Study On The Inhibition Effect And The Mechanism Of Antimicrobial Photodynamic Inactivationon(aPDI)on Food-borne Staphylococcus Aureus

Objective:Food safety receives widespread attention on a global scale.Currently,hundreds of millions of people get sick and a proportion of them die from eating contaminated food each year.Due to abusive use,antibiotics for foodborne pathogens are also gradually losing their effectiveness,owing to the emergence of antibiotics-resistant strains.Staphylococcus aureus is one of the most prevalent foodborne pathogens,whereas a collection of strains exhibit resistance against almost all clinically available antibiotics.Antimicrobial photodynamic inactivation(aPDI)serves as a new approach to control the growth of foodborne bacteria.This paper aims to investigate the inactivation effect of photosensitizers,based on hypocrellin B,on S.aureus and to discover the mechanisms underlying this novel sterilization.The experiment was designed as four groups:blank control(S-L-),negative control(S-L+,S+L-)and experimental group(S+L+).The best one was selected from the three photosensitizers,namely chlorophyll derivative,curcumin and hypocrellin B,and measured the changes of bacterial characterization and gene in the aPDI process,followed by mechanistic insight with cellular and molecular aspects leading to photokilling potency.The new mutant strains were obtained by overexpression treatment of the target genes,and the influence of the altered genes on aPDI was determined,from which key molecules mediating the studied photodynamic course were identified and dissected.Results:1.Chlorophyll derivative,Curcumin and Hypocrellin B can all mediate the inactivation effect of aPDI.And among them,HB exhibited the greatest potency to control the growth of S.aureus.The photokilling efficacy is highly dependent on the conditions used.When 100 n M,5 J/cm~2 was applied,the bacterial survival can be reduced by 2-3orders of magnitude.2.HB-based aPDI disrupted the membrane integrity of staphylococcal cells,leading to changes in cell morphology,leakage of intracellular substances,changes in the activities of enzymes(SOD,CAT)in the cell.Most importantly,it can disrupt the bacterial biofilm and compromise its formation.3.Under the photodynamic treatment,a transcriptome-scale remodeling arises from oxidative stress response,manifested by multiple key regulatory pathways proposed,indicating that cell wall metabolism is implicated in the sterilization effect.4.Overexpression of LrgA gene can improve the survival rate of S.aureus treated with aPDI and increase the formation of bacterial biofilm.5.The HB-mediated photodynamic efficacy was potentiated by the addition of curcumin with a sublethal dose.This dual-photon synergy effect can be induced at 100 n M and 9 J/cm~2,which might be accounted for by the increased type I/type II ratio of ROS.6.aPDI can reduce the microbial contamination of apple slices while maintaining its quality,showing a promising application in food storage.This study demonstrated that aPDI had an inactivation effect on S.aureus and that the dual photosensitizer system had a synergistic effect to control the growth of food-borne bacteria.To the best of our knowledge,this is the first practice and mechanistic insight of dual-photon mediated aPDI associated with prototypic foodborne pathogens,providing novel evidence to expand it to a broader situation to control microorganisms and maintain food quality.