Anti-Gram-Positive Bacterial Activity And Mechanism Of Biphenanthrene LF03 From Fibrous Roots Of Bletilla Striata

Objective The anti-Gram-positive bacterial activity and mechanism of biphenanthrene LF03 from fibrous roots of Bletilla striata were studied to provide a basis for the research of natural antimicrobials and utilize Bletilla striata resources.Methods(1)MICs was determined for thirteen clinical isolates by 96 well microdilution method.MIC and MBC were determined for three Staphylococcus aureus by 96 well microdilution method and plate-coating method.Time-kill curve was determined to observe the dynamic sterilization effect of LF03.The MIC was influenced by mouse plasma and BSA concentration which were determined by chequerboard design and 96 well microdilution method.The antibacterial activity of LF03 combined with other compounds was determined by chequerboard design and 96 well microdilution method.(2)The antibacterial mechanism was explored by growth curve of LF03 in different antibiotics.FCM was used to analyze the influence of LF03 on membrane integrity and membrane potential of Staphylococcus aureus and Bacillus subtilis.(3)Plates with antibiotics.was used to obtain drug-resistant mutant strains.The antibacterial activity and growth tendency of drug-resistant mutant strains were determined by 96 well microdilution method and growth curve.Genome extraction kit was used to extract bacterial DNA and then whole genome sequence was measured.PCR was used to verify the accuracy of sequencing gene.The sequencing results were analyzed by bioinformatics software and antibacterial of LF03 and drug resitance mechanism of MRSA was explored.Results(1)LF03 has been reported to exhibit significantly antibacterial activity of thirteen clinical Staphylococcus aureus.The results of MBC/MIC of some gram-positive bacterial was1,this proved LF03 was a fungicide.LF03 could be combined with ampicillin to reduce the dosage of ampicillin,and can be used with EDTA,membrane permeation promoter,to increase its antimicrobial activity against Escherichia coli The antimicrobial activity of LF03 was reduced when it was combined with mouse plasma and BSA.Albumin was the main factor causing the significant decrease in the antibacterial activity of LF03,and the antibacterial activity of LF03 decreased gradually with the increase of BSA concentration.(2)The kinetics behavior of Bacillus subtilis incubated with LF03 was similar to that of polymyxin B and Nisin(a kind of rapid lysis of membrane antibiotics),suggesting that the cell membrane was one of the antibacterial targets of LF03.The results of fluorescent probes showed that LF03 had a destructive effect on the membrane integrity and membrane potential of Staphylococcus aureus and Bacillus subtilis,and verified that the cell membrane was one of the antibacterial targets of LF03.(3)The drug resistance mutation rate of Staphylococcus aureus and Bacillus subtilis was 0when LF03 concentration for 8 × MIC.Three mutant Staphylococcus aureus strains were received randomly from antibiotic tablet.MICs of three mutant Staphylococcus aureus and Bacillus subtilis strains add 1 × MIC and growth curves exhibited no significant changes compared with standard Staphylococcus aureus and Bacillus subtilis strains.The analysis of bioinformatics showed that there was a good linear relationship between 3 Staphylococcus aureus strains and 22 SNP were found.This genes that SNP belongs to were related to family transposase,MFS transporter,16 S ribosomal RNA,DNA-binding response regulator and so on.Conclusion LF03 exhibited significantly anti-Gram-positive bacterial activity and it’s a fungicide.Cell membrane is one of the main antibacterial targets of LF03.LF03 could destroy the cell membrane integrity and membrane.LF03 exhibited low natural resistance mutation rate in this study.Family transposase,MFS transporter and other genes might be related to the antibacterial and drug resistance mechanisms.