| Pseudomonas aeruginosa (P.a) is a common opportunistic pathogen, often causes chronic infection in immunocompromised patients. Resistant to many kinds of common antibiotics, so P.a infections can repeat easily, and it is difficult to be removed. P.a can form and maintain biofilms (BF), synthesize and release a variety of virulence factors. The BF formation and virulence synthesis are under the regulation of P.a quorum sensing (QS) system. There is possible relationship between chronic P.a infections and its QS system. Then, inhibition of QS system may be one of the key points to control BF infections. The preliminary studies of my group show that honeysuckle aqueous extracts can inhibit BF formation by P.a in vitro and destruct the BF of P.a in vitro effectively[58]. Chlorogenic acid (CA) is the main active ingredient of honeysuckle. Therefore, in this study, the inhibitory effect of sub-inhibitory concentration of CA on P.a BF in vitro was detected firstly; then, the inhibitory effect of sub-inhibitory concentration CA on the P.a virulence factors (including alginate, elastase, protease, pyoverdine, peroxidase and rhamnolipids) was studied. To understand the mechanism of CA on P.a BF and virulence factors, the level of the AHLs signaling molecule synthesized by P.a were detected after the intervention of sub-inhibitory concentration CA. Lastly, the BF model of intraperitoneal infection in rats was constructed to explore the role of QS system and the inhibitory effect of CA on the P.a BF in vivo.PART I THE INHIBITORY EFFECT AND MECHANISM OF CHLOROGENIC ACID ON BIOFILM OF PSEUDOMONAS AERUGINOSA IN VITROSection I THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON BIOFILM FORMATION OF PSEUDOMONAS AERUGINOSA IN VITROOBJECTIVES: To observe the inhibitory effect of CA on the BF formation of P.a.METHODS: (1) Detect the minimal inhibitory concentration (MIC) of CA and erythromycin (EM) against PAO1. (2) Constructed the static BF model in vitro, and divided them into control group, EM group and CA group. EM (1/8MIC) or CA (1/4MIC) was added at the beginning of making BF models. After cultivated for 3 days and 7 days, SEM was used to observe the form of BF and crystal violet staining was used for semi-quantitative analysis of the P.a BF.RESULTS: (1) The MIC of CA to P.a is 3000ug/ml, EM is 128ug/ml.(2)After cultivated for 3 days, yong BF can be seen in the control group under SEM. The scale of the BF both in the CA group and EM group formed are smaller than the control group. After cultivated for 7 days, there is a large number of mature BF in the control group, but a few of thin BF can be seen both in the CA group and EM group. Whether it is cultivated for 3 days or 7 days, the semi-quantitative BF by crystal violet staining in CA group is less than the control group (P<0.01) and EM group (P<0.05).CONCLUSIONS: CA can inhibit P.a to form BF in vitro,and is more effective than EM.Section II THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON PSEUDOMONAS AERUGINOSA VIRULENCE FACTORSOBJECTIVES: To observe the inhibitory effect of CA on P.a virulence factors.METHODS: (1) Set up the static BF formation model of P.a in vitro. There are three groups: control group, EM group with 1/8 MIC EM and CA group with 1/4 MIC CA in final concentration. (2)Detect the alginate concentration in culture medium after treatment with EM or CA. The LasB elastolytic activity in the EM or CA treated bacterium was determined by using Elastin-Congo red. The protease activity in the bacterium on 3d and 7d of the static BF formation model were determined by using Azo-casein. Chloroform extraction method was used for assaying pyoverdine level in the bacterium of BF formation model in different periods. The H2O2 sensitivity of planktonic bacteria and BF bacteria were determined after EM or CA intervention. The orcinol-sulfuric acid method was used to directly assess the amount of rhamnolipids.RESULTS: The alginate contents in culture medium in the CA group is not only less than the control group (P<0.01), but also less than the EM group (P<0.05). Compared with the control group, the elastase activity of EM group and CA group were significantly lower (P <0.01), CA group were even reduced to the same as with the EM group (P>0.05). In different periods, the protease activity of EM group and CA group, while comparing with the control group, were significantly lower (P<0.01), on 3d and 7d, the protease activity of CA group was higher than that of the EM group (P <0.05). In various periods of BF formation, the pyoverdine level in culture medium were detected, the EM group and CA group were lower than that of the control group (P<0.01), compared with the EM group, the CA group is higher (P<0.05). Before the logarithmic growth phase (4-6h), the planktonic bacteria H2O2 sensitivity was not different between CA group and control group (P>0.05), but after entering the logarithmic growth phase, the bacteria of the CA group was more sensitive to H2O2 than the control group (P<0.05 ); Both young and mature BF bacteria in EM group and CA group were more sensitive to H2O2 than the control group (P<0.05), the young BF bacteria in CA group were more sensitive to H2O2 than EM group (P<0.05), while the maturity, CA group was lower than that of the EM group (P<0.05). After the intervention of EM or CA for 48h and 72, the rhamnolipids in this two group were less than the control group (P<0.01), in 48h the inhibitory effect of EM was better than CA, while in 72h, they had the same effect.CONCLUSIONS: Sub-inhibitory concentration of CA can inhibit the release of a variety of virulence factors of P.a, weak the functions of the QS system. On the inhibition of protease activity, 48h rhamnolipids, pyocyanin and the mature BF bacteria H2O2 sensitivity, CA was weaker than EM, about the elastase, 72h rhamnolipids, CA and EM had the same effect, while to alginate and the young BF bacteria H2O2 sensitivity, CA was more effective than EM. Section III THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON PSEUDOMONAS AERUGINOSA QS SIGNALING MOLECULESOBJECTIVES: To explore the inhibitory effect of CA on p.a QS signaling molecules.METHODS: (1)The QS signaling molecules in the bacteria intervented by EM or CA were extracted by acidified ethyl acetate. (2) AHLs produced by PAO1 were detected by High performance liquid chromatography - mass spectrometry (HPLC-MS), and the signal molecule 3-oxo-C12-HSL, C4-HSL levels were further tested.RESULTS:(1)PAO1 produced two main AHLs signaling molecules: 3-oxo-C12-HSL and C4-HSL, In addition, the content of C4-HSL is higher than in 3-oxo-C12-HSL (P<0.05) in extracellular AHLs. (2) For C4-HSL and 3-oxo-C12-HSL detected, both EM group and CA group were significantly less than the control group (P <0.05), about the 3-oxo-C12-HSL, the inhibitory effect of EM was stronger than CA (P<0.05), while the concentration of C4-HSL, EM group and the CA group had no difference (P> 0.05).CONCLUSIONS: PAO1 can produced two main AHLs signaling molecules: 3-oxo-C12-HSL and C4-HSL; about the extracellular AHLs, the content of C4-HSL is higher than 3-oxo-C12-HSL, the C4-HSL is easier to diffusion through the cell membrane to the extracellular; CA can inhibit the QS system signaling molecule AHLs production, its inhibitory effect of 3-oxo-C12-HSL was weaker than EM, but for the C4-HSL, EM and CA has the same effect. PART II THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON PSEUDOMONAS AERUGINOSA BIOFILM FORMATION IN VIVOSection I THE EFFECT OF QS SYSTEM OF PSEUDOMONAS AERUGINOSA ON BIOFILM FORMATION IN VIVOOBJECTIVE: Construction of peritoneal P.a infection model to explore the role of QS system played on BF formation.METHODS: (1) Preparation: The PAO1 wild strains andΔlasR rhlR deficient strains (PA-JP3) were cultivated in vitro for 20h to make them adhere to the PVC tube carriers'surface. (2) Construction of peritoneal P.a BF infection model: divide 48 wistar female rats randomly into two groups: the PAO1 group and the PA-JP3 group, then implant the carriers into the wistar rats'intra-abdominal to establish the P.a BF infection model. Histopathological observation of local peritoneal tissues inflamation, SEM observation of the BF on the carriers'surface and the viable bacteria counts on the carriers'surface were carried out on the 3rd, 7th and 14th day respectively.RESULTS: The inflammation response of the local peritoneal tissue in PAO1 wild strains group was more serious than the PA-JP3 group. On the day 3, 7 and 14, the BF formation of PAO1 on the carriers'surface were thicker than the PA-JP3 on the SEM. The viable colony count of PAO1 on the carriers'surface was significantly higher than the PA-JP3 (P<0.05), the colonies of PAO1 on the carriers'surface were cleared more slowly than the PA-JP3 group, and even rebounded on day 7(P<0.05).CONCLUSION: With the PVC tube carrier, the peritoneal PAO1 BF infection model was constructed successfully; the QS system plays an important role on the formation of BF in vivo.Section II THE INHIBITORY EFFECT OF CHLOROGENIC ACID ON PSEUDOMONAS AERUGINOSA BIOFILM FORMATION IN VIVOOBJECTIVE: Explore the role of CA played on P.a BF in rat peritoneal infection.METHODS: Choosing the PAO1 wild strains as the experimental strains, 32 female rats were randomly divided into 2 groups: the control group and the CA group. The rats were given CA 40mg/kg ? d (intraperitoneal injections) for 3 or 7 days, starting just after implantation of the PVC tube carrier. The control group was injected with the same amount of sterile saline. Histopathological observation of local peritoneal tissues inflamation, SEM observation of the BF on the carriers'surface and the viable bacteria counts on the carriers'surface were carried out on the 3rd and 7th day respectively.RESULTS: After CA administration, the adhesion of PAO1 on the carriers'surface reduced more significantly than the control group, and got fewer and thinner BF by SEM. Compared with the control group, the local inflammatory reactions of the CA treatment group were significantly reduced. On day 3 and 7, the viable colony counts on the carriers'surface were significantly less than the control group (P <0.05).CONCLUSION: CA can inhibit the P.a BF formation in vivo.
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