發(fā)布時間：2018-03-08 16:11

  本文選題：白色念珠菌　切入點：藥敏試驗　出處：《山東農(nóng)業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：白色念珠菌(Candida albicans),又稱白假絲酵母,是一種重要的人畜共患條件致病性真菌,當宿主免疫功能低下時不僅可以導(dǎo)致淺表感染(如陰道炎和鵝口瘡),也能導(dǎo)致致命性的深部感染(系統(tǒng)感染)。由于廣譜抗菌素的廣泛使用、器官移植和體內(nèi)導(dǎo)管植入等醫(yī)療技術(shù)的應(yīng)用以及免疫抑制疾病的增多,白色念珠菌感染已成為臨床上和生產(chǎn)上日益嚴重的問題。隨著抗真菌藥物的使用,白色念珠菌對一種或多種抗真菌藥物耐藥的現(xiàn)象日趨增多,體外抗真菌藥物敏感性試驗在指導(dǎo)合理用藥以及檢測病原菌對藥物的耐藥趨勢方面發(fā)揮越來越大的作用。盡管念珠菌病流行且后果嚴重,其致病機制尚未被詳盡闡述。本研究旨在分離不同來源的白色念珠菌,并分析分離株的毒力和表型特性。篩選的強弱毒株可用于研究白色念珠菌的致病機制。此外,本試驗采用的雞系統(tǒng)性感染模型能更好的模擬宿主-病原菌的相互作用,有助于增進對白色念珠菌和宿主共進化的認識和揭示白色念珠菌的感染機理。試驗具體分為以下幾個部分:1.不同來源白色念珠菌的分離鑒定及體外藥敏試驗本研究利用念珠菌顯色培養(yǎng)基和5.8S-ITS序列分析分離了100株白色念珠菌。體外藥敏試驗結(jié)果顯示,分離株對5-氟胞嘧啶和氟康唑敏感性很高(敏感率分別為95.2%、100%);對兩性霉素B的敏感率為59.5%,耐藥率為19.0%。2.白色念珠菌分離株的毒力分析本研究用鼠系統(tǒng)性感染模型,根據(jù)存活率、器官真菌負荷分析了分離株的毒力。結(jié)果顯示,不同菌株毒力不同,其中分離株J1(雞源)和G2(鴿源)毒力差異顯著�？紤]到菌株毒力可能受宿主種類的影響,我們分別用雞/鴿系統(tǒng)性感染模型再次分析分離株J1和G2的毒力,結(jié)果表明J1毒力依舊明顯強于G2。即篩選出強毒株J1和弱毒株G2。3.強弱毒株表型和基因?qū)W特性分析分析了強弱毒株對高溫、酸堿、氧化壓、滲透壓的耐受力等表型特性和部分毒力相關(guān)基因(ALS1、ALS3、HWP1、EFG1、HSP90和SAP1-6)m RNA表達水平的差異。研究結(jié)果顯示,強弱毒株對高溫、酸堿、氧化壓、滲透壓的耐受力相似,但弱毒株G2對剛果紅的敏感性高于J1。G2對剛果紅更易感,表明其細胞壁完整性受損,可能因此更易被宿主免疫系統(tǒng)清除,導(dǎo)致毒力減弱。此外,相對熒光定量的結(jié)果顯示,攻毒后24 h和/或48 h后,除了HSP90、SAP1、SAP4和SAP6,其余基因在強毒株J1中的轉(zhuǎn)錄水平均高于(≥1.5倍)弱毒株G2。ALS1、ALS3、HWP1和EFG1的表達上調(diào)可部分解釋分離株J1毒力強于G2。攻毒24 h和48 h后SAP5在強毒株中的轉(zhuǎn)錄水平顯著高于弱毒株,暗示SAP5的持續(xù)高水平表達可能與毒力增強有關(guān);而SAP1和SAP4轉(zhuǎn)錄水平均低于弱毒株,暗示SAP家族可能有基因冗余或SAP1和SAP4的作用可被其它基因代替。
[Abstract]:Candida albicansa, also known as Candida albicansa, is an important zoonotic condition pathogenic fungus. When the host's immune function is low, it can lead not only to superficial infections such as vaginitis and thrush, but also to fatal deep infections (systemic infections), due to the widespread use of broad-spectrum antibiotics, With the application of medical techniques such as organ transplantation and catheterization in vivo, and the increasing number of immunosuppressive diseases, candida albicans infection has become an increasingly serious problem both clinically and productively. The resistance of Candida albicans to one or more antifungal agents is increasing. Antifungal susceptibility tests in vitro play an increasingly important role in guiding rational drug use and in detecting the drug resistance trends of pathogens, although candidiasis is prevalent and has serious consequences. The pathogenetic mechanism of Candida albicans has not been elucidated in detail. The aim of this study was to isolate Candida albicans from different sources, and to analyze the virulence and phenotypic characteristics of the isolates. The selected strong and weak strains could be used to study the pathogenicity of Candida albicans. The chicken systemic infection model used in this study can better simulate the host-pathogen interaction. It is helpful to improve the understanding of the coevolution of Candida albicans and reveal the infection mechanism of Candida albicans. The experiment is divided into the following parts: 1. Isolation and identification of Candida albicans from different sources and in vitro drug sensitivity test. 100 strains of Candida albicans were isolated from Candida albicans by coloration medium and 5.8S-ITS sequence analysis. The sensitivity of the isolates to 5-fluorocytosine and fluconazole was very high (the sensitivity rates were 95.22.The sensitivity to amphotericin B was 59.5 and the drug resistance rate was 19.0.2.The virulence of the isolates of Candida albicans in this study was analyzed according to the survival rate. The virulence of the isolates was analyzed by organ fungi load. The results showed that the virulence of different strains was different, among which the virulence of isolates J1 (chicken source) and G2 (pigeon source) were significantly different. Considering that the virulence of strains might be affected by host species, We used the chicken / pigeon systemic infection model to analyze the virulence of isolates J1 and G2, respectively. The results showed that the virulence of J1 was still stronger than that of G2.The virulence of the virulent strain J1 and the attenuated strain G2.3.The phenotypic and genetic characteristics of the virulence strain were analyzed, and the effects of the strong strain on high temperature, acid base and oxidation pressure were analyzed. The phenotypic characteristics of osmotic pressure tolerance and the expression levels of HSP90 and SAP1-6)m RNA of ALS1 / ALS3 / HWP1HWP1 / EFG1HSP90 and SAP1-6)m RNA were found to be similar to those of high temperature, acid-base, oxidized pressure and osmotic pressure of the virulence related gene, the results showed that the resistance to high temperature, acid base, oxidation pressure and osmotic pressure was similar to that of HSP90. However, the sensitivity of attenuated strain G2 to Congo red was higher than that of J1.G2 to Congo red, indicating that its cell wall integrity was impaired, and thus it may be more easily cleared by host immune system, resulting in weakened virulence. In addition, the results of relative fluorescence quantitative analysis showed that, 24 hours and / or 48 hours after drug attack, With the exception of HSP90, SAP1, SAP4 and SAP6, the transcriptional level of other genes in virulent strain J1 was higher than that in attenuated strain G2.ALS1ALS3HWP1 and EFG1, which partly explained that J1 was more virulent than G2.The transcription of SAP5 in virulent strain after 24 h and 48 h was higher than that in virulent strain G2. The level was significantly higher than that of the attenuated strain. It suggested that the continued high expression of SAP5 might be related to the enhancement of virulence, while the transcriptional level of SAP1 and SAP4 was lower than that of the attenuated strain, suggesting that the SAP family might have gene redundancy or SAP1 and SAP4 could be replaced by other genes.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.66

【共引文獻】

相關(guān)期刊論文 前8條

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相關(guān)碩士學位論文 前2條

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本文編號：1584610