耐氟康唑白念珠菌临床株ERG11基因突变检测

目的 筛查耐氟康唑白念珠菌ERG11基因突变,探讨其与耐药的关系.方法 用柯玛嘉显色培养基和25S rDNA转座内含子保守区分型方法,收集鉴定白念珠菌临床株.联用微量稀释法和Rosco药敏纸片扩散法体外测定试验菌株对氟康唑的敏感性.以ERG11序列明确的白念珠菌标准耐药株ATCC 76615-19和达令顿株为质控,以标准敏感株C1b和临床敏感株02928为对照,分3段扩增临床耐药株ERG11基因并测序.结果 获得15株A型白念珠菌临床耐药株.ERG11基因测序共发现16处同义突变和11处错义突变.质控株突变与既往报道一致.敏感株共出现9处同义突变和3处错义突变G640A(E165K)、A945C(E266D)、G1609A/G(V488I).耐药临床株中的14株均出现2处共有的错义突变G487T(A114S)和T916C(Y257H),不伴其他突变;另1株出现8处同义突变和4处错义突变R541C(Y132H)、T495A(D116E)、A530C(K128T)、T1493A(F449Y),其中T1493A(F449Y)未见报道.结论不同来源的白念珠菌临床耐药株集中发生G487T(A114S)和T916C(Y257H)突变,暗示这2处突变极可能参与菌株耐药.耐药株可以发生T1493A(F449Y)突变.     

分离自艾滋病患者白念珠菌唑类药物耐药株中Erg11基因突变研究

目的:探讨分离自艾滋病患者的白念珠菌唑类药物耐药株中Erg11基因的突变及其与唑类耐药的关系.方法:分离自艾滋病患者的100株真菌标本,经科玛嘉显色培养鉴定为白念珠菌85株,根据美国临床实验室标准化协会制订的M27-A方案的液体微量稀释法行体外药物敏感试验,共鉴定出27株唑类耐药株(耐氟康唑和/或伊曲康唑和/或伏立康唑).27株耐药株提取基因组DNA后,对其Erg11基因行PCR扩增、测序并与Genebank中敏感株进行比对分析.结果:在27株唑类耐药株中共发现25处不同点突变,其中5处引起氨基酸替换,分别为D116E、E266D、H485T、V447I和V488I,27株均发生H485T突变,7株耐药株定生E266D突变,4株耐药株发生V488I突变,其中1株同时发生E266D和V4881突变,1株发生V4471突变.结论:分离自艾滋病患者的白念珠菌唑类耐药株中Erg11基因的突变热变区和有意义突变与分离自其他宿主的唑类耐药株的突变情况基本一致;分离自艾滋病患者的白念珠菌唑类耐药株Erg11基因突变并非唯一致耐药因素.     

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。     

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。     

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。     

耐唑类抗真菌药白念珠菌标准菌株的建立与评价

目的 建立耐唑类抗真菌药的白念珠菌(C. albicans)标准菌株,参照《病原微生物菌(毒)种国家标准株评价技术标准》(WS/T 812—2022)要求做好病原微生物菌(毒)种保藏工作,为其他真菌标准菌株的候选提供参考方法。方法 通过菌株表型、鉴定、测试9种抗真菌药物敏感性和耐药基因,评估菌株保存状态下的表型稳定性和活性。结果 菌株经内部转录间隔区(ITS)序列分析和质谱仪(MALDI-TOF MS)鉴定为白念珠菌,连续传代十次仍有稳定的分子生物学特征。该菌株对氟康唑、伏立康唑、伊曲康唑、泊沙康唑的最低抑菌浓度(MIC)分别为16 mg/L、0.5 mg/L、0.5 mg/L和0.5 mg/L,耐药性与ERG11基因双点突变(A114S、Y257H)及编码药物外排泵基因突变(A736V)相关。菌株在–80℃表型稳定至少6个月。结论 本研究建立了耐唑类抗真菌药白念珠菌标准菌株,为白念珠菌感染性疾病的综合防治提供了重要的研究对象和物质基础,对于国家生物安全具有重要的战略意义。     

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.     

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。     

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。     

白念珠菌对唑类药物耐药机制的研究进展

白念珠菌是临床最常见的条件致病性真菌,由于抗真菌药物的长期滥用,其对唑类药物的耐药逐年增多,耐药现象已经成为药物治疗的难点。目前,白念珠菌对唑类药物耐药的主要机制包括:ERG11基因突变或过度表达导致的靶位酶的改变、多药转运蛋白过度表达、细胞应激反应和生物膜的形成等。现就白念珠菌对唑类药物的耐药机制作简单归纳。     

Contribution of Clinically Derived Mutations in ERG11 to Azole Resistance in Candida albicans

In Candida albicans, the ERG11 gene encodes lanosterol demethylase, the target of the azole antifungals. Mutations in ERG11 that result in an amino acid substitution alter the abilities of the azoles to bind to and inhibit Erg11, resulting in resistance. Although ERG11 mutations have been observed in clinical isolates, the specific contributions of individual ERG11 mutations to azole resistance in C. albicans have not been widely explored. We sequenced ERG11 in 63 fluconazole (FLC)-resistant clinical isolates. Fifty-five isolates carried at least one mutation in ERG11, and we observed 26 distinct positions in which amino acid substitutions occurred. We mapped the 26 distinct variant positions in these alleles to four regions in the predicted structure for Erg11, including its predicted catalytic site, extended fungus-specific external loop, proximal surface, and proximal surface-to-heme region. In total, 31 distinct ERG11 alleles were recovered, with 10 ERG11 alleles containing a single amino acid substitution. We then characterized 19 distinct ERG11 alleles by introducing them into the wild-type azole-susceptible C. albicans SC5314 strain and testing them for susceptibilities to FLC, itraconazole (ITC), and voriconazole (VRC). The strains that were homozygous for the single amino acid substitutions Y132F, K143R, F145L, S405F, D446E, G448E, F449V, G450E, and G464S had a ≥4-fold increase in FLC MIC. The strains that were homozygous for several double amino acid substitutions had decreased azole susceptibilities beyond those conferred by any single amino acid substitution. These findings indicate that mutations in ERG11 are prevalent among azole-resistant clinical isolates and that most mutations result in appreciable changes in FLC and VRC susceptibilities.     

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.     

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.     

Changes in susceptibility to posaconazole in clinical isolates of Candida albicans

OBJECTIVES: To characterize the molecular mechanisms responsible for reduced susceptibility to azoles in Candida albicans clinical isolates. MATERIALS AND METHODS: Seven sequential C. albicans isolates were cultured from an AIDS patient treated with posaconazole for refractory oropharyngeal candidiasis. Expression levels of the CDR1, CDR2 and MDR1 genes, encoding efflux pumps previously implicated in azole resistance, and ERG11, encoding the azole target site, were monitored using northern blot and real-time PCR. The ERG11 genes from all seven isolates were sequenced. RESULTS: The seven closely related isolates exhibited significant decreases in susceptibility to fluconazole (MIC >or= 32 mg/L) and voriconazole (MIC >or= 2 mg/L) and progressive decreases in susceptibility to both posaconazole (isolates 1-4 MIC 0.25 mg/L, isolates 5-7 MIC 2 mg/L) and itraconazole (isolates 1-4 MIC 1 mg/L, isolates 5-7 MIC > 8 mg/L). None of the isolates exhibited any significant changes in the expression levels of ERG11 or the efflux pump genes. All seven isolates had multiple mutations in ERG11; isolates one through four each had five missense mutations; four of the resultant amino acid changes were previously associated with azole resistance. The fifth isolate had an additional novel mutation in one copy of ERG11, resulting in a Pro-230 to Leu substitution. This mutation was present in both ERG11 genes in the last two isolates. Select ERG11 genes were expressed in Saccharomyces cerevisiae, the ERG11 allele with all six mutations conferred the highest level of posaconazole resistance. CONCLUSIONS: Multiple mutations in ERG11 are required to confer decreased susceptibility to posaconazole.     

Amino Acid Substitutions in the Cytochrome P-450 Lanosterol 14α-Demethylase (CYP51A1) from Azole-Resistant Candida albicans Clinical Isolates Contribute to Resistance to Azole Antifungal Agents

The cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378–2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1 genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1 gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.     

Identification of a Cryptococcus neoformans cytochrome P450 lanosterol 14α-demethylase (Erg11) residue critical for differential susceptibility between fluconazole/voriconazole and itraconazole/posaconazole

Cryptococcus neoformans strains resistant to azoles due to mutations causing alterations in the ERG11 gene, encoding lanosterol 14α-demethylase, have rarely been reported. In this study, we have characterized a C. neoformans serotype A strain that is resistant to high concentrations of fluconazole (FLC). This strain, which was isolated from an FLC-treated patient, contained five missense mutations in the ERG11 gene compared to the sequence of reference strain H99. Molecular manipulations of the ERG11 gene coupled with susceptibility to triazole revealed that a single missense mutation resulting in the replacement of tyrosine by phenylalanine at amino acid 145 was sufficient to cause the high FLC resistance of the strain. Importantly, this newly identified point mutation in the ERG11 gene of C. neoformans afforded resistance to voriconazole (VRC) but increased susceptibility to itraconazole (ITC) and posaconazole (PSC), which are structurally similar to each other but distinct from FLC/VRC. The in vitro susceptibility/resistance of the strains with or without the missense mutation was reflected in the therapeutic efficacy of FLC versus ITC in the animals infected with the strains. This study shows the importance of the Y145F alteration of Erg11 in C. neoformans for manifestation of differential susceptibility toward different triazoles. It underscores the necessity of in vitro susceptibility testing for each FLC-resistant C. neoformans clinical isolate against different groups of azoles in order to assist patient management.