【作者】 徐永豪；

【导师】 李春阳；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 随着广谱抗生素、免疫抑制剂的广泛应用和介入疗法、器官移植等的深入开展,念珠菌感染呈现逐年上升的趋势,而抗真菌药物的广泛应用则加速了菌株的耐药进程。从分子水平研究念珠菌对抗真菌药尤其是唑类药物的耐药机制,解决念珠菌感染的快速诊断和有效治疗问题,是目前的热点。唑类药物的问世为治疗念珠菌感染拓展了全新的空间,但在其药物选择压力的作用下,念珠菌临床分离株中耐药株的比例明显增加。念珠菌耐药问题逐渐引起人们的重视,如何有效治疗耐菌株的感染正在渐渐成为一个新的医学难题,菌种鉴定和药敏试验结果成为影响临床治疗和判断预后的重要因素。白念珠菌是念珠菌感染的首要致病菌。从基因水平研究白念珠菌对唑类药物的耐药机制,国外的报道主要集中于欧美国家,国内研究则较少。目前认为,白念珠菌对唑类药物耐药存在多种机制,主要包括:（1）药物靶酶基因ERG11突变或过度表达。（2）编码多药流出通道的基因（CDR1、CDR2、MDR1、FLU1）过度表达。（3）生物被膜的形成。（4）细胞内液泡封闭药物分子。ERG11是羊毛甾醇14α-去甲基化酶的编码基因,14α-去甲基化酶是念珠菌细胞膜麦角固醇合成途径中的关键酶,对维持细胞膜的正常结构和功能具有重要意义。唑类抗真菌药物分子可与14α-去甲基化酶分子结合,阻断羊毛固醇去甲基化过程,影响麦角固醇合成。ERG11的突变可以改变14α-去甲基化酶的氨基酸序列,当这种改变足以影响酶分子的空间构型时,酶分子与药物分子间的亲和力可能被削弱,导致菌株耐药。理论上,菌株ERG11基因的过度表达可以使14α-去甲基化酶表达增加,在有限浓度的药物作用下,仍然维持良好的麦角固醇生物合成通路,也可以造成菌株耐药。但现在多认为菌株耐药性的形成与ERG11的过度表达无关,ERG11基因的突变才是这一耐药机制的主要方面。迄今报道的ERG11基因错义已逾70种,可以引起14α-去甲基化酶在61个氨基酸残基上发生69种改变。其中,已经实验证实可以导致菌株对氟康唑耐药的氨基酸置换仅有6种,即Y132H、T315A、S405F、G464S、R467K和1471T。在单株耐药菌株中,可以是多种耐药机制并存,共同参与菌株耐药性的形成;也可以是仅有一种机制起作用,例如,单纯ERG11基因T541C点突变即可导致菌株耐药。如果能够利用分子生物学手段检测到可以直接导致菌株耐药的基因突变,即可以在不必进行药敏试验的情况下确定菌株的耐药表型。基因芯片技术的出现和成熟,为这一设想提供了技术平台。基因芯片又被称为DNA芯片（DNA chip）、DNA阵列（DNA array）、DNA微阵列（DNA microarray）等,该技术是近年出现的分子生物学与微电子技术相结合的核酸检测分析技术,采用原位合成或显微点样技术将大量DNA探针有序地固化于支持物表面,然后与带有标记的核酸样品杂交,通过对杂交信号的检测分析,获得样品的基因序列、基因表达等遗传信息。该技术具有高度并行性、多样性、微型化和快速自动化等特点,成为后基因组时代最重要的基因功能分析技术之一。基因芯片技术已经渗透到生物科学领域的许多方面,有人将其应用于念珠菌全基因组多种基因的检测,并用于比较氟康唑敏感株和耐药株间的差别,也有人将其用于包括念珠菌在内的真菌菌种鉴定,认为DNA芯片在菌种鉴定方面具有极高的敏感性和特异性。但在用DNA芯片检测白念珠菌耐药菌株方面,国内外均未见相关文献报道。目的了解念珠菌临床分离株的菌种分布情况和对氟康唑的敏感性。筛查白念珠菌ERG11基因的错义突变,探讨突变与氟康唑耐药之间的关系。制备DNA芯片,鉴定常见的念珠菌种和可导致菌株对氟康唑耐药的ERG11突变,对DNA芯片技术应用于念珠菌菌种鉴定和药敏鉴定进行初步探索。方法收集临床标本,培养并分离念珠菌株,转种于改良SDA斜面上4℃保存。利用CHROMagar显色培养基作为初筛手段,鉴定白念珠菌（含都柏林念珠菌）、热带念珠菌、光滑念珠菌、克柔念珠菌;辅以血清芽管生成试验、蔗糖同化试验、玉米土温80厚壁孢子形成试验来进一步确认白念珠菌和都柏林念珠菌;辅以四氮唑红（TZC）还原试验来进一步确认热带念珠菌;近平滑念珠菌及其它念珠菌以VITEK2自动鉴定系统鉴定;PCR扩增白念珠菌（含都柏林念珠菌）25S rDNA转座内含子保守序列,区分白念珠菌和含都柏林念珠菌并进行白念珠菌种内分型。联合应用微量稀释法和纸片扩散法,体外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,判读结果时,需对照纸片扩散法的结果,如结果相抵触,则重复试验再判读,最终以微量稀释法结果为准。参考白念珠菌ERG11标准序列（GeneBank X13296）设计3对引物,用Pfu高保真DNA多聚酶分段扩增全部白念珠菌临床耐药株、2株标准耐药株、4株标准敏感株、部分临床敏感株的ERG11基因,以其中ERG11序列已明确的2株标准耐药株作为质控。用凝胶回收试剂盒进行PCR产物纯化,将纯化的PCR产物进行测序,检测突变。根据白念珠菌ERG11基因T541C、A1090G、C1361T、G1537A、G1547A、T1559C等6种突变序列,每种突变设计至少2条等位基因特异性探针（PM）和1条错配探针（MM）;根据白念珠菌、都柏林念珠菌、光滑念珠菌、克柔念珠菌、近平滑念珠菌和热带念珠菌等6种真菌的内转录间隔区ITS2保守序列,分别设计1条种特异性探针。用OmniGrid^TM100点样仪制备DNA芯片,每个探针重复三次,形成三个阵列。用双重PCR方法扩增经ERG11测序的白念珠菌和热带念珠菌C2a、光滑念珠菌Y10a、都柏林念珠菌C8a、克柔念珠菌ATCC6258、近平滑念珠菌ATCC22019等5株标准株的ERG11和ITS2种特异性序列。用醋酸钠/乙醇法纯化双重PCR产物,用DNaseⅠ进行酶切片段化,使酶切产物的片段长度在100 bp左右。针对T541C、A1090G、C1361T、G1537A、G1547A、T1559C等6种ERG11突变序列和白念珠菌、都柏林念珠菌、光滑念珠菌、克柔念珠菌、近平滑念珠菌和热带念珠菌等6种常见条件致病性念珠菌的内转录间隔区（IST2）种特异性序列,人工合成12条50 bp互补寡核苷酸链。用脱氧核苷酸末端转移酶（TDT）对片段化的双重PCR产物和50 bp寡核苷酸链进行Cy3荧光素标记后,进行芯片杂交。杂交后的芯片用GenePix 4000B共聚焦激光扫描仪进行扫描,利用GenePix Pro提取每条探针的荧光信号强度值,做出菌种鉴定和突变检测。结果共收集临床念珠菌426株,其中263株分离自VVC病人的阴道分泌物标本,101株分离自痰液标本,22株分离自尿液标本,16株分离自皮屑或甲屑标本,7株分离自菌血症病人的血液标本,6株分离自口腔刮片标本,其余11株来自其它部位。分离白念珠菌293株（68.8%）,热带念珠菌52株（12.2%）,光滑念珠菌46株（10.8%）,近平滑念珠菌14株（3.3%）,克柔念珠菌10株（2.3%）,都柏林念珠菌5株（1.2%）,其它念珠菌6株（1.4%）。念珠菌临床株对氟康唑的敏感率为84.5%（360/426）,剂量依赖敏感率为7.0%（30/426）,耐药率为8.5%（36/426）。白念珠菌对氟康唑的敏感率89.4%（262/293）,剂量依赖敏感率为5.5%（16/293）,耐药率为5.1%（15/293）;热带念珠菌对氟康唑的敏感率92.3%（48/52）,剂量依赖敏感率为5.8%（3/52）,耐药率为1.9%（1/52）;光滑念珠菌对氟康唑的敏感率56.5%（26/46）,剂量依赖敏感率为23.9%（11/46）,耐药率为19.6%（9/46）。15株白念珠菌耐药株均为A型。共有29株白念珠菌ERG11基因进行了PCR扩增和测序,包括上述15株临床耐药株、8株临床敏感株和6株标准株。共发现37种突变,其中19种为错义突变,另外18种属于同义突变。在耐药株中发现8种错义突变,在敏感株中发现13种错义突变。标准耐药株的ERG11测序结果与已知序列相同。在14株耐药株ERG11中,均同时检测到G487T和T916C,且不伴有其它任何突变。在另1株耐药株GZ04中,检测到4种错义突变T495A、A530C、T541C、T1493A,在标准敏感株中检测到T495A、A504C、G820T、A945C等纯合突变和T495A/C、T566G/T、G630T/G、G635C/G、C1271A/C、G1289T/G等杂合突变。在临床敏感株C261、0461、2855.中未检测到错义突变。在另外5株临床敏感株C522、C1307、0920、2827、2928中检测到纯合突变T495A、A530C、G640A、G820C、A945C和杂合突变A945C/A、G1609A/G。进行芯片杂交的菌株共34株。芯片杂交正确鉴定出上述34株试验菌株的菌种和其中29株白念珠菌ERG11基因中可致唑类耐药的已知突变,正确鉴定了12条人工合成的50 bp ERG11突变序列和ITS2种特异性序列。敏感性和特异性均为100%。结论（1）白念珠菌仍是最主要的致病性念珠菌,白念珠菌的分离比例和念珠菌对氟康唑的敏感性均与既往报道相仿。（2）ERG11突变G487T（A114S）、T916C（Y257H）可能参与菌株对氟康唑耐药。A504C（K119N）、G820T（D225Y）、G820C（D225H）、G640A（E165K）等突变不能导致菌株对氟康唑耐药。（3）用DNA芯片进行念珠菌菌种鉴定和白念珠菌ERG11已知突变筛查,结果可靠。更多还原

【Abstract】 Candidiasis is becoming more common,following the widespread application ofbroad-spectrum antibiotics,immunodepressants,therapeusis of intervention and organ transplantation.And the utilization of antifungal agents promotes the process of resistance formation.More and more researchers pay their attention to the molecular mechanisms of candidal resistance to antifungal agents,especially to azoles,in order to find the way to rapid diagnosis and effective treatment of candidasis.Azoles opened a new world of treatment in candidasis,but in the same time, azole-resistant isolates are more frequently confronted with under the drug selection pressure.Candidal resistance is an arising medical problem,attracting people’s notice. Identification of pathomycetes and susceptibility test play an important role in the clinical treatments and prognostic decisions.Candida albicans is the most important pathomycete of candiasis.Overseas researches on molecular mechanisms of C.albicans to azoles have been carried out. Up to now,there are 4 major hypotheses on azole-resistance mechanisms of Candida spp..The first is based on spatial configuration changes of the target enzyme 14 alpha-demethylase（Erg11p）due to mutations in the encoding gene ERG11.Erg11p is a key enzyme in the ergosterol synthesis pathway of C.albicans.Ergosterol is essential for maintaining the integrity and function of C.albicans membrane.Azoles can bind with the enzyme and block ergosterol synthesis.If one or more mutations in ERG11 result in changes in the Erg11p spatial configuration,a decrease in the affinity between the azole and protein occurs.This altered phenotype often makes isolates resistant to azole.In addition,over expression of ERG11 has been thought to increase resistance,although recent data indicate that over expression is unrelated to azole resistance in C.albicans.Amino acid substitutions have been described in 61 residues of the Erg11p due to missense mutations in ERG11 gene.Erg11p mutations Y132H, T315A,S405F,G464S,R467K and I471T have been shown to cause azole resistance.Multidrug transporters also contribute to drug resistance in Candida species. Over expression of candida drug resistance genes CDR1,CDR2 as well as the multiple drug resistance gene MDR1 can decrease the intracellular drug concentration effectively.Also,biofilms may mediate antifungal resistance.The C.albicans biofilm like that of other species is a highly heterogeneous bilayered structure,composed of cellular and noncellular elements and a matrix consisting of carbohydrate,protein and other components.Biofilms represent a niche for microorganisms where they are thought to be protected from the host immune system and antimicrobial therapies. Finally,fluconazole sequestration within intracellular vacuoles may be a novel mechanism of resistance.Multiple mechanisms of fluconazole resistance can arise in a single C.albicans isolate,and there also are chances that even a single base change in ERG11 can increase resistance to azoles.Microarray technology can provide a platform for us to identify such mutations in ERG11 and to get information of isolates’ susceptibility to fluconazole,without any susceptibility test.There are different names for the microarrays,like DNA/RNA Chips,BioChips or GeneChips.The array can be defined as an ordered collection of microspots,each spot containing a single defined species of a nucleic acid.The microarray technique is based on hybridisation of nucleic acids.In this technique,sequence complementarity leads to the hybridisation between two single-stranded nucleic acid molecules,one of which is immobilised on a matrix,such as membranes,glass or silicon chips. Microarrays are fabricated either by in situ light-directed chemical synthesis or by conventional synthesis followed by immobilisation on a glass substrate.The major feature of this technique is that it allows one to perform a simultaneous analysis of a great number of DNA sequences.The microarray technology,as one of the most important technologies in post-genomics times,may be employed in diagnostics（mutation detection）,gene discovery,gene expression and mapping.It is used to measure expression levels of genes in bacteria,plant,yeast,animal and human samples. Somebody detected several genes in candidal genomics and compared fluconazoleresistant and -sensitive isolates with microarray.In another study,microarray was used to identify fungal species including candida.They considered microarray as a useful technique with extremely high sensitivity and specificity.However,there was no report concerning identification of resistant isolates of C.albicans with microarray.Objectives:to investigate candida species distribution and susceptibility of candida isolates to fluconazole;to screen mutations ERG11 gene of C.albicans isolates and analyze the relationship between mutations and fluconazole resistance;to fabricate DNA microaray,identify frequent candida species and C.albicans ERG11 mutations resulting in fluconazole-resistance.Methods:Clinical isolates of candida species were collected and stored on modified SDA slants at 4℃after inoculating specimen from patients with candidiasis. CHROMagar medium is used to identify C.albicans（including C.dubliniensis）,C. tropicalis,C.glabrata,and C.krusei.Germ tube formation test,saccharobiose assimilation test,and chlamydospore formation test on Corn meal-tween agar（CMA）,were used to confirm the identification of C.albicans（including C.dubliniensis）. Triphenyltetrazolium chloride（TZC）agar reduction test was used to confirm the identification of C.tropicalis.C.parapsilosis and other species were identified by VITEK2 system.The sequence including the 25S rDNA transposable intron of C. albicans（including C.dubliniensis）isolates was amplified by PCR to differentiate these two species and subtype C.albicans isolates.Fluconazole susceptibility was tested in vitro using microdilution and disc diffusion assays to decide sensitive isolates（S）,dose-dependent sensitive isolates （S-DD）and resistant isolates（R）.The microdilution test was performed referring the M27-A2 broth dilution methods protocol as recommended by the Clinical and Laboratory Standards Institute（CLSI）.The results from disc diffusion assays were referred when the results of microdilution test were read,and if there was any conflict, corresponding susceptibility tests should be repeated.The final results were in accordance with those from microdilution test.Three pair of primers were designed referring C.albicans ERG11 sequence of GenBank X13296 and synthesized to amplify the ERG11 gene of fluconazole-resistant isolates,2 resistant type strains,4 sensitive type strains and several sensitive isolates of C.albicans with Pfu DNA polymerase.The 2 resistant type strains with definite ERG11 sequence were as quality control（QC）.PCR products were purified with gel purification kit and then sequenced.At least two perfectly matching probes and one mismatching probe were designed according to each of the following mutations in ERG11:T541C,A1090G, C1361T,G1537A,G1547A,T1559C.One species-specific probe was designed according to each of the following six fungi’ internal transcribed spacer ITS2 conserved sequence:C.albicans,C.dubliniensis,C.tropicalis,C.glabrata,C.krusei and C.parapsilosis.DNA microarray was fabricated with OmniGrid^TM100.Every probes were applied 3 times.ERG11 ORF and species-specific sequence of ITS2 in type strains C2a（C.tropicalis）,Y10a（C.glabrata）,C8a（C.dubliniensis）,ATCC6258 （C.krusei）,ATCC22019（C.parapsilosis）,as well as in those sequenced,were amplified by double PCR.The products were purified with NaAc/alcohol and then incised with DNaseⅠto form fragments of about 100 bp in length.Six ERG11 oligonucleotides embracing T541C,A1090G,C1361T,G1537A,G1547A,or T1559C respectively and six species-specific oligonucleotides from ITS2 region of C.albicans, C.dubliniensis,C.tropicalis,C.glabrata,C.krusei or C.parapsilosis respectively were synthesized.These twelve 50 bp oligonucleotides and the incised PCR products were labeled with Cy3 fluorescein in a system of terminal deoxynucleotidyl transferase（TDT）and hybridized with DNA microarray.The hybridized microarray was scaned by GenePix 4000B confocal laser scanner.The values of fluorescence signal intensity were read by GenePix Pro.Results of species identification and mutation detection were decided from the values.Results:Totally,426 clinical isolates of candida species were collected.Two hundred and sisty three isolates were from VVC patients,101 isolates from sputum specimen,22 isolates from urine,16 isolates from skin or nail scraping specimen,7 isolates from blood,6 isolates from oral cavity scraping specimen,and another 11 isolates from other sites.C.albicans occupied 68.8%（293/426）,C.tropicalis 12.2% （52/426）,C.glabrata 10.8%（46/426）,C.parapsilosis 3.3%（14/426）,C.krusei 2.3% （10/426）,C.dubliniensis 1.2%（5/426）,and others 1.4%（6/426）.Sensitivity rate of candida isolates to fluconazole is 84.5%（360/426）,dose dependent sensitivity rate is 7.0%（30/426）,and resistance rate is 8.5%（36/426）. Sensitivity rate of C.albicans isolates to fluconazole is 89.4%（262/293）,dose dependent sensitivity rate is 5.5%（16/293）,and resistance rate is 5.1%（15/293）. Sensitivity rate of C.tropicalis isolates to fluconazole is 92.3%（48/52）,dose dependent sensitivity rate is 5.8%（3/52）,and resistance rate is 1.9%（1/52）. Sensitivity rate of C.glabrata isolates to fluconazole is 56.5%（26/46）,dose dependent sensitivity rate is 23.9%（11/46）,and resistance rate is 19.6%（9/46）.All of the 15 resistant isolates of C.albicans are genotype A.ERG11 of those 15 resistant isolates,8 sensitive isolates,and 6 type strains of C. albicans was amplified and sequenced.Thirty seven mutations were detected,of which 19 were missense mutation,and the other 18 were silence mutation.Eight missense mutations existed in the resistant strains or isolates,while 13 missense mutations existed in the sensitive.ERG11 sequence of resistant type strains by sequencing was same with what had been reported.In each of 14 resistant isolates, mutation G487T and mutation T916C were detected simultaneously without any other mutation in ERG11 sequence.In the other resistant GZ04,4 missense mutations T495A,A530C,T541C,and T1493A were detected.In the sequences of sensitive type strains,homozygous T495A,A504C,G820T and A945C and heterozygous T495A/C, T566G/T,G630T/G,G635C/G,C1271A/C and G1289T/G were found.No missense mutation was detected in the sensitive isolates C261,0461 or 2855.Homozygous T495A,A530C,G640A,G820C and A945C and heterozygous A945C/A and G1609A/G were found in the other 5 sensitive isolates.Thirty four strains and 12 oligonucleotides were identified by DNA microarray hybridization and correct results were obtained in species identification and mutation detection.Both sensitivity and specificity are 100%.Conclusions:（1）,C.albicans is still the major cause of candidiasis.Both its proportion in composition of Candida isolates and the sensitivity of Candida spp.to fluconazole are similar with the reported data.（2）,the mutations G487T（A114S）and T916C（Y257H）might participate in the formation of resistance to fluconazole,while the following mutations A504C （K119N）,G820T（D225Y）,G820C（D225H）or G640A（E165K）cannot.（3）,it is a reliable method to identify candida species and ERG11 mutations by DNA microarray hybridization.更多还原