The Study Of Berberine On Pseudomonas Aerugingosa Biofilms From Burns Wounds And Obervation Of Clinical Use Chinese Traditional Medicine (Sanhuangshaoshangling)

Objective:To compare shape and amount of bacterial biofilm between berberine group and the control group through in vitro biological model of pseudomonas aeruginosa; to explore the effects of berberine on pseudomonas aeruginosa biofilm; and to explore the clinical application effects of SANHUANGSHAOSHANGLING based on clinical observation of homemade TCM SANHUANGSHAOSHANGLING.Methods:(1) Positive microbial specimens were collected from burn department from January to June,2014. Strains with positive results in oxidase test were selected and VITEK2 Compact was used to diagnose the strains as pseudomonas aeruginosa. ATCC27853 was used as the quality control strain. Drug sensitive test adopted K-B test. According to the latest breakpoint standard (CLSI), drug-resistant strains were selected as research strains. (2) AB (Alcian blue)-CR (Congo red) staining was used to screen pseudomonas aeruginosa biofilm. (3) PVC sheet carrier was used to replace traditional plate culture. Electric microscope was used to observe morphology of biofilm on 1 day,3 days,5 days and 7 days cells to determine mature time of cell biofilm. (4) Crystal violet staining was used for half-quantitative monitoring on biofilm. The cells were cultured in 96-well plates (guide film was added into plate) in L-B broth. When the cells were matured, guide film was taken out. OD value of the eluant was determined by ELIASA at 490nm. (5) Microdilution method was used to determine minimum bacteriostatic concentration (MIC) of berberine to pseudomonas aeruginosa in clinical. Shapes of bacterial biofilm were compared between the experimental group and the blank group under scanning electron microscopy (SEM). And agar plate was used to count viable bacteria on biofilm. (6) Effects of SANHUANGSHAOSHANGLING on rat burn wounds were analyzed through investigation on the wound healing. (7)Effects of SANHUANGSHAOSHANGLING on patients’burn wounds were analyzed through investigation on the wound healing via clinical questionnaire of SANHUANGSHAOSHANGLING.Results:(1) Drug-resistant rate was 76.7%(115/150) among the positive bacterial specimens. (2) Positive bacterial biofilm rate was 83.5% (96/115) by AB-CR staining. (3) SEM showed formation of bacterial biofilm within 1-3 days. Early biofilm was formed within 3 days. The formation became slowly from the 5 days. Bacterial biofilm was matured on 7 days. (4) The amount of bacteria was increased on 1 d, and OD value was 0.45±0.53; The amount of bacteria was obviously increased and began to form BF on 3 d, and OD value was 1.21±0.49; BF were increased on 5 d, and OD value was 2.07±0.47; BF on 7 d had similar amount as on 5 d, and OD value was 2.12±0.35; and the measured results of reference strains in the corresponding time were:0.43±0.36,0.52±0.38,0.49±0.52, and 0.53±0.39. OD value of the experimental strains was relative greater. (5) MIC of berberine to pseudomonas aeruginosa biofilm was 8692ug/ml.2MIC (17384ug/ml) of berberine had inhibition on bacterial biofilm. There were statistically significant differences between berberine group and the blank control group in biofilm bacteria amount (P<0.05). (6) SANHUANGSHAOSHANGLING improving wound healing.(7)Based on clinical application and observation on SANHUANGSHAOSHANGLINQ it had significant effects on controlling burn wound infection, improving fusion rate of granulation on wound and accelerating wound healing.Conclusion:Multiple drug-resistant pseudomonas aeruginosa are adhered to tissue and biological material surface. It can form a large number of bacterial biofilm and aggregate to gradually form anti-permeation barrier. Drug resistance of pseudomonas aeruginosa is strengthened. Before complete formation of bacterial biofilm (7 days), research is carried out on the inhibition of berberine on pseudomonas aeruginosa biofilm. Berberine is an alkaloid that can effectively delay the bacterial biofilm formation. SANHUANGSHAOSHANGLING can effectively treat the repeated infection on burn wound surface, with emarkable bacteriostasis and sterilization and rot muscle removing effects, which is worth popularization and application in clinical.