细菌生物被膜耐药及形成相关基因调控机制的研究

发布时间：2018-09-09 12:17

【摘要】：目的研究机械通气患者气管导管细菌生物被膜的病原学分布,通过生物被膜菌与相应浮游菌在体外培养的药敏试验进行比较,进而探讨细菌生物被膜对抗生素的耐药性。进一步以临床分离的表皮葡萄球菌生物被膜菌和对应的浮游菌为研究对象,探讨表皮葡萄球菌生物被膜形成相关的基因(操纵子—icaA基因,转录调控子一sigB基因,操纵子调节基因—icaR基因,毒力因子调节系统—agrA基因)的分布及与生物被膜形成的关系。方法 (1)研究了110例施行人工气道(气管插管和气管切开)并机械通气的患者拔除的气管导管,用刚果红培养基筛选出生物被膜菌株和相应的浮游菌株,并对生物被膜菌进行半定量检测,药敏实验分析生物被膜菌株和相应的浮游菌在普通MH培养基上耐药差异以及生物被膜阳性菌在泊洛沙姆(Poloxamer, F-127)培养基和普通MH培养基上耐药性差异进行分析。 (2)收集气管导管中分离的表皮葡萄球菌,按生物被膜阳性表皮葡萄球菌及相应的浮游菌表皮葡萄球菌分为2组。使用RT-PCR分别对生物被膜菌组和浮游菌组细菌的icaA、sigB、icaR、agrA基因辉度比进行检测；利用Real-time PCR分别检测生物被膜菌组和浮游菌组细菌的icaA、sigB、icaR、agrA四种与生物被膜形成相关的基因的表达,并通过2-△△Ct方法进行相对定量。分析表皮葡萄球菌临床菌株中icaA、sigB、icaR、agrA基因转录水平与表皮葡萄球菌生物被膜形成的相关性。结果 (1)110例机械通气患者气管导管中筛选出生物被膜阳性菌61株,阳性率为55.45%。以葡萄球菌属,粪肠球菌及铜绿假单胞菌最常见,生物被膜菌株与相应浮游菌株在普通MH培养基上药敏结果相似,差异无统计学意义(P0.05)；生物被膜菌株在普通MH培养基上和在Poloxamer培养基上药敏结果差异有统计学意义(P0.05)且后者耐药性更强。 (2) RT-PCR中icaA、sigB、icaR、agrA基因的辉度测定值统计分析结果显示生物被膜阳性表皮葡萄球菌及相应的浮游菌表皮葡萄球菌两组细菌间差异无显著性(P0.05),但icaA、sigB基因生物被膜菌组平均秩及秩和明显高于浮游菌组；而icaR、agrA基因生物被膜菌组平均秩及秩和明显小于浮游菌组。 (3) Real-time PCR检测显示表皮葡萄球菌生物被膜菌组icaA, sigB基因表达量分别是浮游菌组的418.8倍与81倍,而icaR, agrA基因在生物被膜菌组中表达量仅为浮游菌组的1/145与1/270。结论 (1)本院机械通气患者气管导管生物被膜病原学分布以葡萄球菌属,粪肠球菌和铜绿假单胞菌为主,生物被膜菌与浮游菌在普通MH培养基上耐药性分析未见明显差异,生物被膜菌在Poloxamer培养基上和在MH培养基上耐药性有明显差异,推测Poloxamer培养基有可能反映生物被膜菌较真实的对药物的耐受情况,其耐受性更强。 (2) icaA、sigB、icaR、agrA基因与表皮葡萄球菌生物被膜形成关系密切,icaA, sigB基因能促进表皮葡萄球菌生物被膜的形成,而icaR, agrA基因能抑制表皮葡萄球菌生物被膜的形成。提示agr系统能调节icaA、sigB、icaR基因的表达,agr系统可以抑制icaA和.sigB这两种基因的表达；促进icaR基因的表达。
[Abstract]:objective
To study the Pathogenic Distribution of bacterial biofilm in tracheal tube of patients with mechanical ventilation, and compare the antibiotic susceptibility test of biofilm bacteria and corresponding plankton bacteria cultured in vitro to explore the antibiotic resistance of bacterial biofilm. Objectives: To investigate the distribution of genes related to biofilm formation of Staphylococcus epidermidis (operon-icaA gene, transcription regulator sigB gene, operon regulator-icaR gene, virulence factor regulator-agrA gene) and their relationship with biofilm formation.
Method
(1) 110 patients with artificial airway (tracheal intubation and tracheotomy) and mechanical ventilation were studied. Biofilm strains and corresponding plankton strains were screened by Congo red medium. Semi-quantitative detection of biofilm bacteria was carried out. Drug susceptibility test showed that biofilm strains and corresponding plankton bacteria were cultured in MH. The difference of drug resistance in culture medium and the difference of drug resistance of biofilm-positive bacteria in Poloxamer (F-127) medium and MH medium were analyzed.
(2) Staphylococcus epidermidis isolated from tracheal cannula was divided into two groups according to biofilm-positive Staphylococcus epidermidis and corresponding planktonic bacteria. The expression of icaA, sigB, icaR and agrA genes in S. epidermidis and S. epidermidis was quantified by 2-delta CT. The correlation between the transcription levels of icaA, sigB, icaR and agrA genes and the biofilm formation of S. epidermidis was analyzed.
Result
(1) 61 strains of biofilm-positive bacteria were screened out from 110 patients with mechanical ventilation, and the positive rate was 55.45%. Staphylococcus, Enterococcus faecalis and Pseudomonas aeruginosa were the most common strains. The drug sensitivity of biofilm-positive bacteria was similar to that of corresponding planktonic bacteria on the common MH medium, but the difference was not statistically significant (P 0.05). The results of susceptibility test on MH medium and Poloxamer medium were significantly different (P 0.05), and the latter was more resistant.
(2) Statistical analysis of the luminosity of icaA, sigB, icaR, agrA genes in RT-PCR showed that there was no significant difference between the two groups (P 0.05), but the average rank and rank of icaA, sigB gene biofilm group were significantly higher than those of plankton group. The average rank and rank of the membranous bacteria group were significantly lower than that of the planktonic bacteria group.
(3) Real-time PCR showed that the expression levels of icaA and sigB genes in S. epidermidis biofilm group were 418.8 and 81 times higher than those in plankton group, while the expression levels of icaR and agrA genes in biofilm group were only 1/145 and 1/270 of that in plankton group.
conclusion
(1) Staphylococcus, Enterococcus faecalis and Pseudomonas aeruginosa were the main pathogens of tracheal tube biofilm in patients with mechanical ventilation in our hospital. There was no significant difference in drug resistance between biofilm bacteria and plankton bacteria on ordinary MH medium. The drug resistance of biofilm bacteria on Poloxamer medium and MH medium was significantly different. Poloxamer medium may reflect the true tolerance of biofilm bacteria to drugs, and it is more tolerant.
(2) icaA, sigB, icaR and agrA genes are closely related to the biofilm formation of S. epidermidis. icaA and sigB genes can promote the biofilm formation of S. epidermidis, while icaR and agrA genes can inhibit the biofilm formation of S. epidermidis. It suggests that AGR system can regulate the expression of icaA, sigB and icaR genes, and agr system can inhibit the formation of icaA and si. GB expression of these two genes promotes icaR gene expression.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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