Staphylococcus Aureus Biofilm Formation Characteristic And Inhibition Removal Research

Staphylococcus aureus(S.aureus), a common typical gram-positive pathogen, is widely distributed and grows in group, which can lead to a variety of food contaminations and cause great threat to public health and food safety. S. aureus is capable of forming a mixture of polysaccharide and protein as well as a multilayer structure surrounded by a variety of chemicals in biofilm. Biofilm is a kind of special growth way for microbial to resist bad environment, which is most common form of microorganisms in nature, thereby bringing heavy burden to food industry. Therefore, it is urgently needed to track S.aureus biofilm formation from different genotype in various stages, diverse processing environment and seek for effective inhibition or removal methods on biofilms. Consequently, aimed at epidemic S.aureus in Southern China, the biofilm formation ability is quantified by CV and XTT assay in this study. In order to discuss the correlation between genotype and formation, S.aureus biofilms carrying different atl, ica, aap, agr genes are analyzed at different stages. Meanwhile, biomass and metabolic activity of S.aureus biofilm formation under external environment(temperature, pH, TSB, glucose, salt) is explored in this paper. Based on former formation characteristics, the separated and combined inhibitive effect of natural product NB, enzyme Lse on early adhesion, middle formation, maturation of S.aureus biofilm inhibition and removal effect, so as to provide theoretical references and scientific basis for prevention and control strategies.1. The biofilm of 257 S.aureus was quantified by CV and XTT method. 75.1%(193/257), 22.6%(58/257), 2.3%(6/257) of the isolates can form a small, moderate, large amount of biofilm respectively, while 77.0%(198/257), 17.9%(46/257), 5.1%(13/257) of the strains can form general active, moderate active, very active biofilm respectively. CV and XTT method are complementary and it is appropriate to combine them to make comprehensive evaluation of biofilm.2. 9 typical S.aureus are selected from the strains above to study S.aureus biofilm formation at 8 h, 16 h, 24 h, 48 h, 3 d, 7 d and 14 d stages. Biomass of S.aureus biofilm is quantified by CV method and it increases consistently to a stable amount during 0 to 14 d; Metabolic activity of S.aureus biofilm is quantified by XTT method and it increases from 0-16 h, reduces gradually after 24 h, tend to be stable until 7 d-14 d; Viable cells in S.aureus biofilm are quantified by CFU method and it is highest level at 16 h(109 cfu/mL), then reduces in magnitude, becomes zero to 14 d. In conclusion, the adhesion period is about 0 to 8 h, development period for 16 h-48 h, maturation period for 3 d-7 d and differentiation period for 7 d-14 d. Moreover, the biomass is identified by CV method to explore the relationship between gene and formation and they are observed by scanning electron microscope. The results show that atl, ica, aap and agr gene play an important role in adhesion, development, maturation, differentiation stage respectively, at the same time, S.aureus biofilm formation is influenced by several genetic factors together.3. S.aureus 4506 biofilm formation under various environmental conditions(temperature, pH, TSB, glucose, NaCl) is investigated by CV and XTT method. 37℃ is the optimum temperature while low/high temperature inhibits the formation; The optimum pH of 7, strong acid/alkali can inhibit biofilm adhesion and development period; 200% TSB promotes biofilm formation before 72 h, while TSB less than 100% inhibits biofilm in every stage of formation; Low concentration of Glucose promotes biofilm formation and 5% and 10% Glucose inhibit it instead; 10% NaCl promotes biofilm formation, while 15% and 20% NaCl decreased the ability.4. Based on the formation characteristics, the inhibition and removal effect of NB and Lse on S.aureus in terms of biofilm adhesion, development, maturation stage was explored by CV and XTT method. As for adhesion(8 h) period, the intensity sequence of different methods is NB + Lse, NB, NB+Cur, Lse; For medium-term(24 h) period, the removal and inhibition efficiency rate fell to 40%-70%, including the most effective removal of NB, the least effective inhibition of NB + Cur; For maturation(72 h) period, the removal and inhibition efficiency rate dropped to 20%-50%, the removal effect of NB + Lse, NB is stronger, while the inhibition effect of Lse is weak. Further studied by optical microscope and scanning electron microscope, the results show consistent with the quantification.