Intervention Effect Of BHL DNA Aptamer On Pseudomonas Aeruginosa Quorum Sensing

Objective Our study aims to research on BHL DNA aptamer if it can effectively interfere the Pseudomonas aeruginosa quorum sensing(QS)then inhibit of biofilm formation and pyocyanin production in vitro.Method 1 Determine if Pseudomonas aeruginosa growth curve could be effect by the BHL DNA aptamer with high concentration in vitro. 2 Interfere the formation of Pseudomonas aeruginosa biofilm in vitro : interfere the Pseudomonas aeruginosa biofilm with high concentration of BHL DNA aptamer in vitro;Optical microscope was used to observe the biofilm morphology; Crystal violet staining method was used for quantitative determination of biofilm. 3 Interfered the generation of pyocyanin in vitro: divided into three groups: aptamer group 、 negative control group and random pool control group. Chloroform extraction method was used for Pseudomonas aeruginosa pyocyanin assay after 48 h in culture. 4 e DNA and aptamer were collaborating on biofilm :Lithium chloride pyrolysis method was used for Pseudomonas aeruginosa genomic DNA extraction; divided into four groups: aptamer group、negative control group、g DNA group and aptamer+g DNA group. After cultured for 48 h,crystal violet staining method was used for quantitative determination of biofilm.Result 1 Growth curve of Pseudomonas aeruginosa with a high concentration of BHL DNA aptamer in the culture was highly consistent with the one that did not contain the aptamer.That means the aptamer did not appear to inhibit or promote the proliferation of bacteria. 2 Interfere the formation of Pseudomonas aeruginosa biofilm in vitro : optical microscope observation of the negative control and random pool control showed large dense membrane structure.But the aptamer group 1,2,16,46 showed a single cell distribution or loose network structure. The growth of biofilm was almost destroyed by the aptamer 16. Quantitative determination of biofilm by crystal violet staining showed both the negative control group and the random pool group were higher than the value of aptamer group(P < 0.05); but no significant difference between the negative control group and the random pool group. 3 Interfered the generation of pyocyanin in vitro : the pyocyanin value of the aptamer group was lower than the negative control group and the random pool group(P < 0.05), but no significant differences between the negative control group and the random pool group. 4 e DNA and aptamer were collaborating on biofilm:the negative control group was higher than the aptamer group in biofilm quantitative determination(P < 0.05), but lower than the g DNA and g DNA+aptamer group(P<0.05); aptamer group determination value was lower than the group of g DNA and g DNA+aptamer group(P < 0.05); the g DNA group was higher than the g DNA+aptamer group(P < 0.05).Conclusion 1 The BHL DNA adapter does not affect the growth of bacteria. 2 The BHL DNA adapter can effectively interfere on the formation of Pseudomonas aeruginosa biofilm in vitro. 3 The BHL DNA aptamer can effectively inhibit Pseudomonas aeruginosa pyocyanin biosynthesis in vitro. 4 BHL DNA aptamer can weaken the e DNA’s role in promoting formation of biofilm;it can still inhibit the formation of biofilm in a certain extent when coexistence with e DNA.