Antibacterial properties of distilled cow’s urine on bacterial species from clinical specimens

Introduction and Aim: In Ayurveda, cow's urine has been reported to have many therapeutic values. The raising trend in antibacterial resistance is a cause of concern and demands an alternative therapeutic agent. Hence, the present study aims to know the antibacterial properties of urine from cows on clinical and reference strains of bacteria. Materials and Methods: The present in vitro experimental study, used agar dilution method to know the Minimum Inhibitory Concentration (MIC) of cow’s urine distillate (CUD) on few reference bacterial strains and Enterococcus faecalis, Staphylococcus aureus , Salmonella Typhi, Escherichia coli , and S . Paratyphi A isolated from clinical samples. Resistance and sensitivity of these isolates to routinely used antibiotics was studied by Kirby Bauer’s disk diffusion method. Results: MIC of CUD for different standard bacterial strains varied from 250 to 500μl/ml. Among the clinical isolates, all Salmonella Typhi, 85% of S. Paratyphi A, 60% of E. coli , 80% of the S. aureus , 64.28% S. aureus were resistant to methicillin (MRSA) and 24% of E. faecalis were killed by 500μl/ml of CUD. Clinical strains of bacteria, susceptible to routinely used antibiotics, were also found to be susceptible to CUD and showed a concentration-dependent inhibitory effect. Multi drug resistant strains of nine MRSA , one E. faecalis and two E.coli were also susceptible to 500μl/ml concentration of CUD. Conclusion: CUD can be considered as an alternative anti-bacterial agent for multi-drug resistant bacterial pathogens as it showed concentration dependent inhibitory effect on both Gram-positive and Gram-negative pathogenic bacteria.


INTRODUCTION
ncreasing prevalence of communicable diseases caused by multiple drug resistant pathogens and shortage of new antimicrobial agents is a significant health concern, which necessitated a quest for new antimicrobial agents (1). Nature has always been a source of bioactive compounds which have cured many human ailments. Ayurveda, the Indian system of medicine has been practiced as a natural alternative medicine since ancient times and is considered as a standard health care system in India (2). Cow urine (gomutra) has an exceptional place in Ayurvedic medicine.
'Sushruta Samhita' and 'Ashtanga' describes cows' urine as an effective secretion of animal with many beneficial healing effects. It is highly efficacious against stem borers and acts as a bioenhancer (3)(4)(5). The cow's urine distillate (CUD) is considered having activity enhancing and facilitator property for biologically dynamic molecules. CUD has been used as anti-infective and anti-cancer agent (6). It is known to possess immunomodulatory, anti-diabetic, antioxidant, and anti-fungal effects (7)(8)(9)(10). Further, enhanced wound healing, delayed aging and curative effects on skin lesions have also been reported (11). Medicinal value of cow's urine was appreciated long ago as its efficacy and mechanism of antibacterial effect remain elusive, thorough scientific validation is required to prove its efficacy. Few studies from different Indian states like Madhya Pradesh, Kerala, Karnataka, Gujarat, and New Delhi, have reported the antibacterial effect of cow's urine (12)(13)(14)(15)(16)(17). Interestingly, urine of most of the native cow exhibit antimicrobial properties but not the urine of buffalo, goat and crossbred cows (18). Thus, antimicrobial property of urine of cows is dependent on cow breed and geographical area. Anti-bacterial action of CUD on clinical bacterial isolates is not reported from Mangalore India. Moreover, rise in drug resistance demands for an alternative easily available cost-effective agent. Hence, we attempted to study the antibacterial effect of CUD on reference bacterial strains and common Gram-positive and Gram-negative bacteria isolated from patient samples.

MATERIALS AND METHODS
This is an in vitro experimental study, where antibacterial action of CUD was tested on reference bacterial strains and common Gram-positive and Gram-negative bacteria cultured from patient samples. Institutional Ethics committee clearance was obtained before commencing the study (Reference No: IEC KMC MLR, 02-14/66).

Source of distilled cow's urine
Distillate of cow's urine was procured from a specific local breed (Kapila) of healthy cow, confined to Southern Karnataka, reared at a cow yard located at Surabhivana, Kompadavu, Mangalore.

Bacterial strains used in the study
Standard reference strains of Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were obtained from departmental stock. Clinical isolates (n=120) comprised of Gram-positive cocci: S. aureus (n=25), E. faecalis (n= 25) and Gram-negative bacilli: E. coli (n=25), S. Typhi (n=25) and S. Paratyphi A (n=20). The culture media and chemicals utilised in our study were obtained from Hi-Media Laboratories Pvt Ltd. Mumbai, India. Standard biochemical tests were performed to identify the bacterial isolates (19).

Sterility check of distillate of cow's urine
Sterility check was performed by inoculating 9 ml of Brain Heart Infusion (BHI) broth with one millilitre of CUD and incubating the same at 37 0 C for four weeks. Subculture of BHI broth was done on blood agar, MacConkey's agar and Sabouraud's dextrose agar on 2 nd , 4 th , 7 th , 14 th , 21 st and 30 th day of incubation. Inoculated culture media were incubated at 37 0 C for 48h and observed for any growth (19). Only those distilled cow's urine samples that showed no detectable bacterial or fungal growth were used in the study.

MIC detection of cow's urine on reference and clinical bacterial strains
Agar dilution technique was used to study the MIC of sterile CUD on reference strains of S. aureus ATCC 25923, E. faecalis ATCC 29212, P. aeruginosa ATCC 27853, E. coli ATCC 25922 and bacterial isolates from clinical samples (20). Different concentrations of CUD ranging from 200 to 500μl/ml were added to molten sterile Muller Hinton agar (MHA) and allowed to solidify. Reference and clinical strains were grown in Muller Hinton broth (MHB) for 6 hrs. Turbidity of culture was maintained at 0.5 Mac Farland standard (10 6 CFU/ml). Two microliters of this turbidity adjusted culture was used to inoculate MHA with varying concentration of CUD (200 to 500μl/ml) and MHA without CUD. The culture plates were incubated at 37°C for 24hr. Testing was repeated twice, and reproducible results were obtained. The maximum dilution of the CUD that did not show noticeable growth on MHA plates with CUD was considered as MIC. MHA without CUD acted as the growth control.

Antibiotic susceptibility testing of clinical isolates
S. aureus (n=25) isolated from pus, E. faecalis and E.coli (n=25 each) isolated from urine samples, S. Typhi (n=25) and S. Paratyphi A (n=20) cultured from patient's blood sample were identified by Vitek-2 system. The Kirby Bauer disk diffusion technique was used to check for the antimicrobial resistance of the bacterial strains to routinely used antibiotics and interpreted as per CLSI guidelines (21,22). Different antibiotic discs (Himedia laboratories Ltd, Mumbai, India), as mentioned in Tables (2,(4)(5)(6) were used in the study. E. coli ATCC 25922, S. aureus ATCC 25923, E. faecalis ATCC 29212 and P. aeruginosa ATCC 27853 were used as control strains.

Sterility of CUD
BHI inoculated with CUD did not show any observable growth of bacteria or fungi during the prolonged incubation period of 30 days. This sterile CUD was used in all our experiments.

Effect CUD on standard bacterial strains
The standard reference strains of E. coli ATCC 25922, S. aureus ATCC 25923, E. faecalis ATCC 29212, P. aeruginosa ATCC 27853 were treated with varying concentration of CUD to know the MIC and the outcome is depicted in Table 1.

Susceptibility of Salmonella isolates to routinely used antibiotics and CUD
Isolates of S. Typhi (n=25) and S. Paratyphi A (n=20) obtained from the blood culture of patients suffering from enteric fever were found to be susceptible to chloramphenicol, cefuroxime, ceftriaxone, cotrimoxazole, ciprofloxacin, and ofloxacin, which are the routinely used antibiotics for treating the patients. However, nalidixic acid resistance was around 80% in S. Typhi and 85% in S. Paratyphi A ( Table 2). 100% of the S. Typhi and 85% of the S. Paratyphi A tested were inhibited by 500μl/ml of CUD. It was also observed that CUD had concentration-dependent or dose-dependent action as shown in Table 3.
05 (20) 20 (80) 03 (15) 17 (85)   Susceptibility of E. coli isolates to routinely used antibiotics and CUD E. coli (n=25) obtained from patients with urinary tract infection were found to be resistant to most of the routinely used antibiotics. 92% of the isolates were susceptible to netillin and ciprofloxacin whereas 96% of the E. coli isolates were sensitive to cefazolin. (Table 4). Fifteen strains (60%) of E. coli were inhibited by 500μl/ml concentrate of CUD. However, 10 (40%) of the isolates were resistant to CUD as depicted in Table 3.

Susceptibility of S. aureus to routinely used antibiotics and CUD
Antibiotic susceptibilities of S. aureus (n=25) got from pus samples of patients with wound infection is shown in Table 5. All S. aureus tested were sensitive to vancomycin, linezolid, teicoplanin, netillin, and chloramphenicol, whereas 56% of the S. aureus isolates were MRSA. 20% of the strains were resistant to CUD. However, 80% of the strains were inhibited by 500μl/ml concentration of CUD as shown in Table 3.

Susceptibility of E. faecalis isolates to routinely used antibiotics and CUD
Antibiotic susceptibility pattern of E. faecalis (n=25) isolated from pus and urine samples of patients is shown table 6. All the isolates were found to be susceptible to teicoplanin as shown in Table 2. 12% of the strains were suppressed by 200μl/ml concentration of CUD, and 24% of the strains were suppressed by 500μl/ml concentration of CUD as shown in Table 3.

DISCUSSION
The study was undertaken to know the effect of CUD on clinical bacterial isolates. We selected four different bacterial species, which are the common cause of human infections in this part of the country. Distilled cow's urine was initially tested for sterility and was found to be sterile as observed in the earlier studies (16,17). Sterile CUD was tested on the standard strain of E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 25923, E. faecalis ATCC 29212, to know the MIC. It was surprising to note that standard strains of Gram-negative bacilli were more sensitive (lower MIC) to CUD than the standard strains of Gram-positive cocci ( Table 1). Our findings indicate that CUD has a higher antibacterial effect on standard strains of Gram-negative bacteria.  Tables 3 and 7. It is interesting to note that 500μl/ml concentration of CUD could kill 100% of S. Typhi, 85% of S. Paratyphi A, and 60% of E. coli (Table 3). Our results highlight the fact that CUD has concentrationdependent action on clinical isolates of bacteria. Similar effect was observed for Candida spp. isolated from clinical samples (23). Our study shows CUD to be very effective against S. Typhi when compared to E. coli. Our observation of maximum strains of S. Typhi being sensitive to CUD is in line with a study conducted by Muthaiya Research Foundation in Tamil Nadu, India (10). However, they have studied the effect of CUD only on standard strains of bacteria and not on the clinical bacterial isolates.
Among the Gram-positive clinical strains studied 80% of the S.aureus, and only 24% of E. faecalis were killed by 500μl/ml concentration of CUD (Table 3). Further, we also observed that most of the strains which were susceptible to routinely used antibiotics (100% strains of S. aureus and Salmonella and 66.67% of E. coli) were also susceptible to CUD (Tables 2-5 and 7). In an earlier study, standard strains of S. aureus and E. coli which were sensitive to ofloxacin were also sensitive to CUD, and S. aureus was more susceptible than E. coli (12). However, their study has not checked the effect of CUD on clinical isolates as has been done in our study.
A research study from Gujarat, India by Shah et al., concluded that owing to the variation in the cell wall structure, cows' urine has better bactericidal effect on Gram-positive bacteria when equated to Gramnegative bacteria (16). However, the present study does not support these findings, as S. Typhi was 100% inhibited by CUD, followed by S. Paratyphi A, S. aureus then E. coli and lastly Enterococcus (Table 3). Difference in the findings of our study may be attributed to the fact that we have used agar dilution method and studied the effect of CUD on standard and clinical bacterial isolates whereas, earlier study from Gujarat have examined the antibacterial effect by disk diffusion method on the standard strains only. Alternatively, there could also be differences in the genotype of clinical isolates and the nature of cow's urine which is dependent on the cow's breed and their feeding habits. Sharma et al., have detected the antimicrobial peptides belonging to defensin family in cows' urine using Mass Spectrometry (24). Further studies are necessary to know whether excretion of these antimicrobial peptides vary in different breeds and depends on the diet of the cattle. Additional studies are also necessary to identify the best suited method for MIC determination of CUD on clinical isolates of bacteria.
In the present study clinical isolates of Enterococcus spp. were found to be more resistant to CUD than other clinical bacterial isolates (Tables 3 and 6). Earlier workers have observed invitro antibacterial effect of photo activated cow's urine on standard strains of E. faecalis inoculated into root canals of teeth (25). However, to compare our results, studies focusing on the effect of CUD on clinical isolates of Enterococcus spp. are lacking.
Another notable observation was the antibacterial effect of CUD on MDR bacterial isolates. Nine strains of MRSA were susceptible to 500μl/ml concentration of CUD (Table 7). We did not come across any report on the effect of CUD on MRSA. Hence, in the era of emerging MRSA, cow's urine might show some promise in treating infections caused by MRSA after performing a clinical trial. Further, one MDR E. faecalis and two MDR E.coli were also susceptible to 500μl/ml concentration of CUD. Additional studies involving large number of these MDR Enterococcus and E. coli strains are necessary to draw any inference.
It has been reported that urea, aurum hydroxide, creatinine, phenols, calcium, present in CUD impart antimicrobial property to it. Further, bioenhancer property of CUD is more than cow urine and hence CUD enhances the antimicrobial effect of drugs (9). Recent study has identified antimicrobial peptides in cows' urine using mass spectrometry (24). Thus, remarkable inhibition of pathogenic clinical bacterial isolates by CUD provides an incentive to perform more studies to discover the exact constituent and deduce its mechanism of action which can further be exploited to reduce increasing dependence on antibiotics. Cow's urine offers potential to be explored as an antibacterial for the broader spectrum of bacterial strains thereby establishing scientifically its long-known healing power.

CONCLUSION
The present study highlights the remarkable dosedependent effects of cow's urine distillate as an antibacterial agent, exhibited maximally for S. Typhi and minimally for E. faecalis. CUD was shown to inhibit the growth of most bacterial strains which were either sensitive or resistant to routinely used antibiotics, indicating that the antibacterial effect is almost comparable to antibiotics. Further studies are necessary to characterize the different antimicrobial peptides present in CUD of different breed of cattle and their antibacterial action on different clinical bacterial isolates. Thus, antimicrobial peptides in CUD when characterized fully may be a promising alternative to treat drug resistant bugs.

ACKNOWLEDGMENT
Authors thank the Manipal Academy of Higher Education, Manipal, and Department of Microbiology, Kasturba Medical College, Mangalore for allowing them to conduct the study. The authors are grateful to founder & trustees of Surabhivana, Kompadavu, Mangaluru for providing the cow's urine distillate for this study.