Vibrio Anguillarum Biofilm:the Formation And Intervention Effect

To establish an in vitro model and investigate the structural characteristics of Vibrio anguillarum biofilm,the biofilm of V.anguillarum was developed on glass slide and observed by confocal laser scanning microscope.The activity of biofilm was measured and quantitatively analyzed by CCK-8（Cell Counting Kit-8）to detect the process.Following the culture for some hours,the cover slide was removed for subsequent staining with the iodidepyridine（PI）.This was followed by determination of the formation and characteristics of V.anguillarum biofilm by using CLSM.The CLSM images of biofilm formation at different time points were captured,suggesting that the biofilm was able to develop more efficiently on glass slide.The OD450 value of V.anguillarum biofilm developed quickly before 24 hours and stabilized after 48 hours.To investigate the characteristics of biofilm formation of pathogenic V.anguillarum BYK0638,and to provide a reference for further study on the mechanism and pathogenesis of biofilm formation of V.anguillarum.The characteristics of biofilm formation of V.anguillarum BYK0638 were investigated by modified microtiter-plate test,and the viability of V.anguillarum in the biofilms was detected by CCK-8.The results showed that V.anguillarum BYK0638 developed stable and evident biofilm on the surface of the polystyrene microtiter plate,The OD450 value of V.anguillarum biofilm reached the peak after 24 h and stabilized after 48 h.Within the 107-108 CFU /mL,the OD450 vaule of biofilm was significantly higher than the others（P<0.05）.The OD450 value of biofilm recorded at 25℃ was significantly higher than those recorded at other temperatures（P<0.05）.In the range of pH4-11,when pH value is 7,the amounts of bacterial biofilm was the highest,and at pH 3 and 12,no evident biofilm was found.Biofilm formation of V.anguillarum played no significant role when 0.03 to 2.00mmol/L MgCl₂ was added.However when more than 0.12 mmol/L CaCl2 was added,itwas promoted.Addition of 0.03% MgCl2 significantly promoted the bacterial biofilm formation while CaCl2 played no significantly role.Within 5% NaCl,the OD450 value of biofilm was the highest.The biofilms developed on the surface of the microtiter plate coated by the skin mucus,liver,foregut and hindgut tissue extract of large yellow croaker was significantly higher than the others（P<0.05）.Pathogenic V.anguillarum BYK0638 can develop stable and obvious biofilm.The biofilm formation is closely related to the change of environmental factors,pH,temperature,Mg²⁺,salinity and other environmental factors can significantly affect the formation of biofilm of V.anguillarum.To explore the effects of Rheum palmatum,Andrographis paniculata and Rhus chinensis on V.anguillarum and its biofilm.The minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of Rheum palmatum,Andrographis paniculata and Rhus chinensis to planktonic cell of V.anguillarum were measured by microdilution broth method.V.anguillarum biofilms were treated by 1/8、1/4、1/2、1、2、4 and 8MIC Rheum palmatum,Andrographis paniculata and Rhus chinensis,then all results were measured by the CCK-8（cell counting Kit-8）.The results showed that,compared with other groups,it played significant role when the biofilms were treated by Rhus chinensis,and the growth of the biofilm of BYK0638 could be completely inhibited at 2MIC concentration in 4 hours,and the concentration of SMIC50 was 2MIC.Recommended that actual dosage of prevention treatment is 6.25 mg / mL.