Antimicrobial activity of a natural product from callistemon rigidus and its mechanism of action against staphylococcus aureus

Abstract

The present work was oriented to evaluate the antiand#8208;staphylococcal potential of methanolic extract of leaves of Callistemon rigidus R.Br. The test panel comprised of 34 clinical and 4 standard isolates of S.aureus. Crude methanolic extract of C. rigidus exhibited a MIC in range of 5and#8208;40 and#956;g/ml against the test panel isolates by in vitro microbroth dilution assay. Bioactive fraction from the methanol extract of leaves was isolated by bioassay guided liquidand#8208; liquid fractionation protocol. The bioactive fraction tested positive only for alkaloids phytochemically and hence designated as ABF. The ABF induced 3and#8208;log reduction in colony counts in the test panel isolates at 3.125 and#956;g/ml concentration only. The yield of the ABF was improved by 4% by using microwave oven assisted treatment of the pulverized leaves of C. rigidus. Further TLC fractionation of the ABF yielded ten alkaloidal fractions designated as CSS1and#8208;CSS10. CSS1and#8208;CSS10 were evaluated individually against a limited panel of test isolates to determine their MIC by using in vitro microbroth assay based on visual dye reduction method. CSS1, CSS6 and CSS8 exhibited potential to be taken up further for their antimicrobial properties based on their MIC, MIC50 and MIC90 values. The MIC values of CSS1, CSS6 and CSS8 are 26.69 and#956;g/ml, 16.8 and#956;g/ml and 23.78 and#956;g/ml. Further CSS1, CSS6, and CSS8 were tested against the extended panel of test isolates and gave a MIC of 26.69 and#956;g/ml, 7.93 and#956;g/ml, and 22.45 and#956;g/ml respectively. The time kill kinetics revealed that CSS1 was bacteriostatic while CSS6 and CSS8 were bactericidal as well as bacteriostatic. SEM studies indicated that at MIC values CSS6 and CSS8 induced cell lysis, cell shrinkage and irregular shapes. Expression of enzymes viz lipase, protease and SOD which are the virulence factors in S. aureus were found to be inhibited at the MIC and suband#8208;MIC levels of CSS6 and CSS8 thereby causing susceptibility. Hence CSS6 and CSS8 were multiand#8208;targeting.