不同来源白色念珠菌的分离鉴定及其致病性分析

发布时间：2018-03-08 16:11

本文选题：白色念珠菌　切入点：药敏试验　出处：《山东农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：白色念珠菌(Candida albicans),又称白假丝酵母,是一种重要的人畜共患条件致病性真菌,当宿主免疫功能低下时不仅可以导致浅表感染(如阴道炎和鹅口疮),也能导致致命性的深部感染(系统感染)。由于广谱抗菌素的广泛使用、器官移植和体内导管植入等医疗技术的应用以及免疫抑制疾病的增多,白色念珠菌感染已成为临床上和生产上日益严重的问题。随着抗真菌药物的使用,白色念珠菌对一种或多种抗真菌药物耐药的现象日趋增多,体外抗真菌药物敏感性试验在指导合理用药以及检测病原菌对药物的耐药趋势方面发挥越来越大的作用。尽管念珠菌病流行且后果严重,其致病机制尚未被详尽阐述。本研究旨在分离不同来源的白色念珠菌,并分析分离株的毒力和表型特性。筛选的强弱毒株可用于研究白色念珠菌的致病机制。此外,本试验采用的鸡系统性感染模型能更好的模拟宿主-病原菌的相互作用,有助于增进对白色念珠菌和宿主共进化的认识和揭示白色念珠菌的感染机理。试验具体分为以下几个部分:1.不同来源白色念珠菌的分离鉴定及体外药敏试验本研究利用念珠菌显色培养基和5.8S-ITS序列分析分离了100株白色念珠菌。体外药敏试验结果显示,分离株对5-氟胞嘧啶和氟康唑敏感性很高(敏感率分别为95.2%、100%);对两性霉素B的敏感率为59.5%,耐药率为19.0%。2.白色念珠菌分离株的毒力分析本研究用鼠系统性感染模型,根据存活率、器官真菌负荷分析了分离株的毒力。结果显示,不同菌株毒力不同,其中分离株J1(鸡源)和G2(鸽源)毒力差异显著。考虑到菌株毒力可能受宿主种类的影响,我们分别用鸡/鸽系统性感染模型再次分析分离株J1和G2的毒力,结果表明J1毒力依旧明显强于G2。即筛选出强毒株J1和弱毒株G2。3.强弱毒株表型和基因学特性分析分析了强弱毒株对高温、酸碱、氧化压、渗透压的耐受力等表型特性和部分毒力相关基因(ALS1、ALS3、HWP1、EFG1、HSP90和SAP1-6)m RNA表达水平的差异。研究结果显示,强弱毒株对高温、酸碱、氧化压、渗透压的耐受力相似,但弱毒株G2对刚果红的敏感性高于J1。G2对刚果红更易感,表明其细胞壁完整性受损,可能因此更易被宿主免疫系统清除,导致毒力减弱。此外,相对荧光定量的结果显示,攻毒后24 h和/或48 h后,除了HSP90、SAP1、SAP4和SAP6,其余基因在强毒株J1中的转录水平均高于(≥1.5倍)弱毒株G2。ALS1、ALS3、HWP1和EFG1的表达上调可部分解释分离株J1毒力强于G2。攻毒24 h和48 h后SAP5在强毒株中的转录水平显著高于弱毒株,暗示SAP5的持续高水平表达可能与毒力增强有关;而SAP1和SAP4转录水平均低于弱毒株,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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.66

【共引文献】