Antimicrobial Photodynamic Therapy For Multidrug Resistant Pseudomonas Aeruginosa In Vitro

Background and Purpose:Pseudomonas aeruginosa is an opportunistic gram-negative bacteria that can easily colonize burns and other wounds where the skin barrier is damaged.This local infection can lead to systemic sepsis,especially for severe burn patients,which is often closely related to high morbidity and mortality.With the abuse of antibiotics,the emergence of multi-drug resistant strains forces people looking for new antibacterial technologies.Antimicrobial photodynamic therapy is a sterilization mode independent of antibiotics,which has little damage to the host and can not induce bacterial resistance after repeated treatment.Therefore,it is considered to be a promising method for the treatment of bacterial infections,especially for infections caused by multi-drug resistant bacteria.This study analyzed the killing effect of red light with a wavelength of 635±5nm combined with toluidine blue O on standard Pseudomonas aeruginosa strains and clinically isolated multi-drug resistant strains in vitro.To explore the potentiate effect of potassium iodide and efflux pump inhibitor carbonyl cyanide metachlorophenylhydrazone on the efficiency of TBO-mediated APDT killing Pseudomonas aeruginosa.Observe the changes in microscopic morphology and sensitivity to antibiotics of Pseudomonas aeruginosa before and after TBO-APDT,and detect the changes of multi-drug resistant Pseudomonas aeruginosa（MDR P.aeruginosa）before and after TBO-APDT treatment,so as to analyze the mechanism of APDT antibacterial.So as to make a good research foundation for clinical application.Approach:Multi-drug resistant Pseudomonas aeruginosa was isolated from one clinical patient with burn wounds was studied,and standard strain from ATCC（NO.27853）was used as control.The bacteria were divided into control group,photosensitizer group,light group,and APDT group.The bacterial survival rate was determined by colony counting method.The bacterial survival rate was calculated by the colony serial dilution method.The effects of KI（100m M）or CCCP（0.1μM）on the killing efficiency of Pseudomonas aeruginosa by TBO-APDT were studied respectively,and the differences in the killing efficiency of TBO-APDT on Pseudomonas aeruginosa by different administration methods of KI or CCCP were compared.Morphological alterations of bacteria were observed with laser confocal electron microscope（CLSM）,transmission electron microscope（TEM）and scanning electron microscope（SEM）.After APDT,changes of minimum inhibitory concentrations（MICs）of several antibiotics were detected,including piperacillin/tazobactam,ceftazidime,cefepime,imipenem,meropenem,etc.;Expression of drug-resistant genes of multi-drug resistant Pseudomonas aeruginosa were evaluated using RT-q PCR before and after APDT.Results:1.When TBO concentration was in the range of 2.5μM～10μM and light dose was in the range of 10J/cm²～40J/cm²,the killing efficiency of TBO-APDT on the two Pseudomonas aeruginosa strains improved with the increase of TBO and light dose（P<0.05）.In addition,the killing efficiency of TBO-APDT on multi-drug-resistant Pseudomonas aeruginosa and wild-type Pseudomonas aeruginosa has no significant difference under the same conditions（P>0.05）.2.When the TBO concentration was in the range of 2.5μM～10μM,the light dose was10J/cm²,and the KI concentration was 100m M,the experimental method of incubating the bacteria,TBO,and KI together for 30 minutes and then illuminating had a significantly higher killing efficiency on Pseudomonas aeruginosa compared with TBO-APDT alone（P<0.05）,the sterilization efficiency can be increased by 2 Log10～6 Log10.Under the same conditions,the experimental method of adding Pseudomonas aeruginosa immediately after irradiation of TBO and KI can also improve the sterilization efficiency（P<0.05）compared with TBO-APDT,and the sterilization efficiency can be increased by 3 Log10 at the highest.3.When the TBO concentration was in the range of 2.5μM～10μM,the light dose was10J/cm²,and the CCCP concentration was 0.1μM,the experimental method of incubating the bacteria,TBO,and KI together for 30 minutes and then illuminating had a significantly higher killing efficiency on Pseudomonas aeruginosa compared with TBO-APDT alone（P<0.05）,in which the killing of WT strain can increase by 3.5 Log10,and the killing of Mex AB-Opr M strain can increase by 6.5 Log10.Under the same conditions,the experimental method of adding bacteria immediately after irradiation of TBO and CCCP can also increase the sterilization efficiency of bacteria compared with TBO-APDT（P<0.05）,and the Mex AB-Opr M strain could increase 3 Log10,the WT strain could increase 2 Log10;4.After TBO-APDT treatment,the bacteria were stained with SYTO^?9 green fluorescence and propidium iodide,and then a large number of red-stained bacteria can be observed through CLSM.SEM showed that the two strains of bacteria had different degrees of depression,shrinkage,fracture and other changes,as well as holes and cracks on the surface of bacteria.TEM found that the outermost fimbriae of the bacterial cell wall disappeared,the continuity of the membrane structure was interrupted,and the cell wall and cytoplasmic membrane were separated.The most significant changes were the appearance of transparent vacuoles in the cytoplasm of WT strain and the appearance of bundle like structure with high electron density in the cytoplasm of Mex AB-Opr M strain.5.Before and after sublethal TBO-APDT treatment,the MIC value of Mex AB-Opr M strain changed significantly,Piperacillin/tazobactam is reduced from 128μg/m L to 16μg/m L,Cefepime is reduced from 64μg/m L to 4μg/m L,Imipenem is reduced from 8μg/m L to 2μg/m L,and Meropenem is reduced from 4μg/m L to 0.25μg/m L,Amikacin is reduced from 64μg/m L to 2μg/m L,Tobramycin is reduced from 32μg/m L to 1μg/m L,and Ciprofloxacin is reduced from 4μg/m L to 2μg/m L.RT-PCR was used to detect the expression of the efflux pump gene of the Mex AB-Opr M strain and found that compared with the control group,mex B was 0.08（P<0.001）,mex D was 0.83（P<0.05）,mex Y was 0.69（P<0.05）,mex F was 0.93（P>0.05）.Conclusions:TBO-APDT has good killing efficiency against Pseudomonas aeruginosa.KI can promote the killing efficiency of TBO-APDT against Pseudomonas aeruginosa.CCCP can improve the killing efficiency of TBO-APDT to Pseudomonas aeruginosa with high expression of efflux pump.In addition,TBO-APDT destroys the structural integrity of the bacterial cell wall,resulting in increased membrane permeability.The ocytoplasmic organelles were also attacked by TBO-APDT,and damage the DNA of the Mex AB-Opr M strain,thereby affecting the expression of drug resistance genes,which in turn leads to increased sensitivity to antibiotics.The research results show that TBO-APDT has great prospects for the treatment of multi-drug resistant Pseudomonas aeruginosa,but further animal model experiments and clinical studies need to be carried out to ensure the reliability and safety in clinical practice.