Proteus Mirabilis Biofilm Formation And Its Bactericidal Effect Of Photodynamic Technology

Proteus mirabilis(P.mirabilis)is widely distributed in nature and easily contaminates food and causes bacterial food poisoning.P.mirabilis can form a more resistant biofilm,leading new challenges to the prevention and control of food safety.In this study,the crystal violet staining was used to evaluate the effects of different culture conditions on biofilm formation.Plate counting and fluorescence spectrophotometry were used to explore the photodynamic inactivation effect of alcohol-soluble curcumin on P.mirabilis biofilm in compared with three common photosensitizers.The mechanism of curcumin mediated photodynamic inactivation on P.mirabilis was studied by using polymerase chain reaction(PCR),agarose gel electrophoresis,sodium dodecyl polyacrylamide gel electrophoresis sulfate(SDS-PAGE),transmission electron microscopy(TEM).Ultraviolet-visible spectrophotometry,colony counting and scanning electron microscopy(SEM)were used to investigate the application of photodynamic technology in food,especially on contact materials.The main results are as follows:1.P.mirabilis showed the ability to rapid form biofilms.Optical microscopy,following crystal violet staining,was used to observe the kinetics of P.mirabilis biofilm formation.After 24 h of culture,when the initial bacterial concentration was10~2～10~8 CFU/m L,obvious amount of biofilm formation was observed.The aerobic condition at 37℃was adaptable for the formation of P.mirabilis biofilm.The culture medium composition with 2.0%Na Cl and 1.0%maltose at pH8.0 and double quantity of the medium promoted obviously the formation of P.mirabilis biofilm.2.Alcohol-soluble curcumin mediated photodynamic technology had a significant bactericidal effect on P.mirabilis and its biofilm.A 3.09 log reduction in the concentration of viable biofilm cells was obtained with 120μM curcumin and blue light irradiation for 20 min(31.25 J/cm~2)for biofilm.It was more effective to achieve the same light dose by extending the light time than increasing the light intensity.Incubation at 37℃for 20 min achieved optimal adsorption of curcumin.The bactericidal effects of rose bengal(RB)combined with photocatalysis-xenon lamp ranked the strongest,followed by toluidine blue(TBO)and methylene blue(MB),and alcohol-soluble curcumin(C)ranked the weakest.3.Agarose gel electrophoresis,PCR and SDS-PAGE showed that photodynamic technology caused damage to the outer membrane proteins and cannot damage certain virulence genes of P.mirabilis.The bacteria observed by TEM showed morphological changes,the cytoplasmic membrane was severely damaged and leakage of cytoplasm,cytoplasm was leaked,indicating the cause of bacteria death.4.Water-soluble curcumin combined with photodynamic technology mediated by 420 nm LED blue light could apply to the food industry for food safety.A reduction of>3 log(CFU/m L)was obtained both two kinds of curcumin(100μM)at20 min of light.The inactivation efficiency at low temperatures(-2,4°C)was lower than higher temperatures(15,25°C).There was no significant change in the inactivation of P.mirabilis at initial pH between 5.0 and 10.0.Rough wooden surface was most likely to cause P.mirabilis to adhere,followed by silica gel,polypropylene plastic(PP)and stainless-steel surfaces.After photosensitization treatment,the best inactivation effect for biofilm on stainless-steel,the number of colonies was reduced by 3.1 log(CFU/m L).The results showed that P.mirabilis can easily form highly resistant biofilms,curcumin mediated photodynamic technology could effectively control the contamination of P.mirabilis and its biofilm by damaging protein,cell membrane and morphological structure of bacteria,and it could be applied in food processing line,especially in food contact materials frequently possible contaminated by P.mirabilis.