| Antibiotics plays a significant role in the process of resistant bacterial infection.But as a result of abuse the antibiotics, cause a serious problem of bacterial resistance.So we need to find new ways and new targets, Quorum sensing inhibitor(QSI) inhibit transfering information of signaling molecule, regulating target genes and affecting virulence factor and other secondary metabolites. Pseudomonas aeruginosa have a strong infection power which serious threaten to human health. The Quorum sensing(QS) system regulating the virulence factors and the formation of biofilms. This study utilize the screening systems of gram-negative bacteria with QS, to screening effect QSI from extracts of Chinese medicine and studying on the QS effect and mechanism of Pseudomonas aeruginosa.Through the QS reporter bacteria Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136 monitor QS inhibitory activity. Measure and comparison diameter of bacteriostatic circle and pigment circle on report plate, By MIC method to detect the drugs, minimal inhibitory concentrations on two biosensor strains, drug concentration gradient is equal or lesser than MIC. Ultimately we regard Plantain Herb as a potential QSI, and effective concentration range of MIC-1/4MIC(63ug/mL-16ug/mL).Because of the Plantain extracts contain a large of tannic acid and ester, etc. The CV026 biosensor screen components on report plate after extraction and purified by silica gel column. Discovering the PE12, Plantain ethyl acetate parts, is a potential QSI components.Determined MIC of PE12 to Pseudomonas aeruginosa PA01, and be selected the MIC,1/2 MIC, 1/4 MIC(16 ug/mL, 8 ug/mL, 4 ug/mL) as treatment groups. Treatment group and the control group are detected by MTT method on affecting growth curve of PA01, and then applied to QS virulence factors such as pyocyanin, alginate, rhamnose, proteolytic enzymes and oxidative stress. Laboratory finding that the MIC was significantly inhibit the growth of PA01 and virulence factor. 8ug/mL MIC has no affect on bacteriostasis, but inhibit the action of the virulence factors. For 1, 5, 7 day, Pyocyanin production of 8 ug/mL drug group respectively inhibits 8.33%, 3.10%, 11.01%, 7.14% than control group,P<0.05, same as the function of 16 ug/mL drug group,P>0.05; Production of alginate hc-positie 1,3,5,7 day, 8 ug/mL drug group respectively inhibits 92.15%, 89.41%, 70.28%,46.73% than control group, P<0.01, similar to the 16 ug/mL drug group, P>0.05; Rhamnose content within 24 h and 72 h test respectively, 8 ug/mL drug group respectively inhibits60.96%, 43.66% than the control group, P<0.01, the effect of 8ug/mL less than 16 ug/mL drug group,P<0.05; Exotoxin A in 5.5h and 24 h, 8ug/mL drug group respectively decreased 39.58%, 60.09% than the control group, P<0.01, close to the 16 ug/mL drug groups, P>0.05.In 4 ug/mL drug concentration, the inhibition decreased significantly, only the rhamnose, and pseudomonas bacteria known as part of the role.Examining three concentrations of PE12 affect biofilm formation in 7 days. by semi-quantitative method, silver staining and scanning electron microscopy(SEM) method.Results show that the 16ug/mLcan inhibit bacteria and the formation of bio-film, 8ug/mL drug gruop can inhibit bio-film, For 1,3,5,7 day, 8 ug/mL drug group respectively inhibits37.32%, 51.89%, 67.60%, 35.38% than control group, P<0.01。the control group with 8ug/mL drugs Group for the semi-quantitative method in 96 h, 8 ug/mL drug concentrations significantly enhanced inhibit biofilm formation at 42 h, 8 ug/mL drug group than the control group decreased from(1.95±0.06) to(1.22±0.1), P < 0.01.bacteria is dispersed and Rarely gathered, emplastic is relatively thin under microscope and SEM. 1/4(4ug/mL)MIC and the control group, large numbers of bacteria are linked together and the gunk bio-films is clearly visible around cloud.We select MIC and 1/2 MIC of PE12 as the experimental group, and extract total RNAto reverse transcription, then detect by real-time fluorescent quantitative PCR. Monitoring the change of related gene lasR, LasI and rhlR. Results show that the 1/2 MIC(8ug/mL) of signal molecules synthetase lasI gene has significant inhibitory effect,8 ug/mL drug group inhibits 38.02% than the control group,P<0.01. 8 ug/mL drug group has 47.93% inhibitory effect on las R gene. 8 ug/mL drug group has 25.76% inhibitory effect on rhl R gene. similar to the 16 ug/mL drug group, P<0.01.This study has established the complete biological methods to screening QSI. And obtained Plantain PE12 which has an obvious effect of QS inhibition. It can inhibit pseudomonas aeruginosa PA01 QS that associated several virulence factors and biofilm at8ug/ml concentration. The mechanism of PE12 inhibiting effect is mainly affected the lasI gene. This research has an significant application prospect and innovative theory, we explore new targets for bacterial resistance and provide the systematic idea and approach for anti-bacterial resistance. |
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