Study On The Role And Mechanism Of Bacillus Subtilis In Staphylococcus Aureus Infectious Osteomyelitis

Objective:Explore the effect and molecular mechanism of Bacillus subtilis supernatant on the biofilm production and drug sensitivity of drug-resistant Staphylococcus aureus,evaluate the role of Bacillus subtilis supernatant in the treatment of osteomyelitis,and provide ideas for the prevention and treatment of bone infections.Methods:Collect the supernatant of B.subtilis and co-culture with S.aureus to detect its growth curve,biofilm formation,and sensitivity to antibiotics.Extract bacterial RNA and bacterial membrane surface protein,detect bacterial gene changes and protein synthesis.Fourier transform infrared spectroscopy and transmission electron microscopy were used to observe the morphological changes of bacteria.Establish implant-related osteomyelitis model in mice,and design two experimental programs to evaluate the colonization ability of S.aureus and the sensitivity to antibiotics on the mouse model.Finally,the antibacterial protein was found through the purification of the protein and the UPLC-MS/MS analysis.Results:Here we report that the cell-free supernatant of Bacillus subtilis(B.subtilis CFS)kills planktonic and biofilm S.aureus,and increases S.aureus susceptibility to penicillin and gentamicin as well.Further study shows that B.subtilis CFS suppresses the expression of genes involving in adhesive molecules(cna and clfA),Virulence factor hla,quorum sensing(argA,argB and RNA?),Penicillin resistance(merA)and biofilm formation(ica and sarA)in S.aureus.Additionally,Transmission electron microscopy(TEM),Fourier transform infrared spectroscopy(FTIR)and other experiments show that B.subtilis CFS changes the membrane components,and increases membrane permeabilization of S.aureus.Finally,the minimum duration for killing 90%population(MDK90)demonstrate that B.subtilis CFS considerably increases the susceptibility of S.aureus to penicillin,and effectively reduces S.aureus burdens in a mouse model of implant-related osteomyelitis by two treatment modes.Finally,lantibiotic subtilin was identified by UPLC-MS/MS.Conclusion:The B.subtilis CFS inhibits the biofilm synthesis by inhibiting the biofilm-related genes of S.aureus,and changes the structure of the bacterial membrane to restore sensitivity to antibiotics and reduce the colonization of S.aureus in mice.These data support that the antibacterial protein Lantibiotic subtilin derived from B.subtilis CFS is a potential drug for the treatment of S.aureus osteomyelitis.