携带G487T和T916C突变的白念珠菌耐药基因表达与氟康唑耐药关系的研究
|
摘要
念珠菌属是引起机会性真菌感染的常见病原体,也是医院真菌感染的重要病原菌之一。近年来,由于糖皮质激素、免疫抑制剂、广谱抗生素的广泛应用,器官移植的普遍开展,导管等生物材料应用的增多,原发及继发性免疫功能低下的人群扩大,真菌感染率呈不断上升的趋势。尤以白念珠菌的感染最为常见。
唑类药物是目前应用最广的抗真菌药物。特别是氟康唑,自从20世纪80年代后期问世以来,由于其抗菌谱广、安全和可静脉给药,吸收好,毒性小等优点,成为目前临床治疗真菌感染的首选药物。但是,随着抗真菌药物的广泛使用,加之病人长时间的药物治疗和使用较低剂量的抗真菌药物,为耐药菌株的产生创造了条件。1980年Rosenblatt等人首次报道在应用酮康唑治疗慢性皮肤念珠菌病人身上出现药物耐药现象。此后有关耐药的报道越来越多。
麦角固醇是真菌细胞膜中特有的脂质。它维持着细胞膜的正常结构和功能。14α-去甲基化酶(Ergllp)是念珠菌细胞膜麦角固醇合成途径中的关键酶,ERGll是14α-去甲基化酶的编码基因。大量研究认为这个基因的点突变或过度表达均与唑类耐药相关。ERG11的突变可以改变14α-去甲基化酶的氨基酸序列,从而影响唑类药物与该酶的亲和力,导致菌株耐药。过度表达的外排泵基因CDR1和CDR2以及MDR1基因是第二个与耐药相关的原因。CDR1和CDR2基因属于ABC转运蛋白超家族(ATP-binding cassette transporters, ABC transporters),二者的同源性超过80%,在耐药机制中可发挥协同作用。MDR1基因属于易化扩散载体超家族(major facilitator supeffamily, MFS),目前认为它的表达也与氟康唑耐药相关。近年来发现FLU1基因也与唑类耐药相关。该基因与MDR1基因同源性较高,在耐氟康唑白念珠菌的研究中发现这个基因被敲除后可增加菌株对唑类药物的敏感性。此外,生物膜形成是近年来与耐药相关的一个新发现。生物膜耐药也是导致临床上许多系统性感染难以根除的重要因素。最近的研究认为细胞内液泡封闭药物分子也可能引发菌株耐药。
耐药的发生是一个多水平多因素参与的复杂过程。耐药白念珠菌的流行病学调查发现,85%的耐药株外排泵过度表达；35%的耐药株为药物靶酶的氨基酸改变或过度表达；75%的耐药株为多因素联合耐药。唑类药物对念珠菌耐药机制的研究已经成为当今医学研究的热点。但国内目前在念珠菌耐药性方面的研究仍然较少。
前期研究中我们通过测序发现14株临床耐氟康唑白念珠菌的ERG11基因集中发生G487T和T916C突变,且不伴随有其他突变。目前尚未见过临床菌株存在该突变点的报道。在白念珠菌耐药的发生发展过程中,多种机制均可在不同程度上促进菌株耐药的形成。我们尚不清楚是否有其它耐药机制(如MDR1、CDR1/2和FLU1)介入了这一系列临床株的耐药形成过程。目前也未见该方面的研究报道。该系列菌株在耐氟康唑机制中还存在其它的共同点么?耐药相关基因的表达在这些白念珠菌中又起了什么作用呢?本课题首次对每一株携带G487T和T916C突变的耐氟康唑白念珠菌的耐药相关基因的表达及外排泵功能进行了研究,进一步探讨该系列菌株的基因表达在氟康唑耐药中的作用,从而为明确该系列菌株的耐氟康唑机制提供有力的实验数据,并为后续进一步研究菌株耐药现象提供必要的数据分析及实验支持。
目的
1.了解携带G487T和T916C突变的耐氟康唑白念珠菌在不同培养液的生长情况,使用若丹明6G作为示踪剂检测该系列菌株的外排能力。
2.通过实时定量PCR方法检测携带G487T和T916C突变的耐氟康唑白念珠菌多个耐药相关基因mRNA表达水平。
3.通过蛋白印迹方法检测携带G487T和T916C突变的耐氟康唑白念珠菌Cdrlp和Cdr2p蛋白的表达。
方法
1.院内念珠菌鉴定
收集山东大学齐鲁医院检验科及皮肤科门诊的临床标本,包括痰液、尿液、血液及分泌物等。将标本接种于血平板,挑选可疑菌落涂片革兰染色确认为酵母样真菌后,转种于改良SDA平板分离纯化,通过科玛嘉念珠菌显色培养基鉴定,观察结果。应用微量稀释法和纸片扩散法测定氟康唑对试验菌株的MIC,确定敏感株(S)、剂量依赖敏感株(S-DD)和耐药株(R)。微量稀释法按照美国临床试验标准协会(CLSI)制定的M27-A2方案实施,ATCC 6258和ATCC22019被作为质控菌株。每次试验重复三次。
2.菌株生长及若丹明6G实验
实验菌株：携带G487T和T916C突变的耐氟康唑白念珠菌14株,敏感控制株ATCC102311株。将保存在SDA培养基的菌株经过两次活化,调整菌液浓度,将活化后的耐氟康唑白念珠菌分别转入lml RPMI1640培养液,lmlYEPD培养液和lml 64μg／ml氟康唑的YEPD培养液中,37℃静置培养,倒置相差显微镜下观察耐氟康唑白念珠菌的增殖情况。
使用若丹明6G作为示踪剂,通过测定荧光强度(激发波长529nm,吸收波长553nm)来计算细胞外液若丹明的浓度,进而检测若丹明6G在携带G487T和T916C突变的耐氟康唑白念珠菌中的外排情况。
3.实时定量PCR实验
实验菌株：氟康唑敏感白念珠菌14株(来自第一部分的收集菌株),携带G487T和T916C突变的耐氟康唑白念珠菌14株,敏感株ATCC102311株。根据各耐药基因的基因序列：ERG11(GeneBank号：X13296,下同),CDR1(X77589), CDR2(U63812), MDR1(Y14703), FLU1(AF188621)设计相应的引物。内参照选用看家基因18SrRNA。标准株ATCC 10231被作为敏感控制株。采用酵母RNA试剂盒分别提取各菌株RNA,逆转录成cDNA后,在LightCycler Real Time PCR扩增仪上进行实时定量PCR反应。PCR产物进行凝胶电泳,在凝胶成像系统下观察是否为目的条带,有无杂带。将获得的域值循环数(Ct值)采用2-ΔΔCt方法转换为目的基因的相对表达量,检测多个耐药基因的表达情况,比较该系列菌株CDR1、CDR2、MDR1、ERG11和FLU1基因的表达差异。
4.蛋白印迹实验
实验菌株：携带G487T和T916C突变的耐氟康唑白念珠菌14株,内参照菌株Saccharomyces cerevisiae AD/CDR1和AD/CDR2。提取菌株的质膜蛋白,BCA蛋白浓度测定试剂盒测定蛋白含量,通过7.5%SDS-聚丙烯酰胺凝胶电泳,转膜,蛋白印记,ECL试剂盒显色等步骤,检测菌株CDR1和CDR2基因的表达情况。抗Cdr1p抗体(1：500),抗Cdr2p抗体(1：1000),二抗选用HRP标记的山羊抗兔二抗(抗Cdrlp抗体1：5000；抗Cdr2p抗体1：10000)。应用数字凝胶成像系统FluorChem 9900-50型进行扫描并记录图像,用Quantity One software软件进行半定量分析,计算各组目的蛋白相对于内参的相对灰度值作为各蛋白的相对含量。
结果
1.共收集酵母样菌161株,在161株菌中白念珠菌比例为69.57%,热带念珠菌为19.88%,近平滑念珠菌为4.97%,克柔念珠菌为2.48%,光滑念珠菌为1.86%,其他念珠菌为1.24%。痰标本最多,101份,占所有标本的62.73%。其次为尿液标本,20份(12.42%),来源于阴道分泌物的标本比例为11.18%,血液来源的标本占4.35%,其余(咽拭子、大便、导管分泌物、透析液、胆汁、脐分泌物、支气管肺泡冲洗液等)占9.32%。这些标本中,来自呼吸科的标本占的比例最大,妇科、ICU、血液科次之。112株白念珠菌中,通过微量稀释法筛选出108株敏感株,占96.43%,纸片扩散法试验筛选出109株敏感株(97.32%)。两种方法经卡方检验P>0.05,二者无明显统计学差异。
2.在RPMI1640培养液中,该系列白念珠菌可形成大量菌丝；在YEPD培养液中白念珠菌的菌丝形成数目明显减少；在64μg／ml氟康唑的培养液中它们仍表现活跃。
若丹明6G外排实验结果显示：敏感控制株ATCC10231主动外排若丹明6G能力在所有受试菌株中表现最弱。携带G487T和T916C突变的耐氟康唑白念珠菌在能量供给下可高效主动外排若丹明6G。随着时间的延长该外排能力增强,但60min之后转运能力减弱至平衡或下降。
3.实时定量PCR方法显示携带G487T和T916C突变的耐药组CDR1和CDR2基因的表达明显高于敏感组；而两组的ERG11,MDR1和FLU1基因在mRNA表达水平上差异无统计学意义。与敏感控制株ATCC10231相比,14株携带G487T和T916C突变的耐氟康唑白念珠菌的主动外排泵CDR1和CDR2基因的mRNA表达水平都是明显增高的,分别达到1.6-8倍和3.7-52倍。其中菌株J4266的CDR1 mRNA表达水平最高,而菌株J4263的CDR1mRNA表达水平在14株中最低,但仍然是敏感株的1.7倍。菌株GZ18的CDR2基因mRNA表达是最高的。J4263的CDR2基因mRNA表达水平在14株中最低。在MDR1,FLU1和ERG1l基因中,各个菌株的表达水平是高低不一的。MDR1的表达在4株菌中表达是增高的,分别是菌株GZ17、GZ03、J4266和J592。菌株GZ23的MDR1基因的mRNA表达水平与敏感控制株相同。6株菌中发现ERG11基因过度表达,分别是GZ03、J4266、GZ17、J592、GZ23和GZ51。6株菌的FLU1基因过度表达,分别是J4266、J592、GZ17、GZ03、GZ23和GZ34。
4.蛋白印迹实验显示：内参照Saccharomyces cerevisiae AD/CDR1可以稳定表达Cdr1p, Saccharomyces cerevisiae AD/CDR2菌株可以稳定表达Cdr2p。在这些携带G487T和T916C突变的耐氟康唑白念珠菌中,所有菌株的CDR1基因均有明显的表达,半定量分析结果示菌株J4266表达量最高。这与实时定量mRNA结果相符。但在菌株GZ16、GZ34和GZ51,无法检测到CDR2基因的完整表达。半定量分析结果示菌株J592的Cdr2p蛋白表达最高。而在实时定量PCR比较中它的mRNA表达水平要低于GZ18。这可能提示该系列菌株CDR2基因的蛋白翻译并不是十分稳定的。
结论
1.携带G487T和T916C突变的耐氟康唑白念珠菌均具有主动外排若丹明6G的能力,且该能力随着时间延长而增强,但在60min之后逐渐下降至平衡。
2.氟康唑敏感白念珠菌也可具有主动外排若丹明6G的能力,但该能力远低于携带G487T和T916C突变的耐氟康唑菌株。
3.主动外排泵基因CDR1和CDR2的过度表达存在于每一株携带G487T和T916C突变的白念珠菌。主动外排泵基因CDR1和CDR2在携带G487T和T916C突变的白念珠菌耐氟康唑机制中可能发挥重要作用,而易化扩散载体蛋白MDR1基因的作用可能并不明显。
4.在携带G487T和T916C突变的耐氟康唑白念珠菌中,CDR2基因转录后的蛋白翻译并不十分稳定,Cdr2p蛋白翻译的稳定性可能弱于Cdr1p。
The genus Candida (especially Candida albicans) is the important cause of illness that leads to opportunistic mycoses and one of the main pathogens of nosocomial fungal infections. Following the widespread application of immunodepressants, glucocorticoid, broad-spectrum antibiotics, therapeusis of intervention, organ transplantation and the expanding of population with immunity inhibition, the infection rate of fungi is adscendent tendency in the lately years. The infection caused by pathomycetes candida albicans was the most common.
The azole agents is the most extensively used in the treatment of fungal infections. Especially fluconazole, as the first line azole derivatives, because of broad antibacterial spectrum, strong antibacterial activity, few side effect, safetyly for intravenous injection and so on, is commonly used to treat various candidiasis in clinic since the advent of fluconazole in the late 1980s. However, With the mass use of antifungal agents, resistant strains emerge more frequently under the drug selection pressure and prolonged administration of broad-spectrum antibacterial agents or prophylactic treatment. In 1980, Rosenblatt et al. had reported azole drugs resistance in treating chronic mucocutaneous candidids with ketoconazole for the first time. After that, the phenomenon of drugs resistance are increasing gradually.
Ergosterol is typical lipidd in cellular membrane of fungi. It can maintain the integrity and function of Candida albicans (C. albicans) membrane.14 alpha-demethylase (Erg11p) is a key enzyme in the ergosterol synthesis pathway of C. albicans. The coding gene is ERG11. Mutations in ERG11 can result in changes in the Ergllp spatial configuration, affect the affinity between the azole drugs and the enzyme, and makes isolates resistant to azole finally. The other is that increased expression of the ERG11 gene associated with antifungal drug resistance. The next possible mechanism for drug resistance is over-expression of the CDR1 and CDR2 genes, encoding transporters of the ABC family, and the MDR1 gene, coding for a major facilitator transporter. The homology of CDR1 and CDR2 is greater than 80%, they can have a synergistic effect in azole drug resistance. Another major facilitator gene, FUU1, has also been identified in C. albicans. The FLU1 gene, similar to MDR1, is related with azole resistance. It is reported that this gene can increase azole susceptibility when it is deleted in the study about fluconazole-resistant candida albicans. Besides that, biofilms may be a new mechanism that correlated with resistance development in C. albicans. Biofilms may be associated with poor prognosis in untreated systemic infections. And finally, vesicular vacuoles from resistant isolates may act as a novel fluconazole resistance mechanism.
Occurrence of drug resistance is a complex process involving many different factors. In an analysis about the prevalence of mechanisms of resistance in C. albicans clinical isolates showed that multiple mechanisms of resistance were combined in 75% of resistant isolates, overexpression of genes encoding efflux pumps were detected in 85% of isolates, amino acid substitutions in ERG11 were found in 65% of isolates,35% of the isolates showed overexpression of ERG11. The studies on azole-resistance mechanisms of Candida spp are becoming a very active subject in the world. But there is a few works about Candida drug-resistant in our national.
In a previous investigation, we found G487T and T916C existed simultaneously in a collection of fluconazole-resistant C. albicans by sequencing in ERG11 gene. The two mutations coexisted in fluconazole-resistant isolates without any other mutations in the ERG11 gene. In clinical isolates with the two mutations remain unreported at the present time. In the process of drug resistance in C. albicans, multiple mechanisms can induce resistance in varying degrees. But it is not clear in the gene expression (such as MDR1, CDR1/2 and FLU1) of these clinical isolates. There is no report about this at present. Are there any other similar points associated with fluconazole resistance in these isolates? And what about the expression of resistance genes? The aim of this study was to study expression of resistant genes and efflux pumps in every fluconazole-resistant C. albicans with G487T and T916C for the first time, and further to explore the role of gene expression in fluconazole-resistant isolates, thus provide potent experimental datas for determining mechanisms of drug resistance in these isolates, and provide essential data analysis and experiment background for drug resistance research in future.
Objectives
1. To investigate biological features of fluconazole-resistant C. albicans with mutations G487T and T916C, and to determine the efflux of Rhodamine 6G in these isolates.
2. To detect the mRNA expression of multidrug resistant genes in fluconazole-resistant C. albicans with mutations G487T and T916C by RT-PCR.
3. To detect Cdrlp and Cdr2p expression in fluconazole-resistant C. albicans with mutations G487T and T916C by Western blot.
Methods
1.Indentification of Candida spp. in the hospital Clinical isolates of candida species were collected, including sputum, urine, blood, pus, etc. inoculating specimen on blood plate, choosing dubious specimen and yeast-like strains were seperating by Garm's stain. Then separating and purificating on modified SDA slants, identifing C. albicans by CHROMagar medium. Fluconazole susceptibility was tested in vitro using microdilution and disc diffusion assays. The sensitive isolates (S), dose-dependent sensitive isolates (S-DD) and resistant isolates (R) were decided. M27-A2 broth dilution method were recommended by CLSI. Strains ATCC 6258 (C. krusei) and ATCC22019 (C. parapsilosis) were used as controls. Repeated three times for every test.
2. The isolates growth and Rhodamine 6G experiment
Experiment samples:14 fluconazole-resistant C. albicans isolates with mutations G487T and T916C and the control isolate ATCC10231. After activating twice, adjusting concentration of culture, yeast cells were transfered to RPMI1640 culture solution, YEPD culture solution and YEPD culture solution containing 64ug/ml fluconazole. Being cultured at 37℃, then observed growth of isolates by inverted phase contrast microscope.
Rhodamine 6G was used as tracer agent and the fluorescence densities of samples were measured at an excitation wavelength of 529 nm and an emission wavelength of 553 nm. The efflux of Rhodamine 6G was determined in the strains with mutations G487T and T916C.
3. Real-Time PCR experiment
Experiment samples:14 fluconazole-susceptible C. albicans isolates (from the first part),14 fluconazole-resistant C. albicans isolates with mutations G487T and T916C, the fluconazole-susceptible isolate ATCC 10231. The PCR primers to amplify of C. albicans were designed by referring C. albicans ERG11 sequence of GenBank X13296, CDR1(X77589), CDR2(U63812), MDR1(Y14703) and FLU1(AF188621). The housekeeping gene 18SrRNA was used as control. Total cellular RNA was extracted using the E.Z.N.A.TM Yeast RNA kit by manufacturer's description. The cDNA of each strain were synthesized in a two-step SYBR RT-PCR Kit. The reaction were performed at LightCyclerReal Time PCR machine. ATCC 10231 was used as a control isolate. The amplification products were separated using 2% agarose gel electrophoresis, visualized and photographed with an ImageMaster Video Documentation Systemto further verify their specificity. Using the 2-△△Ct method, one popular calculation formula to evaluate the expression levels of ERG11, CDR1, CDR2, FLU1 and MDR1 of C. albicans.
4. Western Blot experiment
Experiment samples:14 fluconazole-resistant C. albicans isolates with mutations G487T and T916C, Saccharomyces cerevisiae AD/CDR1 and AD/CDR2 were used as controls. Extracting plasma membrane proteins of samples, The concentration of protein were determined by BCA kit. The extracted proteins were electrophoresed on a 7.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The blots were visualized with enhanced chemiluminescene (ECL). The blots were incubated with a 1:500 dilution of anti-Cdrlp antibody, and a 1:1000 dilution of anti-Cdrlp antibody. For anti-Cdrlp antibody, immunoreactivity was detected with horseradish peroxidase-labeled goat anti-rabbit IgG antibodies. The Saccharomyces cerevisiae AD/CDR1 and AD/CDR2 were used as controls. Densitometric analysis of the band intensity was carried out using Quantity One software.
Results
1. Totally,161 clinical isolates of candida species were collected. Among the 161 strains of yeasts, Candida albicans was the most frequently species (69.57 %), followed by C.tropicalis (19.88%), C.parapsilosis (4.97%), C.krusei (2.48%), C.glabrata (1.86%)and others (1.24%).101 isolates from sputum specimen(62.73%),20 isolates from urine (12.42%),18 isolates from Vaginal secretions (11.18%),7 isolates from blood (4.35%) and 15 isolates from other sites (9.32%). The samples from department of respiratory tract were the most, the second is department of gynecology, the third is ICU and department of hematology. Among 112 candida albicans,108 sensitive isolates were detected (97.32%) by microdilution assay, and 109 sensitive isolates were detected (97.32%) by disc diffusion assay. The chi-square criterion displayed that there was no significant difference between them (P>0.05).
2. In all the fluconazole-resistant C.albicans with mutations, mycelial growth had notably increase in RPMI1640 culture, decreased in YEPD. And they were still quite lively in YEPD containing 64μg/ml fluconazole.
The experiment about Rhodamine 6G efflux showed that the efflux of Rhodamine 6G in ATCC10231 was weakest. All the fluconazole-resistant C. albicans isolates with mutations G487T and T916C can active efflux Rhodamine 6G, the efflux ability of Rhodamine 6G was enhanced as time prolonging. The efflux ability will weaken after 60 minutes.
3. Expression of the efflux gene CDR1 and CDR2 in 14 fluconazole-resistant isolates was upregulated 1.6-to 8-fold. Likewise, a 3.7-to 52-fold increase in CDR2 transcript levels was observed relative to the susceptible control strain ATCC 10231. Isolate J4266 had the highest CDR1 expression level, the lowest CDR1 expression level is isolate J4263, and 1.7 times as the control strain. The isolate GZ18 had the highest expression level and J4263 had the lowest expression level in CDR2. The expression of MDR1, FLU1 and ERG11 varied in the different C. albicans isolates compared with ATCC 10231. MDR1 overexpression was observed in four isolates, and isolate GZ23 had the same MDR1 transcript level. Upregulation of the ERG11 gene were observed in six isolates, respectively were GZ03, J4266, GZ17, J592, GZ23 and GZ51. Overexpression of the FLU1 gene were observed in six isolates, respectively were J4266, J592, GZ17, GZ03, GZ23 and GZ34.
4. The results of Western blot showed that expressed bands can be detected in the control strain Saccharomyces cerevisiae AD/CDR1 and AD/CDR2. Cdrlp were all detected in 14 fluconzazole-resistant C. albicans with G487T and T916C mutations, The analysis of the band intensity showed expression of J4266 was maxlmum, corresponding to the result of RT-PCR. But Cdr2p could not be detected in some isolates:GZ16, GZ34 and GZ51. The analysis of the band intensity showed expression of J592 was maximum, but the mRNA expression level of this isolate was inferior to GZ18 by RT-PCR. These results may suggest that stability of protein translation in CDR2 gene is uncertain.
Conclusions
1. The efflux pumps exist in all the fluconazole-resistant C. albicans with mutations G487T and T916C, and efflux of Rhodamine 6G will enhance as time prolonging, but the efflux ability will weaken after 60 minutes.
2. The efflux pumps also exist in fluconazole-susceptible C. albicans, but the efflux ability is far inferior to fluconazole-resistant C. albicans.
3. The expression of efflux gene CDR1 and CDR2 can be detected in fluconazole-resistant C. albicans with mutations G487T and T916C. The efflux gene CDR1 and CDR2 may play an important role in fluconazole resistance, whereas MDR1 play a minor role in fluconazole resistance in these isolates.
4. It may suggest that stability of protein translation in CDR2 gene is uncertain and decreased Cdr2p protein stability compared to Cdrlp in fluconazole-resistant C. albicans with mutations G487T and T916C.
唑类药物是目前应用最广的抗真菌药物。特别是氟康唑,自从20世纪80年代后期问世以来,由于其抗菌谱广、安全和可静脉给药,吸收好,毒性小等优点,成为目前临床治疗真菌感染的首选药物。但是,随着抗真菌药物的广泛使用,加之病人长时间的药物治疗和使用较低剂量的抗真菌药物,为耐药菌株的产生创造了条件。1980年Rosenblatt等人首次报道在应用酮康唑治疗慢性皮肤念珠菌病人身上出现药物耐药现象。此后有关耐药的报道越来越多。
麦角固醇是真菌细胞膜中特有的脂质。它维持着细胞膜的正常结构和功能。14α-去甲基化酶(Ergllp)是念珠菌细胞膜麦角固醇合成途径中的关键酶,ERGll是14α-去甲基化酶的编码基因。大量研究认为这个基因的点突变或过度表达均与唑类耐药相关。ERG11的突变可以改变14α-去甲基化酶的氨基酸序列,从而影响唑类药物与该酶的亲和力,导致菌株耐药。过度表达的外排泵基因CDR1和CDR2以及MDR1基因是第二个与耐药相关的原因。CDR1和CDR2基因属于ABC转运蛋白超家族(ATP-binding cassette transporters, ABC transporters),二者的同源性超过80%,在耐药机制中可发挥协同作用。MDR1基因属于易化扩散载体超家族(major facilitator supeffamily, MFS),目前认为它的表达也与氟康唑耐药相关。近年来发现FLU1基因也与唑类耐药相关。该基因与MDR1基因同源性较高,在耐氟康唑白念珠菌的研究中发现这个基因被敲除后可增加菌株对唑类药物的敏感性。此外,生物膜形成是近年来与耐药相关的一个新发现。生物膜耐药也是导致临床上许多系统性感染难以根除的重要因素。最近的研究认为细胞内液泡封闭药物分子也可能引发菌株耐药。
耐药的发生是一个多水平多因素参与的复杂过程。耐药白念珠菌的流行病学调查发现,85%的耐药株外排泵过度表达；35%的耐药株为药物靶酶的氨基酸改变或过度表达；75%的耐药株为多因素联合耐药。唑类药物对念珠菌耐药机制的研究已经成为当今医学研究的热点。但国内目前在念珠菌耐药性方面的研究仍然较少。
前期研究中我们通过测序发现14株临床耐氟康唑白念珠菌的ERG11基因集中发生G487T和T916C突变,且不伴随有其他突变。目前尚未见过临床菌株存在该突变点的报道。在白念珠菌耐药的发生发展过程中,多种机制均可在不同程度上促进菌株耐药的形成。我们尚不清楚是否有其它耐药机制(如MDR1、CDR1/2和FLU1)介入了这一系列临床株的耐药形成过程。目前也未见该方面的研究报道。该系列菌株在耐氟康唑机制中还存在其它的共同点么?耐药相关基因的表达在这些白念珠菌中又起了什么作用呢?本课题首次对每一株携带G487T和T916C突变的耐氟康唑白念珠菌的耐药相关基因的表达及外排泵功能进行了研究,进一步探讨该系列菌株的基因表达在氟康唑耐药中的作用,从而为明确该系列菌株的耐氟康唑机制提供有力的实验数据,并为后续进一步研究菌株耐药现象提供必要的数据分析及实验支持。
目的
1.了解携带G487T和T916C突变的耐氟康唑白念珠菌在不同培养液的生长情况,使用若丹明6G作为示踪剂检测该系列菌株的外排能力。
2.通过实时定量PCR方法检测携带G487T和T916C突变的耐氟康唑白念珠菌多个耐药相关基因mRNA表达水平。
3.通过蛋白印迹方法检测携带G487T和T916C突变的耐氟康唑白念珠菌Cdrlp和Cdr2p蛋白的表达。
方法
1.院内念珠菌鉴定
收集山东大学齐鲁医院检验科及皮肤科门诊的临床标本,包括痰液、尿液、血液及分泌物等。将标本接种于血平板,挑选可疑菌落涂片革兰染色确认为酵母样真菌后,转种于改良SDA平板分离纯化,通过科玛嘉念珠菌显色培养基鉴定,观察结果。应用微量稀释法和纸片扩散法测定氟康唑对试验菌株的MIC,确定敏感株(S)、剂量依赖敏感株(S-DD)和耐药株(R)。微量稀释法按照美国临床试验标准协会(CLSI)制定的M27-A2方案实施,ATCC 6258和ATCC22019被作为质控菌株。每次试验重复三次。
2.菌株生长及若丹明6G实验
实验菌株：携带G487T和T916C突变的耐氟康唑白念珠菌14株,敏感控制株ATCC102311株。将保存在SDA培养基的菌株经过两次活化,调整菌液浓度,将活化后的耐氟康唑白念珠菌分别转入lml RPMI1640培养液,lmlYEPD培养液和lml 64μg／ml氟康唑的YEPD培养液中,37℃静置培养,倒置相差显微镜下观察耐氟康唑白念珠菌的增殖情况。
使用若丹明6G作为示踪剂,通过测定荧光强度(激发波长529nm,吸收波长553nm)来计算细胞外液若丹明的浓度,进而检测若丹明6G在携带G487T和T916C突变的耐氟康唑白念珠菌中的外排情况。
3.实时定量PCR实验
实验菌株：氟康唑敏感白念珠菌14株(来自第一部分的收集菌株),携带G487T和T916C突变的耐氟康唑白念珠菌14株,敏感株ATCC102311株。根据各耐药基因的基因序列：ERG11(GeneBank号：X13296,下同),CDR1(X77589), CDR2(U63812), MDR1(Y14703), FLU1(AF188621)设计相应的引物。内参照选用看家基因18SrRNA。标准株ATCC 10231被作为敏感控制株。采用酵母RNA试剂盒分别提取各菌株RNA,逆转录成cDNA后,在LightCycler Real Time PCR扩增仪上进行实时定量PCR反应。PCR产物进行凝胶电泳,在凝胶成像系统下观察是否为目的条带,有无杂带。将获得的域值循环数(Ct值)采用2-ΔΔCt方法转换为目的基因的相对表达量,检测多个耐药基因的表达情况,比较该系列菌株CDR1、CDR2、MDR1、ERG11和FLU1基因的表达差异。
4.蛋白印迹实验
实验菌株：携带G487T和T916C突变的耐氟康唑白念珠菌14株,内参照菌株Saccharomyces cerevisiae AD/CDR1和AD/CDR2。提取菌株的质膜蛋白,BCA蛋白浓度测定试剂盒测定蛋白含量,通过7.5%SDS-聚丙烯酰胺凝胶电泳,转膜,蛋白印记,ECL试剂盒显色等步骤,检测菌株CDR1和CDR2基因的表达情况。抗Cdr1p抗体(1：500),抗Cdr2p抗体(1：1000),二抗选用HRP标记的山羊抗兔二抗(抗Cdrlp抗体1：5000；抗Cdr2p抗体1：10000)。应用数字凝胶成像系统FluorChem 9900-50型进行扫描并记录图像,用Quantity One software软件进行半定量分析,计算各组目的蛋白相对于内参的相对灰度值作为各蛋白的相对含量。
结果
1.共收集酵母样菌161株,在161株菌中白念珠菌比例为69.57%,热带念珠菌为19.88%,近平滑念珠菌为4.97%,克柔念珠菌为2.48%,光滑念珠菌为1.86%,其他念珠菌为1.24%。痰标本最多,101份,占所有标本的62.73%。其次为尿液标本,20份(12.42%),来源于阴道分泌物的标本比例为11.18%,血液来源的标本占4.35%,其余(咽拭子、大便、导管分泌物、透析液、胆汁、脐分泌物、支气管肺泡冲洗液等)占9.32%。这些标本中,来自呼吸科的标本占的比例最大,妇科、ICU、血液科次之。112株白念珠菌中,通过微量稀释法筛选出108株敏感株,占96.43%,纸片扩散法试验筛选出109株敏感株(97.32%)。两种方法经卡方检验P>0.05,二者无明显统计学差异。
2.在RPMI1640培养液中,该系列白念珠菌可形成大量菌丝；在YEPD培养液中白念珠菌的菌丝形成数目明显减少；在64μg／ml氟康唑的培养液中它们仍表现活跃。
若丹明6G外排实验结果显示：敏感控制株ATCC10231主动外排若丹明6G能力在所有受试菌株中表现最弱。携带G487T和T916C突变的耐氟康唑白念珠菌在能量供给下可高效主动外排若丹明6G。随着时间的延长该外排能力增强,但60min之后转运能力减弱至平衡或下降。
3.实时定量PCR方法显示携带G487T和T916C突变的耐药组CDR1和CDR2基因的表达明显高于敏感组；而两组的ERG11,MDR1和FLU1基因在mRNA表达水平上差异无统计学意义。与敏感控制株ATCC10231相比,14株携带G487T和T916C突变的耐氟康唑白念珠菌的主动外排泵CDR1和CDR2基因的mRNA表达水平都是明显增高的,分别达到1.6-8倍和3.7-52倍。其中菌株J4266的CDR1 mRNA表达水平最高,而菌株J4263的CDR1mRNA表达水平在14株中最低,但仍然是敏感株的1.7倍。菌株GZ18的CDR2基因mRNA表达是最高的。J4263的CDR2基因mRNA表达水平在14株中最低。在MDR1,FLU1和ERG1l基因中,各个菌株的表达水平是高低不一的。MDR1的表达在4株菌中表达是增高的,分别是菌株GZ17、GZ03、J4266和J592。菌株GZ23的MDR1基因的mRNA表达水平与敏感控制株相同。6株菌中发现ERG11基因过度表达,分别是GZ03、J4266、GZ17、J592、GZ23和GZ51。6株菌的FLU1基因过度表达,分别是J4266、J592、GZ17、GZ03、GZ23和GZ34。
4.蛋白印迹实验显示：内参照Saccharomyces cerevisiae AD/CDR1可以稳定表达Cdr1p, Saccharomyces cerevisiae AD/CDR2菌株可以稳定表达Cdr2p。在这些携带G487T和T916C突变的耐氟康唑白念珠菌中,所有菌株的CDR1基因均有明显的表达,半定量分析结果示菌株J4266表达量最高。这与实时定量mRNA结果相符。但在菌株GZ16、GZ34和GZ51,无法检测到CDR2基因的完整表达。半定量分析结果示菌株J592的Cdr2p蛋白表达最高。而在实时定量PCR比较中它的mRNA表达水平要低于GZ18。这可能提示该系列菌株CDR2基因的蛋白翻译并不是十分稳定的。
结论
1.携带G487T和T916C突变的耐氟康唑白念珠菌均具有主动外排若丹明6G的能力,且该能力随着时间延长而增强,但在60min之后逐渐下降至平衡。
2.氟康唑敏感白念珠菌也可具有主动外排若丹明6G的能力,但该能力远低于携带G487T和T916C突变的耐氟康唑菌株。
3.主动外排泵基因CDR1和CDR2的过度表达存在于每一株携带G487T和T916C突变的白念珠菌。主动外排泵基因CDR1和CDR2在携带G487T和T916C突变的白念珠菌耐氟康唑机制中可能发挥重要作用,而易化扩散载体蛋白MDR1基因的作用可能并不明显。
4.在携带G487T和T916C突变的耐氟康唑白念珠菌中,CDR2基因转录后的蛋白翻译并不十分稳定,Cdr2p蛋白翻译的稳定性可能弱于Cdr1p。
The genus Candida (especially Candida albicans) is the important cause of illness that leads to opportunistic mycoses and one of the main pathogens of nosocomial fungal infections. Following the widespread application of immunodepressants, glucocorticoid, broad-spectrum antibiotics, therapeusis of intervention, organ transplantation and the expanding of population with immunity inhibition, the infection rate of fungi is adscendent tendency in the lately years. The infection caused by pathomycetes candida albicans was the most common.
The azole agents is the most extensively used in the treatment of fungal infections. Especially fluconazole, as the first line azole derivatives, because of broad antibacterial spectrum, strong antibacterial activity, few side effect, safetyly for intravenous injection and so on, is commonly used to treat various candidiasis in clinic since the advent of fluconazole in the late 1980s. However, With the mass use of antifungal agents, resistant strains emerge more frequently under the drug selection pressure and prolonged administration of broad-spectrum antibacterial agents or prophylactic treatment. In 1980, Rosenblatt et al. had reported azole drugs resistance in treating chronic mucocutaneous candidids with ketoconazole for the first time. After that, the phenomenon of drugs resistance are increasing gradually.
Ergosterol is typical lipidd in cellular membrane of fungi. It can maintain the integrity and function of Candida albicans (C. albicans) membrane.14 alpha-demethylase (Erg11p) is a key enzyme in the ergosterol synthesis pathway of C. albicans. The coding gene is ERG11. Mutations in ERG11 can result in changes in the Ergllp spatial configuration, affect the affinity between the azole drugs and the enzyme, and makes isolates resistant to azole finally. The other is that increased expression of the ERG11 gene associated with antifungal drug resistance. The next possible mechanism for drug resistance is over-expression of the CDR1 and CDR2 genes, encoding transporters of the ABC family, and the MDR1 gene, coding for a major facilitator transporter. The homology of CDR1 and CDR2 is greater than 80%, they can have a synergistic effect in azole drug resistance. Another major facilitator gene, FUU1, has also been identified in C. albicans. The FLU1 gene, similar to MDR1, is related with azole resistance. It is reported that this gene can increase azole susceptibility when it is deleted in the study about fluconazole-resistant candida albicans. Besides that, biofilms may be a new mechanism that correlated with resistance development in C. albicans. Biofilms may be associated with poor prognosis in untreated systemic infections. And finally, vesicular vacuoles from resistant isolates may act as a novel fluconazole resistance mechanism.
Occurrence of drug resistance is a complex process involving many different factors. In an analysis about the prevalence of mechanisms of resistance in C. albicans clinical isolates showed that multiple mechanisms of resistance were combined in 75% of resistant isolates, overexpression of genes encoding efflux pumps were detected in 85% of isolates, amino acid substitutions in ERG11 were found in 65% of isolates,35% of the isolates showed overexpression of ERG11. The studies on azole-resistance mechanisms of Candida spp are becoming a very active subject in the world. But there is a few works about Candida drug-resistant in our national.
In a previous investigation, we found G487T and T916C existed simultaneously in a collection of fluconazole-resistant C. albicans by sequencing in ERG11 gene. The two mutations coexisted in fluconazole-resistant isolates without any other mutations in the ERG11 gene. In clinical isolates with the two mutations remain unreported at the present time. In the process of drug resistance in C. albicans, multiple mechanisms can induce resistance in varying degrees. But it is not clear in the gene expression (such as MDR1, CDR1/2 and FLU1) of these clinical isolates. There is no report about this at present. Are there any other similar points associated with fluconazole resistance in these isolates? And what about the expression of resistance genes? The aim of this study was to study expression of resistant genes and efflux pumps in every fluconazole-resistant C. albicans with G487T and T916C for the first time, and further to explore the role of gene expression in fluconazole-resistant isolates, thus provide potent experimental datas for determining mechanisms of drug resistance in these isolates, and provide essential data analysis and experiment background for drug resistance research in future.
Objectives
1. To investigate biological features of fluconazole-resistant C. albicans with mutations G487T and T916C, and to determine the efflux of Rhodamine 6G in these isolates.
2. To detect the mRNA expression of multidrug resistant genes in fluconazole-resistant C. albicans with mutations G487T and T916C by RT-PCR.
3. To detect Cdrlp and Cdr2p expression in fluconazole-resistant C. albicans with mutations G487T and T916C by Western blot.
Methods
1.Indentification of Candida spp. in the hospital Clinical isolates of candida species were collected, including sputum, urine, blood, pus, etc. inoculating specimen on blood plate, choosing dubious specimen and yeast-like strains were seperating by Garm's stain. Then separating and purificating on modified SDA slants, identifing C. albicans by CHROMagar medium. Fluconazole susceptibility was tested in vitro using microdilution and disc diffusion assays. The sensitive isolates (S), dose-dependent sensitive isolates (S-DD) and resistant isolates (R) were decided. M27-A2 broth dilution method were recommended by CLSI. Strains ATCC 6258 (C. krusei) and ATCC22019 (C. parapsilosis) were used as controls. Repeated three times for every test.
2. The isolates growth and Rhodamine 6G experiment
Experiment samples:14 fluconazole-resistant C. albicans isolates with mutations G487T and T916C and the control isolate ATCC10231. After activating twice, adjusting concentration of culture, yeast cells were transfered to RPMI1640 culture solution, YEPD culture solution and YEPD culture solution containing 64ug/ml fluconazole. Being cultured at 37℃, then observed growth of isolates by inverted phase contrast microscope.
Rhodamine 6G was used as tracer agent and the fluorescence densities of samples were measured at an excitation wavelength of 529 nm and an emission wavelength of 553 nm. The efflux of Rhodamine 6G was determined in the strains with mutations G487T and T916C.
3. Real-Time PCR experiment
Experiment samples:14 fluconazole-susceptible C. albicans isolates (from the first part),14 fluconazole-resistant C. albicans isolates with mutations G487T and T916C, the fluconazole-susceptible isolate ATCC 10231. The PCR primers to amplify of C. albicans were designed by referring C. albicans ERG11 sequence of GenBank X13296, CDR1(X77589), CDR2(U63812), MDR1(Y14703) and FLU1(AF188621). The housekeeping gene 18SrRNA was used as control. Total cellular RNA was extracted using the E.Z.N.A.TM Yeast RNA kit by manufacturer's description. The cDNA of each strain were synthesized in a two-step SYBR RT-PCR Kit. The reaction were performed at LightCyclerReal Time PCR machine. ATCC 10231 was used as a control isolate. The amplification products were separated using 2% agarose gel electrophoresis, visualized and photographed with an ImageMaster Video Documentation Systemto further verify their specificity. Using the 2-△△Ct method, one popular calculation formula to evaluate the expression levels of ERG11, CDR1, CDR2, FLU1 and MDR1 of C. albicans.
4. Western Blot experiment
Experiment samples:14 fluconazole-resistant C. albicans isolates with mutations G487T and T916C, Saccharomyces cerevisiae AD/CDR1 and AD/CDR2 were used as controls. Extracting plasma membrane proteins of samples, The concentration of protein were determined by BCA kit. The extracted proteins were electrophoresed on a 7.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The blots were visualized with enhanced chemiluminescene (ECL). The blots were incubated with a 1:500 dilution of anti-Cdrlp antibody, and a 1:1000 dilution of anti-Cdrlp antibody. For anti-Cdrlp antibody, immunoreactivity was detected with horseradish peroxidase-labeled goat anti-rabbit IgG antibodies. The Saccharomyces cerevisiae AD/CDR1 and AD/CDR2 were used as controls. Densitometric analysis of the band intensity was carried out using Quantity One software.
Results
1. Totally,161 clinical isolates of candida species were collected. Among the 161 strains of yeasts, Candida albicans was the most frequently species (69.57 %), followed by C.tropicalis (19.88%), C.parapsilosis (4.97%), C.krusei (2.48%), C.glabrata (1.86%)and others (1.24%).101 isolates from sputum specimen(62.73%),20 isolates from urine (12.42%),18 isolates from Vaginal secretions (11.18%),7 isolates from blood (4.35%) and 15 isolates from other sites (9.32%). The samples from department of respiratory tract were the most, the second is department of gynecology, the third is ICU and department of hematology. Among 112 candida albicans,108 sensitive isolates were detected (97.32%) by microdilution assay, and 109 sensitive isolates were detected (97.32%) by disc diffusion assay. The chi-square criterion displayed that there was no significant difference between them (P>0.05).
2. In all the fluconazole-resistant C.albicans with mutations, mycelial growth had notably increase in RPMI1640 culture, decreased in YEPD. And they were still quite lively in YEPD containing 64μg/ml fluconazole.
The experiment about Rhodamine 6G efflux showed that the efflux of Rhodamine 6G in ATCC10231 was weakest. All the fluconazole-resistant C. albicans isolates with mutations G487T and T916C can active efflux Rhodamine 6G, the efflux ability of Rhodamine 6G was enhanced as time prolonging. The efflux ability will weaken after 60 minutes.
3. Expression of the efflux gene CDR1 and CDR2 in 14 fluconazole-resistant isolates was upregulated 1.6-to 8-fold. Likewise, a 3.7-to 52-fold increase in CDR2 transcript levels was observed relative to the susceptible control strain ATCC 10231. Isolate J4266 had the highest CDR1 expression level, the lowest CDR1 expression level is isolate J4263, and 1.7 times as the control strain. The isolate GZ18 had the highest expression level and J4263 had the lowest expression level in CDR2. The expression of MDR1, FLU1 and ERG11 varied in the different C. albicans isolates compared with ATCC 10231. MDR1 overexpression was observed in four isolates, and isolate GZ23 had the same MDR1 transcript level. Upregulation of the ERG11 gene were observed in six isolates, respectively were GZ03, J4266, GZ17, J592, GZ23 and GZ51. Overexpression of the FLU1 gene were observed in six isolates, respectively were J4266, J592, GZ17, GZ03, GZ23 and GZ34.
4. The results of Western blot showed that expressed bands can be detected in the control strain Saccharomyces cerevisiae AD/CDR1 and AD/CDR2. Cdrlp were all detected in 14 fluconzazole-resistant C. albicans with G487T and T916C mutations, The analysis of the band intensity showed expression of J4266 was maxlmum, corresponding to the result of RT-PCR. But Cdr2p could not be detected in some isolates:GZ16, GZ34 and GZ51. The analysis of the band intensity showed expression of J592 was maximum, but the mRNA expression level of this isolate was inferior to GZ18 by RT-PCR. These results may suggest that stability of protein translation in CDR2 gene is uncertain.
Conclusions
1. The efflux pumps exist in all the fluconazole-resistant C. albicans with mutations G487T and T916C, and efflux of Rhodamine 6G will enhance as time prolonging, but the efflux ability will weaken after 60 minutes.
2. The efflux pumps also exist in fluconazole-susceptible C. albicans, but the efflux ability is far inferior to fluconazole-resistant C. albicans.
3. The expression of efflux gene CDR1 and CDR2 can be detected in fluconazole-resistant C. albicans with mutations G487T and T916C. The efflux gene CDR1 and CDR2 may play an important role in fluconazole resistance, whereas MDR1 play a minor role in fluconazole resistance in these isolates.
4. It may suggest that stability of protein translation in CDR2 gene is uncertain and decreased Cdr2p protein stability compared to Cdrlp in fluconazole-resistant C. albicans with mutations G487T and T916C.
引文
1. Pfaller M, Wenzel R. Impact of the changing epidemiology of fungal infections in the 1990s. Eur J Clin Microbiol Infect Dis.1992 Apr;11(4):287-91.
2. Ghannoum MA, Rex JH, Galgiani JN. Susceptibility testing of fungi:current status of correlation of in vitro data with clinical outcome. Journal of clinical microbiology.1996 Mar;34(3):489-95.
3. 吴绍熙.现代医学真菌检验手册.北京：北京医科大学、中国协和医科大学联合出版社,1998：327-39.
4. Jarvis WR. Epidemiology of nosocomial fungal infections, with emphasis on Candida species. Clin Infect Dis.1995 Jun;20(6):1526-30.
5. 钟南山,叶枫.深部真菌感染：新的挑战与展望.中华结核和呼吸杂志,2006,29(5)：289-90.
6. 吴安华,任南,文细毛等.159所医院医院感染现患率调查结果与分析,中国感染控制杂志,2005,4(1)：12-7.
7. Rangel-Frausto MS, Wiblin T, Blumberg HM, et al. National epidemiology of mycoses survey (NEMIS):variations in rates of bloodstream infections due to Candida species in seven surgical intensive care units and six neonatal intensive care units. Clin Infect Dis.1999 Aug;29(2):253-8.
8. Hajjeh RA, Sofair AN, Harrison LH, et al. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. Journal of clinical microbiology.2004 Apr;42(4):1519-27.
9. Pfaller MA, Messer SA, Hollis RJ, Jones RN, Diekema DJ. In vitro activities of ravuconazole and voriconazole compared with those of four approved systemic antifungal agents against 6,970 clinical isolates of Candida spp. Antimicrobial agents and chemotherapy.2002 Jun;46(6):1723-7.
10. 吴绍熙,廖万清,郭宁如等.中国致病真菌10年动态流行病学研究.临床 皮肤科杂志,1999,28(1)：1-5.
11. Abelson JA, Moore T, Bruckner D, Deville J, Nielsen K. Frequency of fungemia in hospitalized pediatric inpatients over 11 years at a tertiary care institution. Pediatrics.2005 Jul;116(1):61-7.
12. Cowen LE, Sanglard D, Calabrese D, Sirjusingh C, Anderson JB, Kohn LM. Evolution of drug resistance in experimental populations of Candida albicans. Journal of bacteriology.2000 Mar;182(6):1515-22.
13. Magaldi S, Mata S, Hartung C, et al. In vitro susceptibility of 137 Candida sp. isolates from HIV positive patients to several antifungal drugs. Mycopathologia.2001;149(2):63-8.
14. Sangeorzan JA, Bradley SF, He X, et al. Epidemiology of oral candidiasis in HIV-infected patients:colonization, infection, treatment, and emergence of fluconazole resistance. The American journal of medicine.1994 Oct;97(4):339-46.
15. Graybill JR. Changing strategies for treatment of systemic mycoses. Braz J Infect Dis.2000 Apr;4(2):47-54.
16. Rosenblatt HM, Byrne W, Ament ME, Graybill J, Stiehm ER. Successful treatment of chronic mucocutaneous candidiasis with ketoconazole. The Journal of pediatrics.1980 Oct;97(4):657-60.
17. Redding S, Smith J, Farinacci G, et al. Resistance of Candida albicans to fluconazole during treatment of oropharyngeal candidiasis in a patient with AIDS:documentation by in vitro susceptibility testing and DNA subtype analysis. Clin Infect Dis.1994 Feb;18(2):240-2.
18. Moran GP, Sullivan DJ, Henman MC, et al. Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro. Antimicrobial agents and chemotherapy.1997 Mar;41(3):617-23.
19. Rocco TR, Reinert SE, Simms HH. Effects of fluconazole administration in critically ill patients:analysis of bacterial and fungal resistance. Arch Surg. 2000 Feb；135(2)：160-5.
20. 张秀珍.深部真菌感染及耐药变迁.中国处方药,2004,24(3)：65-8.
21. Diekema DJ, Messer SA, Brueggemann AB, et al. Epidemiology of candidemia:3-year results from the emerging infections and the epidemiology of Iowa organisms study. Journal of clinical microbiology.2002 Apr;40(4):1298-302.
22. Wroblewska MM, Swoboda-Kopec E, Rokosz A, Krawczyk E, Marchel H, Luczak M. Epidemiology of clinical isolates of Candida albicans and their susceptibility to triazoles. International journal of antimicrobial agents.2002 Dec;20(6):472-5.
23. 杨蓬勃,胡云建.住院患者真菌感染及耐药性分析.中华医院感染学杂志.2007,17(6)：729-31.
24. 刘彦慧,赖志刚,张丽华.念珠菌检测与药敏分析.中国皮肤性病学杂志.2005,19(7)：411-13.
25. 常东,蒋伟,黄志红,等.真菌感染及耐药性分析.中华医院感染学杂志.2005,15(3)：345-47.
26. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG 11 mutations. The Journal of antimicrobial chemotherapy.2008 Apr;61(4):798-804.
27. 吴绍熙,郭宁如,廖万清.现代真菌病诊断治疗学.北京：北京医科大学、中国协和医科大学联合出版,1997：182-206.
28. White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clinical microbiology reviews. 1998 Apr;11(2):382-402.
29. White TC. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans. Antimicrobial agents and chemotherapy. 1997 Jul;41(7):1488-94.
30. Marichal P, Koymans L, Willemsens S, et al. Contribution of mutations in the cytochrome P450 14alpha-demethylase (Ergllp, Cyp51p) to azole resistance in Candida albicans. Microbiology (Reading, England).1999 Oct;145 (Pt 10):2701-13.
31. Podust LM, Poulos TL, Waterman MR. Crystal structure of cytochrome P450 14alpha-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proceedings of the National Academy of Sciences of the United States of America.2001 Mar 13;98(6):3068-73.
32. Revankar SG, Kirkpatrick WR, McAtee RK, et al. Detection and significance of fluconazole resistance in oropharyngeal candidiasis in human immunodeficiency virus-infected patients. The Journal of infectious diseases. 1996 Oct;174(4):821-7.
33. Sanglard D, Ischer F, Koymans L, Bille J. Amino acid substitutions in the cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrobial agents and chemotherapy.1998 Feb;42(2):241-53.
34. Goldman GH, da Silva Ferreira ME, dos Reis Marques E, et al. Evaluation of fluconazole resistance mechanisms in candida albicans clinical isolates from HIV-infected patients in Brazil. Diagnostic microbiology and infectious disease.2004 Sep;50(1):25-32.
35. Wang WL, Li RY, Wang DL, et al. Resistant mechanisms of Candida albicans to fluconazole. Chin Med J.1999,112(5):466-471.
36. Lamb DC, Kelly DE, Schunck WH, et al. The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. The Journal of biological chemistry.1997 Feb 28;272(9):5682-8.
37. White TC. Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrobial agents and chemotherapy.1997 Jul;41(7):1482-7.
38. Albertson GD, Niimi M, Cannon RD, Jenkinson HF. Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrobial agents and chemotherapy.1996 Dec;40(12):2835-41.
39. Franz R, Kelly SL, Lamb DC, Kelly DE, Ruhnke M, Morschhauser J. Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains. Antimicrobial agents and chemotherapy.1998 Dec;42(12):3065-72.
40. Nakayama H, Ikebe T, Beppu M, Shirasuna K. High expression levels of nuclear factor kappaB, IkappaB kinase alpha and Akt kinase in squamous cell carcinoma of the oral cavity. Cancer.2001 Dec 15;92(12):3037-44.
41. White TC, Holleman S, Dy F, Mirels LF, Stevens DA. Resistance mechanisms in clinical isolates of Candida albicans. Antimicrobial agents and chemotherapy.2002 Jun;46(6):1704-13.
42. Kelly SL, Lamb DC, Kelly DE, et al. Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol delta5,6-desaturation. FEBS letters.1997 Jan 2;400(1):80-2.
43. Kelly SL, Lamb DC, Kelly DE, Loeffler J, Einsele H. Resistance to fluconazole and amphotericin in Candida albicans from AIDS patients. Lancet. 1996 Nov 30;348(9040):1523-4.
44. Miyazaki Y, Geber A, Miyazaki H, et al. Cloning, sequencing, expression and allelic sequence diversity of ERG3 (C-5 sterol desaturase gene) in Candida albicans. Gene.1999 Aug 5;236(1):43-51.
45. Wolfger H, Mamnun YM, Kuchler K. Fungal ABC proteins:pleiotropic drug resistance, stress response and cellular detoxification. Research in microbiology.2001 Apr-May; 152(3-4):375-89.
46. Prasad R, De Wergifosse P, Goffeau A, Balzi E. Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals. Current genetics.1995 Mar;27(4):320-9.
47. Sanglard D, Ischer F, Monod M, Bille J. Cloning of Candida albicans genes conferring resistance to azole antifungal agents:characterization of CDR2, a new multidrug ABC transporter gene. Microbiology (Reading, England).1997 Feb;143(Pt 2):405-16.
48. Morschhauser J. The genetic basis of fluconazole resistance development in Candida albicans. Biochimica et biophysica acta.2002 Jul 18; 1587 (2-3):240-8.
49. Goldway M, Teff D, Schmidt R, Oppenheim AB, Koltin Y. Multidrug resistance in Candida albicans:disruption of the BENr gene. Antimicrobial agents and chemotherapy.1995 Feb;39(2):422-6.
50. Calabrese D, Bille J, Sanglard D. A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole. Microbiology (Reading, England).2000 Nov;146 (Pt 11):2743-54.
51. Hawser SP, Douglas LJ. Resistance of Candida albicans biofilms to antifungal agents in vitro. Antimicrobial agents and chemotherapy.1995 Sep; 39 (9):2128-31.
52. Baillie GS, Douglas LJ. Role of dimorphism in the development of Candida albicans biofilms. Journal of medical microbiology.1999 Jul;48(7):671-9.
53. Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, McCormick T, Ghannoum MA. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. Journal of bacteriology.2001 Sep; 183(18):5385-94.
54. Donlan RM, Costerton JW. Biofilms:survival mechanisms of clinically relevant microorganisms. Clinical microbiology reviews.2002 Apr; 15 (2):167-93.
55. Maebashi K, Kudoh M, Nishiyama Y, et al. A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient. Microbiology and immunology. 2002;46(5):317-26.
56. Loffler J, Einsele H, Hebart H, Schumacher U, Hrastnik C, Daum G Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains. FEMS microbiology letters.2000 Apr 1; 185(1):59-63.
57. Kohli A, Smriti, Mukhopadhyay K, Rattan A, Prasad R. In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry. Antimicrobial agents and chemotherapy.2002 Apr;46(4):1046-52.
58. 卫生部医政司.全国临床检验操作规程第二版.南京：东南大学出版社,1997：452-71.
59. Pinjon E, Sullivan D, Salkin I, Shanley D, Coleman D. Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. Journal of clinical microbiology.1998 Jul;36(7):2093-5.
60. National committee for clinical laboratory standards. Reference method for broth dilution antifungal susceptibility testing of yeasts, Approved standard-Second Edition. NCCLS document M27-A2.2002,22:1-29.
61. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.2001 Dec;25(4):402-8.
62. Prasad R, Panwar SL, Smriti. Drug resistance in yeasts-an emerging scenario. Advances in microbial physiology.2002;46:155-201.
63. 韦莉萍,桂希恩,杨志成,等.医院内真菌深部感染调查及其危险因素分析.中华医院感染学杂志,1998,8(1)：28-30.
64. 刘自贵,裘雁秋,谭明珍,等.院内念珠菌感染及其药物敏感性研究.中华医院感染学杂志,2000,10(4)：304-05.
65. Schoofs A, Odds FC, Colebunders R, Ieven M, Goossens H. Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients. Eur J Clin Microbiol Infect Dis.1997 Apr; 16(4):296-300.
66. Jabra-Rizk MA, Brenner TM, Romagnoli M, et al. Evaluation of a reformulated CHROMagar Candida. Journal of clinical microbiology.2001 May;39(5):2015-6.
67. Berrouane YF, Herwaldt LA, Pfaller MA. Trends in antifungal use and epidemiology of nosocomial yeast infections in a university hospital. Journal of clinical microbiology.1999 Mar;37(3):531-7.
68. 吴爱武.院内感染标本分离的260株念珠菌调查与分析.上海医学检验杂志,2000,15(3)：172-73.
69. 方芳,伊和姿,吴丽桂.危重病人203例深部真菌感染的调查研究.皮肤病与性病,2005,27(1)：6-7.
70. Raad I, Hanna H, Boktour M, et al. Management of central venous catheters in patients with cancer and candidemia. Clin Infect Dis.2004 Apr 15; 38(8):1119-27.
71. Klotz SA, Drutz DJ, Zajic JE. Factors governing adherence of Candida species to plastic surfaces. Infection and immunity.1985 Oct;50(1):97-101.
72. Wingard JR, Merz WG, Rinaldi MG, Johnson TR, Karp JE, Saral R. Increase in Candida krusei infection among patients with bone marrow transplantation and neutropenia treated prophylactically with fluconazole. The New England journal of medicine.1991 Oct 31;325(18):1274-7.
73. Saville SP, Lazzell AL, Monteagudo C, Lopez-Ribot JL. Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection. Eukaryotic cell.2003 Oct;2(5):1053-60.
74. Whiteway M, Oberholzer U. Candida morphogenesis and host-pathogen interactions. Current opinion in microbiology.2004 Aug;7(4):350-7.
75. Haynes K. Virulence in Candida species. Trends in microbiology.2001 Dec;9(12):591-6.
76. Ha KC, White TC. Effects of azole antifungal drugs on the transition from yeast cells to hyphae in susceptible and resistant isolates of the pathogenic yeast Candida albicans. Antimicrobial agents and chemotherapy.1999 Apr;43(4):763-8.
77. Sanglard D, Kuchler K, Ischer F, Pagani JL, Monod M, Bille J. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrobial agents and chemotherapy.1995 Nov;39(11):2378-86.
78. Maesaki S, Marichal P, Vanden Bossche H, Sanglard D, Kohno S. Rhodamine 6G efflux for the detection of CDR1-overexpressing azole-resistant Candida albicans strains. The Journal of antimicrobial chemotherapy.1999 Jul;44(1):27-31.
79. 郭慧君,夏忠弟,吴高莉,等.耐药白念珠菌主动外排泵蛋白的功能与基因表达.中南大学学报(医学版).2007,32(1)：183-7.
80. 孙伟玮,高平挥,王英,等.ABC转运蛋白与临床白念珠茵耐药.第二军医大学学报.2008,29(9)：1038-41.
81. 饶勇,曾振灵,陈杖榴.抗生素耐药性的主动外排机制.国外医药·抗生素分册,2002,23(3)：109-114.
82. Balan I, Alarco AM, Raymond M. The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter. Journal of bacteriology.1997 Dec; 179 (23):7210-8.
83. Franz R, Michel S, Morschhauser J. A fourth gene from the Candida albicans CDR family of ABC transporters. Gene.1998 Oct 5;220(1-2):91-8.
84. St Georgiev V. Membrane transporters and antifungal drug resistance. Current drug targets.2000 Nov;l(3):261-84.
85. Schuetzer-Muehlbauer M, Willinger B, Krapf G, Enzinger S, Presterl E, Kuchler K. The Candida albicans Cdr2p ATP-binding cassette (ABC) transporter confers resistance to caspofungin. Molecular microbiology.2003 Apr;48(1):225-35.
86. Albertson GD, Niimi M, Cannon RD, Jenkinson HF. Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrobial agents and chemotherapy.1996 Dec;40(12):2835-41.
87. Del Sorbo G, Schoonbeek H, De Waard MA. Fungal transporters involved in efflux of natural toxic compounds and fungicides. Fungal Genet Biol.2000 Jun;30(1):1-15.
88. Smriti, Krishnamurthy SS, Prasad R. Membrane fluidity affects functions of Cdrlp, a multidrug ABC transporter of Candida albicans. FEMS microbiology letters.1999 Apr 15;173(2):475-81.
89. Ramage G, Bachmann S, Patterson TF, Wickes BL, Lopez-Ribot JL. Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. The Journal of antimicrobial chemotherapy.2002 Jun;49(6):973-80.
90. Niimi M, Niimi K, Takano Y, et al. Regulated overexpression of CDR1 in Candida albicans confers multidrug resistance. The Journal of antimicrobial chemotherapy.2004 Dec;54(6):999-1006.
91. Perea S, Lopez-Ribot JL, Kirkpatrick WR, et al. Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrobial agents and chemotherapy.2001 Oct;45(10):2676-84.
92. O'Connor L, Lahiff S, Casey F, Glennon M, Cormican M, Maher M. Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR using hybridisation probes on the LightCycler. Molecular and cellular probes.2005 Jun; 19(3):153-62.
93. Morschhauser J, Barker KS, Liu TT, Bla BWJ, Homayouni R, Rogers PD. The transcription factor Mrrlp controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS pathogens.2007 Nov;3(11):e164.
94. Chau AS, Mendrick CA, Sabatelli FJ, Loebenberg D, McNicholas PM. Application of real-time quantitative PCR to molecular analysis of Candida albicans strains exhibiting reduced susceptibility to azoles. Antimicrobial agents and chemotherapy.2004 Jun;48(6):2124-31.
95. Park S, Perlin DS. Establishing surrogate markers for fluconazole resistance in Candida albicans. Microbial drug resistance (Larchmont, NY.2005 Fall; 11(3):232-8.
96. Holmes AR, Lin YH, Niimi K, et al. ABC transporter Cdrlp contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates. Antimicrobial agents and chemotherapy.2008 Nov;52(11):3851-62.
97. Nakamura K, Niimi M, Niimi K, et al. Functional expression of Candida albicans drug efflux pump Cdrlp in a Saccharomyces cerevisiae strain deficient in membrane transporters. Antimicrobial agents and chemotherapy. 2001 Dec;45(12):3366-74.
98. Lyons CN, White TC. Transcriptional analyses of antifungal drug resistance in Candida albicans. Antimicrobial agents and chemotherapy.2000 Sep; 44(9):2296-303.
1. Foxman B, Marsh JV, Gillespie B et al (1998) Frequency and response to vaginal symptoms among white and African American women:results of a random digit dialing survey. J Womens Health 7:1167-1174
2. Lanchares JL, Hermandez ML (2000) Recurrent vaginal candidiasis changes in etiopathogenical patterns. Int Gynaecol Obstet 71 Suppl 1:S29-35
3. Landers DV, Wiesenfeld HC, Heine RP et al (2004) Predictive value of the clinical diagnosis of lower genital tract infection in women. Am J Obstet Gynecol 190:1004-1010
4. Nyirjesy P, Seeney SM, Grody MH et al (1995) Chronic fungal vaginitis:the value of cultures. Am J Obstet Gynecol 173:820-823
5. Sobel JD, Chaim W (1997) Treatment of Torulopsis glabrata vaginitis: retrospective review of boric acid therapy. Clin Infect Dis 24:649-652
6. Spinillo A, Capuzzo E, Gulminetti R et al (1997) Prevalence of and risk factors for fungal vaginitis caused by non-albicans species. Am J Obstet Gynecol 176:138-141
7. Boschman CR, Bodnar UR, Tornatore MA et al (1998) Thirteen-year evolution of azole resistance in yeast isolates and prevalence of resistant strains carried by cancer patients at a large medical center. Antimicrob Agents Chemother 42:734-738
8. Sobel JD, Zervos M, Reed BD et al (2003) Fluconazole susceptibility of vaginal isolates obtained from women with complicated Candida vaginitis:clinical implications. Antimicrob Agents Chemother 47:34-38
9. Bauters TG, Dhont MA, Temmerman MI et al (2002) Prevalence of vulvovaginal candidiasis and susceptibility to fluconazole in women. Am J Obstet Gynecol 187:569-574
10. Calabrese D, Bille J, Sanglard D (2000) A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole. Microbiology 146 (Pt 11):2743-2754
11. Perea S, Lopez-Ribot JL, Kirkpatrick WR, et al (2001) Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 45(10):2676-2684
12. Andes D, Nett J, Oschel P et al (2004) Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect and Immun 72:6023-6031
13. Maebashi K, Kudoh M, Nishiyama Y et al (2002) A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient. Microbiol Immunol 46:317-326
14. CLSI. (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard-Second Edition. CLSI document M27-A2, Pennsylvania, USA.
15. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods 25:402-408
16. Yang YL, Cheng HH, Ho YA et al (2003) Fluconazole resistance rate of Candida species from different regions and hospital types in Taiwan. J microbiol Immunol Infect 36:187-191
17. Richter SS, Galask RP, Messer SA et al (2005) Antifungal Susceptibilities of Candida Species Causing Vulvovaginitis and Epidemiology of Recurrent Cases. J Clin Microbiol 43:2155-2162
18. Sojakova M, Liptajova D, Borovsky M et al (2004) Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia. Mycopathologia 157:163-169
19. Lamb DC, Kelly DE, Schunck WH et al (1997) The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. J Biol Chem 272:5682-5688
20. Kelly SL, Lamb DC, Kelly DE (1999) Y132H substitution in Candida albicans sterol 14alpha-demethylase confers fluconazole resistance by preventing binding to haem. FEMS Microbiol Lett 180:171-175
21. Kelly SL, Lamb DC, Loeffler J, et al (1999) The G464S amino acid substitution in Candida albicans sterol 14alpha-demethylase causes fluconazole resistance in the clinic through reduced affinity. Biochem Biophys Res Commun 262:174-179
22. Lamb DC, Kelly DE, White TC et al (2000) The R467K amino acid substitution in Candida albicans sterol 14alpha-demethylase causes drug resistance through reduced affinity. Antimicrob Agents Chemother 44:63-67
23. Xu YH, Chen LM, Li CY (2008) Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG 11 mutations. J Antimicrob Chemother 61:798-804
24. Perea S, Lopez-Ribot JL, Kirkpatrick WR et al (2001) Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 45:2676-2684
25. White TC, Holleman S, Dy F et al (2002) Resistance mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother 46:1704-1713
26. Goldman GH, da Silva Ferreira ME, dos Reis Marques E et al (2004) Evaluation of fluconazole resistance mechanisms in candida albicans clinical isolates from HIV-infected patients in Brazil. Diagn Microbiol Infec Dis 50:25-32
27. Holmes AR, Lin YH, Niimi K et al (2008) ABC transporter Cdrlp contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates. Antimicrob Agents Chemother 52:3851-3862
28. Lyons CN, White TC (2000) Transcriptional analyses of antifungal drug resistance in Candida albicans. Antimicrob Agents Chemother 44:2296-2303
1 Dixon DM, McNeil MM, Cohen ML, et al: Fungal infections:a growing threat. Public Health Rep 1996; 111:226-235.
2 White TC, Marr KA, Bowden RA:Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 1998; 11:382-402.
3 Sanglard D, Odds FC:Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2002; 2:73-85.
4 Sanglard D, Ischer F, Koymans L, et al: Amino acid substitutions in the cytochrome P-450 lanosterol 14a-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrob Agents Chemother 1998; 42:241-253.
5 Jia XM, Ma ZP, Jia Y, et al: RTA2, a novel gene involved in azole resistance in Candida albicans. Biochem Biophys Res Commun 2008; 373:631-636.
6 Calabrese D, Bille J, Sanglard D:A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole. Microbiology 2000; 146:2743-2754.
7 Chandra J, Kuhn DM, Mukherjee PK, et al:Biofilm formation by the fungal pathogen Candida albicans:development, architecture, and drug resistance. J Bacteriol 2001; 183:5385-5394.
8 Maebashi K, Kudoh M, Nishiyama Y, et al: A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient. Microbiol Immunol 2002; 46:317-326.
9 Xu Y, Chen L, Li C:Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations. J Antimicrob Chemother 2008; 61:798-804.
10 National Committee for Clinical Laboratory Standards (NCCLS):Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast:Approved Standard,2nd edn. Wayne, PA:NCCLS,2002; Vol 28:pp M27-A3.
11 National Committee for Clinical Laboratory Standards (NCCLS):Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts:Approved Guideline. Wayne, PA:NCCLS,2004; Vol 29:ppM44-A2.
12 McCullough MJ, Clemons KV, Stevens DA:Molecular and phenotypic characterization of genotypic Candida albicans subgroups and comparison with Candida dubliniensis and Candida stellatoidea. J Clin Microbiol 1999; 37:417-421.
13 Livak KJ, Schmittgen TD:Analysis of relative gene expression data using real-time quantitative PCR and the 2△△Ct method Methods 2001; 25:402-408.
14 Giulietti A, Overbergh L, Valckx D, et al: An overview of real-time quantitative PCR:applications to quantify cytokine gene expression. Methods 2001; 25:386-401.
15 Morschhauser J, Barker KS, Liu TT, et al: The transcription factor Mrrlp controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog 2007; 3:e164.
16 O'Connor L, Lahiff S, Casey F, et al: Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR using hybridisation probes on the LightCycler. Mol Cell Probes 2005; 19:153-162.
17 Akbar DH, Tahawi AT:Candidemia at a University Hospital:epidemiology, risk factors and predictors of mortality. Ann Saudi Med 2001; 21:178-182.
18 St-Germain G, Laverdiere M, Pelletier R, et al:Prevalence and antifungal susceptibility of 442 Candida isolates from blood and other normally sterile sites: results of a 2-year (1996 to 1998) multicenter surveillance study in Quebec, Canada. J Clin Microbiol 2001; 39:949-953.
19 Bedini A, Venturelli C, Mussini C, et al: Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiarycare hospital. Clin Microbiol Infect 2006; 12:75-80.
20 Marichal P, Koymans L, Willemsens S, et al:Contribution of mutations in the cytochrome P450 14α-demethylase (Ergllp, Cyp51p) to azole resistance in Candida albicans. Microbiology 1999; 145:2701-2713.
21 Podust LM, Poulos TL, Waterman MR:Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proc Natl Acad Sci USA 2001; 98:3068-3073.
22 Lamb DC, Kelly DE, Schunck WH, et al:The mutation T315A in Candida albicans sterol 14a-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. JBiol Chem 1997; 272:5682-5688.
23 Kelly SL, Lamb DC, Kelly DE:Y132H substitution in Candida albicans sterol 14 a-demethylase confers fluconazole resistance by preventing binding to haem. FEMS Microbiol Lett 1999; 180:171-175.
24 Kelly SL, Lamb DC, Loeffler J, et al:The G464S amino acid substitution in Candida albicans sterol 14a-demethylase causes fluconazole resistance in the clinic through reduced affinity. Biochem Biophys Res Commun 1999; 262:174-179.
25 Lamb DC, Kelly DE, White TC, et al:The R467K amino acid substitution in Candida albicans sterol 14a-demethylase causes drug resistance through reduced affinity. Antimicrob Agents Chemother 2000; 44:63-67.
26 Chen SH, Sheng CQ, Xu XH, et al:Identification of Y118 amino acid residue in Candida albicans sterol 14a-demethylase associated with the enzyme activity and selective antifungal activity of azole analogues. Biol Pharm Bull 2007; 30:1246-1253.
27 Jiang WS, Tan SS, Jiang GY, et al:Synergistic effect of terbinafine combined with fluconazole or itraconazole on stable fluconazole-resistant Candida albicans induced by fluconazole in vitro. Chin JMicrobiol Immunol 2006; 26:360-364 [in Chinese].
28 Perea S, Lopez-Ribot JL, Kirkpatrick WR, et al: Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 2001; 45:2676-2684.
29 Goldman GH, da Silva Ferreira ME, dos Reis Marques E, et al: Evaluation of fluconazole resistance mechanisms in Candida albicans clinical isolates from HIV-infected patients in Brazil. Diagn Microbiol Infect Dis 2004; 50:25-32.
30 Ribeiro MA, Paula CR:Up-regulation of ERG11 gene among fluconazole-resistant Candida albicans generated in vitro:is there any clinical implication? Diagn Microbiol Infect Dis 2007; 57:71-75.
31 White TC, Holleman S, Dy F, et al:Resistance mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother 2002; 46:1704-1713.
32 Morschhauser J:The genetic basis of fluconazole resistance development in Candida albicans. Biochim Biophys Acta 2002; 1587:240-248.
33 Holmes AR, Lin YH, Niimi K, et al:ABC transporter Cdrlp contributes more than Cdr2p does to fluconazole efflux in fluconazoleresistant Candida albicans clinical isolates. Antimicrob Agents Chemother 2008; 52:3851-3862.
34 White TC:Increased mRNA levels of ERG 16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 1997; 41:1482 1487.
35 Marr KA, Lyons CN, Rustad TR, et al:Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR. Antimicrob Agents Chemother 1998; 42:2584-2589.
36 Lyons CN, White TC:Transcriptional analyses of antifungal drug resistance in Candida albicans. Antimicrob Agents Chemother 2000; 44:2296-2303.
2. Ghannoum MA, Rex JH, Galgiani JN. Susceptibility testing of fungi:current status of correlation of in vitro data with clinical outcome. Journal of clinical microbiology.1996 Mar;34(3):489-95.
3. 吴绍熙.现代医学真菌检验手册.北京：北京医科大学、中国协和医科大学联合出版社,1998：327-39.
4. Jarvis WR. Epidemiology of nosocomial fungal infections, with emphasis on Candida species. Clin Infect Dis.1995 Jun;20(6):1526-30.
5. 钟南山,叶枫.深部真菌感染：新的挑战与展望.中华结核和呼吸杂志,2006,29(5)：289-90.
6. 吴安华,任南,文细毛等.159所医院医院感染现患率调查结果与分析,中国感染控制杂志,2005,4(1)：12-7.
7. Rangel-Frausto MS, Wiblin T, Blumberg HM, et al. National epidemiology of mycoses survey (NEMIS):variations in rates of bloodstream infections due to Candida species in seven surgical intensive care units and six neonatal intensive care units. Clin Infect Dis.1999 Aug;29(2):253-8.
8. Hajjeh RA, Sofair AN, Harrison LH, et al. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. Journal of clinical microbiology.2004 Apr;42(4):1519-27.
9. Pfaller MA, Messer SA, Hollis RJ, Jones RN, Diekema DJ. In vitro activities of ravuconazole and voriconazole compared with those of four approved systemic antifungal agents against 6,970 clinical isolates of Candida spp. Antimicrobial agents and chemotherapy.2002 Jun;46(6):1723-7.
10. 吴绍熙,廖万清,郭宁如等.中国致病真菌10年动态流行病学研究.临床 皮肤科杂志,1999,28(1)：1-5.
11. Abelson JA, Moore T, Bruckner D, Deville J, Nielsen K. Frequency of fungemia in hospitalized pediatric inpatients over 11 years at a tertiary care institution. Pediatrics.2005 Jul;116(1):61-7.
12. Cowen LE, Sanglard D, Calabrese D, Sirjusingh C, Anderson JB, Kohn LM. Evolution of drug resistance in experimental populations of Candida albicans. Journal of bacteriology.2000 Mar;182(6):1515-22.
13. Magaldi S, Mata S, Hartung C, et al. In vitro susceptibility of 137 Candida sp. isolates from HIV positive patients to several antifungal drugs. Mycopathologia.2001;149(2):63-8.
14. Sangeorzan JA, Bradley SF, He X, et al. Epidemiology of oral candidiasis in HIV-infected patients:colonization, infection, treatment, and emergence of fluconazole resistance. The American journal of medicine.1994 Oct;97(4):339-46.
15. Graybill JR. Changing strategies for treatment of systemic mycoses. Braz J Infect Dis.2000 Apr;4(2):47-54.
16. Rosenblatt HM, Byrne W, Ament ME, Graybill J, Stiehm ER. Successful treatment of chronic mucocutaneous candidiasis with ketoconazole. The Journal of pediatrics.1980 Oct;97(4):657-60.
17. Redding S, Smith J, Farinacci G, et al. Resistance of Candida albicans to fluconazole during treatment of oropharyngeal candidiasis in a patient with AIDS:documentation by in vitro susceptibility testing and DNA subtype analysis. Clin Infect Dis.1994 Feb;18(2):240-2.
18. Moran GP, Sullivan DJ, Henman MC, et al. Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro. Antimicrobial agents and chemotherapy.1997 Mar;41(3):617-23.
19. Rocco TR, Reinert SE, Simms HH. Effects of fluconazole administration in critically ill patients:analysis of bacterial and fungal resistance. Arch Surg. 2000 Feb；135(2)：160-5.
20. 张秀珍.深部真菌感染及耐药变迁.中国处方药,2004,24(3)：65-8.
21. Diekema DJ, Messer SA, Brueggemann AB, et al. Epidemiology of candidemia:3-year results from the emerging infections and the epidemiology of Iowa organisms study. Journal of clinical microbiology.2002 Apr;40(4):1298-302.
22. Wroblewska MM, Swoboda-Kopec E, Rokosz A, Krawczyk E, Marchel H, Luczak M. Epidemiology of clinical isolates of Candida albicans and their susceptibility to triazoles. International journal of antimicrobial agents.2002 Dec;20(6):472-5.
23. 杨蓬勃,胡云建.住院患者真菌感染及耐药性分析.中华医院感染学杂志.2007,17(6)：729-31.
24. 刘彦慧,赖志刚,张丽华.念珠菌检测与药敏分析.中国皮肤性病学杂志.2005,19(7)：411-13.
25. 常东,蒋伟,黄志红,等.真菌感染及耐药性分析.中华医院感染学杂志.2005,15(3)：345-47.
26. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG 11 mutations. The Journal of antimicrobial chemotherapy.2008 Apr;61(4):798-804.
27. 吴绍熙,郭宁如,廖万清.现代真菌病诊断治疗学.北京：北京医科大学、中国协和医科大学联合出版,1997：182-206.
28. White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clinical microbiology reviews. 1998 Apr;11(2):382-402.
29. White TC. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans. Antimicrobial agents and chemotherapy. 1997 Jul;41(7):1488-94.
30. Marichal P, Koymans L, Willemsens S, et al. Contribution of mutations in the cytochrome P450 14alpha-demethylase (Ergllp, Cyp51p) to azole resistance in Candida albicans. Microbiology (Reading, England).1999 Oct;145 (Pt 10):2701-13.
31. Podust LM, Poulos TL, Waterman MR. Crystal structure of cytochrome P450 14alpha-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proceedings of the National Academy of Sciences of the United States of America.2001 Mar 13;98(6):3068-73.
32. Revankar SG, Kirkpatrick WR, McAtee RK, et al. Detection and significance of fluconazole resistance in oropharyngeal candidiasis in human immunodeficiency virus-infected patients. The Journal of infectious diseases. 1996 Oct;174(4):821-7.
33. Sanglard D, Ischer F, Koymans L, Bille J. Amino acid substitutions in the cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrobial agents and chemotherapy.1998 Feb;42(2):241-53.
34. Goldman GH, da Silva Ferreira ME, dos Reis Marques E, et al. Evaluation of fluconazole resistance mechanisms in candida albicans clinical isolates from HIV-infected patients in Brazil. Diagnostic microbiology and infectious disease.2004 Sep;50(1):25-32.
35. Wang WL, Li RY, Wang DL, et al. Resistant mechanisms of Candida albicans to fluconazole. Chin Med J.1999,112(5):466-471.
36. Lamb DC, Kelly DE, Schunck WH, et al. The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. The Journal of biological chemistry.1997 Feb 28;272(9):5682-8.
37. White TC. Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrobial agents and chemotherapy.1997 Jul;41(7):1482-7.
38. Albertson GD, Niimi M, Cannon RD, Jenkinson HF. Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrobial agents and chemotherapy.1996 Dec;40(12):2835-41.
39. Franz R, Kelly SL, Lamb DC, Kelly DE, Ruhnke M, Morschhauser J. Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains. Antimicrobial agents and chemotherapy.1998 Dec;42(12):3065-72.
40. Nakayama H, Ikebe T, Beppu M, Shirasuna K. High expression levels of nuclear factor kappaB, IkappaB kinase alpha and Akt kinase in squamous cell carcinoma of the oral cavity. Cancer.2001 Dec 15;92(12):3037-44.
41. White TC, Holleman S, Dy F, Mirels LF, Stevens DA. Resistance mechanisms in clinical isolates of Candida albicans. Antimicrobial agents and chemotherapy.2002 Jun;46(6):1704-13.
42. Kelly SL, Lamb DC, Kelly DE, et al. Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol delta5,6-desaturation. FEBS letters.1997 Jan 2;400(1):80-2.
43. Kelly SL, Lamb DC, Kelly DE, Loeffler J, Einsele H. Resistance to fluconazole and amphotericin in Candida albicans from AIDS patients. Lancet. 1996 Nov 30;348(9040):1523-4.
44. Miyazaki Y, Geber A, Miyazaki H, et al. Cloning, sequencing, expression and allelic sequence diversity of ERG3 (C-5 sterol desaturase gene) in Candida albicans. Gene.1999 Aug 5;236(1):43-51.
45. Wolfger H, Mamnun YM, Kuchler K. Fungal ABC proteins:pleiotropic drug resistance, stress response and cellular detoxification. Research in microbiology.2001 Apr-May; 152(3-4):375-89.
46. Prasad R, De Wergifosse P, Goffeau A, Balzi E. Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals. Current genetics.1995 Mar;27(4):320-9.
47. Sanglard D, Ischer F, Monod M, Bille J. Cloning of Candida albicans genes conferring resistance to azole antifungal agents:characterization of CDR2, a new multidrug ABC transporter gene. Microbiology (Reading, England).1997 Feb;143(Pt 2):405-16.
48. Morschhauser J. The genetic basis of fluconazole resistance development in Candida albicans. Biochimica et biophysica acta.2002 Jul 18; 1587 (2-3):240-8.
49. Goldway M, Teff D, Schmidt R, Oppenheim AB, Koltin Y. Multidrug resistance in Candida albicans:disruption of the BENr gene. Antimicrobial agents and chemotherapy.1995 Feb;39(2):422-6.
50. Calabrese D, Bille J, Sanglard D. A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole. Microbiology (Reading, England).2000 Nov;146 (Pt 11):2743-54.
51. Hawser SP, Douglas LJ. Resistance of Candida albicans biofilms to antifungal agents in vitro. Antimicrobial agents and chemotherapy.1995 Sep; 39 (9):2128-31.
52. Baillie GS, Douglas LJ. Role of dimorphism in the development of Candida albicans biofilms. Journal of medical microbiology.1999 Jul;48(7):671-9.
53. Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, McCormick T, Ghannoum MA. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. Journal of bacteriology.2001 Sep; 183(18):5385-94.
54. Donlan RM, Costerton JW. Biofilms:survival mechanisms of clinically relevant microorganisms. Clinical microbiology reviews.2002 Apr; 15 (2):167-93.
55. Maebashi K, Kudoh M, Nishiyama Y, et al. A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient. Microbiology and immunology. 2002;46(5):317-26.
56. Loffler J, Einsele H, Hebart H, Schumacher U, Hrastnik C, Daum G Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains. FEMS microbiology letters.2000 Apr 1; 185(1):59-63.
57. Kohli A, Smriti, Mukhopadhyay K, Rattan A, Prasad R. In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry. Antimicrobial agents and chemotherapy.2002 Apr;46(4):1046-52.
58. 卫生部医政司.全国临床检验操作规程第二版.南京：东南大学出版社,1997：452-71.
59. Pinjon E, Sullivan D, Salkin I, Shanley D, Coleman D. Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. Journal of clinical microbiology.1998 Jul;36(7):2093-5.
60. National committee for clinical laboratory standards. Reference method for broth dilution antifungal susceptibility testing of yeasts, Approved standard-Second Edition. NCCLS document M27-A2.2002,22:1-29.
61. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.2001 Dec;25(4):402-8.
62. Prasad R, Panwar SL, Smriti. Drug resistance in yeasts-an emerging scenario. Advances in microbial physiology.2002;46:155-201.
63. 韦莉萍,桂希恩,杨志成,等.医院内真菌深部感染调查及其危险因素分析.中华医院感染学杂志,1998,8(1)：28-30.
64. 刘自贵,裘雁秋,谭明珍,等.院内念珠菌感染及其药物敏感性研究.中华医院感染学杂志,2000,10(4)：304-05.
65. Schoofs A, Odds FC, Colebunders R, Ieven M, Goossens H. Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients. Eur J Clin Microbiol Infect Dis.1997 Apr; 16(4):296-300.
66. Jabra-Rizk MA, Brenner TM, Romagnoli M, et al. Evaluation of a reformulated CHROMagar Candida. Journal of clinical microbiology.2001 May;39(5):2015-6.
67. Berrouane YF, Herwaldt LA, Pfaller MA. Trends in antifungal use and epidemiology of nosocomial yeast infections in a university hospital. Journal of clinical microbiology.1999 Mar;37(3):531-7.
68. 吴爱武.院内感染标本分离的260株念珠菌调查与分析.上海医学检验杂志,2000,15(3)：172-73.
69. 方芳,伊和姿,吴丽桂.危重病人203例深部真菌感染的调查研究.皮肤病与性病,2005,27(1)：6-7.
70. Raad I, Hanna H, Boktour M, et al. Management of central venous catheters in patients with cancer and candidemia. Clin Infect Dis.2004 Apr 15; 38(8):1119-27.
71. Klotz SA, Drutz DJ, Zajic JE. Factors governing adherence of Candida species to plastic surfaces. Infection and immunity.1985 Oct;50(1):97-101.
72. Wingard JR, Merz WG, Rinaldi MG, Johnson TR, Karp JE, Saral R. Increase in Candida krusei infection among patients with bone marrow transplantation and neutropenia treated prophylactically with fluconazole. The New England journal of medicine.1991 Oct 31;325(18):1274-7.
73. Saville SP, Lazzell AL, Monteagudo C, Lopez-Ribot JL. Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection. Eukaryotic cell.2003 Oct;2(5):1053-60.
74. Whiteway M, Oberholzer U. Candida morphogenesis and host-pathogen interactions. Current opinion in microbiology.2004 Aug;7(4):350-7.
75. Haynes K. Virulence in Candida species. Trends in microbiology.2001 Dec;9(12):591-6.
76. Ha KC, White TC. Effects of azole antifungal drugs on the transition from yeast cells to hyphae in susceptible and resistant isolates of the pathogenic yeast Candida albicans. Antimicrobial agents and chemotherapy.1999 Apr;43(4):763-8.
77. Sanglard D, Kuchler K, Ischer F, Pagani JL, Monod M, Bille J. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrobial agents and chemotherapy.1995 Nov;39(11):2378-86.
78. Maesaki S, Marichal P, Vanden Bossche H, Sanglard D, Kohno S. Rhodamine 6G efflux for the detection of CDR1-overexpressing azole-resistant Candida albicans strains. The Journal of antimicrobial chemotherapy.1999 Jul;44(1):27-31.
79. 郭慧君,夏忠弟,吴高莉,等.耐药白念珠菌主动外排泵蛋白的功能与基因表达.中南大学学报(医学版).2007,32(1)：183-7.
80. 孙伟玮,高平挥,王英,等.ABC转运蛋白与临床白念珠茵耐药.第二军医大学学报.2008,29(9)：1038-41.
81. 饶勇,曾振灵,陈杖榴.抗生素耐药性的主动外排机制.国外医药·抗生素分册,2002,23(3)：109-114.
82. Balan I, Alarco AM, Raymond M. The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter. Journal of bacteriology.1997 Dec; 179 (23):7210-8.
83. Franz R, Michel S, Morschhauser J. A fourth gene from the Candida albicans CDR family of ABC transporters. Gene.1998 Oct 5;220(1-2):91-8.
84. St Georgiev V. Membrane transporters and antifungal drug resistance. Current drug targets.2000 Nov;l(3):261-84.
85. Schuetzer-Muehlbauer M, Willinger B, Krapf G, Enzinger S, Presterl E, Kuchler K. The Candida albicans Cdr2p ATP-binding cassette (ABC) transporter confers resistance to caspofungin. Molecular microbiology.2003 Apr;48(1):225-35.
86. Albertson GD, Niimi M, Cannon RD, Jenkinson HF. Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrobial agents and chemotherapy.1996 Dec;40(12):2835-41.
87. Del Sorbo G, Schoonbeek H, De Waard MA. Fungal transporters involved in efflux of natural toxic compounds and fungicides. Fungal Genet Biol.2000 Jun;30(1):1-15.
88. Smriti, Krishnamurthy SS, Prasad R. Membrane fluidity affects functions of Cdrlp, a multidrug ABC transporter of Candida albicans. FEMS microbiology letters.1999 Apr 15;173(2):475-81.
89. Ramage G, Bachmann S, Patterson TF, Wickes BL, Lopez-Ribot JL. Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. The Journal of antimicrobial chemotherapy.2002 Jun;49(6):973-80.
90. Niimi M, Niimi K, Takano Y, et al. Regulated overexpression of CDR1 in Candida albicans confers multidrug resistance. The Journal of antimicrobial chemotherapy.2004 Dec;54(6):999-1006.
91. Perea S, Lopez-Ribot JL, Kirkpatrick WR, et al. Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrobial agents and chemotherapy.2001 Oct;45(10):2676-84.
92. O'Connor L, Lahiff S, Casey F, Glennon M, Cormican M, Maher M. Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR using hybridisation probes on the LightCycler. Molecular and cellular probes.2005 Jun; 19(3):153-62.
93. Morschhauser J, Barker KS, Liu TT, Bla BWJ, Homayouni R, Rogers PD. The transcription factor Mrrlp controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS pathogens.2007 Nov;3(11):e164.
94. Chau AS, Mendrick CA, Sabatelli FJ, Loebenberg D, McNicholas PM. Application of real-time quantitative PCR to molecular analysis of Candida albicans strains exhibiting reduced susceptibility to azoles. Antimicrobial agents and chemotherapy.2004 Jun;48(6):2124-31.
95. Park S, Perlin DS. Establishing surrogate markers for fluconazole resistance in Candida albicans. Microbial drug resistance (Larchmont, NY.2005 Fall; 11(3):232-8.
96. Holmes AR, Lin YH, Niimi K, et al. ABC transporter Cdrlp contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates. Antimicrobial agents and chemotherapy.2008 Nov;52(11):3851-62.
97. Nakamura K, Niimi M, Niimi K, et al. Functional expression of Candida albicans drug efflux pump Cdrlp in a Saccharomyces cerevisiae strain deficient in membrane transporters. Antimicrobial agents and chemotherapy. 2001 Dec;45(12):3366-74.
98. Lyons CN, White TC. Transcriptional analyses of antifungal drug resistance in Candida albicans. Antimicrobial agents and chemotherapy.2000 Sep; 44(9):2296-303.
1. Foxman B, Marsh JV, Gillespie B et al (1998) Frequency and response to vaginal symptoms among white and African American women:results of a random digit dialing survey. J Womens Health 7:1167-1174
2. Lanchares JL, Hermandez ML (2000) Recurrent vaginal candidiasis changes in etiopathogenical patterns. Int Gynaecol Obstet 71 Suppl 1:S29-35
3. Landers DV, Wiesenfeld HC, Heine RP et al (2004) Predictive value of the clinical diagnosis of lower genital tract infection in women. Am J Obstet Gynecol 190:1004-1010
4. Nyirjesy P, Seeney SM, Grody MH et al (1995) Chronic fungal vaginitis:the value of cultures. Am J Obstet Gynecol 173:820-823
5. Sobel JD, Chaim W (1997) Treatment of Torulopsis glabrata vaginitis: retrospective review of boric acid therapy. Clin Infect Dis 24:649-652
6. Spinillo A, Capuzzo E, Gulminetti R et al (1997) Prevalence of and risk factors for fungal vaginitis caused by non-albicans species. Am J Obstet Gynecol 176:138-141
7. Boschman CR, Bodnar UR, Tornatore MA et al (1998) Thirteen-year evolution of azole resistance in yeast isolates and prevalence of resistant strains carried by cancer patients at a large medical center. Antimicrob Agents Chemother 42:734-738
8. Sobel JD, Zervos M, Reed BD et al (2003) Fluconazole susceptibility of vaginal isolates obtained from women with complicated Candida vaginitis:clinical implications. Antimicrob Agents Chemother 47:34-38
9. Bauters TG, Dhont MA, Temmerman MI et al (2002) Prevalence of vulvovaginal candidiasis and susceptibility to fluconazole in women. Am J Obstet Gynecol 187:569-574
10. Calabrese D, Bille J, Sanglard D (2000) A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole. Microbiology 146 (Pt 11):2743-2754
11. Perea S, Lopez-Ribot JL, Kirkpatrick WR, et al (2001) Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 45(10):2676-2684
12. Andes D, Nett J, Oschel P et al (2004) Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect and Immun 72:6023-6031
13. Maebashi K, Kudoh M, Nishiyama Y et al (2002) A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient. Microbiol Immunol 46:317-326
14. CLSI. (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard-Second Edition. CLSI document M27-A2, Pennsylvania, USA.
15. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods 25:402-408
16. Yang YL, Cheng HH, Ho YA et al (2003) Fluconazole resistance rate of Candida species from different regions and hospital types in Taiwan. J microbiol Immunol Infect 36:187-191
17. Richter SS, Galask RP, Messer SA et al (2005) Antifungal Susceptibilities of Candida Species Causing Vulvovaginitis and Epidemiology of Recurrent Cases. J Clin Microbiol 43:2155-2162
18. Sojakova M, Liptajova D, Borovsky M et al (2004) Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia. Mycopathologia 157:163-169
19. Lamb DC, Kelly DE, Schunck WH et al (1997) The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. J Biol Chem 272:5682-5688
20. Kelly SL, Lamb DC, Kelly DE (1999) Y132H substitution in Candida albicans sterol 14alpha-demethylase confers fluconazole resistance by preventing binding to haem. FEMS Microbiol Lett 180:171-175
21. Kelly SL, Lamb DC, Loeffler J, et al (1999) The G464S amino acid substitution in Candida albicans sterol 14alpha-demethylase causes fluconazole resistance in the clinic through reduced affinity. Biochem Biophys Res Commun 262:174-179
22. Lamb DC, Kelly DE, White TC et al (2000) The R467K amino acid substitution in Candida albicans sterol 14alpha-demethylase causes drug resistance through reduced affinity. Antimicrob Agents Chemother 44:63-67
23. Xu YH, Chen LM, Li CY (2008) Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG 11 mutations. J Antimicrob Chemother 61:798-804
24. Perea S, Lopez-Ribot JL, Kirkpatrick WR et al (2001) Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 45:2676-2684
25. White TC, Holleman S, Dy F et al (2002) Resistance mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother 46:1704-1713
26. Goldman GH, da Silva Ferreira ME, dos Reis Marques E et al (2004) Evaluation of fluconazole resistance mechanisms in candida albicans clinical isolates from HIV-infected patients in Brazil. Diagn Microbiol Infec Dis 50:25-32
27. Holmes AR, Lin YH, Niimi K et al (2008) ABC transporter Cdrlp contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates. Antimicrob Agents Chemother 52:3851-3862
28. Lyons CN, White TC (2000) Transcriptional analyses of antifungal drug resistance in Candida albicans. Antimicrob Agents Chemother 44:2296-2303
1 Dixon DM, McNeil MM, Cohen ML, et al: Fungal infections:a growing threat. Public Health Rep 1996; 111:226-235.
2 White TC, Marr KA, Bowden RA:Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 1998; 11:382-402.
3 Sanglard D, Odds FC:Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2002; 2:73-85.
4 Sanglard D, Ischer F, Koymans L, et al: Amino acid substitutions in the cytochrome P-450 lanosterol 14a-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrob Agents Chemother 1998; 42:241-253.
5 Jia XM, Ma ZP, Jia Y, et al: RTA2, a novel gene involved in azole resistance in Candida albicans. Biochem Biophys Res Commun 2008; 373:631-636.
6 Calabrese D, Bille J, Sanglard D:A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole. Microbiology 2000; 146:2743-2754.
7 Chandra J, Kuhn DM, Mukherjee PK, et al:Biofilm formation by the fungal pathogen Candida albicans:development, architecture, and drug resistance. J Bacteriol 2001; 183:5385-5394.
8 Maebashi K, Kudoh M, Nishiyama Y, et al: A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient. Microbiol Immunol 2002; 46:317-326.
9 Xu Y, Chen L, Li C:Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations. J Antimicrob Chemother 2008; 61:798-804.
10 National Committee for Clinical Laboratory Standards (NCCLS):Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast:Approved Standard,2nd edn. Wayne, PA:NCCLS,2002; Vol 28:pp M27-A3.
11 National Committee for Clinical Laboratory Standards (NCCLS):Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts:Approved Guideline. Wayne, PA:NCCLS,2004; Vol 29:ppM44-A2.
12 McCullough MJ, Clemons KV, Stevens DA:Molecular and phenotypic characterization of genotypic Candida albicans subgroups and comparison with Candida dubliniensis and Candida stellatoidea. J Clin Microbiol 1999; 37:417-421.
13 Livak KJ, Schmittgen TD:Analysis of relative gene expression data using real-time quantitative PCR and the 2△△Ct method Methods 2001; 25:402-408.
14 Giulietti A, Overbergh L, Valckx D, et al: An overview of real-time quantitative PCR:applications to quantify cytokine gene expression. Methods 2001; 25:386-401.
15 Morschhauser J, Barker KS, Liu TT, et al: The transcription factor Mrrlp controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog 2007; 3:e164.
16 O'Connor L, Lahiff S, Casey F, et al: Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR using hybridisation probes on the LightCycler. Mol Cell Probes 2005; 19:153-162.
17 Akbar DH, Tahawi AT:Candidemia at a University Hospital:epidemiology, risk factors and predictors of mortality. Ann Saudi Med 2001; 21:178-182.
18 St-Germain G, Laverdiere M, Pelletier R, et al:Prevalence and antifungal susceptibility of 442 Candida isolates from blood and other normally sterile sites: results of a 2-year (1996 to 1998) multicenter surveillance study in Quebec, Canada. J Clin Microbiol 2001; 39:949-953.
19 Bedini A, Venturelli C, Mussini C, et al: Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiarycare hospital. Clin Microbiol Infect 2006; 12:75-80.
20 Marichal P, Koymans L, Willemsens S, et al:Contribution of mutations in the cytochrome P450 14α-demethylase (Ergllp, Cyp51p) to azole resistance in Candida albicans. Microbiology 1999; 145:2701-2713.
21 Podust LM, Poulos TL, Waterman MR:Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proc Natl Acad Sci USA 2001; 98:3068-3073.
22 Lamb DC, Kelly DE, Schunck WH, et al:The mutation T315A in Candida albicans sterol 14a-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. JBiol Chem 1997; 272:5682-5688.
23 Kelly SL, Lamb DC, Kelly DE:Y132H substitution in Candida albicans sterol 14 a-demethylase confers fluconazole resistance by preventing binding to haem. FEMS Microbiol Lett 1999; 180:171-175.
24 Kelly SL, Lamb DC, Loeffler J, et al:The G464S amino acid substitution in Candida albicans sterol 14a-demethylase causes fluconazole resistance in the clinic through reduced affinity. Biochem Biophys Res Commun 1999; 262:174-179.
25 Lamb DC, Kelly DE, White TC, et al:The R467K amino acid substitution in Candida albicans sterol 14a-demethylase causes drug resistance through reduced affinity. Antimicrob Agents Chemother 2000; 44:63-67.
26 Chen SH, Sheng CQ, Xu XH, et al:Identification of Y118 amino acid residue in Candida albicans sterol 14a-demethylase associated with the enzyme activity and selective antifungal activity of azole analogues. Biol Pharm Bull 2007; 30:1246-1253.
27 Jiang WS, Tan SS, Jiang GY, et al:Synergistic effect of terbinafine combined with fluconazole or itraconazole on stable fluconazole-resistant Candida albicans induced by fluconazole in vitro. Chin JMicrobiol Immunol 2006; 26:360-364 [in Chinese].
28 Perea S, Lopez-Ribot JL, Kirkpatrick WR, et al: Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 2001; 45:2676-2684.
29 Goldman GH, da Silva Ferreira ME, dos Reis Marques E, et al: Evaluation of fluconazole resistance mechanisms in Candida albicans clinical isolates from HIV-infected patients in Brazil. Diagn Microbiol Infect Dis 2004; 50:25-32.
30 Ribeiro MA, Paula CR:Up-regulation of ERG11 gene among fluconazole-resistant Candida albicans generated in vitro:is there any clinical implication? Diagn Microbiol Infect Dis 2007; 57:71-75.
31 White TC, Holleman S, Dy F, et al:Resistance mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother 2002; 46:1704-1713.
32 Morschhauser J:The genetic basis of fluconazole resistance development in Candida albicans. Biochim Biophys Acta 2002; 1587:240-248.
33 Holmes AR, Lin YH, Niimi K, et al:ABC transporter Cdrlp contributes more than Cdr2p does to fluconazole efflux in fluconazoleresistant Candida albicans clinical isolates. Antimicrob Agents Chemother 2008; 52:3851-3862.
34 White TC:Increased mRNA levels of ERG 16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 1997; 41:1482 1487.
35 Marr KA, Lyons CN, Rustad TR, et al:Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR. Antimicrob Agents Chemother 1998; 42:2584-2589.
36 Lyons CN, White TC:Transcriptional analyses of antifungal drug resistance in Candida albicans. Antimicrob Agents Chemother 2000; 44:2296-2303.