Studies Of The Inhibition Effects And Mechanisms Of Photodynamic Therapy Against Multidrug-resistant Acinetobacter Baumannii Using Two Different Photosensitizers In Vitro

Objective:To evaluate the efficacy of in-vitro antimicrobial photodynamic therapy against multidrug-resistant Acinetobacter baumannii（MDR-AB）mediated by New Methylene Blue（NMB）and MLSiPcI4,and explore the inhibition mechanisms of those photosensitizing agents.Methods:1.Set up four groups with following conditions:group E with both photosensitizing agent and irradiation（PS+L+）,group PS with photosensitizing agent only（PS+L-）,group L with irradiation only（PS-L+）,and group C subject to no photosensitizing agent and irradiation（PS-L-）.The MDR-AB in groups E and PS were incubated with various concentrations of NMB or MLSiPcI4 for 30 min,respectively.As a substitute for photosensitizer agent,0.9%NaCl solution was utilized to incubate the MDR-AB in groups L and C for 30 min,individually.To activate the photosensitizing agents,infrared light with a wavelength of 660 nm was utilized as light treatment.After incubation with NMB,the bacteria from Groups E were exposed to infrared light for 240s with a fluence of 42J/cm²,while the ones from group L were treated similarly for comparison.Meanwhile,the bacteria from Groups E mediated by MLSiPcI4 were activated by light treatment for 1200s with a fluence of 720J/cm²,while group L was also treated similarly.However,the group PS and group C were not subject to irradiation.After serial dilutions,10μl of bacterial suspensions would be extracted from each group and inoculated on blood Agar plates,which would be incubated for 24 hours.Then the colony number on the plates from each group would be counted and the difference in colony number would be investigated through variance analysis.2.The morphological changes of MDR-AB in each group after treatments mentioned above would be observed via transmission electron microscope.3.The concentrations of peptidoglycan,AKP,Mg²⁺and K⁺in bacteria suspensions from each group were detected by the corresponding kits,and the statistical analysis would be performed to analyze the difference in those 4 substances across the bacterial suspensions.4.The DNA from wild-type MDR-AB would be extracted using DNA kit and divided into 4 groups,such as groups E,PS,L,and C,due to different treatments on the DNA.The DNAs from groups E and PS were incubated with NMB for 30 min,respectively,while the DNAs in groups L and C were incubated with 1xPBS instead for 30 min,individually.Next,DNAs from groups E and L were exposed to infrared light with a wavelength of 660nm and a fluence of 42J/cm²,while the other 2 groups were not.By gel electrophoresis,the size and resolution of DNA from each group will be analyzed.Results:1.When 5μM of NMB was utilized as photosensitizing agent,the colony number from group E was significantly less than ones from the other 3 groups（group E-147±35;group PS-318±103;group L-349±91;group C-336±85;P<0.05）.When the concentration of NMB increased to 10μM,the colony number from group E was also significantly less（group E-88±395;group PS-202±65;group L-195±49;group C-245±84;P<0.05）.Under either concentration of NMB,no statistically difference in the colony numbers from groups PS,L,and C were found by pairwise comparison.To test the effects of the other photosensitizing agent,0.5μM of MLSiPcI4 was utilized and the colony numbers from group E and PS were significantly lower than one from group L and C（group E-16±17;group PS-19±14;group L-219±166;group C-196±120;P<0.05）.However,there was no significantly difference in colony number between groups E and PS（p=0.964）,as well as between groups L and C（p=0.726）.When the concentration of MLSiPcI4 was reduced to 0.25μM,there was no significantly difference in colony numbers across 4 groups（P>0.05）.2.Comparing to the other 3 groups,the cell wall of bacteria from group E was damaged more distinctly under transmission electron microscope.3.The content of peptidoglycan in suspension from group E was higher than that from the rest of group（group E-6.8294±1.2456;group PS-5.9623±0.9698:group L-5.6738±0.7346;group C-5.6778±1.0667;P<0.05）.But no significantly difference in peptidoglycan concentration from the groups PS,L,and C was observed by pairwise comparison（P>0.05）.Furthermore,no significant variance was found in the content of AKP,K⁺and Mg²⁺across 4 groups（P>0.05）.4.The gel electrophoresis showed that the DNA band from group E was blurrier than ones from the other three groups.Even with adjustment of resolution to 75%,the DNA band from group E remained undetectable,while the DNA bands from the other three groups clearly displayed on the gel.Conclusion:1.The antibacterial photodynamic therapy mediated by NMB and activated by infrared light displays strong inhibitions against MDR-AB in vitro.The results from this study suggested that the antibacterial photodynamic therapy mediated by MLSiPcI4 and activated by infrared light didn’t exhibit strong suppressions against the growth of MDR-AB in vitro.2.The mechanism of strong inhibitions against MDR-AB in vitro by destroying the cell wall of bacteria.More researchs are needed to conclude that NMB-mediated photodynamic therapy led to the death of MDR-AB via directly destroying bacterial DNA.