Study On The Synergistic Antibacterial Effect And Mechanism Of Photodynamic Combined With Gentamicin

Antibiotics have effectively cured many bacterial infections,and the quality of human life has been significantly improved.It is worrisome that the widespread use or overuse of antibiotics around the world has greatly increased the selection pressure of bacteria,and the emergence of bacterial resistance has seriously hindered the efficacy of antibiotics and produced a variety of toxic and side effects.Since the development of new drugs cannot keep up with the emergence of bacterial resistance,it is important to use mature antibiotics appropriately and explore the combination with other strategies to improve their functional effectiveness.In the 1970s,gentamicin(GEN)was used to deal with serious infectious diseases.It has broad-spectrum antibacterial activity which passively diffuses through the outer membrane porins,and then enters the cell via the transport system.It irreversible binds to the 3 0S subunit of the ribosome,blocks the synthesis of bacterial protein,and destroys the integrity of the bacterial cell membrane,eventually causing the death of the bacteria.Now GEN is still widely used in clinic and preclinical research.Photodynamic antimicrobial chemotherapy(PACT)is a promising way of antimicrobial therapy.In the exist of oxygen,photosensitizers(PS)are excited by light of appropriate wavelength to produce singlet oxygen,hydroxyl radicals,superoxide anion and other reactive oxygen species(ROS),which can inactivate bacteria.PACT destroys the biological macromolecules of bacteria through multiple targets,and repeated treatments are unlikely to cause bacterial resistance.PS is the key factor affecting the therapeutic effect of PACT.As a phenothiazine cationic photosensitizer widely used in clinic,toluidine blue(TB)has a high affinity to the cell membrane of Gram-negative bacteria and Gram-pasitive bacteria,and showing excellent antibacterial effects in PACT.However,there are few researches on the treatment of clinically resistant bacterial isolates using TB-PACT,and it is worthy of further research in the aspect of combating bacterial resistance.Preliminary studies have confirmed that a single target is not as effective as multi-molecule targets of drug.Therefore,the combination of the existing drugs or therapies provides a valuable route for solving bacterial resistance.On the one hand,the ingeniously designed combinational therapy is not easy to induce bacterial resistance.On the other hand,it can expand the clinical application of antibiotics and reduce the side effects.Therefore,we developed the combination of GEN and TB-PACT to investigate their synergistic antibacterial effect on S.aureus and MDR S.aureus,and explore the possible synergistic antibacterial mechanism from the production of ROS,changes in cell membrane fluidity and permeability,biofilm formation ability and oxidative stress response.We also evaluated the therapeutic effects of GEN?TB-PACT in vivo by using two mouse scald infection models.Research methods and resultsPart 1 Bacteriostatic effect of TB-PACT and GEN on S.aureus/MDR S.aureusThe sensitivity of S.aureus/MDR S.aureus to different kinds of antibiotics was analyzed by plate coating method.The enrichment of TB in two strains of bacteria was explored by combining alkaline lysis and fluorescence microscopy.The plate coating method was used to verify the antibacterial effects of TB-PACT and TB-PACT?GEN on S.aureus,MDR S.aureus and P.aeruginosa.The post-antibiotic effect of TB-PACT?GEN on S.aureus and MDR S.aureus was investigated with the aid of microplate reader.The results found that with the increase of the incubation time,the relative fluorescence intensity of TB in the bacteria gradually increased,reaching a maximum at 45 min.The TB concentration selected in the experiment had no effect on the survival rate of the two bacteria.TB-PACT results showed that the bacteriostatic effect was positively correlated with light dose and TB concentration.TB-PACT?GEN synergistically inhibited bacterial proliferation which could eliminate 4.6-log S.aureus and 2.67-log MDR S.aureus,and produce 3.0-log killing of P.aeruginosa.The combined treatment inhibited the reproliferation of S.aureus and MDR S.aureus within 12 h and 11h,respectively.Part 2 Antibacterial mechanism of TB-PACT?GENThe production of intracellular and extracellular ROS before and after TB-PACT?GEN treatment was detected by H2DCFH-DA,SOSG with flow cytometry and UV-spectrophotometer.The effect of NAC on the efficiency of synergistic sterilization was counted by the plate coating method.DiIC18 and PI probes combined with fluorescence microscopy were used to evaluate changes in bacterial cell membrane fluidity and permeability.The morphology of bacteria was observed with the field emission scanning electron microscope(FESEM).The expression of oxidative stress related genes in bacteria before and after TB-PACT?GEN treatment was compared by quantitative real-time PCR(qRT-PCR).The ability of bacterial biofilm formation and related genes expression were evaluated by quantitative method of crystal violet,SEM and qRT-PCR.The results showed that the ROS production during the combined treatment was significantly higher than either single treatment.After adding NAC,the survival rate of bacteria increased,suggesting that ROS is one of the cooperative antibacterial mechanisms.After TB-PACT?GEN treatment,the fluidity and permeability of bacterial cell membrane were significantly improved,and the bacterial surface was collapsed and damaged,indicating that the treatment caused damage to the bacterial cell membrane.After the combined treatment,the expression of AgrA,AgrB and PSM decreased significantly,the expression of oxidative stress genes was up-regulated,and the ability of bacterial biofilm formation decreased.Part 3 Therapeutic effect of TB-PACT?GEN on scald infection in miceTwo kinds of scald infection models of Balb/C mice were established to explore the therapeutic effect of TB-PACT?GEN from the aspects of wound healing,bacterial colonization,inflammatory factors(IL-6,TNF-?)and growth factors(TGF-?1,VEGF)expression,H?E staining of skin,body weight and blood biochemical indexes of mice.The study found that after TB-PACT?GEN treatment,the number of bacteria colonization in the wound of mice was effectively reduced,the content of skin inflammatory factors was significantly reduced,and the number of growth factors was increased,which was conducive to tissue repair.H?E staining showed that complete epidermal layer and hair follicle appeared earlier in the combined treatment group,suggesting that TB-PACT?GEN could promote wound healing.After TB-PACT?GEN treatment,the weight of mice was relatively stable and the blood biochemical indexes were in the normal range,indicating that TB-PACT?GEN treatment was relatively safe.ConclusionsIn summary,TB-PACT?GEN synergistically inhibited the proliferation of S.aureus and MDR S.aureus.During the treatment,a large number of ROS were produced,which damaged bacterial cell membrane,affected oxidative stress response and biofilm formation.TB-PACT?GEN could effectively promote wound healing by reducing the number of bacteria colonization and the content of inflammatory factors,and increasing the expression of growth factors in the wound.