| Vibrio parahaemolyticus is one of the leading bacterial of seafood-derived foodborne pathogen which is halophilic and usually widely distributed in estuarine and marine environment in warm and tropical areas. In this study, the formation and characterization of Vibrio parahaemolyticus biofilm and the detachment under ultrasound treatment were investigated; The effect of anti-microbial photodynamic technology(APDT) against V. parahaemolyticus biofilm under different concentration of methylene blue(MB) and irradiation time were measured. The influence of D-tyrosine on biofilm formation and bacterial inactivation were also studied.Confocal laser scanning microscope(CLSM) was used to visualize the effect of inactivation treatment against V. parahaemolyticus biofilm. The main results are as follows:1. V. parahaemolyticus biofilms formed on glass surfaces can be easily observed by crystal violet staining method. During the process of biofilm formation from planktonic bacteria cells to sessile biofilms, the biomass was growing gradually. The bacterial aggregates made the biofilm structure more complex and more mature and the number of bacterial cells wrapped by extracellular polymeric substances(EPS) was increasing with the prolonging of incubation time.At the initial incubation stage from 0 h to 24 h, biofilm biomasses increased dramatically and reached the max value at 24 h and then a equilibrium state. After the biofilm became matured,the production of biomass ceased and even started to fall.2. The influences of environmental factors on biofilm formation ability were analyzed by biomass values using enzyme-linked immunosorbent assay(ELISA) plate reader under 595 nm.Results showed that when the medium salinity was 3%, rotate speed 70 r/min and incubation time 24 h, the biofilm was mature and became strong also the V. paraheamolyticus bacterial concentration reached 2.56×107CFU/cm2. Data also showed that the salinity of culture medium,shear stress and incubation time has great influence on V. parahaemolyticus biofilm formation and maturation. In addition, by counting colony-forming method, the optimized condition for detaching the biofilm from glass surface under ultrasound treatment were obtained. Under the pulsed ultrasound treatment of frequency at 50 kHz for 4 min, the biofilm could detach from theglass surface and kept the viability of the bacteria at the same time.3. The efficacy to inactivate V. parahaemolyticus biofilm by APDT mainly depends on the MB concentration and illumination time. When V. parahaemolyticus biofilm was exposed to 10μg/mL MB for 1 min illumination, the inactivation rate was 99.51%, while 20 min the inactivation rate was 99.97%. The inactivation rate was 74.29% by 0.1 μg/mL MB after 20 min irradiation. Exposure to 1000 μg/mL MB for 20 min resulted in almost completely inactivation of the bacteria in biofilm matrix and the death rate was 99.99%. V. parahaemolyticus biofilms showed 10-100 fold tolerate ability differences under APDT compared with planktonic forms.CLSM can be efficiently used to visualize the effect of APDT based on SYT09/PI staining, and the inactivation rate can be easily analyzed by the change of the red or green color occur on CLSM images.4. D-tyrosine significantly inhabited V. parahaemolyticus biofilm formation by reducing extracellular polymeric substances formation without influencing bacterial growth, meanwhile,D-tyrosine disrupted mature biofilm evidenced by the reduction of biofilm biomass. APDT effect was enhanced by adding D-tyrosine due to the weakening of the protection of EPS to bacterial cells engaged within biofilm matrix. The combination of APDT and D-tyrosine can enhance the inactivation activity against V. parahaemolyticus biofilm and may be widely used in the food industry to control the pathogen biofilms. |
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