Antibacterial activity of Withania somnifera against Gram-positive isolates from pus samples

Abstract

Background:

Withania somnifera is an important medicinal plant that has been used in Ayurvedic and indigenous medicine since ancient times. In the view of its varied therapeutic potential, it has also been the subject of considerable modern scientific attention. Attention has been drawn to antibacterial activity of the plant and its metabolites due to the challenge on growing antibacterial resistant pathogens.

Aim:

To examine the antimicrobial potential of leaf extract of W. somnifera against Gram-positive cocci.

Materials and Methods:

In this study, leaf extract of W. somnifera was used to examine their antimicrobial potential against Gram-positive cocci (n = 20) from pus samples of patients admitted in Government Medical College, Haldwani. Agar well diffusion method was used by taking methanolic leaf extract of W. somnifera.

Results:

It was observed that the methanolic leaf extract of W. somnifera was very effective in inhibiting the test pathogens including methicillin resistant Staphylococcus aureus and Enterococcus spp., with an average zone of inhibition of 20.6 mm and 19.4 mm at 2 mg/ml (100 μl) concentration, respectively.

Conclusion:

These results indicate that the antimicrobial property of W. somnifera leaf supports the traditional use of the plant in therapeutic use against microbial infections.

Introduction

Vancomycin-resistant Enterococcus and methicillin resistant Staphylococcus aureus (MRSA) are associated with increase rates of illness and death. Both these organisms are now endemic in many health care institutions.[1] These pathogens are now increasingly exhibiting resistance to the commonly used antibiotics due to widespread and inappropriate use of antibiotics in human and veterinary medicine.[2,3] MRSA is resistant to all beta-lactam antibiotics. Vancomycin had long been considered for the treatment and cure of MRSA infections. However, now vancomycin-resistant strains have been also reported.[3] To substitute synthetic antibiotics, many of the modern and effective drugs have their origin in traditional folk medicine. Therapeutic efficacy of many indigenous plants for several disorders has been described by practitioners of traditional medicine.[4]

Withania somnifera (L.) Dunal also known as “Aswagandha” belongs to the family Solanaceae and is widely used in Ayurvedic medicine. It is an ingredient in many formulations prescribed for a variety of musculoskeletal conditions (e.g. arthritis, rheumatism), and as a general tonic to increase energy, improve overall health and longevity, and prevent disease in athletes, and elderly.[5,6,7] Recently, the antimicrobial activity of W. somnifera was studied by several authors on control strains (microbial type culture collection).[5,7,8,9] However, there is no report of antibacterial activity of leaf extract of W. somnifera on human pathogenic Gram-positive bacterial strains from soft tissue infection. Hence, this study was planned to examine the antimicrobial potential of leaf extract of W. somnifera against Gram-positive cocci (n = 20) from pus samples of patients suffering from soft tissue infection.

Materials and Methods

Plant description

Withania somnifera is an erect, herbaceous, and evergreen tomentose shrub. The leaf base is cuneate and is densely hairy beneath. The flowers are yellow and berries orange-red.

Plant collection

The leaves of W. somnifera were collected from the Central Institute of Medicinal and Aromatic Plants (CIMAP), Pantnagar (Uttarakhand).

Preparation of extract

Extraction procedure was followed as Owais et al.[8] with slight modification at the research and development laboratory in CIMAP, Pantnagar. Fresh leaves were washed thoroughly 2–3 times with running water followed by sterile distilled water. Washed leaves were air dried under the shade at room temperature and then pulverized by mortar pestle. Shade dried leaf powder was shaken overnight in methanol and then extracted successively in Soxhlet apparatus. Extract was filtered by Whatman number one filter paper and filtered solution was evaporated under reduced pressure with the help of rotary evaporator. The dried leaf extract was dissolved in methanol to final concentration of 1 mg/ml and 2 mg/ml.

Microorganisms

Gram-positive cocci obtained from clinical samples of pus from patients admitted to Government Medical College, Haldwani were used to find out the antimicrobial potential of leaf extract of W. somnifera. A total of 20 isolates including S. aureus (MRSA and methicillin sensitive staphylococcus aureus [MSSA]), Enterococcus and Streptococcus spp. were examined against methanolic extract of leaf of W. somnifera.

Sensitivity testing

The antibacterial susceptibility test was carried out using the agar diffusion method.[10] Muller Hinton agar (MHA) was used for antibacterial susceptibility testing. For diffusion method, Petri plates were prepared by pouring 20 ml of MHA. Stock bacterial solution was thawed and immediately suspended in peptone water and incubated for 2–3 h at 37°C. After matching the turbidity with 0.5 Mc Farland, the inoculum of each isolate was spread on two MHA plates and was allowed to dry for 10 min. Four wells of 9 mm diameter each were punched in each plate using a sterile borer. Plant extract with concentration of 1 mg/ml and 2 mg/ml with different volumes of 20 μl, 50 μl, and 100 μl were poured in each of the 3 wells of MHA plates containing bacterial inoculum. Fourth (control well) was filled with 50 μl methanol. The plates were kept for 1 h at room temperature to allow the diffusion into the medium and then incubated aerobically at 37°C for 18 h. The inhibition zones formed around the wells were measured in millimeters [Figure 1]. For each concentration, the zones of inhibition produced by different strains of a species were averaged.

Figure 1.

Zone of inhibition (in mm) by Withania somnifera leaf extract in concentration of 1 mg/ml by using different volumes of 20 µl, 50 µl, and 100 µl and 2 mg/ml by using different volumes of 20 µl, 50 µl, and 100 µl on pathogenic isolates of -Staphylococcus aureus and Streptococcus spp. C-negative control (containing 100 µl methanol)

Results

Methanolic extract of W. somnifera showed potent antibacterial activity against Gram-positive clinical isolates that have been reported to be more resistant against routinely used first line of antibiotics such as ampicillin, co-trimoxazole, and erythromycin. At the concentration of 2 mg/ml (100 μl) average zone size of inhibition for MRSA and Enterococcus spp. was 20.6 and 19.8 mm, respectively. Whereas, for MSSA and Streptococcus spp. average zone of inhibition were 22.4 and 22.2 mm, respectively. At the concentration of 1 mg/ml (100 μl) average zone size of inhibition for MRSA and Enterococcus spp. was 19.2 and 19 mm, respectively. Whereas, for MSSA and Streptococcus spp. zone of inhibition were 20.8 and 21.2 mm, respectively. Overall increase of zone of inhibition was noticed with a higher concentration of leaf extract [Table 1 and Figure 1].

Table 1.

Zone of inhibition (in mm) for different concentration of W. somnifera leaf extract

Discussion

W. somnifera leaf extract showed good inhibition of MRSA and Enterococcus spp. which were resistant against routinely used first line antibiotics. As in the previous study,[9] methanolic root extracts of W. somnifera revealed 4 mm inhibitory zone against Escherichia coli and 10 mm inhibitory zone at 10 μg/ml against Enterococcus, which is very less than that observed in our study. This difference may be due to a different part of the plant extract being used. Owais et al.,[8] observed 22 mm zone of inhibition against S. aureus at a concentration of 20 mg/ml of W. somnifera leaf extract. In another study,[5] 15 mm zone of inhibition at a concentration of 100 μg/ml against S. aureus and E. coli by W. somnifera leaf extract was reported. In present study good inhibitory zone against Enterococcus spp., and S. aureus at the concentration of 2 mg/ml was observed. Difference in the zone of inhibition might be attributed to the difference in bacterial strains used, methodology employed, and different geographical area from where the plant was obtained.

Reference standard institutes like Clinical Laboratory Standard Institute or European Committee on Antibiotic Susceptibility have guidelines for interpretation of disc diffusion and minimum inhibitory concentration for antibiotics, but there is no such standard zone chart or guideline available for W. somnifera extract. In the present study, convincing zone of inhibition was found by W. somnifera leaf extract in pathogenic Gram-positive cocci isolates which were resistant to routinely used first line antibiotics (with no zone of inhibition). However, sample size of the present study was low; hence, this requires a much more exhaustive in vitro and in vivo study involving identification and isolation of specific W. somnifera compounds acting against a resistant strain.

Conclusion

This finding supports the use of leaf extract of W. somnifera in the treatment of multidrug-resistant pathogens by alternative systems of medicine. Clinical trials with Withania somnifera for its activity against bacterial infections should be conducted.