臨床感染持留菌的篩選及其感染免疫機制研究
發(fā)布時間：2019-06-25 15:20
【摘要】：持留菌(Persisters)是存在于某個細菌群體中的表型異化小亞群�？梢阅褪苤滤罎舛鹊目咕幬�,國內(nèi)外學者認為其與慢性感染的反復發(fā)作及細菌的生物被膜感染等密切相關。肺炎克雷伯菌是重要的醫(yī)院和社區(qū)獲得性革蘭陰性機會致病菌,可導致患者發(fā)生尿路感染、肺炎、菌血癥及肝膿腫等多種疾病。本課題針對重要的臨床耐藥問題,利用實驗室感染病原樣本庫,從建立微生物快速診斷技術和臨床樣本持留菌篩選入手,研究頑固性難治性感染相關的持留菌的感染免疫調(diào)控機制。通過觀察持留菌流行病學特征和持留特性,從感染免疫角度闡述單核巨噬細胞與持留菌之間的相互作用,解釋免疫逃避機制,為免疫干預治療提供理論依據(jù)。第一部分臨床細菌感染現(xiàn)狀分析及分子診斷檢驗本部分研究對臨床細菌感染現(xiàn)狀進行分析,對醫(yī)院相關感染進行流行病學調(diào)查;分析耐藥的分子基礎,形成分子診斷方案,為臨床感染控制提供預警。建立了基于多重PCR體系的主要耐藥菌快速鑒定和耐藥機制檢測的分子生物學方法,可以很好的作為傳統(tǒng)微生物檢驗方法的補充,有利于對臨床耐藥菌株進行耐藥機制的系統(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,對臨床菌株進行了持留菌的篩選并對其持留特性和耐藥性進行了研究。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,對氨芐西林100%耐藥。對哌拉西林/他唑巴坦等耐藥率均超過75%,對環(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濃度時可以實現(xiàn)對持留菌的殺滅。這一現(xiàn)象提示我們,不能通過判讀臨床常規(guī)藥敏試驗結果來區(qū)分持留菌,持留菌由于缺乏明確的特異標志物而難以嚴格區(qū)分和鑒定,這是持留菌研究中的一個難點和值得挖掘的研究切入點;此外,雖然體外實驗中抗生素濃度達到一定值時可以殺滅持留菌,但實際上體內(nèi)不可能達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,擬進一步探討持留菌的持留機制或者其感染免疫機制,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-單核巨噬細胞間相互作用及機制研究針對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胞與持留菌之間的相互作用。首先通過體外實驗發(fā)現(xiàn)單核細胞對持留菌的吞噬作用降低,進一步通過芯片檢測、生物信息學分析和實驗驗證來確定單核巨噬細胞中免疫相關基因的表達及其效應信號通路。發(fā)現(xiàn)持留菌感染單核巨噬細胞,可以誘導SOCS-1蛋白在感染初期即大量表達,且部分依賴于TLR4信號通路;SOCS-1蛋白可以下調(diào)MAL的水平進而影響TLR4下游信號通路的活化,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產(chǎn)生的SOCS-1負調(diào)控炎癥因子的表達,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胞與持留菌之間的相互作用,探討了持留菌的免疫逃避機制,為免疫干預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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R515
本文編號：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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R515
【相似文獻】
相關期刊論文 前4條
1 潘毓萱;結核分枝桿菌持留性和持留菌的界定[J];中華結核和呼吸雜志;2004年04期
2 姜北;黎庶;胡曉梅;;持留菌研究方法的新進展[J];醫(yī)學研究生學報;2014年06期
3 吳艷霞;熊靜遠;田蓮田;裴曉方;Yunrong Chai;;微生物持留菌與醫(yī)療衛(wèi)生[J];現(xiàn)代預防醫(yī)學;2014年05期
4 陸宇;高孟秋;趙偉杰;王彬;馬麗萍;朱莉貞;;結核分枝桿菌持留狀態(tài)體內(nèi)外模型的建立與檢測[J];中華結核和呼吸雜志;2007年03期
相關會議論文 前1條
1 趙雁林;趙冰;端木宏謹;李傳友;;建立結核分枝桿菌持留菌模型和比較蛋白質(zhì)組學研究方法[A];2004年中國防癆協(xié)會臨床基礎專業(yè)委員會學術會議論文集[C];2004年
相關博士學位論文 前2條
1 秦琴;臨床感染持留菌的篩選及其感染免疫機制研究[D];第二軍醫(yī)大學;2016年
2 韓儉;金黃色葡萄球菌持留菌和L-型形成機制研究[D];蘭州大學;2014年
相關碩士學位論文 前3條
1 王慧;臨床微生物樣本中持留菌的篩選及對肺炎克雷伯桿菌持留株持留機制的研究[D];第二軍醫(yī)大學;2015年
2 馬亞男;鮑曼不動桿菌耐藥持留菌的形成機制研究[D];安徽大學;2015年
3 姜北;金黃色葡萄球菌轉(zhuǎn)座子突變文庫構建與持留性突變株研究[D];第三軍醫(yī)大學;2014年
，本文編號：2505786
本文鏈接：http://www.sikaile.net/yixuelunwen/chuanranbingxuelunwen/2505786.html
最近更新
教材專著
