Antibacterial mechanisms of endophytic fungi Alternaria alternata of ethyl acetate extract on Escherichia coli / 中草药

ABSTRACT

Objective To study the antibacterial mechanisms of ethyl acetate extract (B06e) from the fermentation liquid of an endophytic fungus Alternaria spp. Alternaria spp isolated from the medicinal plant Humata tyermanni. Methods Double dilution method was adopted to measure the minimum inhibitory concentration (MIC) of B06e against Escherchia coli. Then, the changes of electric conductivity of bacterial culture, nucleic acid and protein concentration before and after treated by B06e were analysed, respectively. Besides, flow cytometry, scanning electron microscopy, gel retardation experiments, circular dichroism spectrum and Real-time quantitative PCR were introduced to study the antimicrobial mechanisms of B06e against E. coli. Results The results showed that MIC value of B06e against E. coli was 25 μg/mL. The electric conductivity of 3 × MIC treatment group was 1.01 times the value of the control group. The β-galactosidase activity of 3 × MIC treatment group was 11.6 times more than the value of the control group. Flow cytometry analysis showed that PI positive cells ratio of 3 × MIC treatment group was 286.5 times the value of the control group. Scanning electron microscopy showed that cell surface becomes rough after the treatment of B06e, a large number of cell membrane collapse. These results suggested that B06e can increase the permeability of cell membrane, destroy the integrity of cell membrane. The results of gel retardation experiments and circular dichroism spectrum applied that B06e can be inserted into DNA structure at particular position, however, can not cause DNA degradation. Real-time quantitative PCR results showed that the expressions of recA and recN genes were both up-regulated with the values of 2.9 and 3.7 times the value of the control group, respectively. This result suggested that B06e can destroy the DNA structure, which force E. coli to initiate SOS repair. Conclusion B06e can kill E. coli cell by destroying the cell membrane and damaging DNA structure.