Evaluations of biocidal potential of Euclea crispa stem bark extract and ability to compromise the integrity of microbial cell membrane

Abstract

This study evaluates biocidal effect of Euclea crispa stem bark extract and determines time-kill kinetics of its potent fractions as well as membrane attack capability. Susceptibility of the bacterial cells and minimum inhibitory concentrations (MICs) were determined via agar-well diffusion and broth micro-dilution respectively, time-kill kinetics were evaluated at different concentrations over 2-h period and membrane attack potential was evaluated via scanning electron microscopy imaging and measurement of the amount of proteins and nucleic acids released from the cells. Maximum zone of inhibition (22 ± 0.58 mm) was observed for n-butanol and ethyl acetate fractions at 10 mg/ml. The lowest MIC exhibited by n-butanol (0.31 mg/ml) was against Enterococcus faecalis while that of ethyl acetate, n-hexane and aqueous fractions was 0.63 mg/ml against a number of test isolates. After 120 min of contact time at 1 × MIC, ethyl acetate fraction achieved total mortality against Listeria sp. and S. Typhimurium selected as representative isolates and when the concentration was increased to 2 × MIC n-butanol and aqueous fractions achieved total killing against Listeria sp. The SEM images revealed a level of anatomical disruption in the membrane of test isolates. Maximum amount of proteins released from Listeria sp. (0.63 ± 0.004 μg/ml) was by ethyl acetate fraction, while n-hexane fraction released maximum amount (0.79 ± 0.001 μg/ml) from S. Typhimurium under the same concentration of 3 × MIC after 120 min of exposure. The maximum amount of nucleic acids (47.9 ± 0.12 μg) released from Listeria sp. was by n-butanol fraction at 3 × MIC after 120 min, hence, membrane disruption is confirmed as one of the mechanisms behind the biocidal action of E. crispa extracts.