Study On The Antibacterial Effect And Mechanism Of S-PS-aPDT On S.aureus&MDR S.aureus Planktonic Bacteria And Its Bacterial Biofilm

Background and purposeIt is reported that one-third of the global mortality rate is due to bacterial infections,which can cause a wide range of diseases.Among them,Staphylococcus aureus is the main bacterium causing widespread clinical infections,including infective endocarditis and osteoarthritis,skin and soft tissue inflammation,pneumonia,and instrument infection.Staphylococcus aureus has high toxicity,and it is easy to produce drug resistance,which makes the traditional treatment ineffective and increases the difficulty in clinical treatment of bacterial infections.At present,the traditional method of treating bacterial infections is still antibiotic therapy.The advent of antibiotics has saved most patients' lives.However,with the development of modernization,the abuse of antibiotics increases the number of resistant bacteria in worldwide.There are many mechanisms for explaining bacteria resistance to antibiotics,the presence of efflux pumps and the formation of bacterial biofilm are two important factors among them.Efflux pumps belong to the transmembrane efflux systems,which serve to pump noxious compounds from microbial cell.At present,Major facilitator superfamily(MFS)and Multi-anfimicrobial extrusion(Mate)are two reported efflux pumps in MDR S.aureus which depend on the changes of ion potential energy to efflux harmful substances that absorbed by MDR S.aureus.Bacterial biofilm(BF)refers to the way in which bacteria adhere to the surface of non-living or active tissues and is wrapped in a heterogeneous polymeric matrix produced by themselves.The main components of BF include polysaccharide intercellular adhesin(PIA),extracellular DNA(eDNA)and the like.Among them,eDNA supports the structure of biofilm,and PIA plays an adhesion role in the BF.BF builds a natural barrier protecting bacteria from external stresses.So the occurrence of BF and bacterial drug-resistance account for the unsatisfactory therapeutic efficacy of clinical infections.It is a top priority that exploring new approaches to anti-bacteria without producing drug-resistance.In recent years,various antibacterial strategies have been developed to treat bacterial infections,such as metal nanoparticles,cationic polymers,peptidoglycans,nanocarriers,photothermal therapy(PTT)and photodynamic therapy(PDT).PDT is a potential treatment for antibacterial or antitumor treatment by injecting a photosensitizer locally or systemically under a certain wavelength of light excitation.Based on PDT,antibacterial therapy that kills pathogenic bacteria through the synergistic action of light,photosensitizer,and oxygen is called antimicrobial photodynamic therapy(aPDT).The damage mechanism of aPDT is based on the oxidative damage of reactive oxygen species(ROS)produced by photosensitizer(PS)during the process of being stimulated by light.ROS can attack multiple targets of cells,including membranes,lipids,proteins and DNA.,Leading to cytoplasmic leakage,degradation and cell death,which has a killing effect on bacterial cells.Moreover,the non-specific targets of ROS make bacteria difficult to develop resistance to aPDT,activate multiple death signaling pathways,and produce killing effects independent of antibiotic resistance.In addition,the repeated use of aPDT is highly spatially controllable,has no cumulative toxicity,and is superior to traditional anti-infective therapies.PS is an important part of aPDT.Previous studies have shown a variety of PSs exhibit obvious antibacterial effects,but there still exist many shortcomings,such as low water solubility,serious side effects and so on.The photosensitizer S-Porphin sodium(S-PS)used in this study is a novel photosensitizer developed by optimizing HPPH(FDA-approved PS).It overcomes the poor solubility of HPPH in physiological environment and could be an ideal PS with high water solubility and low dark-toxicity.Importantly,S-PS has the characteristics of high purity and easy extraction.It previously showed that S-PS exhibited significant antitumor effect under photo-stimulation,showing high potential photoactivity.Therefore,the purpose of this study was to explore the possible antibacterial effects and mechanisms of S-PS mediated aPDT(S-PS-aPDT)in the planktonic bacteria and biofilms of S.aureus as well as in MDR S.aureus,hoping to provide evidence for using S-PS-aPDT as an alternative strategy for combating antibiotic resistance in bacterial infections.Methods and ResultsPart 1 Antibacterial effect of S-Porphin sodium photodynamic therapy on the planktonic bacteria of S.aureus/MDR S.aureusThe experiment measures the uptake of S-PS in S.aureus/MDR S.aureus and the light/dark toxicity of S-PS,and further explores the antibacterial effect and mechanism of S-PS-aPDT on the planktonic bacteria of S.aureus/MDR S.aureus through EPR detection and AFM observation.The results showed that the enrichment amount of S-PS in S.aureus/MDR S.aureus reached the maximum at 40 min,and S-PS itself did not produce toxicity,but after irradiation,it could produce significant killing effect on plankton of S.aureus/MDR S.aureus.In addition,due to the presence of efflux pumps in MDR S.aureus,the sensitivity of MDR S.aureus to S-PS-aPDT is much lower than S.aureus.However,the susceptibility of MDR S.aureus to S-PS-aPDT can be improved to some extent after the efflux pump inhibitor is added.S-PS-aPDT can damage bacterial cell walls/membrane by generating ROS,leaking out its contents,creating depressions,thereby killing bacteria.Part 2 Antibacterial effect of S-Porphin sodium photodynamic therapy on the biofilms of S.aureus/MDR S.aureus.A certain number of planktonic bacteria will gradually form a bacterial biofilm after aggregation.In order to investigate the antibacterial effect and mechanism of S-PS-aPDT on S.aureus/MDR S.aureus biofilm,the paper first studied the biofilm formation ability of two strains of S.aureus/MDR S.aureus Congo red plate streaking method.The enrichment of S-PS in S.aureus/MDR S.aureus biofilm was observed using CLSM.Based on this,the antibacterial effect of S-PS-aPDT on S.aureus/MDR S.aureus biofilm was studied,and RT-PCR was used to detect changes in the expression of S.aureus/MDR S.aureus biofilm-related genes(ica operon)before and after S-PS-aPDT treatment.The results show that under certain culture conditions,S.aureus/MDR S.aureus has the ability to form bacterial biofilms.CLSM observations found that S-PS can be enriched in S.aureus/MDR S.aureus biofilms,and the enrichment reached the maximum at 2h and 3h,respectively;the observation results of the plate coating method and CLSM 3D modeling showed that S-PS-aPDT had a certain killing effect on the biofilm,and could damage the bacteria in the membrane to a certain extent;RT-PCR test results showed that after treatment with S-PS-aPDT,the expression of ica operon was significantly reduced.In addition,the treatment of S-PS-aPDT can significantly inhibit the content of PI A.Part 3 CMCS-OSA@DNase I&S-PS-liposome disintegrates MDR S.aureus biofilm to sensitive S-PS-aPDTIn order to improve the anti-MDR S.aureus biofilm effect of S-PS-aPDT,a degradable hydrogel which can damage the bacterial biofilm extracellular matrix was designed.First,sodium alginate(NaIO4)was used to achieve the injectability and degradability of oxidize sodium alginate.The degree of oxidation was determined by the iodometric method.The most oxidized sodium alginate(OSA)and CMCS were selected to mix in different proportions,and then test their gelation time,stability,injectability and degradability,respectively.On this basis,DNase I and S-PS-liposome were added into CMCS-OSA hydrogel and detect the release of DNase I in PBS at 37?.The results show that the oxidation of OSA is directly proportional to the NalO4 inputs.The hydrogel which formed by 10%OSA and 5%CMCS with a volume ratio of 1:1 has rapid gelling ability,good injectability and self-degrading.CMCS-OSA@DNase I&S-PS-liposome has the function of continuous release of DNase I.After 12 h release,the amount of DNase I tends to be stable.In addition,the sustained release of DNase I effectively destroyed the structure of the MDR S.aureus biofilm.After 12 h incubation with the biofilm,the MDR S.aureus biofilm basically disintegrated,and the bacteria showed a single free state.It lays the foundation for the subsequent research combining aPDT antibacterial biofilm.ConclusionIn summary,the research on the antibacterial effect and mechanism of S-PS-aPDT on S.aureus&MDR S.aureus phytoplankton and biofilm was studied,and it was confirmed that S-PS-aPDT has a good antibacterial effect on S.aureus/MDR S.aureus phytoplankton and biofilm.Active oxygen is the main reason for the antibacterial activity of S-PS-aPDT.In addition,the CMCS-OSA@DNase I&S-PS-liposome designed and synthesized in this paper can effectively destroy the structure of the MDR S.aureus biofilm,resulting in the disintegration of the bacterial biofilm and exposing the free bacteria in the BF.It laid a foundation for the use of CMCS-OSA@DNase I&S-PS-liposome in combination with aPDT to enhance its antibacterial effect.The research results provide important theoretical basis and application value for promoting the clinical application of S-PS-aPDT.