to the presence of some compounds that possess antibiotic action such as tannins that are present in high concentration (25%) in Punica granatum^15-18.

 

Muthuswamy et al.¹⁹ reported that ethanolic extract of Cinnamomum zeylanicum bark (2% w/v) could inhibit E. coli O157:H7 by 94%.

Pandey et al.²⁰ found that the ethanolic extract of Ruta graveolens produced moderate antibacterial activity against E. coli O157:H7. This was consistent with our results. On the other hand, Ivanova et al.²¹ found that the Ethanolic extract of Ruta graveolens had no antibacterial activity against E. coli O157:H7.

 

Allium cepa (1% w/v) exhibited antibacterial effect against E. coli O157²². This may be due to the isothiocyanates²³ that may inactivate extracellular enzymes through the oxidative cleavage of disulphide bonds²⁴. Isothiocyanate-mediated growth inhibitory activity of Raphanus sativus extracts against E. coli was reported by Beevi et al²⁵.

 

Bnyan et al.²⁶ study on the effect of ethanolic extract of Citrullus colocynthis against E. coli showed a significant inhibitory effect. Similar results were found with the ethanolic extract of Glycyrrhiza glabra L.²⁷

 

Brandi et al.²⁸ reported the inhibitory effect of the juice of Brassica oleracea on verotoxigenic E. coli O157:H7. Moreover, Hafidh et al.²⁹ found that the methanolic extract of Brassica oleracea exerted a bactericidal activity against E .coli O157:H7.

 

Wong and Kitts³⁰ proved the ability of the methanolic extract of Petroselinum crispum to cause cell damage and inhibit the growth of E. coli by chelation of iron required for bacterial growth.

 

Eugenia aromatica extract was inhibitory to E. coli³¹. Moreover, Eugenol, one of the main constituents of Eugenia aromatica, was found to inhibit the growth of Escherichia coli O157:H7³².

 

Kaur and Arora³³ reported a moderate antibacterial activity of ethanolic extract of Anethum graveolens against E. coli, while Baananou et al.³⁴ found a strong growth inhibitory effect of Apium graveolens oil against E. coli.

 

The extracts of Allium kurrat and Eruca sativa showed the highest antibacterial activity in our study. The reports about the antibacterial efficiency of Allium kurrat and Eruca sativa are few. The crude juices of Allium kurrat and Eruca sativa showed antibacterial activity against E. coli³⁵. Moreover, Khoobchandani et al.³⁶ studied the antimicrobial activity of Eruca sativa extracts and seed oil and found a higher activity of the seed oil compared to that of the methanolic extract. This activity may be attributed to the presence of glucosinolates; the precursors of isothiocyanates³⁷. Allium kurrat contains a phenolic compound; gallic acid which may mediate the antibacterial properties³⁸. Gallic acid was reported to have antibacterial activity against E. coli by affecting the cell membrane causing pores in the membrane through which leakage in the essential cell constituents occurs³⁹. Moreover, Allium kurrat is one of the Allium species that exert antimicrobial properties due to the presence of thiosulfintes⁴⁰.

 

This study suggests the use of the extracts of Allium kurrat and Eruca sativa as promising alternatives for the treatment of E. coli O157:H7 infection.

 

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Accepted on 05.07.2013      © RJPT All right reserved