Introduction
Infectious diseases caused by various types of microorganisms such as bacteria, fungi, viruses and protozoa and it is a major leading cause of death all over the world mainly in developing countries. Bacteria are major causal agent of infection. When the host defense mechanism become impaired then bacteria invade into body and causes infections.1 Urinary tract infections are the most common infectious disease found in human urinary tract system and caused by several bacterial uropathogens.2 Every year 150 million people suffered with UTI globally.2 UTI found in both males and females in all age groups but females are more susceptible for UTI than males and 75% UTI patients were females due to their anatomy and physiology of urinogenital system and risk of infection increases with age. 3 About 50% of females suffered with one symptomatic UTI during their lifetime. The risk of infection also observed in postmenopausal women. 4, 5
UTI’s are most frequently found infectious disease in both hospital and community in developing countries due to lack of awareness, improper sanitation, use of unhygienic public toilets and low economic status. People suffered with hospital acquired UTI during their visit to hospital. They acquired infection by hospital environment and different medical devices. The causal organism associated with hospital acquired UTI are mostly endogenous pathogens but sometimes they originate from the hospital environment. 6 The causal agent responsible for hospital acquired UTI are E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis and Candida and reported as more resistant strain of uropathogens.7, 8 The community acquired UTI is an infection that found in the community and mostly observed in rural area. The most common causative agent of community acquired UTI’s are E. coli, Klebsiella and Enterococcus among them E. coli is the most predominant uropathogen responsible for community acquired UTI.9
The treatment of UTI done by various antibiotics, they prevent growth and multiplication of bacteria. UTI’s are treated with diverse group of antibiotics such as β-lactamase, aminoglycosides, fluroquinolones and many others. The unwanted and nonjudicious use of antibiotics, inappropriate dosage, self-medication by direct purchasing of antibiotic from pharmacist without consulting with physician, selling of fake drug are several factors responsible for the development of antibiotics resistance and associated with emergence of MDR strains of bacteria. The emergence of drug resistance among uropathogens create hurdle in treatment and control of diseases. The other reasons of drug resistance among uropathogens are spontaneous mutation or acquisition of resistant genes among organisms by horizontal transfer.10, 11
The emergence of drug resistance and costly treatment of UTI has motivated researcher to find out new effective alternative and natural bioactive antimicrobial agent for treatment of infection. From ancient time’s large number of tribals, rural and urban community used plants and its parts as a source of medicine to treat different types of infections. Ethnomedicine is still use as a primary and alternative source of medicine in treatment of various infectious diseases because they are cheap and easily available.12, 13
The present study conducted to determine the antibacterial activity of Asparagus racemosus and Withania somnifera against some common bacteria causing urinary tract infection. Asparagus racemosus is a woody climbing plant commonly known as ‘Shatawari’, also called ‘queens of herbs ’used in Ayurvedic medicine and belongs to family Asparagaceae and found in India, Australia, Asia and Srilanka. The roots and stems are used for making medicine and used in treatment of diarrhea, dysentery and urinary tract infection due to its antimicrobial property. It has antioxidant and antibacterial effect and stimulates the immune system. Its root has a good antidiuretic effect. It helps control burning sensation in UTI, it increases urine flow and flush out various harmful toxins and pathogens. It is mainly contains β – Sitosterol which have good antibacterial activity against UTI causing bacteria. This drug is safe without any side effects.14, 15 Withenia somnifera is annual evergreen shrub belong to Solanaceae family commonly known as ‘Ashwagandha’ or ‘Winter Cherry’. It is native of India, Asia and Africa. It has antibacterial, anti-inflammatory, antioxidant and immunobooster activity. It used to relieve bladder pain, burning sensation during urination and help in removal of kidney stone. 16, 17
Materials and Methods
The study was conducted to evaluated antibacterial effect of methanolic root extracts of two plants Asparagus racemosus and Withania somnifera on isolated uropathogens. This study was performed in SRL laboratory Ujjain center. The urine samples collected from different hospitals of Ujjain were processed in lab. Total 120 urine samples from suspected patients were collected from November 2018 to October 2019. The collected 0.5 ml of urine sample was inoculated on three different selective and differential media, which were Blood agar, MacConkey agar and Chrome agar with the help of sterilized loop, by streak plate method. The plates were incubated at 37oC for 24 h and after incubation colony forming unit were counted, if colony count is more than 105 colonies forming unit/ml than it indicated significant bacteriuria and was considered as positive urine culture. The single pure colonies were selected and subjected to morphological, microscopic and biochemical examinations as per the standard procedure for confirmation of isolated uropathogenic bacteria.18 In morphological examination shape, size, color and margin of colony was observed. In microscopic examination gram staining was done for differentiation between gram positive and gram negative bacteria and the shape, color, arrangement of bacteria were also examined. The biochemical tests included Catalase, Oxidase, Coagulase, Indole production, Methyl Red, Voges-Proskauer, Citrate utilization, Triple sugar iron, Urease, Mannitol fermentation, Bile Esculine Hydrolysis test and motility tests.
Antibacterial activity of plant extract
The antibacterial activity of root extract of Asaparagus racemosus and Withania somnifera was determined against isolated uropathogens E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis and S. aureus. For this plant extract of both plants were prepared. The dried powder of plant parts were purchased from Amsar Private Limited, Indore and stored in airtight container. The root part of both plants were taken and their methanolic extract were prepared by dissolving 5 gram of dried root powder in 9 ml of methanol and volume was made up to 10 ml. The extracts were serially diluted to obtain different concentration of plant extracts. The antibacterial activity of this prepared plant extracts were observed on isolated uropathogens by disc diffusion method.19 In this method disc of plant extract was prepared by soaking 6 mm filter paper disc in diluted plant extract. After preparing, the disc fresh culture of isolated bacteria was spread on Mueller-Hinton agar plate surface using sterile swab. The inoculated plates were placed in sterile conditions for 1 hour for stability then disc of prepared plant extracts were placed on agar surface with the help of sterile forceps and incubated at 37oC for 24 h. After incubation, the zone of growth inhibition around the plant extract disc was determined and antibacterial activity of both plant extracts against isolated uropathogens was observed.
Results
Prevalence of UTI and frequency of uropathogens
It was seen that out of 120 urine sample, 72 were positive and its prevalence was 60%. The five uropathogens were isolated from patients, among which four were gram negative and one was gram positive uropathogenic bacteria. The E. coli was most prevalent uropathogen and its frequency was 37.5% which was followed by K. pneumoniae, P. aeruginosa, P. mirabilis and S. aureus and their frequency of occurrence was 25%, 15.2%, 13.8% and 9.7%. The frequency of isolated uropathogens were shown in Figure 1.
Effect of plant extracts
In our study plant extracts of both Asparagus racemosus and Withania somnifera showed antibacterial activity against five isolated uropathogenic bacteria. The antibacterial effects were categorized low, moderate or high based on the size of growth inhibition zone. It was seen that methanolic root extract of Asparagus racemosus had moderate killing activity against K. pneumoniae, P. aeruginosa and low killing activity against P. mirabilis and S. aureus and methanolic root extract of Withania somnifera was effective for killing K. pneumoniae and had moderate killing activity against E. coli, P. aeruginosa, P. mirabilis. The results of antibacterial activity were seen only on sensitive isolates and results are shown inTable 1.
Table 1
Antibacterial activity of Asparagus racemosus and withania somnifera against isolated uropathogens.
Discussion
In our study the overall prevalence of UTI was 60 %. Paul et al., (2021) conducted similar study and investigate prevalence of UTI in primary care center of Aasam, India, found percentage of UTI was 59.6%, and Kumar et al., (2019) found that percentage of UTI was 60.6% in Ambala (Haryana), India. In Bangladesh Akhtar et al., (2017) reported 58.9%. These results are almost similar to results of current study, which suggested that the prevalence of UTI is not much different.20, 21, 22 During the study five uropathogens E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis and S. aureus were isolated and among them E. coli was most frequently isolated uropathogens and its The frequency of occurrence was 37.5%. Higher frequency of E. coli isolates was also correlates with studies done by Agrawal et al., (2021), Mendem et al., (2020) in India and Odoki et al., (2019, reported frequency of E. coli isolates 43.5%, 47.0% and 41.9% respectively. 23, 24, 25
The antibacterial effect of methanolic root extract of Asparagus racemosus and Withania somnifera was determined in our study and both have killing effect against uropathogens. The antibacterial effect of Asparagus racemosus was also observed by Mukhtair Uddin in 2012, they observed killing effect of methanolic root extract against eight bacterial strains of K. pneumoniae, E. coli, P. alkaligens, Proteus spp., Salmonella typhi, Sheigella, V. cholera and S. aureus. In 2014 Chouhan and Pandey studied antibacterial potential of Asparagus racemosus extract against bacteria causing UTI. They reported that aqueous and methanolic extracts of Asparagus racemosus exhibited broad spectrum antibacterial effect against both gram positive and gram negative bacteria in comparison to ethanolic and ethyl acetate extract. Similarly Arya et al., 2018 also reported efficacy of root powder of Asparagus racemosus in reducing UTI, boost the immune system and prevent chances of reinfection.26, 27, 28
The antibacterial effect Withania somnifera also detected by Bokaeian et al., 2015, they showed that ethanol extract of Withania somnifera leaf extract might be used as natural drug for treatment of infectious disease caused by S. aureus. Das et al, 2019 investigated root and stem extract of Withania somnifera plant have antibacterial activity against some human pathogenic bacteria. Beigomi et al., 2021 evaluated antimicrobial effect of Rosemary and Withania somnifera methanol extract prepared by ultrasound waveform on E. coli biofilm isolated from urinary tract infection.29, 30, 31
Conclusion
The treatment and prevention of UTI can be done by plant and its part because they are cost effective, easily available and safe with no side effects. These plant extracts reduces antimicrobial resistance hazards and help to decrease adverse effects of antibiotic because it contains several secondary metabolites and active phytochemicals responsible for their antibacterial activity. The root extracts of both Asparagus racemosus and Withania somnifera showed promising antibacterial activity against the isolated uropathogens. Therefore, isolation and identification of secondary metabolites of plants will help in to develop modulator or precursor for synthesis of new antimicrobial agent as alternative of antibiotics.