Effects Of Pomegranate Peel Extract Tannin On Acinetobacter Baumannii Biofilms

Objective:To explore the effects of tannin which extracted from Pomegranate peel on the formation of biofilm and pre-formed biofilm of Acinetobacter baumannii(Ab), which would provide theoretical and experimental basis for treatment of Ab.Methods:1.The minimal inhibitory concentration(MIC)of the tannin and Ciprofloxacin(CIP)to Ab ATCC19606 were determined by broth dilution method. 2. The concentration of 1/2MIC,1/4MIC and 1/8MIC of Tannin acted on Ab for 4h, 8h, 24 h and 48 h, then used crystal violet stained with detected the Optical density to observe the effects of tannin on the formation of Ab biofilm. Tannin of the abrove different concentration acted on Ab for 48 h also to observe the effects of tannin on the formation of Ab biofilm by the Fluorescence microscope. 3. The concentration of 1/2MIC,1/4MIC and 1/8MIC of Tannin and CIP were made up respectively. And these two drugs were co-cultured with Ab mature biofilm separately or in combination for 4h,8h,24 h and 48 h. The optical density value and the number of colonies were detected separately with the Crystal violet staining assay and flat colony counting method. After acting 24 h by these mentioned abrove,we used fluorescence microscope and confocal laser scanning microscope(CLSM) to explore the effects of tannin to pre-formed biofilm of Ab. 4. The FQ-PCR was adopt to detect the genes Aba I, Bap, Csu E and Omp A, which related to biofilm formation for explicitly indicating the effests of tannin and CIP separately or in combination.Results: 1.The MIC of tannins and CIP to Ab ATCC19606 were 1.95 mg/m L and 0.50 mg/m L respectively. 2. The result of tannins at 1/2 MIC,1/4MIC and 1/8MIC concentration acted on Ab for 4h, 8h, 24 h and 48 h, showed all of them had significant inhibitory effect on the formation of biofilms, while the best effect was at the time of 48h; and when the concentration of tannins increased from 1/4MIC to 1/2 MIC, the Optical density value fell about 2 times. After 48 h, the effects of tannins on biofilms formation increased with the concentration increasely.Which were only a small amount of thin dotted green fluorescence under Fluorescence microscope(FM). 3. After 1/2MIC,1MIC and 2MIC of tannin and CIP separately and joint acted on pre-formed biofilm of Ab for 4h, 8h, 24 h and 48 h, the results showed that the effect of the combination was better than alone,and the best effect was appeared with 2 MIC(tannin + CIP)for 24 h compared with blank group which Optical density values was obviously decrease. And the colony count was also obviously down compared with the blank group:(12±5)×106CFU/m L vs(153±2)×106CFU/m L. It showed that there were only a small amount of thin dotted green fluorescence under FM, but a large number of red fluorescence under CLSM. 4. The result of Real-time fluorescent quantitative PCR showed the relevant expression level of m RNA with Aba I and Bap increased, but the Omp A、Csu E genes’ expression level decreased when treated with tannin and CIP separately or combination on pre-formed biofilm of Ab.Conclusion: 1.The tannin extract from Pomegranate peel had some inhibitory effects on the biofilm formation of Ab, which would be enhanced by increasing the concentration, and it could inhibit above 50% of the biofilm formation. 2. The effect of tannin or CIP separately was weaker than that of the combination on the pre-formed biofilm of Ab. And the combination of them could have some damage and disrupt to the biofilms, of which the combination of 2MIC tannin and 2MIC CIP was the best for removing most of the biofilm. 3.The tannin and CIP may be impact the biofilm fomation by affecting the m RNA expression of Aba I、Bap、Csu E、Omp A gene,which were related to the biofilm formation.