臨床感染持留菌的篩選及其感染免疫機(jī)制研究
發(fā)布時(shí)間：2019-06-25 15:20
【摘要】：持留菌(Persisters)是存在于某個(gè)細(xì)菌群體中的表型異化小亞群。可以耐受致死濃度的抗菌藥物,國內(nèi)外學(xué)者認(rèn)為其與慢性感染的反復(fù)發(fā)作及細(xì)菌的生物被膜感染等密切相關(guān)。肺炎克雷伯菌是重要的醫(yī)院和社區(qū)獲得性革蘭陰性機(jī)會(huì)致病菌,可導(dǎo)致患者發(fā)生尿路感染、肺炎、菌血癥及肝膿腫等多種疾病。本課題針對(duì)重要的臨床耐藥問題,利用實(shí)驗(yàn)室感染病原樣本庫,從建立微生物快速診斷技術(shù)和臨床樣本持留菌篩選入手,研究頑固性難治性感染相關(guān)的持留菌的感染免疫調(diào)控機(jī)制。通過觀察持留菌流行病學(xué)特征和持留特性,從感染免疫角度闡述單核巨噬細(xì)胞與持留菌之間的相互作用,解釋免疫逃避機(jī)制,為免疫干預(yù)治療提供理論依據(jù)。第一部分臨床細(xì)菌感染現(xiàn)狀分析及分子診斷檢驗(yàn)本部分研究對(duì)臨床細(xì)菌感染現(xiàn)狀進(jìn)行分析,對(duì)醫(yī)院相關(guān)感染進(jìn)行流行病學(xué)調(diào)查;分析耐藥的分子基礎(chǔ),形成分子診斷方案,為臨床感染控制提供預(yù)警。建立了基于多重PCR體系的主要耐藥菌快速鑒定和耐藥機(jī)制檢測(cè)的分子生物學(xué)方法,可以很好的作為傳統(tǒng)微生物檢驗(yàn)方法的補(bǔ)充,有利于對(duì)臨床耐藥菌株進(jìn)行耐藥機(jī)制的系統(tǒng)分析和調(diào)查,更好地為感染防控提供依據(jù)。第二部分肺炎克雷伯桿菌持留株的篩選及特性研究本部分研究利用本院建立的菌株樣本庫,對(duì)臨床菌株進(jìn)行了持留菌的篩選并對(duì)其持留特性和耐藥性進(jìn)行了研究。利用生長曲線法,共篩選獲得51株持留菌,其中26株(48.9%)為肺炎克雷伯桿菌。26株肺炎克雷伯桿菌持留株全部產(chǎn)ESBLs,對(duì)氨芐西林100%耐藥。對(duì)哌拉西林/他唑巴坦等耐藥率均超過75%,對(duì)環(huán)丙沙星的耐藥率為60.8%。隨環(huán)丙沙星濃度升高,持留菌比例下降,甚至在50MIC濃度時(shí)可以實(shí)現(xiàn)對(duì)持留菌的殺滅。這一現(xiàn)象提示我們,不能通過判讀臨床常規(guī)藥敏試驗(yàn)結(jié)果來區(qū)分持留菌,持留菌由于缺乏明確的特異標(biāo)志物而難以嚴(yán)格區(qū)分和鑒定,這是持留菌研究中的一個(gè)難點(diǎn)和值得挖掘的研究切入點(diǎn);此外,雖然體外實(shí)驗(yàn)中抗生素濃度達(dá)到一定值時(shí)可以殺滅持留菌,但實(shí)際上體內(nèi)不可能達(dá)到這樣的抗生素濃度,靠加大抗生素劑量的方法治療持留菌感染行不通,因而,擬進(jìn)一步探討持留菌的持留機(jī)制或者其感染免疫機(jī)制,以期為解決這一臨床問題提供理論依據(jù)。第三部分肺炎克雷伯菌持留株-單核巨噬細(xì)胞間相互作用及機(jī)制研究針對(duì)前期篩選出的臨床持留菌株,從感染免疫角度闡述單核巨噬細(xì)胞與持留菌之間的相互作用。首先通過體外實(shí)驗(yàn)發(fā)現(xiàn)單核細(xì)胞對(duì)持留菌的吞噬作用降低,進(jìn)一步通過芯片檢測(cè)、生物信息學(xué)分析和實(shí)驗(yàn)驗(yàn)證來確定單核巨噬細(xì)胞中免疫相關(guān)基因的表達(dá)及其效應(yīng)信號(hào)通路。發(fā)現(xiàn)持留菌感染單核巨噬細(xì)胞,可以誘導(dǎo)SOCS-1蛋白在感染初期即大量表達(dá),且部分依賴于TLR4信號(hào)通路;SOCS-1蛋白可以下調(diào)MAL的水平進(jìn)而影響TLR4下游信號(hào)通路的活化,降低TNF-α、IL-6、IL-1β等炎癥因子的分泌。證明肺炎克雷伯菌持留株感染可以通過誘導(dǎo)產(chǎn)生的SOCS-1負(fù)調(diào)控炎癥因子的表達(dá),從而逃逸宿主免疫攻擊。本課題從感染免疫角度闡述單核巨噬細(xì)胞與持留菌之間的相互作用,探討了持留菌的免疫逃避機(jī)制,為免疫干預(yù)治療提供了理論依據(jù)。
[Abstract]:Persorters are a small subset of the phenotype that is present in a certain bacterial population. The anti-bacterial drug with lethal concentration can be tolerated, and the domestic and foreign scholars believe that it is closely related to the repeated attack of the chronic infection and the biological membrane infection of the bacteria. Klebsiella pneumoniae is an important hospital and community-acquired Gram-negative opportunistic pathogen, which can lead to a variety of diseases such as urinary tract infection, pneumonia, bacteremia, and liver abscess. Aiming at the important clinical drug-resistance problem, using the laboratory-infected pathogen sample library, the paper starts with the establishment of the micro-organism rapid diagnosis technology and the screening of the clinical sample holding-keeping bacteria, and studies the infection-immune regulation mechanism of the bacteria-retaining bacteria associated with the intractable intractable infection. In order to provide the theoretical basis for the treatment of immune intervention, by observing the epidemiological characteristics and the holding characteristics of the retained bacteria, the interaction between the mononuclear macrophages and the retaining bacteria is described from the angle of infection, and the immune escape mechanism is explained. The present situation of clinical bacterial infection in the first part and the analysis of the present part of the molecular diagnosis and examination of the present part of the clinical bacteria infection are analyzed, and the epidemiological investigation on the infection of the hospital is carried out. The molecular basis of the drug resistance is analyzed, and the molecular diagnosis scheme is formed, and the early warning is provided for the control of clinical infection. A molecular biological method based on the rapid identification of the main drug-resistant bacteria and the detection of the drug resistance mechanism based on the multiple PCR system is established, which can be used as a supplement to the traditional microbial test method, and is beneficial to the system analysis and investigation of the drug-resistant mechanism of the clinical drug-resistant strain, And provides a basis for better prevention and control of infection. The screening and characterization of the second part of Klebsiella pneumoniae (Klebsiella pneumoniae) were studied in this part. The strain samples library established in our hospital were used to screen and study the retention characteristics and drug resistance of the strains. The results showed that 26 strains (48.9%) were Klebsiella pneumoniae and 26 strains (48.9%) were Klebsiella pneumoniae.26 strains of Klebsiella pneumoniae were all ESBLs, which were resistant to 100% of methicillin. The drug resistance rate was over 75%, and the resistance rate of ciprofloxacin to ciprofloxacin was 60.8%. With the increase of the concentration of ciprofloxacin, the proportion of the retained bacteria is decreased, and the killing of the retaining bacteria can be realized even at the concentration of 50 MIC. This phenomenon suggests that we can not distinguish the remaining bacteria from the results of the clinical routine drug sensitivity test, which is difficult to distinguish and identify by the lack of specific specific markers, which is a difficult and worthwhile research entry point in the study of retained bacteria; in addition, Although the concentration of the antibiotic in the in vitro experiment reaches a certain value, the stay-keeping bacteria can be killed, It is proposed to study the retention mechanism or the immune mechanism of the retained bacteria in order to provide a theoretical basis for the solution of this clinical problem. The interaction and mechanism of the third part of klebsiella pneumoniae (klebsiella pneumoniae)-mononuclear macrophages (kleklebsiella) on the pre-screened clinical retention strains, the interaction between the mononuclear macrophages and the retaining bacteria was described from the angle of infection. First, by in vitro experiments, the phagocytosis of the mononuclear macrophages was reduced, and the expression of the immune-related genes in the mononuclear macrophages and their effect signal pathways were further determined by chip detection, bioinformatics analysis and experimental verification. It is found that the SOCS-1 protein can be expressed in the early stage of infection, and the part of the SOCS-1 protein is dependent on the TLR4 signal pathway. The SOCS-1 protein can down-regulate the level of the MAL and then influence the activation of the signal pathway downstream of the TLR4, and the secretion of the inflammatory factors such as TNF-1, IL-6, IL-1 and the like can be reduced. It is proved that the infection of the strain of Klebsiella pneumoniae can control the expression of the inflammatory factor by inducing the generated SOCS-1 to escape the host immune attack. In this paper, the interaction between the mononuclear macrophages and the retaining bacteria is described from the angle of infection, and the immune escape mechanism of the retaining bacteria is discussed, which provides a theoretical basis for the treatment of immune intervention.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R515
本文編號(hào)：2505786
[Abstract]:Persorters are a small subset of the phenotype that is present in a certain bacterial population. The anti-bacterial drug with lethal concentration can be tolerated, and the domestic and foreign scholars believe that it is closely related to the repeated attack of the chronic infection and the biological membrane infection of the bacteria. Klebsiella pneumoniae is an important hospital and community-acquired Gram-negative opportunistic pathogen, which can lead to a variety of diseases such as urinary tract infection, pneumonia, bacteremia, and liver abscess. Aiming at the important clinical drug-resistance problem, using the laboratory-infected pathogen sample library, the paper starts with the establishment of the micro-organism rapid diagnosis technology and the screening of the clinical sample holding-keeping bacteria, and studies the infection-immune regulation mechanism of the bacteria-retaining bacteria associated with the intractable intractable infection. In order to provide the theoretical basis for the treatment of immune intervention, by observing the epidemiological characteristics and the holding characteristics of the retained bacteria, the interaction between the mononuclear macrophages and the retaining bacteria is described from the angle of infection, and the immune escape mechanism is explained. The present situation of clinical bacterial infection in the first part and the analysis of the present part of the molecular diagnosis and examination of the present part of the clinical bacteria infection are analyzed, and the epidemiological investigation on the infection of the hospital is carried out. The molecular basis of the drug resistance is analyzed, and the molecular diagnosis scheme is formed, and the early warning is provided for the control of clinical infection. A molecular biological method based on the rapid identification of the main drug-resistant bacteria and the detection of the drug resistance mechanism based on the multiple PCR system is established, which can be used as a supplement to the traditional microbial test method, and is beneficial to the system analysis and investigation of the drug-resistant mechanism of the clinical drug-resistant strain, And provides a basis for better prevention and control of infection. The screening and characterization of the second part of Klebsiella pneumoniae (Klebsiella pneumoniae) were studied in this part. The strain samples library established in our hospital were used to screen and study the retention characteristics and drug resistance of the strains. The results showed that 26 strains (48.9%) were Klebsiella pneumoniae and 26 strains (48.9%) were Klebsiella pneumoniae.26 strains of Klebsiella pneumoniae were all ESBLs, which were resistant to 100% of methicillin. The drug resistance rate was over 75%, and the resistance rate of ciprofloxacin to ciprofloxacin was 60.8%. With the increase of the concentration of ciprofloxacin, the proportion of the retained bacteria is decreased, and the killing of the retaining bacteria can be realized even at the concentration of 50 MIC. This phenomenon suggests that we can not distinguish the remaining bacteria from the results of the clinical routine drug sensitivity test, which is difficult to distinguish and identify by the lack of specific specific markers, which is a difficult and worthwhile research entry point in the study of retained bacteria; in addition, Although the concentration of the antibiotic in the in vitro experiment reaches a certain value, the stay-keeping bacteria can be killed, It is proposed to study the retention mechanism or the immune mechanism of the retained bacteria in order to provide a theoretical basis for the solution of this clinical problem. The interaction and mechanism of the third part of klebsiella pneumoniae (klebsiella pneumoniae)-mononuclear macrophages (kleklebsiella) on the pre-screened clinical retention strains, the interaction between the mononuclear macrophages and the retaining bacteria was described from the angle of infection. First, by in vitro experiments, the phagocytosis of the mononuclear macrophages was reduced, and the expression of the immune-related genes in the mononuclear macrophages and their effect signal pathways were further determined by chip detection, bioinformatics analysis and experimental verification. It is found that the SOCS-1 protein can be expressed in the early stage of infection, and the part of the SOCS-1 protein is dependent on the TLR4 signal pathway. The SOCS-1 protein can down-regulate the level of the MAL and then influence the activation of the signal pathway downstream of the TLR4, and the secretion of the inflammatory factors such as TNF-1, IL-6, IL-1 and the like can be reduced. It is proved that the infection of the strain of Klebsiella pneumoniae can control the expression of the inflammatory factor by inducing the generated SOCS-1 to escape the host immune attack. In this paper, the interaction between the mononuclear macrophages and the retaining bacteria is described from the angle of infection, and the immune escape mechanism of the retaining bacteria is discussed, which provides a theoretical basis for the treatment of immune intervention.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R515
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