本文選題：銅綠假單胞菌 + 群集運(yùn)動(dòng)　； 參考：《華中科技大學(xué)》2011年博士論文

【摘要】：目的：觀察銅綠假單胞菌pvdQ基因?qū)θ杭\(yùn)動(dòng)(Swarming motility)中抗生素抗性的影響及作用機(jī)制的研究。構(gòu)建銅綠假單胞菌酰基酶編碼基因pvdQ基因的高表達(dá)株和突變株,觀察pvdQ基因?qū)θ杭\(yùn)動(dòng)的影響及在群集運(yùn)動(dòng)條件下對(duì)抗生素抗性的改變及其作用的機(jī)制。 方法：利用電穿孔的方法構(gòu)建pvdQ基因的空質(zhì)粒株、高表達(dá)株和突變株,并用熒光定量PCR比較上述三種菌株在銅綠假單胞菌PAO1野生株中的表達(dá)。將PAO1野生株、pvdQ基因的空質(zhì)粒株、高表達(dá)株和突變株分別置于群集運(yùn)動(dòng)瓊脂培養(yǎng)基中過夜儀培養(yǎng)觀察比較群集運(yùn)動(dòng)直徑的大小。用E試驗(yàn)法測(cè)PAO1野生株和pvdQ基因的高表達(dá)株的MIC值。根據(jù)MIC值將PAO1野生株和pvdQ基因的高表達(dá)株置于含有不同濃度抗生素的培養(yǎng)皿中過夜培養(yǎng),比較兩者群集運(yùn)動(dòng)直徑的大小。將PAO1野生株和高表達(dá)株在電鏡下觀察群集運(yùn)動(dòng)細(xì)胞的形態(tài)。采用結(jié)晶紫染色法,觀察比較PAO1野生株和pvdQ基因的高表達(dá)株培養(yǎng)24h,24h和72h的生物膜的生長(zhǎng)情況。在激光掃描共聚焦顯微鏡F觀察PAO1野生株和pvdQ基因的高表達(dá)株生長(zhǎng)24h,24h和72h時(shí)生物膜的厚度。用熒光染料NPN在熒光分光光度計(jì)中比較PAO1野生株和pvdQ高表達(dá)株對(duì)膜通透性的改變。采用熒光定量PCR法比較PAO1和pvdQ高表達(dá)株多重耐藥外排泵MexAB-OprM的oprM基因和MexGHI-opmD的1nexI基因的表達(dá)。 結(jié)果：熒光定量PCR結(jié)果顯示,成功構(gòu)建pvdQ基因空質(zhì)粒株、高表達(dá)株和突變株。比較四株菌的群集運(yùn)動(dòng)直徑變化：空質(zhì)粒株與PAO1野生株相比較,直徑無顯著變化(P0.05)；突變株與PAO1野生株相比較,直徑減小(P0.05)；高表達(dá)株與PAO1野生株相比較,直徑增大(P0.05)。E試驗(yàn)結(jié)果顯示銅綠假單胞菌對(duì)上述5中抗生素均敏感,而PAO1野生株和pvdQ高表達(dá)株之間差異不明顯。比較PAO1野生株和pvdQ高表達(dá)株兩株菌在含有不同濃度抗生素的培養(yǎng)皿中群集運(yùn)動(dòng)直徑大�。嚎股貪舛瘸杀对黾�,但群集運(yùn)動(dòng)直徑不是成倍下降而是成不規(guī)則增加,說明PAO1野生株和高表達(dá)株均能提高抗生素抗性；PAO1在頭孢他啶濃度8μg/ml,環(huán)丙沙星濃度0.4μg/ml,美羅培南濃度4μg/ml,多粘菌素B濃度16μg/ml,慶大霉素8μg/ml時(shí)群集運(yùn)動(dòng)被抑制；而pvdQ高表達(dá)株是在頭孢他啶濃度32μg/ml,環(huán)丙沙星濃度1.6μg/ml,美羅培南濃度16μg/ml,多粘菌素B濃度64μg/ml,慶大霉素濃度64μg/ml時(shí)群集運(yùn)動(dòng)被抑制。因此,pvdQ高表達(dá)株與PAO1野生株相比抗性提高了2-8倍。電鏡觀察結(jié)果：pvdQ高表達(dá)株分化的群集細(xì)胞與PAO1野生株相比較細(xì)胞變長(zhǎng),核質(zhì)增多。結(jié)晶紫染色的結(jié)果顯示：培養(yǎng)24h PAO1野生株、高表達(dá)株和突變株形成的生物膜厚度差異無統(tǒng)計(jì)學(xué)意義(P0.05),培養(yǎng)48h和72h時(shí)與PAO1野生株相比高表達(dá)株生物膜明顯變薄(P0.05),PAO1野生株與突變株相比較突變株生物膜明顯增厚(P0.05)用激光共聚焦顯微鏡觀察PAO1野生株和pvdQ高表達(dá)株在培養(yǎng)12h,24h和72h時(shí)生物膜的厚度,結(jié)果發(fā)現(xiàn)：72hpvdQ高表達(dá)株較PAO1野生株生物膜明顯變薄。將pvdQ基因與外膜通透性、多重耐藥外排泵基因做了相關(guān)性的研究,研究結(jié)果顯示：pvdQ高表達(dá)株與PAO1野生株相比較,pvdQ高表達(dá)株能夠明顯降低外膜通透性。對(duì)多重耐藥外排泵基因表達(dá)的結(jié)果顯示：oprM基因和mexI基因在群集細(xì)胞中,PAO1野生株和pvdQ高表達(dá)株的表達(dá)明顯高于浮游細(xì)菌細(xì)胞,但群集細(xì)胞之間的比較PAO1野生株和pvdQ高表達(dá)株的oprM基因和mexI基因表達(dá)無明顯差異(P0.05) 結(jié)論：pvdQ高表達(dá)株促進(jìn)群集運(yùn)動(dòng)直徑,突變株則抑制群集運(yùn)動(dòng)直徑,說明pvdQ基因可能參與調(diào)控銅綠假單胞菌PAO1野生株的群集運(yùn)動(dòng)。在群集運(yùn)動(dòng)條件下銅綠假單胞菌pvdQ高表達(dá)株與PAO1野生株相比較能夠?qū)㈩^孢他啶、環(huán)丙沙星、美羅培南、多粘菌素B和慶大霉素的抗性提高2-8倍,細(xì)菌群集運(yùn)動(dòng)被抑制的抗生素濃度均顯著性的高于MIC值。pvdQ基因在浮游細(xì)菌中參與改變抗生素抗性的可能很小,而在群集運(yùn)動(dòng)過程中可能是通過參與群集細(xì)胞的分化,降低外膜通透性來實(shí)現(xiàn)的,但不排除多重耐藥外排泵的協(xié)同參與。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378

【共引文獻(xiàn)】

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