ERG11基因突变与白念珠菌对氟康唑耐药性的初步研究

目的探讨白念珠菌氟康唑作用的靶酶编码基因（ERG11）突变与氟康唑耐药性的关系。方法采用聚合酶链反应（PCR）扩增临床分离的23株白念珠菌ERG11基因,包括2株耐氟康唑株、9株氟康唑剂量依赖敏感株和12株氟康唑敏感株,并进行双向测序。应用B last软件,将测序结果与网上已发表序列（GenBankAY856352）进行比对,以确定是否发生基因突变。结果23株白念珠菌的ERG11基因序列共检出16个同义突变位点和18个错义突变位点。错义突变中Y205E、I437V、A255V、E260V、K487N、G472R、N435V、D502E、K143Q为新发现的突变位点。结论Y205E、I437V、A255V位点突变发现于敏感株,可能与白念珠菌耐药无关;K487N、G472R、N435V、D502E、E260V发现于剂量依赖敏感株,K143Q发现于耐药株,可能与耐药的形成有关。     

3种念珠菌对氟康唑耐药的易感性及耐药机制比较

目的研究比较3种念珠菌对氟康唑耐药的易感性及其体外诱导耐药株的耐药机制。方法选取同一患者中同时分离的对氟康唑敏感的白念珠菌、光滑念珠菌和热带念珠菌各1株,在体外氟康唑的作用下诱导其成为耐药株。采用罗丹明6G试验比较敏感株与耐药株的外排泵作用,RT-PCR检测外排相关基因CDR1、CDR2以及靶酶编码基因ERG11的表达,并对ERG11基因进行PCR扩增和测序,同时通过罗丹明123试验对3种念珠菌的线粒体膜电位(ΔΨm)进行检测。结果光滑念珠菌最易被氟康唑诱导为耐药株,其CDR1的过度表达引起外排泵作用增强。白念珠菌和热带念珠菌的诱导耐药株以ERG11表达增加为主,其中白念珠菌的ERG11存在V437I、A430V、S263L和T128K突变位点。此外,白念珠菌和光滑念珠菌的诱导耐药株表现为呼吸缺陷。结论不同念珠菌对氟康唑耐药的易感性不同,耐药机制也存在差异。     

白念珠菌临床分离株吡咯类耐药机制研究

目的分析女性生殖道感染白念珠菌临床分离株对5种抗真菌药物的耐药率,探讨白念珠菌对吡咯类药物的耐药机制。方法 (1)收集2015年1-12月自复旦大学附属妇产科医院女性生殖道感染患者中分离的白念珠菌1 646株,统计菌株对5种抗真菌药物的耐药情况。(2)收集包括该院和上海市另2所妇产科专科医院微生物室临床分离白念珠菌氟康唑耐药菌株30株、剂量依赖性敏感(S-DD)菌株13株、敏感菌株10株。采用实时荧光定量PCR技术分析吡咯类耐药组、S-DD组和敏感组之间药物外排泵相关基因CDR1、CDR2、MDR1和药物靶酶基因ERG11表达水平的差异。同时,PCR扩增ERG11和ERG3基因并测序,分析ERG11和ERG3基因与耐药相关的突变位点。结果 (1)1 646株白念珠菌对伊曲康唑耐药率最高,为5.2%,对伏立康唑、氟康唑和5-氟胞嘧啶的耐药率分别为3.2%、2.5%和2.1%,所有菌株对两性霉素B均敏感。(2)S-DD组和耐药组ERG11基因表达较敏感组均显著升高,差异有统计学意义(P0.05);而药物外排泵基因CDR1、CDR2和MDR1表达量在敏感组、S-DD组和耐药组间差异无统计学意义。(3)检测到ERG11基因存在13个错义突变位点,其中T123I、P98S和Y286D为新发现的3个氨基酸置换位点;且T123I和Y132H同时出现在26株耐药株中,其中16株为吡咯类药物全耐药;此外,2株吡咯类全耐药菌株中检测到ERG3基因杂合突变。结论外阴阴道念珠菌病患者中分离的白念珠菌对吡咯类药物的耐药率比5-氟胞嘧啶和两性霉素高;ERG11基因突变及其过表达是该病白念珠菌吡咯类耐药的主要分子机制之一。     

尿路感染患者泌尿系统分离菌株中非白念珠菌的分布及其耐药性研究

目的 :分析从尿路感染患者泌尿系统分离的非白念珠菌分布情况及其耐药性,为临床诊疗提供依据。方法:收集2013年至2014年上海市东方医院南院收治的尿路感染患者临床分离的病原菌,采用Vitek2 Compact全自动微生物分析系统和真菌显色平板鉴定,对难以鉴定的菌株用真菌内转录间隔区1~4(internal transcribed spacer4,ITS1-4)测序分析鉴定。用Sensititre誖Yeast-One比色法检测55株非白念珠菌对两性霉素B、卡泊芬净、阿尼芬净、米卡芬净、5-氟胞嘧啶、氟康唑、伊曲康唑、伏立康唑和泊沙康唑这9种抗真菌药物的敏感性。对热带念珠菌和光滑念珠菌进行唑类药物耐药基因ERG11测序,分析潜在的耐药突变位点。结果:临床分离的55株非白念珠菌,包括26株光滑念珠菌,17株热带念珠菌和其他念珠菌12株。药敏试验表明,临床分离菌株对氟康唑、伊曲康唑的耐药率较高,分别为63.64%(35/55)、50.91%(28/55);而对伏立康唑和泊沙康唑的耐药率分别为12.73%(7/55)、7.27%(4/55)。热带念珠菌ERG11基因T1037A位点苯丙氨酸突变为酪氨酸、T1103C位点苯丙氨酸突变为丝氨酸。结论:尿路感染最常见非白念珠菌是光滑念珠菌和热带念珠菌,其构成比超过了白念珠菌,而热带念珠菌ERG11基因T1037A、T1103C位点突变可能是潜在的唑类药物耐药位点。     

白假丝酵母菌耐吡咯类药物的ERG11基因分析

目的 研究白假丝酵母菌耐吡咯类药物的ERG11的变异情况.方法 将93例诊断为真菌性阴道炎的患者的阴道分泌物标本进行真菌培养,筛选白假丝酵母菌菌株,利用纸片扩散法进行氟康唑、酮康唑、咪康唑药敏试验,用加热裂解法提取菌株DNA,扩增ERG11基因,扩增后的PCR产物进行双向测序,测序结果与GenBank中的标准序列（SC5314）比较分析.结果 93例均培养出假丝酵母菌,包括60株白假丝酵母菌,19株热带假丝酵母菌,9株克柔假丝酵母菌和5株光滑假丝酵母菌;白假丝酵母菌对氟康唑、酮康唑、咪康唑耐药率分别为13.33%,20.00％和51.67％;对白假丝酵母菌的ERG11基因测序发现存在25个碱基突变位点,其中13个同义突变,12个错义突变,其中有6个是新变异：V36F,V51L,T123I,E194K,Y257H和K344N.结论 耐吡咯类药物白假丝酵母菌ERG11基因有多个错义突变位点,其中某些位点突变导致的氨基酸变异可能与其耐药性产生有关.     

Erg11基因突变与白念珠菌对唑类药物耐药性的初步研究

目的探讨Erg11基因突变与白念珠菌对唑类药物耐药的关系,以初步了解白念珠菌对唑类药物耐药的机制。方法从临床分离耐唑类药物的白念珠菌,通过PCR扩增Erg11基因并测序,与敏感白念珠菌进行对照,以确定是否发生基因突变。结果2株唑类药物敏感的白念珠菌中,通过Erg11基因测序,未发现有义突变;6株耐唑类药物的白念珠菌中,Erg11基因存在E266D、Y257H、K259E、A114S、F487L突变,其中A114S、Y257H、K259E、F487L为新发现的突变位点。结论白念珠菌对唑类药物耐药与Erg11基因的突变有关,通过Erg11基因突变,可引起Erg11P的蛋白结构发生改变,最终可导致唑类药物与Erg11P亲和力下降而产生耐药。     

白念珠菌MRR2基因错义突变C1409A与氟康唑耐药的相关性

目的 研究锌簇转录因子——多药耐药调节因子2(Mrr2)编码基因MRR2错义突变C1409A与白念珠菌氟康唑耐药的相关性。方法 利用白念珠菌工程菌株SN152构建MRR2基因敲除菌株,通过一步法克隆及定点突变技术构建MRR2基因C1409A突变型表达质粒,再用高效醋酸锂转染法将质粒片段转染入MRR2基因敲除菌株,异位表达于ADE2位点以构建MRR2基因定点突变菌株。通过体外药物敏感性试验及实时荧光定量聚合酶链反应(PCR)分析错义突变C1409A与氟康唑耐药的关系。结果 氟康唑对MRR2基因C1409A定点突变的白念珠菌的最低抑菌浓度(MIC)较对工程菌株SN152的MIC增加4倍,CDR1表达上调约3倍。结论 MRR2基因错义突变C1409A可上调白念珠菌CDR1表达并介导白念珠菌对氟康唑的耐药。     

耐氟康唑热带假丝酵母菌ERG11基因突变分析

目的探讨临床分离的耐氟康唑热带假丝酵母菌的ERG11基因突变情况及其与氟康唑耐药的关系。方法收集临床分离的热带假丝酵母菌,用ATB Fungus 3酵母菌药敏试剂盒测定其对氟康唑的敏感性。对8株耐氟康唑的菌株及随机选取的17株敏感株,提取基因组DNA,扩增ERG11基因并测序,测序结果与Gen Bank中的已知标准序列(M23673)进行比对分析。结果 25株热带假丝酵母菌均扩增到ERG11基因并成功测序,发现7个不同的碱基突变位点。耐药株错义突变位点为Y132F、S154F和K143Q,其中K143Q为首次报道;敏感株无错义突变位点。结论耐氟康唑的热带假丝酵母菌ERG11基因存在多个错义突变位点,且多为多位点突变,可能与其耐药性的产生有关。     

外阴阴道念珠菌病患者菌群调查及其ERG5基因突变研究

目的?了解外阴阴道念珠菌病（VVC）患者白念珠菌分离及ERG5基因突变情况。方法?收集无锡市妇幼保健院2018年6—12月妇产科门诊VVC患者阴道分泌物500例，通过镜下观察菌丝及孢子选取可疑标本，经沙保弱琼脂平板增菌后接种科玛嘉显色平板，进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）鉴定；用真菌快速培养鉴定药敏试剂盒对白念珠菌进行药敏试验；PCR扩增部分白念珠菌ERG5基因并进行测序分析。结果?显色平板鉴定结果显示，VVC病原以白念珠菌最多（54.7%），其次为光滑念珠菌（22.5%）、热带念珠菌（16.5%）、克柔念珠菌（4.2%）和其他真菌（2.1%）；156株显色平板鉴定结果为白念珠菌的菌株中，经MALDI-TOF MS鉴定为白念珠菌共155株。155株白念珠菌对5-氟尿嘧啶均敏感，对各类唑类药物呈现不同水平耐药性；对7株（3株唑类药物耐药菌株，3株唑类药物敏感菌株和1株质控菌株）进行ERG5扩增测序，检出2个突变位点（1个同义突变位点G528A，1个错义突变位点G528C）。结论?外阴阴道念珠菌以白念珠菌为主，并对各类唑类药物表现出不同的耐药率，耐药菌株中ERG5基因在G528C位点突变可能与耐药有关。     

白色假丝酵母菌ERG11基因突变与唑类抗真菌药物耐药的关系

目的探讨白色假丝酵母菌ERG11基因突变与唑类抗真菌药物耐药的关系。方法纸片扩散法初步筛选临床分离的耐唑类抗真菌药物的白色假丝酵母菌,测定初筛耐药株对氟康唑和伊曲康唑的最低抑菌浓度,随机选择10株白色假丝酵母耐药株,提取基因组DNA。利用DNASTAR软件辅助设计3对PCR引物,以提取的目的 DNA为模板,分别从前(P1)、中(P2)、后(P3)3段扩增ERG11基因全序列。扩增后的PCR产物经纯化后测序,将测序结果与GenBank中已知标准序列(X13296)进行比较分析。结果电泳结果显示,获得的3段PCR产物大小与预期结果一致。测序结果显示成功获得10株白色假丝酵母菌ERG11全基因序列。与GenBank中标准株序列(X13296)比较,耐药株均存在同义突变和错义突变,共27个碱基突变位点。15个位点是错义突变,其中F126LI、166N、H183Q、V437I、S453F和N490K为新发现的突变位点。结论耐唑类抗真菌药物的白色假丝酵母菌ERG11基因有多个发生错义突变的位点,且为多点突变,可能与其耐药有关。     

唑类耐药白色念珠菌羊毛甾醇14α-去甲基化酶基因的研究

目的探讨白色念珠菌羊毛甾醇14α-去甲基化酶（CYP51）基因突变与对唑类抗真菌药物耐药的关系,从分子水平了解其耐药机制。方法纸片扩散法和NCCL公布的M-27方案测定耐药株对氟康唑和伊曲康唑的MIC;设计引物,PCR扩增唑类耐药白色念珠菌的CYP51基因;扩增产物测序并与Genbank序列相比较分析。结果扩增产物测序分析表明,成功扩增到白色念珠菌CYP51基因。与X13296株序列相比较,两个耐药株都存在有意义突变和无意义突变。两株菌共有22个碱基突变。与以往报道的相同,突变发生氨基酸替换的有F105L、K128T、Y133H、T199I、R267H、G464S和G467K。其中,两株菌都有Y132H和G467K突变。F71L、W244R、T311N和T352I为新发现的突变,未见报道。同时,也发现了9个未发生氨基酸替换的突变。结论白色念珠菌对唑类抗真菌药物的耐药与CYP51基因突变有关,且为多位点突变。     

Genetic analysis of azole resistance in the Darlington strain of Candida albicans

High-level azole resistance in the Darlington strain of Candida albicans was investigated by gene replacement in C. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C(14)alpha-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.     

Drug resistance genes and trailing growth in Candida albicans isolates

OBJECTIVES: To investigate possible molecular mechanisms of azole resistance among fluconazole-susceptible bloodstream isolates of Candida albicans that displayed the trailing growth phenomenon, and to compare these isolates with bloodstream and mucosal isolates that showed reduced susceptibilities to fluconazole. METHODS: Twelve C. albicans isolates-seven trailing and five susceptible dose dependent (SDD) or resistant (R)-were screened for ERG11 mutations by DNA sequencing and quantification of ERG11, CDR1 and MDR1 expression by RT-PCR using the LightCycler high-speed PCR system. RESULTS: SDD and R isolates possessed more homozygous ERG11 mutations than did the trailing isolates. Two of these, V404I and V509M, have not been described previously and were found exclusively in fluconazole SDD and R isolates. Quantification of ERG11 expression revealed that both trailing and SDD and R isolates were capable of ERG11 up-regulation in response to fluconazole, although the SDD and R isolates showed maximal up-regulation at higher fluconazole concentrations. Quantification of CDR1 and MDR1 revealed that all isolates, regardless of in vitro fluconazole response, were capable of CDR1 and MDR1 up-regulation following fluconazole exposure. Furthermore, the SDD and R isolates expressed higher constitutive levels of CDR1 and MDR1 or CDR1, respectively, in the absence of drug compared with trailing isolates. CONCLUSIONS: Trailing isolates, although susceptible to fluconazole, express the same molecular mechanisms as SDD and R isolates following fluconazole exposure but regulate them differently.     

In vitro antifungal activity of BMS-207147 and itraconazole against yeast strains that are non-susceptible to fluconazole

The activities of itraconazole and the new triazole BMS-207147 were determined against Candida strains that were susceptible-dose dependent (fluconazole MICs 16 to 32 micrograms/mL) or resistant (MICs > or = 64 micrograms/mL) to fluconazole. These strains included clinical isolates of Candida krusei, Candida glabrata, and Candida albicans. In addition, 16 isogenic, genetically characterized isolates of C. albicans, with progressively decreased susceptibility to fluconazole, were tested. BMS-207147 MICs to C. krusei, a species considered intrinsically resistant to fluconazole, were at 0.13 to 0.5 microgram/mL. The population distribution of the fluconazole-nonsusceptible C. glabrata was bimodal with BMS-207147/itraconazole MICs at 0.5 to 2 micrograms/mL and > or = 16 micrograms/mL. The BMS-207147 MICs to the majority of fluconazole-nonsusceptible C. albicans strains tested were < or = 1 microgram/mL. The activity of BMS-207147 was minimally affected by overexpression of the gene encoding the efflux pump MDR1, but MIC increases were observed with changes in ERG11 and with overexpression of the CDR transporter gene. Nonetheless, BMS-207147 can be active against C. albicans mutants containing cumulative resistance mechanisms to azoles. In other words, fluconazole-resistant candidal strains may be susceptible to BMS-207147.     

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

Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles in C. albicans strains displaying high-level fluconazole resistance (MICs, > or =64 microg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14alpha-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.     

Resistance mechanisms in clinical isolates of Candida albicans

Resistance to azole antifungals continues to be a significant problem in the common fungal pathogen Candida albicans. Many of the molecular mechanisms of resistance have been defined with matched sets of susceptible and resistant clinical isolates from the same strain. Mechanisms that have been identified include alterations in the gene encoding the target enzyme ERG11 or overexpression of efflux pump genes including CDR1, CDR2, and MDR1. In the present study, a collection of unmatched clinical isolates of C. albicans was analyzed for the known molecular mechanisms of resistance by standard methods. The collection was assembled so that approximately half of the isolates were resistant to azole drugs. Extensive cross-resistance was observed for fluconazole, clotrimazole, itraconazole, and ketoconazole. Northern blotting analyses indicated that overexpression of CDR1 and CDR2 correlates with resistance, suggesting that the two genes may be coregulated. MDR1 overexpression was observed infrequently in some resistant isolates. Overexpression of FLU1, an efflux pump gene related to MDR1, did not correlate with resistance, nor did overexpression of ERG11. Limited analysis of the ERG11 gene sequence identified several point mutations in resistant isolates; these mutations have been described previously. Two of the most common point mutations in ERG11 associated with resistance, D116E and E266D, were tested by restriction fragment length polymorphism analysis of the isolates from this collection. The results indicated that the two mutations occur frequently in different isolates of C. albicans and are not reliably associated with resistance. These analyses emphasize the diversity of mechanisms that result in a phenotype of azole resistance. They suggest that the resistance mechanisms identified in matched sets of susceptible and resistant isolates are not sufficient to explain resistance in a collection of unmatched clinical isolates and that additional mechanisms have yet to be discovered.