Study On The Activity Of Antibacterial Substance P1 Of Paenibacillus Polymyxa Strain J

Antibiotics have always been the key drug for the treatment of bacterial infections.However,the emergence of pathogens(especially methicillin-resistant Staphylococcus aureus,MRSA)has shown tolerance to many antibiotics.Paenibacillus polymyxa J which isolated and identified by our group has a strong inhibitory effect on pathogenic bacteria and phytopathogenic fungi,especially for multi-drug resistant Staphylococcus aureus.This study mainly separates and traces antibacterial substance which has the function of bactericidal for?Super Bacteria?MRSA.In order to obtain a large amount of antibacterial substance,the fermentation conditions of strain J were optimized by strain selection,single factor test and orthogonal test.Theoptimal group selected:seed age 18h,inoculum volume 6%,temperature 32°C,pH8,liquid volume 75 mL,rotating speed 200r/min,incubation time 24h.The yield of crude fermentation extract was twice the original under the optimal condition.After separation and purification,the antibacterial substance designed P1 was obtained.The result showed that the property of antibacterial substance P1 is extremely unstable and easily decomposes,therefore it loses antibacterial activity.It is most affected by temperature and acid-basematerial.Different solvents have certain effects on its activity,simultaneously the activity loss is great when preparing solids.The result showed antibacterial substance P1 has antibacterial activity against Staphylococcus,Bacillus and Streptococcus sanguis.The minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC)and bactericidal curve were determined.The antibacterial substance P1 has a identical MIC value of 64?g/mL for MRSA,Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus(MSSA)and MBC values of 125?g/mL.Its4×MIC will kill the concentrations of the cells which is 10~5~10~6 CFU/mL at 8h,6h and 8h respectively.The MIC and MBC of Bacillus subtilis were 32?g/mL,64?g/mL,respectively,and Bacillus cereus were 125?g/mL and 250?g/mL.The MIC and MBC of Bacillus amyloliquefaciens were 250?g/mL and 500?g/mL.Three strains of Bacillus sp which concentrations of the cells were 10~5~10~6CFU/mL were completely killed by MIC of 4×MIC at 4h,12h and 12h respectively.Antibacterial substance P1 which is MIC>500?g/mL has bacteriostatic effect on Streptococcus sanguis,but no antibacterial effect on diphtheria bacilli.The results showed that the antibacterial effect of antibacterial substance P1 on bacteria was bactericidal.The results indicated that the antibacterial substance P1 has an inhibitory effect on the biofilm formation of Staphylococcus,Bacillus and Streptococcus sanguis.Meanwhile,antibacterial substance P1 have the function of dissolves to biofilm formed of Staphylococcus.The experiment tested that the biofilm formed of MRSA,MSSA,and S.aureus reduce more than35%.However,the 10×MIC antibacterial substance P1 did not inhibit the production of two major virulence factors?-hemolysin(Hla)and leukocidin(PVL)of S.aureus.The results showed that the cytoxicity of different concentrations of antibacterial substance P1were significantly different compared to maximum LDH releaseaed.The cytoxicity of antibacterial substance(16?g/mL)was 1.2%.The cytoxicity of the antibacterial substance at250?g/mL was 6.5%while the concentration of antibacterial substances increased,the cytoxicity also increased.In order to screen antibacterial substances P1 related genes of strain J,we tried to establish a molecular manipulation system in strain J with pRN5101 and pIC333 plasmids.However,The positive clone have no acquired after experimental verification.Strain J bioinformatics analysis and literature reports strain J has a perfect restriction-modification(RM)system.RM system can cleave exogenous DNA which methylation and demethylation.That makes it difficult to establish a molecular manipulation system in J strain.In summary,the optimization of fermentation technology enhance the yield of antibacterial substance P1,further studies the activity of antibacterial substance P1 through separation and purification,and preliminarily explores the molecular manipulation tool of Paenibacillus polymyxa J from the molecular level.It lays a foundation for the antibacterial substance P1 as a new type of antibiotic and structural analysis,meanwhile lays a foundation for the establishment of J molecular manipulation tools in the future.