Study On The Antibacterial And Antibiotic Susceptibility Effect Of The 5-aminolevulinic Acid Photodynamic Therapy On The Mycobacterium Abscessus In Skin Infection

Backgound and Objective Nontuberculous mycobacteria（NTM）is a collective term for mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae.Mycobacterium abscessus（M.abscessus）belongs to the rapid-growing mycobacteria（RGM）group of nontuberculous mycobacteria,which can invade human lungs,skin soft tissues,bones and joints,lymphnodes and other tissues or organs to cause local or disseminated diseases.In recent years,the incidence of cutaneous NTM disease has been increasing,and the incidence of cutaneous Mycobacterium abscessus infection is also increasing year by year,and even more significantly.Current treatment regime for skin infections caused by Mycobacterium abscessus is generally carried out with reference to the treatment of pulmonary infections,which requires a combination of multiple antibiotics for treatment,with most of the treatment duration being more than 6 months.However,in clinical practice,the results of simple antibiotic treatment are often unsatisfactory,but instead a large number of drug-resistant bacteria emerge,making clinical treatment more fraught with difficulties,and also putting a heavy economic and psychological burden on the patients.Photodynamic therapy（PDT）is administered through local photosensitizer,and then the light matching the wavelength of the photosensitizer is used to irradiate the sick cells or tissues to produce reactive oxygen species（ROS）such as oxygen radicals and singlet oxygen by photochemical reaction,thus promoting oxidative stress to achieve the purpose of treatment.Photodynamic therapy has been widely used for the diagnosis and treatment of condyloma acuminatum,acne and many other skin tumors.In recent years,the killing effect of photodynamic therapy on pathogenic microorganisms has also received increasing attention.Many studies have described the excellent killing effect of photodynamic therapy on Gram-negative bacteria,Gram-positive bacteria,viruses,fungi and parasites.There are also many cases of applying photodynamic therapy in the clinical treatment of diseases caused by mycobacteria and related basic studies in recent years,which have shown the good potential of photodynamic therapy in the treatment of diseases caused by mycobacteria.Thus,photodynamic therapy can be a potentially effective way to treat Mycobacterium abscessus infections of the skin.Our team had successfully treated several patients with Mycobacterium abscessus skin infection using ALA-PDT in combination with antibiotics,so in this experiment,we started from these clinical data and comprehensively reviewed and analyzed the condition,treatment,prognosis and follow-up of these patients,and found that the condition of these patients was quickly controlled after ALA-PDT treatment,and the course of subsequent treatment was significantly shortened with low recurrence rate.Then we collected strains isolated from patients with cutaneous Mycobacterium abscessus infection for drug susceptibility determination and ALA-PDT treatment,analyzed and summarized the drug susceptibility of these strains,and initially obtained the bactericidal properties of ALA-PDT against these strains,and we found that ALA-PDT could indeed play an effective role in killing Mycobacterium abscessus.Then we established an in vitro biofilm model of Mycobacterium abscessus using Mycobacterium abscessus standard strain ATCC19977,and studied the sublethal and lethal doses of ALA-PDT on Mycobacterium abscessus,and investigated the killing effect of ALA-PDT on Mycobacterium abscessus planktonic bacteria and biofilm after treatment.In our study,we found that ALA-PDT at sublethal doses could differentially affect the susceptibility of Mycobacterium abscessus to many antibiotic drugs and investigated the possible mechanisms involved.Methods1.Basic case data were collected from patients with clinical infection of Mycobacterium abscessus who attended our hospital.2.Isolation and cultivation of clinical and standard strains of Mycobacterium abscessus,and construction of Mycobacterium abscessus biofilm models by 96-well plates and cell slides.3.Measurement of MIC values of Mycobacterium abscessus by microbial broth dilution method.4.Treatment of Mycobacterium abscessus and its biofilm with ALA-PDT.5.Measurement of viable changes in Mycobacterium abscessus and its biofilm after treatment with ALA-PDT using the CFU counting method.6.To measure the changes in biofilm activity of Mycobacterium abscessus after ALA-PDT treatment using the crystal violet staining method.7.Observation of structural changes of Mycobacterium abscessus and its biofilm after ALA-PDT treatment using SEM.8.Observation the effect of ALA-PDT on the percentage of live and dead Mycobacterium abscessus biofilms using CLSM.9.Observation the effect of ALA-PDT on the cell wall permeability of Mycobacterium abscessus.10.Construction the phenotypes of Mycobacterium abscessus for inducing drug resistance and treating them with ALA-PDT.11.Measure the effect of ALA-PDT on the m RNA expression of drug resistance genes whi B7,erm（41）and efflux pump genes MAB₁409c,MAB₃142c in Mycobacterium abscessus by RT-q PCR.Result:1.ALA-PDT can significantly relieve the condition of clinical Mycobacterium abscessus infections,shorten the treatment period,and accelerate wound healing.2.Clinically isolated Mycobacterium abscessus is only sensitive to clarithromycin,azithromycin,amikacin,linezolid,cefoxitin and cefmetazole,and has a high resistance rate to isoniazid,rifampin,minocycline,meropenem,ciprofloxacin and moxifloxacin.3.ALA-PDT killed all Mycobacterium abscessus with different drug resistance,but there was no significant relationship between killing effect and source.4.ALA alone and light alone had no killing effect on Mycobacterium abscessus,and the killing effect of ALA-PDT on Mycobacterium abscessus was dose-dependent and increased with the concentration of ALA and the intensity of light.5.The killing effect of ALA-PDT on Mycobacterium abscessus biofilm was also dose-dependent and increased with the dose of ALA concentration.6.The results from SEM showed that ALA-PDT could disrupt the adhesion structure between Mycobacterium abscessus,and also had a disruptive effect on the bacterial structure of Mycobacterium abscessus.7.From the CLSM observation,it can be seen that ALA-PDT can disrupt the structure of Mycobacterium abscessus biofilm,and also increase the number of dead bacteria within the biofilm.8.ALA-PDT at sublethal doses increased the permeability of the cell wall of Mycobacterium abscessus,whereas light and photosensitizer alone had no effect on the permeability of the cell wall of Mycobacterium abscessus.9.After a period of co-incubation of Mycobacterium abscessus with clarithromycin,Mycobacterium abscessus exhibited a significant increase in the expression of drug resistance genes and efflux pump genes that induce drug resistance.10.The m RNA expression of drug resistance genes whi B7,erm（41）and efflux pump genes MAB₁409c,MAB₃142c in Mycobacterium abscessus before and after ALA-PDT.Conclusion:1.ALA-PDT in combination with antibiotic therapy can significantly control the condition of cutaneous M.abscessus infection,shorten the treatment cycle,and reduce the recurrence.The antibiotics can use sensitive drugs such as clarithromycin,cefoxitin,amikacin and linezolid.2.ALA-PDT showed significant killing effect of M.abscessus in plankton and biofilm,and the effect was enhanced with the increasing dose of ALA-PDT.3.The susceptibility of M.abscessus to antibiotics was increased after ALA-PDT treatment,and the induced resistance was also inhibited.4.ALA-PDT affects drug resistance in M.abscessus probably by increasing cell wall permeability and decreasing the expression of related drug resistance genes.