The Antibacterial Effect Of5-aminolevulinic Acid Photodynamic Therapy On Staphylococcus Epidermidis Planktonic And Biofilm Cultures

BackgroundStaphylococcus epidermidis (S.epidermidis) is one of the normal flora whitch settledon our skin and mucous membrane. Its pathogenicity is usually weak. However,withthe increasing of the treatment of the development and the emergence of the aging andlow immunity population,The solidify enzyme negative staphylococcus, especiallyS.epidermidis cause the incidence of nosocomial infection was increased. S.epidermidishas become one of the most important pathogenic bacteria nosocomial infection. Andthis tendency has been more than staphylococcus aureus. According to the reports,S.epidermidis infections has risen to top four of the hospital infection of pathogenicbacteria, because of the bacterial biofilm (BBF)formation. BBF can protect the internalbacteria from bacteriacide and escape from the host immune killer, and BBF constantlyrelease bacteria to enter the bloodstream whitch caused chronic and repeated infection,antibacterial agents is difficult to confront.Photodynamic therapy(PDT)is a new type of non-invasive treatments, through thephotosensitizer selective gathered for target cells, with irradiation under the specificwavelength light, singlet oxygen and free radical can be released from the target cellswhitch can cause selective toxic effect. Before the1990s, PDT was focuses on thediagnosis and treatment of cancer. In nearly20years, with the clinical dermatologistresearch and applications, PDT become mature and improvement. In recent years, with the emergence of new photosensitizer and application, and the improvement of,treatment technique bacterial and viral disease in the treatment of PDT has alsoachieved significant results. In view of the existing abroad to learn to use differentphotosensitizer for PDT antibacterial research, and has obtained good antibacterialeffect, has not been seen ALA-PDT on S.epidermidis planktonic and biofilm cultures.According to the report, because of the different experimental condition, the differentlight source, and the different photosensitizer, PDT parameters are greatly different.This paper base on the ALA fluorescent pharmacokinetic in the body of the bacteria.Evaluate the absorption and transformation of the ALA in S.epidermidis,looking forPpIX peak time, and the best irradiation time.To search for the optimal photosensitizerconcentration and light dose for ALA-PDT to S.epidermidis planktonic and biofilmcells.Objective1、 To investigate ALA pharmacokinetics, To get the optimal ALA incubation time.2、To establish some parameters of ALA-PDT to s.epidermidis planktonic cells. suchas the optimal illumination time, ALA concentration and red light radiation dose．3、To establish bacterial biofilm model. It would be significant for the further study onthe ALA-PDT for bacterial biofilm antibacterial effect.4、To establish some parameters of ALA-PDT to s.epidermidis biofilm. such as theoptimal illumination time,ALA concentration and red light radiation dose．Methods1、To investigate ALA pharmacokinetics, To get the optimal ALA incubation time.This experiment was divided into three groups. In group1, S.epidermidis incubated with 50mmol/l of ALA solution for3,5,8,12,16,18,20and24hours at37℃in dark room,Ingroup2,S.epidermidis incubated with10,20,30,40and50mmol/l of ALA solution for16hours at37℃in dark room. In group3,S.epidermidis incubated with fresh TSB solution（without ALA）for24hours at37℃in dark room. Fluorescence intensity ofProtopophrin IX (PpIX) was examined and conducted quantification analysis withConfocal laser scanning microscopy (CLSM).2、To establish some parameters of ALA-PDT to s.epidermidis planktonic cells. suchas the optimal illumination time, ALA concentration and red light radiation dose. Afterexperiment1, different dose of red light was irradiated in group1,100J/cm2red light wasirradiated in group2, no irradiation in group3.The S.epidermidis planktonic cells growthwere analyzed by colony Forming units (CFUs).3、To establish bacterial biofilm model. Biofilms were grown on coverslips，structureof the biofilm was observed under Confocal laser scanning microscopy (CLSM).4、To establish some parameters of ALA-PDT to s.epidermidis biofilm.The experimentwas divided into different light dose of ALA-PDT treatment group, and control group.Colony Forming units (CFUs) was used to investigate the photodynamic inactivation ofthe biofilm; CLSM was used to observe the vitality of the biofilms; SEM was used toobserved the morphological structure of the biofilms.Results1、In group1，brick red PpIX fluorescence were observed by CLSM at any time point,and the intensity of PpIX fluorescence were increased with incubation period.A higherintensity of PpIX fluorescence was noted in16,18,20and24hours compared with3,5,8and12hours. The difference was significant (P<0.05)． In group2， PpIXfluorescence intensity was increased with the increasing of the concentration of ALA.50 mmol/l of ALA induce a higher intensity of PpIX fluorescence compared with10,20,30and40mmol/l (P<0.05). No brick red PpIX fluorescence were observed in group3.2、With the increasing of irradiation in group1, the survival of S.epidermidis weredecreased, And all of the planktonic cells were killed after100J/cm2red lightirradiation with50mmol/l of ALA. The difference was significant (P<0.05) comparedwith the control.with the increasing of the concentration of ALA in group2, the survivalof S.epidermidis were decreased, the cells was inhibited in50mmol/l of ALA group,andthe difference was significant (P<0.05) compared with with the control.3、16h later,a visible, pale, membranoidsubstance spread in the coverslip. after thestaining wtth fluorescent dye, we can see BBF was composed with a compact structure,including many cells aggregate clouds, and has some matrix setapart by some channelsunder CLSM.Some planktonic cells can be observed on Longitudinal cut and section.we obtained the3D image of S.epidermidis biofilm.we can see bacteria gathered into a ahill, similar to the image of stone forest, some mass was hollow, like a pipe insert intothem. This image shows the BBF has three dimensional structure, and its form wasdiversity，heterogeneity，uneven open, whitch similar to previous reporter. It could bea model for we further study of ALA-PDT S.epidermidis biofilm.4、Compared with controls a significant number of cells within biofilms could beinactivated with the increasing of the light dosage in treatment group, and the differencewas significant (P<0.01). With the increasing of the light dosage the drastic reduction incells survival within biofilms was observed by CLSM, and Dead/Live Ratio was alsoincreased compared with controls, and the difference was significant (P<0.01); Themorphological structure of the biofilms were disruption with the increasing of the lightdosage was observed by SEM. Conclusions1、ALA can be absorpted by S.epidermidis,and can be transformated in to PpIX, PpIXpresent brick red fluorescence under CLSM. and the PpIX fluorescence intensity wasincreased with incubation period and concentration of ALA. S.epidermidis does notcontain the endogenous PpIX.2、ALA-PDT has significant inhibition of S.epidermidis planktonic cells growth. Theoptimal data were50mmol/l ALA, incubation time16hours and100J/cm2red lightdosage.3、S.epidermidis (ATCC35984) biofilm was successful prepared.The biofilm containsa large matrix, the cells proliferated in the matrix,form a membrane structure; Inaddition, BBF has space in three-dimensional structure, can be used as a research model.4、ALA-PDT has antibacterial effect on biofilm cells,It can not only reduce the biofilmvitslity,also can damage biological membrane structure.