ANTIMICROBIAL ACTIVITIES OF (+)–USNIC ACID ON BACILLUS CEREUS AND BACILLUS SUBTILIS IN VITRO AND IN VIVO USING THAI SILKWORM MODEL

Abstract

B. cereus and B. subtilis not only continue to cause serious clinical problems through food poisoning, but also show natural resistance to some antimicrobial drugs. One of the key factors enabling pathogen to resist to antimicrobial drugs is the ability to form biofilm. This study was aimed to evaluate the antimicrobial activity, inhibitory effect of biofilm formation including time to kill B. cereus and B. subtilis of (+)-usnic acid. Antimicrobial activity was determined by broth microdilution method. MIC of (+)-usnic acid for B. cereus and B. subtilis was the same (62.50 µg/ml), whereas MBC of (+)-usnic acid for B. cereus and B. subtilis were 125 and 250 µg/ml, respectively. The antimicrobial activities of (+)-usnic acid against B. cereus was more effective than that on B. subtilis. Time kill studies showed that (+)-usnic acid exerted both bacteriostatic and bactericidal depending on concentration used and incubation time observed against B. cereus and B. subtilis. The result also indicated that bacteriostatic and bactericidal activity of (+)-usnic acid and ampicillin were concentration dependent. Furthermore, (+)-usnic acid had inhibitory effect on biofilm formation in a concentration dependent manner as well ranging from concentration equal or greater than half of MIC value with statistical significance comparing with negative control (p<0.05). Therefore, this study showed that (+)-usnic acid had both antimicrobial and antibiofilm formation activities on B.cereus and B.subtilis. In vivo study, silkworm were utilized for efficacy testing of compounds. ED50 of (+)usnic acid and ampicillin were 3.56 mg/ml and 0.23 mg/ml respectively for B. cereus, whereas ED50 of the(+)-usnic acid and ampicillin for B. subtilis were 3.92 mg/ml and 1.01 mg/ml respectively. Therefore, these findings showed that (+)-usnic acid had antimicrobial activities against B. cereus and B. subtilis both in in vitro and in vivo. Inhibition of biofilm formation may play an important role for the mechanism of action for antimicrobial activity shown.