1. Background
Urinary Tract infection (UTI) is the second most common infectious presentation in community medical practice after respiratory tract infections (1). A complicated UTI includes abnormalities of the urinary tract that impede urine flow. The prevalence of urinary tract infections varies with age and sex. The risk factors for UTI involve colonization with a different uropathogen in cases of recurrent UTI, glucosuria and impaired granulocyte function (2). Drug resistance is a natural biological response of microbes involving mutation and cross genetic transfer. It becomes a major problem only when disease-causing organisms develop the ability to fight off disease-curing drugs when these drugs are partially used or used unnecessarily (3). The increasing resistance pattern to a drug creates pressure to switch to a different drug that is more potent than that prescribed previously. It is worth to mention that just as drug resistance is mainly an acquired property; it can also be lost in course of time (4). The aim of this study was to determine the antibiotic susceptibility pattern of uropathogens and their sensitivity towards W. somnifera. Withania somniferaL. (Dunal) (from the family Solanaceae), which is classically known for its ethnomedicine properties. Many pharmacological studies have been conducted to investigate the properties of Withania somniferaas a multi-purpose medicinal agent (5). The plant is used for the treatment of tuberculosis, rheumatism, inflammatory conditions and as a potential anti-tumor agent (6).
2. Objectives
The aim of this study was to evaluate the antibacterial effect of extracts of W. Somnifera on extended-spectrum beta-lactamase (ESBL) pathogens isolated from UTI patients by the stroke method.
3. Patients and Methods
3.1. Sample Preparation
Early morning urine was collected from 32 positive patients who had clinical symptoms of UTI and were from the Trichy region of Tamilnadu. Thirty-milliliter urine samples were collected in a clean sterile centrifuge tube and centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded aseptically and the sediment in the centrifuge tube was homogenized in 100 μL of supernatant. The homogenized suspension was then used for total bacterial count and identification of uropathogens.
3.2. Isolation and Identification of Uropathogens
Uropathogens were identified through culture, microscopy and biochemical tests. MacConkey agar, blood agar, chocolate agar and Oxoid clarity agar media (Himedia Ltd.) were inoculated with 100 μL of the prepared urine samples, and incubated at 37°C for 24 hours. Bacterial identification was done by phenotypic examination followed by Gram staining, and a series of standard biochemical tests were also performed to identify the bacteria of interest (7).
3.3. Antibiotic Susceptibility Testing
The agar disc diffusion assay was used to determine the antimicrobial susceptibilities of uropathogens. The discs used in this study included amoxicillin 30 μg, amoxiclav 30 μg, cefepime (4th) 30 μg, cefixime (3rd) 5 μg, chloramphenicol 30 μg, clotrimazol 25 μg, amikacin 10 μg, ampicillin 30 μg, ceftriaxone (3rd) 30 μg, ciprofloxacin 5 μg, gentamycin 10 μg, erythromycin 15 μg and penicillin 10 μg. The diameters of the zones of inhibition for individual antimicrobial agents were translated into susceptible, intermediate and resistant categories according to the National Committee for Clinical Laboratory Standards Institute (8, 9).
3.4. Detection of Extended-Spectrum Beta-Lactamase by the Double Disc Diffusion Method
An amoxicillin-clavulanic acid (20 mg and 10 mg) disc was placed on the center of the plate. Ceftazidime (30 mg), ceftriaxone (30 mg), cefotaxime (30 mg) and aztreonam (30 mg) discs were placed peripherally away from the amoxicillin clavulanic acid disc. After 24 hours of incubation at 37°C, band formation between the amoxicillin clavulanic acid disc and any other disc was considered ESBL positive. The ESBL positive strains were further subjected to phenotypic confirmatory tests using sensitivity discs, which contained third-generation cephalosporins both with and without clavulanic acid. The discs used included cefotaxime (30 mg), cefotaxime and clavulanic acid (30 mg and 10 mg). The differences in the zone of inhibition caused by the cephalosporins alone and when combined with clavulanic acid were recorded and if the difference was 5 mm or more, the strains were confirmed as ESBL-producing strains (10).
3.5. Antibacterial Activity of W. somnifera Against ESBL Producers
Well dried finely powdered roots and leaf were extracted with ethylacetate and concentrated by evaporation. The crude extracts were assayed against ESBL E. coli, Pseudomonas aeruginosa and Proteus sp.; about 100 µL of (5 mg/mL) W. somnifera extracts were loaded on sterile discs and were tested against test pathogens. The test pathogens were swabbed on Mueller Hinton agar plates and the sample disc were placed on the center of plates along with a positive control amoxicillin-clavulanic acid and a negative control Ethyl acetate. All the plates were replicated three times and the zone of inhibition was calculated by the mean values.
4. Results
Of the 32 samples, 20 were female and 12 were male and a high frequency of infection was found in the age group between 30-45 years (Table 1). Twenty-six microorganisms have been isolated from urine samples and six different genera were identified. The frequencies of isolates are given in Table 2. Among the 26 isolated UTI pathogens, 42 were ESBL producers belonged to E. coli (23%) Pseudomonas sp (11.5%) and 3.8% of Klebsiella sp and Proteus sp. The use of medicinal plants proved to be economical and effective; in addition, they are easily available and safe to use. Root extracts of W. somnifera had the highest antibacterial activity against ESBL E. coli, (64% relative inhibitory zone) followed by Pseudomonas aeruginosa (56 % relative inhibitory zone). The least relative inhibitory zone was 53% against ESBL Klebsiellasp and Proteus (Table 3). However, antibacterial potentials were also observed against non-ESBL E. coli, Klebsiella, Enterococcus and Staphylococcus. Most of the non-ESBL pathogens were highly sensitive to root extract of W. somnifera and the maximum activity was 20 ± 0.16 mm against Klebsiella sp. This antibacterial activity has been attributed to the presence of some active constituents in the extract. This research finding encourages usage of W. somnifera against UTI infection. Further studies such as purification, nuclear magnetic resonance and mass spectrum are required to determine the pharmaceutical value of W. somnifera.
Table 1.
Frequency of Urinary Tract Infection Among the Collected Samples
| Variable | Frequency | Value a | |
|---|---|---|---|
| Male | Female | ||
| Age, y | |||
| 0-15 | 0 | 2 | 6.25 |
| 15-30 | 2 | 4 | 18.75 |
| 30-45 | 6 | 8 | 43.75 |
| 45-60 | 3 | 4 | 21.87 |
| Above 60 | 1 | 2 | 9.37 |
| Total | 12 | 20 | 100 |
a Data are presented as %.
Table 2.
| Name of Organisms | Frequency | Number of ESBL Producer |
|---|---|---|
| E. coli | 7 (26.92) | 6 (23) |
| Pseudomonas aeruginosa | 5 (19.23) | 3 (11.5) |
| Enterococcus sp | 4 (15.38) | 0 (0) |
| Klebsiellasp | 4 (15.38) | 1 (3.8) |
| Staphylococcus sp | 2 (7.69) | 0 (0) |
| Proteus sp | 4 (15.38) | 1 (3.8) |
aAbbreviation: ESBL, Extended-Spectrum Beta-Lactamase.
bData are presented as No. (%).
Table 3.
Antimicrobial Study of W. somnifera Against Extended-Spectrum Beta-Lactamase Pathogens a
| Test Pathogen (ESBL) | Zone of Inhibition in mm in diameter, mm × Dm | ||||
|---|---|---|---|---|---|
| Amox/Culv | Ethyl Acetate | Root Extract | Percentage of RIZD | Leaf Extract | |
| E. coli | 17 | 8 | 19 | 64 | 14 |
| Pseudomonas aeruginosa | 16 | 9 | 18 | 56 | 12 |
| Proteus sp | 17 | 9 | 18 | 53 | 15 |
| Klebsiellasp | 16 | 9 | 18 | 53 | 16 |
aAbbreviations: ESBL,Extended-Spectrum Beta-Lactamase;Dm, Diameter; RIZD, Relative Inhibition Zone Diameter.
5. Discussion
In this present investigation it was observed that UTIs are more prevalent in the age group of 30-45 years and are most frequently found in female cases. Among the isolates 15 (58%) isolates were non-ESBL and highly sensitive to cefepime, cefixime, chloramphenicol and clotrimazol. Urinary tract infection is more prevalent in females and there has been many previous reports on this subject (11). In this research, the antibacterial study showed that the root extracts of W. somnifera showed potent antibacterial activity against all ESBL producers. The leaf extracts showed maximum zone of inhibition against ESBL Klebsiellasp (16 mm) and least inhibitory activity against Pseudomonas aeruginosa (12 mm zone of inhibition). Antimicrobial activity of methanolic extracts of W. somnifera against B. subtilis, E. coli, P. Fluorescens and S. aureus has been reported previously (12). Aqueous extract of W. somnifera was more effective against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus earlier reported by Jaina and Varshney (13).