Effects Of ALA-PDT On Biofilm Formation And QS In Pseudomonas Aeruginosa

Background and Objectives Aminolevulinic acid photodynamic therapy(ALA-PDT)is considered to be a promising new method for the elimination of microbial infection.Because of its good efficacy,PDT has been widely used in the treatment of some infectious skin diseases,such as acneand viral warts.In addition,some studie s have found that PDT is also effective in the infection of bacterial fungi and their biofilms(Biofilm,BF),including Staphylococcus aureus,Staphylococcus epidermidis,Pseudomonas aeruginosa,Candida albicans and some resistant bacteria(Methicillin-resistant Staphylococcus aureus).However,there is few research about the mechanism of PDT on the structure and function of biofilm.The quorum sensing(QS)system is the most important regulatory system for the formation and production of virulence factors of Pseudomonas aeruginosa(PA),while the impact of PDT on the QS systems and related genes has never been reported.In this study,the in-vitro biofilm model of Pseudomonas aeruginosa was established to explore the killing effect of ALA-PDT on Pseudomonas aeruginosa and its elimination effect and mechanism,and to provide experimental evidence for the clinical application of ALA-PDT in the treatment of Pseudomonas aeruginosa infection.Methods1.The in vitro biofilm modelof Pseudomonas aeruginosa was established,and each group was proceeded with different concentrations of ALA,light alone,ALA-PDT according to the requirements of the corresponding treatment.2.The transformation of ALA in Pseudomonas aeruginosa was detected by confocal laser scanning microscopy(CLSM).3.The killing effect of ALA-PDT on the bacteria in Pseudomonas aeruginosa biofilm was quantitatively determined by XTT viable assay.4.The effect of ALA-PDT on the viability of biofilm and its effect on the three-dimensional structure of biofilm were observed by CLSM through fluorescence staining of Pseudomonas aeruginosa biofilm.5.The effects of ALA-PDT on the structure and function of bacteria and biofilm in Pseudomonas aeruginosa biofilm were observed by scanning electron microscopy(SEM).6.The effects of ALA-PDT on the secretion of pyocyanin and elastase were detected by the virulence factor test.7.The effect of ALA-PDT on the expression of phage resistance gene(phz H,LasB)and QS-related genes(lasI,las R,rhl I,rhlR)was investigated by real-time quantitative PCR(qRT-PCR).Results1.ALA incubation group showed a lot of brick red fluorescence under CLSM,while the control group showed no obvious fluorescence,indicating that the exogenous photosensitizer ALA in Pseudomonas aeruginosa was converted into Protoporphyrin Ⅸ(PpⅨ).2.The quantitative testing of XTT live bacteria results showed that ALA-PDT could effectively kill live bacteria in Pseudomonas aeruginosa biofilm.The killing effect was positively correlated with the concentration of ALA.There was no significant difference in the production of photodynamic effects among the simple ALA incubation group,thesimpl e light group and the control group.3.The results of CLSM and SEM indicated that ALA-PDT had a positive correlation with the concentration of ALA and that the ALA-PDT could kill the living bacteria in the biofilm and destroy the three-dimensional structure of biofilm.And also no significant differences were observed in the production of photodynamic effects among the simple ALA incubation group,the simple light group and the control group.4.The virulence factor test showed that ALA-PDT can significantly inhibit the secretion of virulence factors of Pseudomonas aeruginosa,which is positively correlated with the concentration of ALA,and no significant differences were observed in the inhibition effect of photodynamic factors among the simple ALA incubation group,the simple light group and the control group.5.The results of q RT-PCR showed that there was no significant change in the expression of Pseudomonas aeruginosa virulence factor and QS-related regulatory genes between the simple ALA incubation and red light irradiation.Only when ALA and red light were simultaneously applied,ALA-PDT can significantly reduce the expression of QS-related genes,and the inhibitory effect on regulation of gene expression was proportional to the concentration of ALA.Conclusions Our study primarily elucidatesthe elimination effect of Pseudomonas aeruginosa biofilm by ALA-PDT and its related mechanisms.The singlet oxygen produced by ALA-PDT can produce toxic effects and destroy the cell membrane,organelle and nucleus of the target bacteria,and destroy the genes and proteins of the QS system.This effect on the QS system further destroys the biofilm and reduces the recurrence rate of biofilm.Our results show that ALA-PDT can effectively kill in-vitro Pseudomonas aeruginosa and its biofilm,and provide further experimental evidence for the clinical use of PDT in the treatment of Pseudomonas aeruginosa infection.PDT is one of the most promising treatments for drug-resistant or refractory bacterial infections,and further researches are needed.