不同唑类药物体外诱导热带念珠菌耐药特征及其耐药机制研究

目的比较体外不同唑类药物诱导热带念珠菌耐药性产生的特点以及耐药机制的不同。方法选取1株临床分离的唑类敏感菌,分别在含16μg/mL氟康唑,2μg/mL伏立康唑,1μg/mL泊沙康唑的液体培养基中进行传代培养。显色微量肉汤稀释法检测每一代菌的抗真菌药物敏感性。对第50代传代菌的唑类作用靶位点基因ERG11进行扩增测序,并对ERG11基因、泵蛋白基因MDR1、CDR1以及线粒体细胞色素b基因CYTb进行荧光定量检测。结果体外氟康唑、伏立康唑暴露的情况下,菌株很快出现耐药性;相比,泊沙康唑并未诱导出耐药菌;氟康唑、伏立康唑诱导耐药性产生的方式呈现不同特征。氟康唑诱导下菌株MIC值逐渐上升,但伏立康唑诱导菌出现了明显的跳跃式升高。耐药机制研究发现,伏立康唑诱导菌ERG11基因出现了与耐药密切相关的G/G1390G/A碱基杂合突变。荧光定量PCR结果显示,仅伏立康唑诱导耐药菌CDR1基因的相对表达量显著升高。结论热带念珠菌在体外氟康唑、伏立康唑暴露情况下会很快出现耐药性,但其出现的特征以及耐药机制有所差别。     

白念珠菌氟康唑耐药株与敏感株SRB1、CDR1、ERG11表达比较分析

目的了解SRB1在白念珠菌氟康唑耐药株和敏感株表达差异,探讨与白念珠菌耐药性的关系。方法使用同一亲本来源的白念珠菌氟康唑耐药株CA-16和敏感株CA-3为研究对象,采用实时荧光定量RT-PCR的方法扩增各菌株的目的基因SRB1、CDR1、ERG11,观察各菌株目的基因表达情况。结果与白念珠菌氟康唑敏感株CA-3相比,在mRNA水平氟康唑耐药株CA-16的SRB1和耐药基因CDR1、ERG11表达升高,SRB1、CDR1、ERG11表达水平差异具有统计学意义(P0.05)。结论白念珠菌SRB1的表达增高和白念珠菌对氟康唑耐药密切相关,SRB1可能是一个新的耐药候选基因。     

ERG11基因与热带念珠菌唑类药物耐药关系的研究

目的探讨临床唑类药物耐药热带念珠菌菌株ERG11基因突变及表达情况。方法连续收集临床分离的热带念珠菌菌株,采用微量肉汤稀释法检测其对氟康唑、伏立康唑及伊曲康唑的药物敏感性,对唑类药物耐药菌株及部分敏感菌株进行ERG11基因测序,同时采用RT-PCR测定ERG11基因表达量。结果临床共分离92株热带念珠菌菌株,其中有29株为唑类药物耐药菌株,耐药率31.52%。40株热带念珠菌(29株耐药菌株和11株敏感菌株)ERG11基因序列共发现2个错义突变(S154F、Y132F)和5个同义突变,其中24株唑类药物耐药菌株同时出现上述两个错义突变位点。实时荧光定量PCR结果显示,在29株唑类药物耐药菌株中有19株其ERG11基因表达量较敏感菌株增高。分析16株对3种唑类药物全耐药菌株及其余13株仅对一种或两种药物耐药菌株,显示前者ERG11基因表达水平高于后者,差异有统计学意义。结论临床热带念珠菌唑类药物耐药与ERG11基因突变及过表达有关,有关热带念珠菌唑类药物的耐药机制还需进一步研究。     

白色念珠菌氟康唑耐药相关基因的差异显示研究

采用差异显示PCR技术 (DifferentialDisplay PCR ,DD PCR)寻找白色念珠菌的氟康唑耐药相关基因。体外用含氟康唑的酵母培养基 (YEPD)诱导培养临床白色念珠菌氟康唑敏感株 4 35 (对咪康唑耐药 ) ,诱导 80d后得到氟康唑耐药子代 4 35 2 (MIC =1 2 8μg mL)。DD PCR比较 4 35 2、4 35分别在含氟康唑、不含药的YEPD液基中的基因表达 ,找到 3个明显差异片段 ,分别与数据库中白色念珠菌的醇脱氢酶基因ADH1、多药耐药基因CDR1及拓扑异构酶基因TOP2有高度同源性。半定量RT PCR中证实了ADH1、CDR1在氟康唑耐药株中的差异表达 ;对已知氟康唑耐药基因MDR1作半定量RT PCR时发现MDR1在氟康唑耐药株 4 35 2中无表达 ,而在氟康唑敏感株 4 35中有表达。结果表明 ,ADH1、CDR1基因的高表达与白色念珠菌氟康唑耐药性形成相关 ,ADH1可能是新的耐药基因 ;MDR1的表达可能在氟康唑敏感株或其它唑类耐药株中也存在     

芒果苷协同氟康唑抗耐药白念珠菌作用研究

目的研究芒果苷与氟康唑合用对唑类耐药白念珠菌协同抗真菌的作用和机制。方法采用棋盘式微量稀释法测试芒果苷协同氟康唑对22株耐药白念珠菌的最小抑菌浓度MIC80;时间-杀菌曲线探究两药联用对4株耐药白念珠菌生长的抑制作用;药物生长抑制实验实验探究不同浓度芒果苷和不同浓度氟康唑协同抗耐药白念珠菌药效;通过实时定量RT-PCR检测两药联用时耐药基因CDR1、CDR2、MDR1表达水平。结果芒果苷联合氟康唑可产生协同抗唑类白念珠菌作用,协同指数(FICI)0.5;两药合用对白念珠菌生长可产生抑制作用;两药合用降低耐药基因CDR1表达水平。结论芒果苷与氟康唑合用可产生协同抗唑类耐药白念珠菌作用。     

卷柏素对唑类药物体外抗念株菌的增效作用

本文研究中药卷柏中炔多酚类成分卷柏素与氟康唑、酮康唑联用对念珠菌的体外抗真菌效果,并初步探讨对氟康唑增效的分子机制。采用棋盘法测定两类药物联用对念珠菌体外抗真菌活性,以FICI法评价结果。实验结果表明,卷柏素与氟康唑、酮康唑合用对白念珠菌、近平滑念珠菌表现协同作用(FICI≤0. 5);对克柔念珠菌表现无关作用(FICI=1)。qRT-PCR检测卷柏素与氟康唑联用对白念株菌相关基因表达的影响显示,卷柏素能显著地逆转氟康唑诱导白念珠菌ERG5、ERG11、CDR1、CDR2、MDR1、FLU1和SIR2表达上调。Western-blot检测结果亦表明,卷柏素逆转组蛋白去乙酰化酶Sir2的上调。卷柏素显示出对氟康唑、酮康唑的抗念珠菌活性的体外增效作用。     

白念珠菌唑类药物耐药相关转录因子研究进展

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

转录因子Pho4参与白念珠菌药物敏感性的调控

目的通过对缺失相应转录因子基因的白念珠菌进行抗真菌药物敏感性的筛选,考察转录因子对白念珠菌耐药性的影响及调控机制。方法通过微量液基稀释法、点板实验(Spot Assay)检测实验菌株对抗真菌药物的敏感性。采用实时定量PCR(RT-PCR)的方法检测白念珠菌耐药性相关MDR1,CDR1以及ERG11的表达,并通过检测菌株对罗丹明6G的外排能力进一步检测菌株对抗真菌药物的外排能力。结果最低抑菌浓度(minimal inhibitory concentration,MIC)测定和Spot Assay实验结果表明,与亲本菌相比,PHO4基因缺失菌对氟康唑、咪康唑的敏感性显著升高。虽然耐药相关基因的表达增加,但对罗丹明6G的外排能力降低,抗氧化应激能力下降。结论转录因子Pho4的缺失可能通过降低白念珠菌的抗氧化应激能力,减弱对药物的外排作用而导致对唑类药物敏感,但其具体的调控机制有待进一步研究。     

转录因子RPN4对白念珠菌药物敏感性的研究

目的利用基因缺失菌库研究转录因子敲除后对白念珠菌药物敏感性的影响,并利用药物敏感性差异菌株初步考察可能的耐药性调控机制。方法微量液基稀释法测定最低抑菌浓度(minimal inhibitory concentration,MIC);点板法(spot assay)和生长曲线法验证菌株对氟康唑的敏感性;实时定量PCR(RT-PCR)法检测药物敏感性差异菌株中多药耐药基因CDR1和MDR1的表达,并通过罗丹明6G外排实验测定外排能力。结果亲本菌SN250对氟康唑表现为耐药,多药耐药基因CDR1和MDR1高表达,MIC80大于16μg/m L,大部分转录因子缺失菌与亲本菌SN250表现出相同的耐药性,但转录因子RPN4缺失菌株对氟康唑的敏感性升高,MIC80降为0.5μg/m L;多药耐药基因CDR1和MDR1的表达均降低,20 min和40min时对罗丹明6G的外排能力降低。结论转录因子RPN4有可能通过促进多药耐药基因CDR1和MDR1表达和菌株外排能力而降低对药物的敏感性,但相关机制有待进一步深入研究。     

河北地区侵袭性酵母样真菌的菌株分布及药敏分析

目的调查河北地区16家三级教学医院侵袭性酵母样真菌的菌株构成、分布及其体外药敏特点,为临床抗真菌药物的合理应用提供依据。方法收集河北地区2016～2017年侵袭性酵母样真菌感染患者的菌株,对菌种构成进行分析,采用Sensititre YeastOne显色药敏板对9种抗真菌药进行药敏试验。结果共检出侵袭性酵母样真菌260株,其中白念珠菌是最主要的致病菌,占42.31%;非白念珠菌占57.69%,主要包括热带念珠菌19.23%、近平滑念珠菌18.08%和光滑念珠菌9.23%。白念珠菌对氟康唑、伏立康唑、伊曲康唑的耐药率分别为2.8%、1.9%和0.9%;热带念珠菌对氟康唑和伏立康唑的耐药率均为12.8%;近平滑念珠菌复合体对氟康唑和伏立康唑的耐药率分别为14.9%和10.6%;白念珠菌、热带念珠菌和近平滑念珠菌复合体对棘白菌素类抗菌物的耐药率几乎0%。结论侵袭性酵母样真菌感染中最常见的是白念珠菌感染,但是非白念珠菌感染已经超过50%。白念珠菌对抗真菌药物仍然有较高的敏感性,但对唑类药物的敏感率在不同种的念珠菌中存在差异。棘白菌素类对白念珠菌、热带念珠菌和近平滑念珠菌有很高的抗菌活性。     

Evolution of drug resistance in experimental populations of Candida albicans

Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast Candida albicans and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes, CDR1 and CDR2; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway, ERG11; and the major facilitator gene, MDR1. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of C. albicans.     

Ibuprofen reverts antifungal resistance on Candida albicans showing overexpression of CDR genes

Several mechanisms may be associated with Candida albicans resistance to azoles. Ibuprofen was described as being able to revert resistance related to efflux activity in Candida . The aim of this study was to uncover the molecular base of antifungal resistance in C. albicans clinical strains that could be reverted by ibuprofen. Sixty-two clinical isolates and five control strains of C. albicans were studied: the azole susceptibility phenotype was determined according to the Clinical Laboratory for Standards Institute, M27-A2 protocol and minimal inhibitory concentration values were recalculated with ibuprofen (100 μg mL⁻¹); synergistic studies between fluconazole and FK506, a Cdr1p inhibitor, were performed using an agar disk diffusion assay and were compared with ibuprofen results. Gene expression was quantified by real-time PCR, with and without ibuprofen, regarding CDR1 , CDR2 , MDR1 , encoding for efflux pumps, and ERG11 , encoding for azole target protein. A correlation between susceptibility phenotype and resistance gene expression profiles was determined. Ibuprofen and FK506 showed a clear synergistic effect when combined with fluconazole. Resistant isolates reverting to susceptible after incubation with ibuprofen showed CDR1 and CDR2 overexpression especially of the latter. Conversely, strains that did not revert displayed a remarkable increase in ERG11 expression along with CDR genes. Ibuprofen did not alter resistance gene expression significantly ( P >0.05), probably acting as a Cdrp blocker.     

The genetic basis of fluconazole resistance development in Candida albicans

Infections by the opportunistic fungal pathogen Candida albicans are widely treated with the antifungal agent fluconazole that inhibits the biosynthesis of ergosterol, the major sterol in the fungal plasma membrane. The emergence of fluconazole-resistant C. albicans strains is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis (OPC) in acquired immunodeficiency syndrome (AIDS) patients. Resistance can be caused by alterations in sterol biosynthesis, by mutations in the drug target enzyme, sterol 14alpha-demethylase (14DM), which lower its affinity for fluconazole, by increased expression of the ERG11 gene encoding 14DM, or by overexpression of genes coding for membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) superfamilies. Different mechanisms are frequently combined to result in a stepwise development of fluconazole resistance over time. The MDR1 gene is not or barely transcribed during growth in vitro in fluconazole-susceptible C. albicans strains, but overexpressed in many fluconazole-resistant clinical isolates, resulting in reduced intracellular fluconazole accumulation. The activation of the gene in resistant isolates is caused by mutations in as yet unknown trans-regulatory factors, and the resulting constitutive high level of MDR1 expression causes resistance to other toxic compounds in addition to fluconazole. Disruption of both alleles of the MDR1 gene in resistant C. albicans isolates abolishes their resistance to these drugs, providing genetic evidence that MDR1 mediates multidrug resistance in C. albicans.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

Evaluation of the V404I and V509M amino acid substitutions of <Emphasis Type="Italic">ERG11</Emphasis> gene in <Emphasis Type="Italic">Candida albicans</Emphasis> isolates by pyrosequencing

The molecular mechanisms underlying fluconazole resistance in C. albicans involve mutations and the overexpression of the ERG11 gene and membrane transport proteins. We examined the relationship between the reduced fluconazole susceptibility of C. albicans and mutations of V404I and V509M in the ERG11 gene in 182 C. albicans clinical isolates using the Pyrosequencing™ method. DNAs from these clinical isolates with different levels of in-vitro fluconazole susceptibility — one resistant, five susceptible dose-dependent (SDD), four trailer, and 172 susceptible — were analyzed. None of the fluconazole-susceptible, SDD, trailer or resistant isolates had mutations of V404I or V509M. Our results showed that no correlation can be found between the V404I or V509M mutation and fluconazole susceptibility in C. albicans.     

A proteomic approach to understanding the development of multidrug-resistant<Emphasis Type="Italic"> Candida albicans</Emphasis> strains

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by the overexpression of genes that encode multidrug efflux pumps (CDR1, CDR2, or MDR1). We have undertaken a proteomic approach to gain further insight into the regulatory network controlling efflux pump expression and drug resistance in C. albicans. Three pairs of matched fluconazole-susceptible and resistant clinical C. albicans isolates, in which drug resistance correlated with stable activation of MDR1 or CDR1/2, were analyzed for differences in their protein expression profiles. In two independent, MDR1-overexpressing, strains, additional up-regulated proteins were identified, which are encoded by the YPR127 gene and several members of the IFD (YPL088) gene family. All are putative aldo-keto reductases of unknown function. These proteins were not up-regulated in a fluconazole-resistant strain that overexpressed CDR1 and CDR2 but not MDR1, indicating that expression of the various efflux pumps of C. albicans is controlled by different regulatory networks. To investigate the possible role of YPR127 in the resistance phenotype of the clinical isolates, we constitutively overexpressed the gene in a C. albicans laboratory strain. In addition, the gene was deleted in a C. albicans laboratory strain and in one of the drug-resistant clinical isolates in which it was overexpressed. Neither forced overexpression nor deletion of YPR127 affected the susceptibility of the strains to drugs and other toxic substances, suggesting that the regulatory networks which control the expression of efflux pumps in C. albicans also control genes involved in cellular functions not related to drug resistance.Communicated by D. Y. Thomas     

The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance

BackgroundFor many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species.AimsIn this study we investigated missense mutations in ERG11 genes of Candida albicans, Candida glabrata and Candida tropicalis isolates previously evaluated by susceptibility testing to fluconazole.MethodsScreening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing.ResultsWe identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p.ConclusionThe L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance.