Effect of Terminalia chebula , Trikatu formulation and their combination on Staphylococcal biofilm

Introduction: The present study evaluates Terminalia chebula, Trikatu formulation and their combination for their inhibitory effect on biofilm formed by Staphylococci. Methodology: Clinical isolates of Staphylococci were screened for their biofilm forming activity by Congo red agar, Tube and Tissue culture plate (TCP) method. Two strongly biofilm forming strains along with a standard biofilm forming strain (ATCC 35984Staph. epidermidis) were selected to study the plant extracts by TCP method. Aqueous extracts of the Terminalia chebula, Trikatu and their combination with various concentrations were added to the inoculate during incubation and also after 24 hrs of incubation in separate assay systems. 24 hrs after incubation with the test drugs, the biofilm was quantified. Results: None of the study drugs showed any effect on Staphylococci in biofilm state. TCh showed significant prevention of biofilm formation at the concentrations of 200, 400 and 800 μg/ml. The Combination showed significant prevention of biofilm formation at the concentrations of TCh and Tr: 100 + 50, 200 + 100 and 400 + 200 μg/ml. The combination showed significantly better biofilm prevention as compared to TCh. Conclusion: Though the study drugs did not show any effect on Staphylococci in biofilm state, TCh and combination could prevent biofilm formation. Tr enhanced the biofilm formation preventing property of TCh, thus showing synergism. Further evaluation with the various constituents of these plant drugs is required. Keyword: Staphylococci, Biofilms, Antibiotics Introduction: Biofilm-mediated infections in the hospital environment have a huge burden on the resources of the health services. Centers for Disease Control and Prevention estimated that biofilms are associated with about 65% of nosocomial infections. Staphylococcal infections are recognized as the most frequent biofilm-associated infections. The effective control of such infections requires strategies that prevent the formation or promote the detachment of biofilms. Many antimicrobial agents fail to act against biofilm forming organisms and result in poor outcome of an infection. Numerous preclinical studies have been reported in the literature which evaluate antibiotics, disinfectants, and plant drugs against biofilm forming organisms; however very few of them have shown promising effect. Clinical studies with adequate sample size are hardly reported and no drug has been marketed as an antibiofilm drug. Indian Journal of Basic and Applied Medical Research; June 2015: Vol.-4, Issue3, P. 375-387 376 www.ijbamr. com P ISSN: 2250-284X , E ISSN : 2250-2858 Ayurveda is a treasure mine of plants and many of these plant drugs have been evaluated to determine their antibacterial potential. Hence the present study was undertaken to evaluate anti-biofilm potential of Ayurvedic plant drugs. Terminalia chebula (TCh) is reported to have antibacterial activity against pathogenic Gram positive and negative bacteria in vitro. Hence it was felt worthwhile to select this plant to determine its ability to act against the microorganisms in biofilm states. Yogavahaktva is an Ayurvedic concept in which one drug facilitates passage of another drug to its site of action. Trikatu(Tr), a combination of Zinziber officinale, Piper longum and Piper nigrum in equal parts, has been mentioned to possess the yogavahaktva property in Ayurvedic text. This combination is claimed to enhance the efficacy of the concurrently administered drugs. It was of interest to find out whether it enhances effects of TCh when given concurrently with it. Hence it was decided to study the effects of abovementioned plant products against CONS and Staphylococcus aureus in the biofilm state and also their ability to prevent the formation of a biofilm. Thus two assay systems were planned in one, it was decided to introduce the test drugs after formation of a biofilm by the organisms and in another before its formation. Material and methods: Prior to the initiation of study, an approval of the Committee for Academic Research Ethics was sought. Test drugsDried aqueous extracts of TCh and Tr formulation were used for the study. TCh was purchased from Natural Remedies Pvt. Ltd., Bangalore. The extractive value of extract of TCh was 20%. Tr formulation containing Zingiber officinalis, Piper longum and Piper nigrum in equal proportion was donated by Shri Dhootapapeshwar Auyvedic Research Foundation. The extractive value of Tr extract was 41.19%. For each drug, 6 concentrations were selected based on the dose range (upper and lower) prescribed in Ayurveda assuming that the drug on oral administration gets completely absorbed from the GI tract, their concentrations in the total body water, extracellular fluid and plasma compartment were computed. To use TCh and Tr in combination, it was decided that the concentrations of both should be in the ratio of 2:1. Hence for this combination also 5 final concentrations were prepared (Table 1). Table 1: Selected concentrations of the test drugs Plant extract Concentrations (mcg expressed as crude powdered drug/ml) Terminalia chebula (TCh) 25, 50, 100, 200, 400 and 800 Trikatu formulation (Tr) 25, 50, 100, 200, 400 and 800 TCh + Tr (TCh + Tr) TCh + Tr: 25 +12.5; TCh + Tr: 50 + 25; TCh + Tr: 100 + 50; TCh + Tr: 200 + 100; TCh + Tr: 400 + 200 Indian Journal of Basic and Applied Medical Research; June 2015: Vol.-4, Issue3, P. 375-387 375 www.ijbamr. com P ISSN: 2250-284X , E ISSN : 2250-2858 Organisms Biofilms producing ATCC 35984Staphylococcus epidermidis was used as a positive control (HiMedia Laboratories Pvt. Ltd India) and nonbiofilm producing ATCC 12228Staphylococcus epidermidis (HiMedia Laboratories Pvt. Ltd India) was used as a negative control for standardization of assay methods. Staphylococci isolated from different clinical specimens i.e. pus, sputum, blood culture, endotracheal tip, central line tip, urine or other samples as a part of routine investigation and were screened for biofilm formation. Strains which demonstrate biofilms forming capacity were used in the study. Congo red Agar method A method described by Freeman, et al was used for screening biofilm formation by Staphylococcus isolates. The isolates were cultured on the agar and incubated for 48 hours. Biofilm formation was indicated by black colonies with a dry crystalline consistency. Weak slime producers usually remained pink (Fig 1). A darkening of the colonies with the absence of a dry crystalline colonial morphology indicated an indeterminate result. Fig. 1: Biofilm forming (left) and non biofilm forming Staphylococci on Congo red agar Tube methodA qualitative assessment of biofilm formation was done as described by Christensen, et al Tryptic soya broth (TSB) with 1% glucose was used as media for growth of organisms. Ten ml of TSB glucose was inoculated with loopful of microorganisms and incubated for 24 hours at 37°C. The tubes were decanted and washed with PBS and stained with crystal violet (0.1%). Excess stain was removed immediately and tubes were washed with deionized water. Tubes were than dried in inverted position. Biofilm formation was considered positive when a visible film lined the wall and bottom of the tube. Ring formation at the liquid interface was not indicative of biofilm formation. (Fig. 2) Indian Journal of Basic & Applied Medical Research