肺炎克雷伯菌耐药分析及其intI1在生物被膜中转录水平研究

发布时间：2018-06-30 03:12

本文选题：肺炎克雷伯菌 + 整合酶　；参考：《大连医科大学》2010年硕士论文

【摘要】： 目的：肺炎克雷伯菌(Klebsiella Pneumoniae,KP)是临床上常见的条件致病菌,具有较高的耐药性。了解肺炎克雷伯菌耐药现状,为临床治疗提供指导；整合子是一种可以移动的基因元件,通过位点特异的基因重组在临床细菌耐药性传播中起重要作用。了解Ⅰ类整合子在肺炎克雷伯菌中的分布状况,分析它在多重耐药中的作用；细菌生物被膜是细菌附着在有生命或无生命的材料表面后,由细菌及其所分泌的胞外多聚物(主要是胞外多糖)共同组成的呈膜状的细菌群体。研究表明,生物被膜菌的性质与浮游菌有所不同,它可以增加对抗生素的抵抗能力。肺炎克雷伯菌形成生物膜是其重要的毒力因子,检测肺炎克雷伯菌Ⅰ类整合酶基因在生物被膜状态和浮游状态表达水平的差异,探讨整合子的作用机制。方法：收集肺炎克雷伯菌共123株,药敏实验了解其耐药情况,设计合成Ⅰ类整合酶、Ⅰ类整合子可变区的上下游引物,煮沸法提取细菌总DNA作为模板,整合子可变区PCR扩增产物纯化后测序,所得序列在Genbank数据库进行比对分析。将Ⅰ类整合酶阳性的肺炎克雷伯菌分别进行普通液相培养和生物被膜培养,提取各菌株两种状态下的总RNA。通过RT-PCR半定量方法,以细菌的16sRNA为内参照,分别测定整合酶基因在生物被膜状态和浮游状态下的各自表达量。结果：1.收集的肺炎克雷伯菌株中,经过药敏实验发现其对β-内酰胺类、喹诺酮类、氨基糖苷类、磺胺类药物抗生素有较高的耐药率：哌拉西林(100%)、头孢曲松(76%)、左氧氟沙星(60%)、妥布霉素(60%)、复方新诺明(63%),所有菌株均对美罗培南敏感。 2.Ⅰ类整合酶基因在临床分离的肺炎克雷伯菌中较为常见,123株细菌中检出Ⅰ类整合酶阳性菌47株,检出率为38.2%。Ⅰ类整合子阳性菌株对8种抗菌药物的耐药率与阴性菌株有统计学差异(x2检验,P＜0.05)。47株整合酶阳性菌株,进行Ⅰ类整合子可变区扩增,其中23株有阳性扩增产物,阳性扩增产物片段大小含680bp,1600bp,2400bp三类。主要为aadA2和dfrA12基因,aadA2基因编码链霉素3'-腺苷酰基转移酶,介导对链霉素和壮观霉素抗药性；dfrA12基因编码蛋白介导甲氧苄氨嘧啶的抗药性。dfrA12-orfF-aadA2是最常见的基因盒排列方式。 3.试验检测到肺炎克雷伯浮游菌和生物被膜菌均有intⅠ1的表达。菌株在两种状态下intⅠ1 mRNA的表达量不同,生物膜细菌intⅠ1基因的表达水平较高。在普通液态培养组Ⅰ类整合酶基因表达量为0.08±0.05,而在生物被膜状态组Ⅰ类整合酶基因表达量则为0.28±0.004。生物膜状态整合酶基因表达量约为浮游状态的3倍。结论：药敏实验证实KP临床菌株对β-内酰胺类、喹诺酮类、氨基糖苷类、磺胺类药物抗生素有较高的耐药率。Ⅰ类整合子在临床分离的肺炎克雷伯菌株中广泛存在(整合酶检出率38.2%),耐药情况较为严峻(16种抗生素中有9种耐药率超50%)。整合子中的耐药基因盒与其抗药性密切相关。Ⅰ类整合酶基因在生物被膜状态下mRNA转录水平上调,提示肺炎克雷伯菌生物被膜的形成有利于整合子及相关耐药基因的表达,整合子在生物膜细菌中可进行更加活跃的基因盒捕获和积累,整合子和细菌生物膜的形成是导致细菌对抗生素耐药的重要机制。
[Abstract]:Objective: Klebsiella Pneumoniae (KP) is a common clinical pathogen with high drug resistance. Understanding the current drug resistance of Klebsiella pneumoniae to provide guidance for clinical treatment. The integron is a kind of mobile gene element that can be used in the spread of clinical bacterial resistance through gene recombination in loci. Important role. To understand the distribution of class I integrons in Klebsiella pneumoniae, and to analyze its role in multidrug resistance; bacterial biofilm is a membrane like bacterial colony formed by bacteria and their secreted extracellular polymers (mainly extracellular polysaccharides) after they are attached to the surface of life or inanimate materials. The study shows that the nature of biofilm is different from that of planktonic bacteria. It can increase the resistance to antibiotics. Klebsiella pneumoniae is an important virulence factor to detect the difference of the level of the integrase gene of Klebsiella pneumoniae type I integrase gene in the biofilm state and the floating state, and to explore the mechanism of the integron.
Methods: 123 strains of Klebsiella pneumoniae were collected and the drug sensitivity test was used to understand the drug resistance. The first class integrase was synthesized, the upper and downstream primers of class I integron variable region were primed, the total DNA of bacteria was extracted by boiling method as a template, and the PCR amplification product of the integron variable region was purified and sequenced. The sequence of the obtained sequence was compared and analyzed in the Genbank database. The co enzyme positive Klebsiella pneumoniae were cultured in common liquid phase and biofilm culture respectively. The total RNA. in two states of each strain was extracted by RT-PCR semi quantitative method, and the 16sRNA of bacteria was used as internal reference to determine the respective expression of integrase gene in the state of biofilm and floating state respectively.
Results: 1. of the klebber strains collected, the drug sensitivity tests showed that they had high resistance rates to beta lactam, quinolones, aminoglycosides, sulfonamide antibiotics: piperacillin (100%), ceftriaxone (76%), levofloxacin (60%), tufomycin (60%), and compound novamamine (63%). All strains were sensitive to meropenem. Feeling.
2. class I integrase gene was more common in clinical isolates of Klebsiella pneumoniae, and 47 strains of integrase positive bacteria were detected in 123 bacteria. The detection rate was that the resistance rate of 38.2%. type I integron positive strains to 8 kinds of antibiotics and negative strains were statistically different (x2 test, P < 0.05).47 strain integrase positive strain, and carried out class I whole The mutable region of the zygote was amplified, 23 of which had positive amplification products, the size of positive amplification products contained 680bp, 1600bp, 2400bp three, mainly aadA2 and dfrA12 gene, aadA2 gene encoding streptomycin 3'- adenosine acyltransferase, mediating resistance to streptomycin and splendenin; dfrA12 gene encoding protein mediated methoxyl pyrimidine. Sex.DfrA12-orfF-aadA2 is the most common way of gene cassette arrangement.
The 3. test detected the expression of int i 1 of Klebsiella pneumoniae and biofilm bacteria. The expression of int i 1 mRNA was different in two states, and the expression level of int i 1 gene was higher in the biofilm bacteria. The expression of class I integrase gene was 0.08 + 0.05 in the ordinary liquid culture group, and the type I integrase in the biofilm state group The expression level of gene was 0.28 + 0.004., and the expression of integrase gene in biofilm was 3 times that of planktonic state.
Conclusion: the drug sensitivity test confirmed that KP clinical strains have high resistance to beta lactam, quinolones, aminoglycosides, sulfonamide antibiotics. Class I integron exists widely in clinical isolates of Klebsiella pneumoniae (integrase detection rate 38.2%), and the drug resistance is severe (9 kinds of antibiotics among 16 kinds of antibiotics are over 50%). The resistance gene box in the zygote is closely related to its resistance to drug resistance. Class I integrase gene is up-regulated at the mRNA transcriptional level in the biofilm state, suggesting that the formation of Klebsiella pneumoniae biofilm is beneficial to the expression of integrons and related resistance genes, and the integron can capture and accumulate more active gene boxes in biofilm bacteria. The formation of zygote and bacterial biofilm is an important mechanism leading to the antibiotic resistance of bacteria.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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