Screening Of Anti-MRSA Active Ingredients Based On Soil Microorganism And Preliminary Study On Mechanism Of Action

At present,Methicillin-resistant Staphylococcus aureus(MRSA)has posed great threats to human beings and animals due to no efficacious drug for treatment of MRSA derived diseases.Therefore,it is urgently needed to accelerate the research and development of new anti-MRSA drugs.Recently,soil secondary metabolites under extreme conditions are considered to be an important source for new drug development.Since Changbaishan area is characterized by extreme climate,the soil secondary metabolites there are of great research value.In this study,a Bacillus velezensis strain CB6 of which the purified metabolite CB6-C with 31.405k Da and strong anti-MRSA activity was successfully isolated by partial altitude soil analysis of Changbaishan.The main research contents are as follows:1.Distribution characteristics of secondary metabolite-producing bacteria in some areas of Changbaishan.270 soil samples collected from partial area in Changbaishan were taken as research objects.The soil eDNA was extracted.Then,the 16S rRNA V3-V4hypervariable region,PKS KS domain and NRPS AD domain gene fragments were all sequenced for library construction through the MiSeq PE300 platform.The results showed that secondary metabolites,rich in altitudes of 1100-1600 m and higher than1800 m,are more possible to originated from Bacillus,Streptomyces and unrecognized bacteria.2.Screening of anti-MRSA strains and whole genome sequencing analysis.Isolation and identification of secondary metabolites rich Bacillus,Streptomyces and unrecognized bacteria were performed using MRSA as indicator strain.84 strains with antagonistic action against MRSA were isolated.After hydrogen oxide re-screening,a Bacillus velezensis strain,CB6 with strong anti-MRSA effect was finally screened and analyzed.Results of whole genome sequencing showed that the genome of CB6 was 3963507 bp in length.It has 12 secondary metabolite gene clusters that accounts for 20.947%of the entire genome.3.Isolation,identification and physicochemical property study of antibacterial protein CB6-C.The antimicrobial compounds from CB6 fermentation supernatant were separated and purified by ammonium sulfate precipitation,ultrafiltration,Sephadex G-75 gel chromatography column and QAE-Sephadex A-25 ion exchange column.After identification by SDS-PAGE and LC-MS/MS,the antibacterial protein CB6-C with higher purity was obtained.The protein has stronger bacteriostatic effect on gram-positive bacteria than gram-negative bacteria,good pH stability,organic reagent stability and safety.Moreover,metal ions CO²⁺K⁺and Ni⁺can improve the antibacterial activity of CB6-C.4.Study on mechanism of the antibacterial protein CB6-C safety and action.The test of CB6-C action was measured by bactericidal kinetics and the combined with antibiotics.The results showed that CB6-C has a good inhibitory effect on MRSA and has a synergistic effect with ampicillin.The mechanism of CB6-C action was preliminarily studied by plasmolysis assays and competitive inhibition test.The results showed that the target of CB6-C was lipophoteichoic acid on MRSA cell wall.Additionally,mouse model test revealed that CB6-C had a therapeutic effect on MRSA infected mice.