Study On The Mechanism Of Alizarin, An Active Ingredient Of Traditional Chinese Medicine, Inhibiting The Resistance Mutation Of Escherichia Coli

The selective pressure of antibiotics is the dominant cause of mutations in bacterial resistance.The excessive use of antibiotics has caused serious pollution of antibiotic resistance worldwide,posing a huge threat to public health safety and human health.Traditional Chinese medicine(TCM),an alternative to antibiotics widely used in China,is inevitably taken concurrently by humans with antibiotic drugs and remains in the environment.However,there is no information about the effect and mechanisms on the antibiotic resistance of effective ingredients of TCM to bacteria.In this study,wild-type(non-resistant)Escherichia coli K12(MG1655)was selected to explore the effect of Alizarin,an effective component of TCM,on the resistance mutation induced by various antibiotics.The multi-antibiotic resistance of 30 d mutants was also investigated.Finally,in the case of tetracycline(Tet),high-throughput sequencing technology was used to analyze the mutation sites and genetic changes,as well as the expression of related functional genes to reveal the mechanism of Alizarin inhibiting antibiotic resistance.The results showed that the selective pressure of Tet could induce 100% E.coli K12 to produce Tet resistance within 10 days.Alizarin significantly reduced the mutation frequency of E.coli K12 during 7-30 days without affecting the bactericidal properties of Tet.The mutation frequency induced by Alizarin self was extremely low,and the inhibition had no dose-response relationship.Meanwhile,Alizarin could inhibit the mutation frequency of E.coli K12 induced by erythromycin and other antibiotics as well.The mutants induced by 30-day exposure in different treatment groups all showed multi-antibiotic resistance,among which the resistance to the quinolone antibiotic ciprofloxacin increased the most.Mutants have undergone a variety of mutations(e.g.,deletion,insertion,and substitution)in information storage and processing,cell metabolism,mobile genetic elements(such as transposase and prophage),and putative protein.The mutation of mgt A gene,which is mainly involved in cell membrane transport,promotes the development of genetic resistance.Besides,Alizarin significantly reduced the reactive oxygen species(ROS)production and cell membrane permeability of E.coli K12 induced by Tet,which can reduce the chance of bacterial gene mutation.Furthermore,Alizarin significantly down-regulated the overexpression of genes involved in the regulation of oxidative stress and SOS response,cell membrane and efflux pump system,DNA repair,and other important biological processes that are stimulated by Tet in E.coli K12,thereby inhibiting bacteria exhibiting antibiotic resistance.