铜绿假单胞菌pvdQ基因对群集运动中抗生素抗性的影响及作用机制的研究

发布时间：2018-04-27 23:53

  本文选题：铜绿假单胞菌 + 群集运动　； 参考：《华中科技大学》2011年博士论文

【摘要】：目的：观察铜绿假单胞菌pvdQ基因对群集运动(Swarming motility)中抗生素抗性的影响及作用机制的研究。构建铜绿假单胞菌酰基酶编码基因pvdQ基因的高表达株和突变株,观察pvdQ基因对群集运动的影响及在群集运动条件下对抗生素抗性的改变及其作用的机制。 方法：利用电穿孔的方法构建pvdQ基因的空质粒株、高表达株和突变株,并用荧光定量PCR比较上述三种菌株在铜绿假单胞菌PAO1野生株中的表达。将PAO1野生株、pvdQ基因的空质粒株、高表达株和突变株分别置于群集运动琼脂培养基中过夜仪培养观察比较群集运动直径的大小。用E试验法测PAO1野生株和pvdQ基因的高表达株的MIC值。根据MIC值将PAO1野生株和pvdQ基因的高表达株置于含有不同浓度抗生素的培养皿中过夜培养,比较两者群集运动直径的大小。将PAO1野生株和高表达株在电镜下观察群集运动细胞的形态。采用结晶紫染色法,观察比较PAO1野生株和pvdQ基因的高表达株培养24h,24h和72h的生物膜的生长情况。在激光扫描共聚焦显微镜F观察PAO1野生株和pvdQ基因的高表达株生长24h,24h和72h时生物膜的厚度。用荧光染料NPN在荧光分光光度计中比较PAO1野生株和pvdQ高表达株对膜通透性的改变。采用荧光定量PCR法比较PAO1和pvdQ高表达株多重耐药外排泵MexAB-OprM的oprM基因和MexGHI-opmD的1nexI基因的表达。 结果：荧光定量PCR结果显示,成功构建pvdQ基因空质粒株、高表达株和突变株。比较四株菌的群集运动直径变化：空质粒株与PAO1野生株相比较,直径无显著变化(P0.05)；突变株与PAO1野生株相比较,直径减小(P0.05)；高表达株与PAO1野生株相比较,直径增大(P0.05)。E试验结果显示铜绿假单胞菌对上述5中抗生素均敏感,而PAO1野生株和pvdQ高表达株之间差异不明显。比较PAO1野生株和pvdQ高表达株两株菌在含有不同浓度抗生素的培养皿中群集运动直径大小：抗生素浓度成倍增加,但群集运动直径不是成倍下降而是成不规则增加,说明PAO1野生株和高表达株均能提高抗生素抗性；PAO1在头孢他啶浓度8μg/ml,环丙沙星浓度0.4μg/ml,美罗培南浓度4μg/ml,多粘菌素B浓度16μg/ml,庆大霉素8μg/ml时群集运动被抑制；而pvdQ高表达株是在头孢他啶浓度32μg/ml,环丙沙星浓度1.6μg/ml,美罗培南浓度16μg/ml,多粘菌素B浓度64μg/ml,庆大霉素浓度64μg/ml时群集运动被抑制。因此,pvdQ高表达株与PAO1野生株相比抗性提高了2-8倍。电镜观察结果：pvdQ高表达株分化的群集细胞与PAO1野生株相比较细胞变长,核质增多。结晶紫染色的结果显示：培养24h PAO1野生株、高表达株和突变株形成的生物膜厚度差异无统计学意义(P0.05),培养48h和72h时与PAO1野生株相比高表达株生物膜明显变薄(P0.05),PAO1野生株与突变株相比较突变株生物膜明显增厚(P0.05)用激光共聚焦显微镜观察PAO1野生株和pvdQ高表达株在培养12h,24h和72h时生物膜的厚度,结果发现：72hpvdQ高表达株较PAO1野生株生物膜明显变薄。将pvdQ基因与外膜通透性、多重耐药外排泵基因做了相关性的研究,研究结果显示：pvdQ高表达株与PAO1野生株相比较,pvdQ高表达株能够明显降低外膜通透性。对多重耐药外排泵基因表达的结果显示：oprM基因和mexI基因在群集细胞中,PAO1野生株和pvdQ高表达株的表达明显高于浮游细菌细胞,但群集细胞之间的比较PAO1野生株和pvdQ高表达株的oprM基因和mexI基因表达无明显差异(P0.05) 结论：pvdQ高表达株促进群集运动直径,突变株则抑制群集运动直径,说明pvdQ基因可能参与调控铜绿假单胞菌PAO1野生株的群集运动。在群集运动条件下铜绿假单胞菌pvdQ高表达株与PAO1野生株相比较能够将头孢他啶、环丙沙星、美罗培南、多粘菌素B和庆大霉素的抗性提高2-8倍,细菌群集运动被抑制的抗生素浓度均显著性的高于MIC值。pvdQ基因在浮游细菌中参与改变抗生素抗性的可能很小,而在群集运动过程中可能是通过参与群集细胞的分化,降低外膜通透性来实现的,但不排除多重耐药外排泵的协同参与。
[Abstract]:Objective: To observe the effect of Pseudomonas aeruginosa pvdQ gene on antibiotic resistance in cluster movement (Swarming motility) and the mechanism of its action. To construct high expression strain and mutant of Pseudomonas aeruginosa acylase encoding gene pvdQ gene, and to observe the effect of pvdQ gene on the cluster movement and to antibiotic resistance under the condition of cluster motion. The mechanism of the change and its effect.
Methods: an empty plasmid strain of pvdQ gene was constructed by electroporation, and the expression of the above three strains in the wild strains of Pseudomonas aeruginosa in the wild strain of Pseudomonas aeruginosa was compared with the fluorescence quantitative PCR. The empty plasmid of the PAO1, the pvdQ gene, the high expression strain and the mutant were placed in the clustered exercise agar medium for the night, respectively. The size of the cluster movement diameter was observed by the instrument. The MIC value of the high expression strain of the PAO1 wild plant and the pvdQ gene was measured by the E test. The high expression strain of the PAO1 wild strain and the pvdQ gene was placed in a Petri dish containing different concentrations of antibiotics on MIC value for the night culture, and the size of the group movement diameter was compared. The wild strain and the high table of the PAO1 were compared. The morphology of the clustered motor cells was observed under the electron microscope. The growth of the biofilms of 24h, 24h and 72h was observed by crystal violet staining, and the growth of the biofilms of 24h, 24h and 72h in the wild strain and the pvdQ gene were observed. The thickness of the membrane in 24h, 24h and 72h of the PAO1 wild strain and pvdQ gene was observed by the laser scanning confocal microscope F. The changes in membrane permeability of PAO1 wild strain and pvdQ high expression strain were compared with fluorescent dye NPN in the fluorescence spectrophotometer. The expression of oprM gene and MexGHI-opmD 1nexI gene of MexAB-OprM in PAO1 and pvdQ high expression strains were compared by fluorescence quantitative PCR method.
Results: the results of fluorescence quantitative PCR showed that the pvdQ gene empty plasmid strain, high expression strain and mutant strain were successfully constructed. Compared with the wild strain of the four strains, the diameter of the empty plasmid was not significantly changed (P0.05), the diameter of the mutant was compared with the wild strain of PAO1 (P0.05), and the high expression strain and the wild strain of the PAO1 strain were compared with that of the wild strain of PAO1. Comparison, the diameter enlargement (P0.05).E test showed that Pseudomonas aeruginosa was sensitive to all 5 of the above antibiotics, but the difference between the PAO1 wild strain and the pvdQ high expression strain was not obvious. Compared with the PAO1 wild strain and the pvdQ high expression strain, the two strains of bacteria in the culture dish containing different concentrations of antibiotics were the size of the cluster motion: the concentration of antibiotics increased doubly. However, the diameter of the clustered motion was not doubly down but increased irregularly, indicating that both the PAO1 wild plant and the high expression strain could improve antibiotic resistance; PAO1 was 8 u g/ml in ceftazidime, 0.4 g/ml in ciprofloxacin, 4 micron g/ml in meropenem, 16 g/ml in the concentration of polymyxin B, and inhibition of cluster motion when gentamicin 8 u g/ml; and pvdQ The high expression strain was 32 mu g/ml in ceftazidime, 1.6 g/ml in ciprofloxacin, 16 micron g/ml in meropenem, 64 mu in the concentration of polymyxin B and 64 micron g/ml in the concentration of gentamicin. Therefore, the resistance of the pvdQ high expression strain to the wild strain of PAO1 was 2-8 times higher. Compared with the wild strain of PAO1, the cells became longer and the nuclear quality increased. The result of crystal violet staining showed that there was no significant difference in the thickness of the biofilm (P0.05) in the cultivation of the 24h PAO1 wild strain and the high expression strain and the mutant strain (P0.05), and the biofilm of the high expression strain of 48h and 72h was obviously thinner (P0.05), PAO1 wild plant and mutation. The biomembrane of the mutant strain was obviously thickened (P0.05) with the laser confocal microscope to observe the thickness of the PAO1 wild strain and the pvdQ high expression strain in the culture of 12h, 24h and 72h. The results showed that the 72hpvdQ high expression strain was obviously thinner than the wild strain of the PAO1, and the pvdQ gene and the membrane permeability, the multidrug resistant efflux pump gene were made. The results showed that the high expression of pvdQ was significantly lower in the outer membrane permeability compared with the PAO1 wild strain, and the expression of the multidrug resistant efflux pump gene showed that the expression of the oprM and mexI genes in the cluster cells, the expression of the PAO1 wild strain and the pvdQ high expression strain was significantly higher than that of the planktonic bacterial cells. However, there was no significant difference in the expression of oprM gene and mexI gene between PAO1 wild strain and pvdQ high expression strain (P0.05).
Conclusion: pvdQ high expression strain promotes the cluster movement diameter, and the mutant strain inhibits the cluster movement diameter, indicating that the pvdQ gene may participate in the control of the cluster movement of the wild strains of Pseudomonas aeruginosa (PAO1). The high expression of Pseudomonas aeruginosa pvdQ strain can be compared with the wild strains of PAO1 under the clustered motion conditions, which can be used to make ceftazidime, ciprofloxacin and meropenem. The resistance of polymyxin B and gentamicin increased by 2-8 times. The concentration of antibiotics inhibited by bacterial colonization was significantly higher than that of MIC value.PvdQ genes involved in the change of antibiotic resistance in planktonic bacteria, which may be achieved by participating in the differentiation of cluster cells and reducing the permeability of the outer membrane during the cluster exercise. However, it does not exclude the synergistic participation of multidrug-resistant efflux pumps.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378

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