In Vitro Bactericidal Effect Of Povidone Iodine Solution On Bacterial Biofilm Of Staphylococcus Aureus

ObjectiveThrough the establishment of Staphylococcus Aureus bio-film model in titanium plate surface, the structure of bacterial bio-film and bacterial survival rate in middle layer and bottom layer of bacterial bio-film was observed under con-focal laser scanning microscope; in vitro bactericidal effect of povidone iodine solution in concentration of 0.5%, 0.25% was investigated on bacterialbio-film of Staphylococcus aureus.MethodsStaphylococcus aureus preserved in slope was selected and inoculated in tryptic soy broth(TSB), then cultured in 37。C incubator(5% CO2) for 16 hours. The concentration of bacteria was adjusted to 1 x 106 CFU/ml by turbidimetry. 15 clean, passive, sterilized titanium plates were divided into 3 of 25cm2 cell culture flask randomly; each cell culture flask included five titanium plates, 1ml prepared bacterial suspension, 20 ml of TSB; titanium plates were immersed in bacterial suspension and cultivated for 24 hours in 37。C incubator(5% CO2) to form mature bio-film. Then 15 pieces of titanium plate were taken from the 3 of cell culture flask randomly after rinsed three times by PBS, 10 of them were stained by SYTO9/PI dye in lucifugal environment for 20 minutes, the bio-film structure and bacteria survival rate was observed under con-focal laser scanning microscope, 5 other pieces of titanium plate was used for bacterial cultivation after deal with by Vortex-Genie、Ultrasonication、centrifuge.According to the modeling method above, Staphylococcus aureus bio-film models were established in the 45 pieces of titanium plate surface, and then all titanium plates were taken off from the cell culture bottles, rinsed three times by PBS; 45 pieces of titanium plate were divided randomly into 3 groups(15 for each group); among them two groups were Immersed in 0.5% and 0.25% of povidone iodine solution respectively for 5 minutes and then the iodine residue was removed from titanium plate surface by neutralizing agent which was composed of sodium thiosulfate and Twain 80 and lecithin. 10 pieces of titanium plate of that two group were stained by SYTO9/PI dye in lucifugal environment for 20 minutes, and the bio-film structure and bacteria survival rate was observed under con-focal laser scanning microscope, 5 other pieces of titanium plate was used for bacterial cultivation after deal with by Vortex-Genie、Ultrasonication、centrifuge. 10 pieces of titanium plate of the third group was as the blank control group,5 other pieces of titanium plate was used for bacterial cultivation after deal with by Vortex-Genie、Ultrasonication、centrifuge、dilution.Results①Bacterial bio-film expressed as a highly organized structure of multi-cell population under con-focal laser scanning microscope. The image of bacterial bio-film under con-focal laser scanning microscope was composed of orange and red and green fluorescent areas, The orange fluorescent area indicated the viable and dead bacteria superposed in the same position, the green fluorescence indicated living bacteria and red fluorescence indicated dead bacteria.②The bacterial bio-film under con-focal laser scanning microscope is conglomerated polymers. Living bacteria were wrapped in the middle of the bacteria bio-film, but the dead bacteria ware remained at the periphery of bacterial bio-film.③After 24 hours of incubation, living bacteria rate in the middle layer of bio-film was(67.35 ± 1.06)%, in the bottom living bacteria rate was( 39.42 ± 2.23)%. The live bacteria ratio of bacterial bio-film between the middle layer and bottom layer of was different significantly statistically(P< 0.01).④Bio-film middle living bacteria rate was(66.65 ±1.37)% and the bottom living bacteria rate of bio-film was(38.22±3.15)% after effect of 0.5% concentrations of povidone iodine solution.⑤Bio-film middle living bacteria rate was( 66.53±1.00)% and the bottom living bacteria rate was(39.13±2.41)% after effect of 0.25% concentrations of povidone iodine solution.⑥biofilm middle living bacteria rate and the bottom living bacteria rate are without statistical significance respectively composed with 0.25% and 0.5% concentrations of povidone iodine solution and the blank control group(p<0.05).⑦bacterial cultivation of model group 、0.25% and 0.5% concentrations of povidone iodine solution in blood agar culture showed Staphylococcus aureus colony forming,They are identified as Staphylococcus aureus.Conclusions①In this experiment the establishment of Staphylococcus aureus BBF model method is relatively simple and repeatable and can be applied to research on the bio-film forming ability of the same bacteria on different implant surface.②In bacterial bio-film, the survival rate of bacteria in the middle layer was higher than the bottom layer, dead bacteria ware remained at the periphery of bacterialbio-film.③Povidone iodine solution in concentration of 0.5%, 0.25% showed no significant bactericidal effect on bacterial bio-film.