呼吸机相关性肺炎新生儿气管导管生物膜菌群及其相互作用研究

发布时间：2018-03-02 12:40

本文选题：VAP　切入点：机械通气新生儿　出处：《重庆医科大学》2016年博士论文　论文类型：学位论文

【摘要】：第一部分呼吸机相关性肺炎新生儿气管导管生物膜菌群分析目的气管导管生物膜是呼吸机相关性肺炎(ventilator-associated pneumonia,VAP)发生的重要危险因素。本研究旨在对机械通气新生儿气管导管生物膜菌群进行分析,以探讨VAP发生相关的生物膜细菌。方法收集重庆医科大学附属儿童医院2014年1月31日至2015年1月31日诊断为呼吸窘迫综合征(neonatal respiratory distress syndrome,NRDS)和肺炎的机械通气48小时以上的新生儿的气管导管标本。对气管导管生物膜细菌DNA进行提取,并扩增16s r RNA基因V3-V4区,进行Miseq测序。将测序得到的原始数据进行拼接、质控获得有效序列,计算覆盖度指数及绘制稀释曲线来评价Miseq测序深度与覆盖度。分析并计算菌群多样性指数,同时根据97%的相似水平将序列划分为不同的可操作分类单元(operational taxonomy unit,OTU),进行OTU分析得到细菌种属信息。结果基于Miseq测序和生物信息学研究发现机械通气新生儿气管导管生物膜均为多种细菌共存,且菌群多样性明显。不同基础疾病的患儿气管导管生物膜菌群多样性及组成存在明显差异(P0.05)。Spearman相关性分析显示气管导管生物膜上链球菌的检出可能与VAP的发生具有相关性(P0.05)。虽然差异没有达到统计学意义(P0.05),但是导管生物膜链球菌的检出者所需机械通气时间更长,白细胞水平更低。结论链球菌属在VAP新生儿气管导管生物膜中检出率明显升高,提示其可能与VAP的发生具有一定相关性。第二部分呼吸机相关性肺炎新生儿气管导管生物膜菌群与咽拭子、导管吸取物菌群关系研究目的出于对患儿安全的考虑,无法取得机械通气过程中的气管导管标本,使得在机械通气过程中特别是怀疑发生VAP时的导管生物膜菌群无法获知,需要寻找其他类型标本来代表导管生物膜菌群。方法同时收集重庆医科大学附属儿童医院2014年1月31日至2015年1月31日诊断VAP的新生儿气管导管、咽拭子及导管吸取物标本。对以上三种类型标本进行细菌DNA提取,扩增16s r RNA基因V3区,进行变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)。根据DGGE图谱以进行菌群多样性及聚类分析。对DGGE条带进行切割克隆测序,将序列与Gen Bank比对,获得细菌种属信息。比较三种类型标本菌群多样性及组成特征。结果合并有VAP的机械通气新生儿导管生物膜细菌多样性与相应的导管吸取物标本类似,而与咽拭子标本存在明显差异(P0.05)。导管生物膜、咽拭子及导管吸取物标本总体细菌构成存在明显差异(P0.05)。导管吸取物标本葡萄球菌的检出能很好的预测其在导管生物膜的存在,灵敏度为85.7%,特异度为83.3%。咽拭子中假单胞菌属的检出对预测其在导管生物膜中的存在有一定的帮助,特别是在早发VAP患者中灵敏度为60.0%,特异度为100%。结论虽然不能完全反映出导管生物膜的菌群情况,但是运用咽拭子及导管吸取物的细菌检测可以帮助预测相应导管生物膜细菌,特别是VAP致病菌如葡萄球菌属和假单胞菌属,所以其在VAP的疾病监控中具有一定的应用价值。第三部分导管生物膜链球菌的分离鉴定及其对铜绿假单胞菌生物膜形成影响的体外研究目的进一步分离及鉴定导管生物膜临床链球菌,并探讨其对VAP常见致病菌铜绿假单胞菌生物膜形成的影响。方法收集重庆医科大学附属儿童医院2014年1月31日至2015年1月31日诊断VAP的新生儿气管导管标本。对气管导管生物膜进行细菌培养,根据菌落形态、革兰染色及接触酶试验初步鉴定出链球菌,进一步提取细菌DNA进行链球菌管家基因扩增并测序。将序列结果与链球菌数据库比对,MEGA6绘制进化树了解临床链球菌种属信息。将其与铜绿假单胞菌PAO1(ATCC BAA-47)进行共培养生物膜,并与铜绿假单胞菌PAO1单独培养的生物膜对比,比较生物膜形成情况及铜绿假单胞菌群体感应系统关键基因las I,las R,rhl,rhl R和藻酸盐形成关键基因agl D的表达。结果在所有导管生物膜标本中,分离到一株链球菌,经过进化树亲缘分析鉴定为血链球菌。其可促进铜绿假单胞菌PAO1生物膜的形成,并可促进铜绿假单胞菌PAO1的生长。临床血链球菌显著上调铜绿假单胞菌PAO1群体感应las及rhl系统关键基因,及藻酸盐合成关键基因的表达(P0.05)。结论临床分离血链球菌可促进VAP致病菌铜绿假单胞菌生物膜的形成,这可能在VAP的致病中起到一定作用。第四部分导管生物膜链球菌临床分离株及铜绿假单胞菌混合培养生物膜对人肺上皮细胞的影响目的探讨临床分离链球菌与铜绿假单胞菌共培养生物膜对人肺上皮细胞活性及炎症因子释放的影响。方法分别将临床分离链球菌和铜绿假单胞菌PAO1共培养生物膜培养液、铜绿假单胞菌PAO1生物膜培养液及临床分离链球菌生物膜的培养液干预BEAS-2B肺上皮细胞(ATCC 9609)。干预24小时后,显微镜下观察不同处理组细胞的变化,CCK-8检测细胞活性,Hochest/PI进行细胞凋亡与坏死染色。并在处理1、3、6、9、12和24小时后分别收集不同生物膜培养液干预组的细胞上清液,ELISA检测IL-8水平。结果临床血链球菌与铜绿假单胞菌共培养生物膜对BEAS-2B细胞的活性损伤明显小于铜绿假单胞菌单独培养生物膜(P0.05)。ELISA检测结果说明,干预3小时后IL-8达到最高峰,且共培养组IL-8水平低于铜绿假单胞菌单独培养生物膜组(P0.05)。结论临床血链球菌可能参与调控宿主对铜绿假单胞菌生物膜感染的固有免疫反应。
[Abstract]:The first part of ventilator-associated pneumonia in the endotracheal tube biofilm flora analysis to endotracheal tube biofilm is ventilator-associated pneumonia (ventilator-associated pneumonia, VAP) an important risk factor. The purpose of this study was to mechanical ventilation for neonatal tracheal catheter biofilm bacteria were analyzed to investigate the occurrence of VAP biofilm bacteria related to children's hospital. Methods from January 31, 2014 to January 31, 2015 affiliated to Medical University Of Chongqing diagnosed respiratory distress syndrome (neonatal respiratory distress syndrome, NRDS) of mechanical ventilation and tracheal catheter were pneumonia 48 hours of newborns. The endotracheal tube biofilm bacterial DNA extraction and R amplification of 16S RNA gene V3-V4 region, Miseq sequencing. The original data will be obtained by sequencing mosaic, quality control effective sequence, calculating coverage index and drawing dilute Release curve to evaluate the Miseq sequencing depth and coverage. The analysis and calculation of the bacterial diversity index, and according to the similarity level of 97% sequence is classified as operational taxonomic units (operational taxonomy unit, different OTU), OTU analysis of bacterial species information. Based on the results of Miseq sequencing and bioinformatics research found mechanical ventilation in neonatal endotracheal tube biofilm were polymicrobial coexistence, and bacterial diversity significantly. Different basic diseases in children with tracheal catheter biofilm bacteria diversity and composition has obvious difference (P0.05).Spearman correlation analysis showed that there is correlation between detection and VAP endotracheal tube biofilm of Streptococcus (P0.05) although the differences did not reach statistical significance (P0.05), but the detection of ductal biofilm Streptococcus required mechanical ventilation longer, lower levels of white blood cells. Conclusion. The detection rate of bacteria was significantly higher in the VAP of neonatal tracheal catheter in biological membranes, suggesting that it may have a certain correlation with the occurrence of VAP. The second part of ventilator-associated pneumonia in neonatal endotracheal tube biofilm bacteria and throat swab, catheter draw object for children with bacteria research group relationship between security considerations, unable to obtain the endotracheal tube specimen machine the process of ventilation, the mechanical ventilation process especially suspected catheter biofilm flora occurred when VAP is not informed, to find other types of samples to represent the catheter biofilm bacteria. Neonatal tracheal catheter method and collected from January 31, 2014 to January 31, 2015 for diagnosis of VAP children's Hospital Affiliated to Medical University Of Chongqing, throat swabs and catheter aspiration specimens for the above three types of specimens for bacterial DNA extraction, 16S amplification of R RNA gene V3 region by denaturing gradient gel electrophoresis (D Enaturing gradient gel electrophoresis, DGGE). According to DGGE patterns for bacterial diversity and cluster analysis. The DGGE strip cutting cloning and sequencing, sequence and Gen Bank comparison, get the species information. Comparison of three types of specimens of flora diversity and composition characteristics. The results with VAP in neonates with mechanical ventilation the catheter biofilm bacteria diversity and corresponding catheter aspiration specimens, and throat swab specimens have obvious difference (P0.05). The catheter biofilm, throat swab and catheter aspiration specimens of bacterial composition has obvious difference (P0.05). The catheter aspiration specimens of Staphylococcus aureus detection can well predict the in the presence of catheter biofilm, the sensitivity was 85.7%, specificity was 83.3%. throat swabs from Pseudomonas detection will help to predict the existence of the catheter in the biofilm, especially in early VAP patients The sensitivity was 60%, specificity was 100%. conclusion although not completely reflect the flora of catheter biofilm, but the use of throat swab and catheter suck bacteria detected can help predict the corresponding catheter biofilm bacteria, especially VAP pathogenic bacteria such as Staphylococcus and Pseudomonas spp., so its application value in disease surveillance in VAP. Objective to study in vitro the third portion of the catheter biofilm of Streptococcus and isolation and identification of Pseudomonas aeruginosa biofilm formation further isolation and identification of clinical catheter biofilm of Streptococcus, and investigate its influence on the formation of common pathogenic bacteria of Pseudomonas aeruginosa biofilm on VAP. Methods a newborn. Samples were collected from January 31, 2014 to January 31, 2015 the catheter diagnosis of VAP children's Hospital Affiliated to Medical University Of Chongqing. The endotracheal tube biofilm were cultured according to the bacteria, Fall morphology, Gram staining and identification of catalase test Streptococcus, further extracting bacterial DNA gene was amplified and sequenced. Streptococcus and Streptococcus results sequence database, MEGA6 phylogenetic tree of Streptococcus species. To understand the clinical information and the Pseudomonas aeruginosa PAO1 (ATCC BAA-47) were co cultured with biofilm that biofilm compared and cultured separately with Pseudomonas aeruginosa PAO1, biofilm formation and Pseudomonas aeruginosa quorum sensing system of key genes Las I, Las R, RHL, R and RHL expression of key genes AGL D alginate formation. Results in all the catheter biofilm samples, isolated strains of Streptococcus through the genetic analysis of the phylogenetic tree, identified as Streptococcus sanguis. It can promote the formation of Pseudomonas aeruginosa biofilm in PAO1, and can promote the growth of Pseudomonas aeruginosa PAO1. Clinical Streptococcus sanguis significantly up-regulated P. The key gene of Pseudomonas PAO1 quorum sensing LAS and RHL system, and the expression of key genes in the synthesis of alginate (P0.05). Conclusion Streptococcus isolated from clinical blood formation can promote VAP pathogenic bacteria Pseudomonas aeruginosa biofilm, which may play a role in the pathogenesis of VAP. The fourth part is the introduction of clinical isolates of Streptococcus tube biofilm strains of Pseudomonas aeruginosa and the effect of mixed culture biofilm on human lung epithelial cells objective to investigate the effect of co culture of biofilm on the release of active and inflammatory factors in human lung epithelial cells in clinical isolates of Streptococcus and Pseudomonas aeruginosa respectively. Methods the clinical isolates of Streptococcus pneumoniae and Pseudomonas aeruginosa biofilm PAO1 co culture medium. The biofilm of Pseudomonas aeruginosa cultured in PAO1 and clinical isolates of Streptococcus biofilm medium intervention BEAS-2B lung epithelial cells (ATCC 9609). After 24 hours of treatment, were observed under the microscope at Changes of cells, CCK-8 cell activity detection, Hochest/PI staining and cell apoptosis and necrosis. In the treatment of 1,3,6,9,12 cell supernatant and 24 hours respectively after collecting different biofilm culture medium in the intervention group, ELISA to detect the level of IL-8. The clinical results of Streptococcus sanguis and Pseudomonas aeruginosa were cultured on BEAS-2B cell membrane damage of biological activity significantly less than that of Pseudomonas aeruginosa biofilm (P0.05).ELISA test results show that, after 3 hours of treatment IL-8 reached the peak, and the co culture group level of IL-8 was lower than that of Pseudomonas aeruginosa biofilm alone group (P0.05). Conclusion the clinical Streptococcus sanguis may be involved in the regulation of the innate immune response of host Pseudomonas aeruginosa biofilm infection.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R722.135

【相似文献】