反转录转座子TCA4与白念珠菌耐药性的研究

目的研究高温刺激下白念珠菌中反转录转座子的表达情况与耐药性产生的关系,探寻白念珠菌耐药的分子机制。方法微量液基稀释法测定氟康唑对高温诱导的白念珠菌的最低抑菌浓度（minimal inhibitory concentration,MIC）;斑点法（spotassay）考察诱导菌株对药物的耐受能力;实时定量PCR（RT-PCR）方法检测诱导菌株中反转录转座子TCA4中开放阅读框的表达水平。结果长期高温刺激能降低白念珠菌对氟康唑（16μg／mE）的耐受能力;高温诱导菌株中反转录转座子TCA4中Orf19．2668和Orf19．2669的表达水平相比于亲本菌ATCC10231发生高表达。结论高温刺激能使反转录转座子TCA4发生转座激活,反转录转座子TCA4的转座激活与白念珠菌耐药性形成相关,与此同时可能还有其他机制参与白念珠菌耐药性的形成。     

CCH1或MID1基因缺失对白念珠菌药物耐受性及致病性的影响

CCH1和MID1基因编码的钙闸门是外源钙进入细胞内的重要通道,Ca2+作为细胞内重要的第2信使分子,其浓度的升高可激活相应的途径参与各种细胞反应过程。该研究将利用钙通路CCH1和MID1基因的单缺失菌株,并构建其相应的回补菌株,研究CCH1或MID1基因缺失后对白念珠菌药物耐受性和致病性的影响作用。通过药物平板敏感性试验和微量液基稀释法比较不同菌株对唑类药物敏感性的变化,进一步添加钙通道阻滞剂和钙离子螯合剂来分析钙离子浓度变化对药物作用的影响,结果发现CCH1或MID1基因的缺失明显对氟康唑和伊曲康唑表现出敏感性,且药物作用受到钙离子浓度变化的调节。最后建立小鼠感染模型分析不同菌株的毒力变化差异,确定CCH1或MID1基因的缺失显著减弱了白念珠菌的致病性。     

白念珠菌耐药机制新进展

近年来对于深部真菌感染的研究报道越来越多,其已日益成为一些重要疾病临床治疗过程中的常见并发症,其中,白念珠菌病的发病率仍居高不下.虽然目前有多种抗真菌药物应用于临床,但其耐药现象愈来愈严重,给临床治疗带来了极大的挑战.近来有关白念珠菌耐药机制的研究有了较新的进展.该文就新发现的白念珠菌的耐药机制,作一概述.     

白假丝酵母菌耐药与抗氰呼吸的相关性研究

目的探讨白假丝酵母菌的耐药情况及其与抗氰呼吸的相关性。方法用真菌药敏测定试剂盒测定从临床分离出来的37株白假丝酵母菌的耐药性,并从中选出5株耐药菌和5株敏感菌进行抗氰呼吸的研究。结果白假丝酵母菌对益康唑的耐药率最高,达54.1%,耐药白假丝酵母菌的抗氰呼吸速率均值为(17.56±6.75)nmol/(min.A620),敏感白假丝酵母菌的抗氰呼吸速率均值为(7.99±5.80)nmol/(min.A620),耐药白假丝酵母菌的抗氰呼吸速率明显升高,且耐药菌株抗氰呼吸速率占总呼吸的比例明显高于敏感菌株(P0.05),差异具有显著性。结论兰州市区白假丝酵母菌对益康唑耐药性较高,且白假丝酵母菌的耐药与抗氰呼吸途径相关。     

白念珠菌对特比萘芬耐药性的体外梯级诱导和回复研究

目的以浓度梯级倍增的特比萘芬在体外诱导白念珠菌标准株获得耐药子代菌株,并观察其耐药稳定性,从细胞水平研究白念珠菌对特比萘芬耐药前后生物学特性的变化,为进一步采用基因芯片在基因表达水平上研究特比萘芬对白念珠菌的药理作用及其诱导耐药机制提供理想的实验模型。方法将白念珠菌ATCC90028株在特比萘芬浓度逐渐梯级倍增的YPD液体培养基中分别转种传代,直到最后转种至含1024μg／ml特比萘芬的YPD液体培养基中培养,分别测定诱导后形成的各子代菌株的MIC值;选用以1024μg／ml特比萘芬诱导形成的耐药菌株,在不含特比萘芬的YPD液体培养基中连续传代10次后,测定其MIC值,观察其耐药表型的稳定性;并分别用肉眼、光镜和电镜观察白念珠菌耐药性产生前后的形态学特征。结果特比萘芬MIC值为8μg／ml的白念珠菌母本菌株（白念珠菌ATCC90028）成功地被诱导成特比萘芬MIC值为≥512μg/ml的子代菌株,进一步的耐药稳定性实验说明诱导后形成的子代菌株的表型是相对稳定的,诱导后的子代耐药菌株与其母本相比,生长繁殖速度减慢,细胞形态不规则,部分细胞胞膜不完整。结论通过在药物浓度梯级倍增的培养基中连续传代培养的方法可成功建立相同基因型的对特比萘芬敏感的白念珠菌母本和对特比萘芬耐药的子代模型,为获取有亲本的耐药白念珠菌菌株提供了一个有效的实验方法,是在基因水平研究白念珠菌对特比萘芬耐药机制的理想实验模型。     

耳念珠菌耐药机制研究进展

耳念珠菌感染及其耐药问题已愈演愈烈,其临床耐药机制的研究已刻不容缓。本文整理了国内外耳念珠菌临床菌株对三大类抗真菌药物的耐药情况,并从外排泵异常表达、靶标蛋白突变、生物被膜和基因组可塑性4个方面总结了耳念珠菌已报道的或潜在的耐药机制,并与其他念珠菌耐药机制进行了对比。     

Comprehensive genetic analysis of adhesin proteins and their role in virulence of Candida albicans

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.     

重症监护病房白念珠菌耐药性8年变化趋势

目的调查上海长征医院重症监护室(ICU)近8 a中临床分离白念珠菌的耐药性变化,为临床治疗提供参考。方法上海长征医院ICU 2002～2009年从414例患者中首次分离出414株白念珠菌,对其中277株进行药敏试验。采用Cox-Stuart趋势检验回顾性分析临床分离真菌中白念珠菌所占比例变化趋势和白念珠菌对常用抗真菌药物耐药率的变化趋势。结果 2002～2009年间,上海长征医院ICU白念珠菌分离株数从2002年的34株增加至2009年的92株,但白念珠菌占总真菌分离株数的百分比维持在34.6%～55.7%,P=0.03。白念珠菌对于5-氟胞嘧啶和两性霉素B平均耐药率分别为4.0%和0.7%,对其他常用抗真菌药的耐药率依次为咪康唑47.0%、酮康唑10.8%、伊曲康唑19.9%、特比萘芬42.6%、氟康唑14.6%及伏立康唑13.0%。白念珠菌对5-氟胞嘧啶和两性霉素B和伊曲康唑耐药率的8年变化无统计学差异。结论上海长征医院ICU近8 a来白念珠菌仍然为临床较为常见的真菌分离株,但白念珠菌占总分离株数的百分比有逐渐减少的趋势。白念珠菌对常用抗真菌药物耐药性均无明显变化趋势。     

白假丝酵母CFL1基因通过转录调控参与氧化压力应答

【背景】CFL1基因是白假丝酵母高铁还原酶基因,介导胞外铁离子的还原,在白假丝酵母胞内铁稳态的维持方面发挥着重要作用。【目的】研究CFL1基因调节氧化压力应答的分子机制。【方法】采用液体培养及巨噬细胞模型,测定CFL1缺失对氧化压力耐受性和杀伤巨噬细胞能力的影响;使用羟基自由基清除剂二甲基亚砜(DMSO)分析其对缓解氧化压力敏感性的影响;采用实时荧光定量PCR分析CFL1缺失对氧化压力应答基因表达的影响;采用过氧化氢酶(CAT)活性测定方法研究CFL1缺失对CAT1基因表达的影响;通过构建WT-CAT1-GFP和cfl1Δ/Δ-CAT1-GFP菌株分析过氧化氢酶基因过表达对cfl1Δ/Δ氧化压力敏感性的影响。【结果】白假丝酵母CFL1基因的缺失会造成杀伤巨噬细胞能力的减弱,氧化压力应答基因表达的下降。过氧化氢酶基因的过表达则能恢复与野生型几乎一致的氧化压力水平。【结论】CFL1基因通过转录调控参与白假丝酵母氧化压力应答过程。     

Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

Biofilm of Candida albicans: formation,regulation and resistance

Candida albicans is the most common human fungal pathogen, causing infections that range from mucous membranes to systemic infections. The present article provides an overview of C. albicans, with the production of biofilms produced by this fungus, as well as reporting the classes of antifungals used to fight such infections, together with the resistance mechanisms to these drugs. Candida albicans is highly adaptable, enabling the transition from commensal to pathogen due to a repertoire of virulence factors. Specifically, the ability to change morphology and form biofilms is central to the pathogenesis of C. albicans. Indeed, most infections by this pathogen are associated with the formation of biofilms on surfaces of hosts or medical devices, causing high morbidity and mortality. Significantly, biofilms formed by C. albicans are inherently tolerant to antimicrobial therapy, so the susceptibility of C. albicans biofilms to current therapeutic agents remains low. Therefore, it is difficult to predict which molecules will emerge as new clinical antifungals. The biofilm formation of C. albicans has been causing impacts on susceptibility to antifungals, leading to resistance, which demonstrates the importance of research aimed at the prevention and control of these clinical microbial communities.     

光滑念珠菌的生物学、流行病学以及耐药机制研究进展

随着HIV感染患者的增多、器官移植、放疗化疗及抗真菌药物的广泛使用,近年来全世界范围内念珠菌感染趋势发生了明显变化,除白念珠菌外,光滑念珠菌在临床上的检出率逐年增加,在部分国家和地区已成为第二常见的侵袭性念珠菌。光滑念珠菌临床分离株通常对一线抗真菌药物高度耐药,由于目前治疗策略匮乏,其造成的系统感染死亡率可高达50%。为了进一步加深人们对光滑念珠菌的认识,研发遏制其感染的诊疗策略,本文综述了近年来光滑念珠菌的流行病学、毒力因子以及耐药机制等方面的进展,为国内同行深入探究其耐药特性和致病机理提供参考。     

白念珠菌唑类药物耐药相关转录因子研究进展

近年来白念珠菌的感染率呈逐年上升趋势,随着唑类药物的广泛应用,耐药菌株不断增多,已成为临床治疗的一大难题.白念珠菌的耐药机制主要与ERG 11基因的突变和过表达、药物外排泵相关基因表达增多及生物膜的形成等有关,由于转录因子是耐药基因表达的关键调节因子,关于锌簇转录因子与耐药关系的研究越来越多,如TAC 1、MRR 1、MRR 2、UPC 2、NDT 80等,其点突变可引起某些耐药基因的过表达而介导耐药,该领域研究已成为热点,该文就此研究进展做一概述.     

血培养分离出212株念珠菌的菌种分布及耐药性分析

目的分析血培养分离出的念珠菌的菌种分布及耐药分析,为临床念珠菌血症的诊治提供理论依据。方法回顾性分析2017年1月—2020年12月期间,本院血培养分离出的念珠菌的菌种分布、药敏试验结果及患者血培养分离出念珠菌前后96 h内的G试验结果。结果血培养中分离出念珠菌314例,阳性率2.1%,其中非重复分离株212例。检出率最高的是近平滑念珠菌(72株,34.0%),其次是白念珠菌(55株,25.9%)和光滑念珠菌(28株,13.2%)。念珠菌检出率最高的科室为ICU(62株,29.2%),其次是新生儿科(39株,18.4%)和血液科(20株,9.4%)。检出的212株念珠菌除一株近平滑念珠菌为两性霉素B的非野生株,其余均为两性霉素B的野生株。白念珠菌对唑类药物的敏感率超过90%。但光滑念珠菌和热带念珠菌对唑类药物的敏感性较低。血培养分离出念珠菌的前后96 h内,G试验的阳性率为73.7%。结论本院念珠菌检出率前3位分别是近平滑念珠菌、白念珠菌和光滑念珠菌。光滑念珠菌、热带念珠菌对唑类药物敏感性比较低,在经验用药时需要合理选择抗真菌药物。G试验对念珠菌血症有较高的价值,需要对结果进行动态监测。     

MAPK转导通路在白念珠菌菌体抗氧化应激中的作用

目的探讨MAPK通路在念珠菌抗氧化应激中的作用。方法采用不同浓度过氧化氢刺激白念珠菌,通过流式细胞仪检测念珠菌的凋亡率,并计算其增殖指数;通过实时荧光定量PCR检测MAPK通路中8种基因的表达水平。结果随着过氧化氢的刺激浓度增高,白念珠菌的凋亡率逐渐升高,而其增殖指数下降。在不同的过氧化氢浓度刺激下,MAPK通路中各基因表达水平基本一致,即在较低的过氧化氢浓度刺激下,各基因表达水平均有一定的上升,而随着浓度增高,在高浓度的过氧化氢刺激下,各基因表达水平趋于稳定。结论在低浓度的过氧化氢刺激下,白念珠菌的凋亡率虽有所上升,但其相应的增殖指数也有所上升,即生长加快。这可能与其MAPK通路中各基因表达增强有一定的关系。     

呼吸内科患者白色念珠菌痰培养阳性的危险因素及耐药性变迁分析

目的分析呼吸内科患者白色念珠菌痰培养阳性的危险因素及对常用抗真菌药物的耐药性,为临床预防、早期诊断及有效治疗提供依据。方法对2007年1月至2009年12月浙江大学医学院附属第一医院呼吸内科白色念珠菌痰培养阳性患者的临床资料进行回顾性调查,统计其年龄分布、可能感染因素、基础疾病、被感染前抗菌药物的使用及近3年对抗真菌药物的耐药性变迁。结果呼吸内科白色念珠菌痰培养阳性的相关因素多,老年、气管插管/切开、机械通气、慢性阻塞性肺疾病及长期多种抗菌药物的使用患者中感染几率明显增高,且3年来,白色念珠菌的耐药性有所升高。结论临床上细心操作、抗菌药谨慎使用是目前减少白色念珠菌感染的重要途径。     

Oxidative stress in cervical cancer pathogenesis and resistance to therapy

Cervical cancer (CC) is one of the most common cancers among females, and it is most notable in developing countries. The exact etiology of CC is poorly understood; but, smoking, oral contraceptives, immunosuppression, and infection with human papillomavirus (HPV) may increase the risk of CC. There is also an association between CC and oxidative stress. Oxidative stress is caused by a disturbed oxidant-antioxidant balance in favor of the former, leading to an excessive generation of free radicals, particularly reactive oxygen species (ROS), and subsequently to biological damages. Thus, redox enzymatic and nonenzymatic regulators are required to maintain the redox homeostasis. Dysregulated antioxidants system and the pathogenic role of oxidative stress in CC have been investigated in several clinical and preclinical studies. In this study, we reviewed studies that have addressed the cross-talk between oxidative stress and CC pathogenesis and resistance to therapy.     

The carboxylic acid transporters Jen1 and Jen2 affect the architecture and fluconazole susceptibility of Candida albicans biofilm in the presence of lactate

Candida albicans has the ability to adapt to different host niches, often glucose-limited but rich in alternative carbon sources. In these glucose-poor microenvironments, this pathogen expresses JEN1 and JEN2 genes, encoding carboxylate transporters, which are important in the early stages of infection. This work investigated how host microenvironments, in particular acidic containing lactic acid, affect C. albicans biofilm formation and antifungal drug resistance. Multiple components of the extracellular matrix were also analysed, including their impact on antifungal drug resistance, and the involvement of both Jen1 and Jen2 in this process. The results show that growth on lactate affects biofilm formation, morphology and susceptibility to fluconazole and that both Jen1 and Jen2 might play a role in these processes. These results support the view that the adaptation of Candida cells to the carbon source present in the host niches affects their pathogenicity.