白色念珠菌氟康唑耐药相关基因研究进展

近 2 0年来由于临床上对癌症患者、器官移植患者大量使用化疗药物、广谱抗生素和免疫抑制剂 ,以及艾滋病 (AIDS)的流行 ,使深部真菌感染的发生率在免疫受损的病人群体中急剧升高。深部真菌感染已日益成为一种常见病、多发病 ,并已逐渐成为这类疾病患者死亡的主要原因之一。深部真菌感染中最常见菌属是念珠菌属 (Candidaspecies) ,其中以白色念珠菌 (Candidaalbicans)为最常见菌种。目前临床上常用的抗真菌药物有两大类 :干扰真菌细胞膜脂质合成的药物(二性霉素B ,唑类药物 )和干扰真菌核酸合成的药物 (5 氟胞嘧啶 )。由于唑类药物中的氟…     

近平滑念珠菌的形态转换观察

目的观察近平滑念珠菌在不同培养基的形态转换现象,以及温度对其形态转换的影响。方法收集近平滑念珠菌正常人皮肤携带株及临床致病株和标准株,接种于改良Lee培养基和含桃红B的YPD培养基,观察其不同形态转换,以及温度变化对光滑(W)与皱褶(O)形态转换的影响。结果近平滑念珠菌在Lee培养基和含桃红B的YPD培养基上,均可以出现多种形态以及一定频率W-O转换现象。在观察W向O形态转换过程中发现,与25℃培养温度相比,37℃条件下光滑菌落形态占更多的比例。结论近平滑念珠菌体外培养时存在形态转换及W-O转换现象,且于37℃时更易保持光滑形态。含桃红B的YPD培养基也可以用于基本的W-O形态转换观察。     

近平滑念珠菌形态多样性及毒力因子的分泌活性研究

目的比较近平滑念珠菌不同临床分离株的菌落和细胞形态及其分泌型天冬氨酸蛋白酶(SAP)酶活性,阐明该病原真菌的形态多样性及毒力差异。方法分别用Lee’s Glc NAc、Lee’s glucose、Spider 3种培养基于37℃培养7株近平滑念珠菌临床分离株,观察各菌株的菌落及细胞形态;用牛血清白蛋白培养基(BSA)于30℃培养7株近平滑念珠菌,通过观察形成晕圈能力的强弱确定其毒力因子(SAP)的分泌活性。结果 7株近平滑念珠菌临床分离株在不同条件下菌落表型有较明显的不同,菌落有光滑型、皱褶型及火山口样型,且染色程度不同,各分离株细胞在形成酵母相或菌丝相的能力方面呈现出不同程度的差异;SAP酶活性观察实验中检测到各菌株水解晕圈大小从2.55 cm到1.55 cm各不相等。结论近平滑念珠菌临床分离株具有形态多样性,各菌株SAP酶活性明显不同,提示菌株间的毒力有明显差异。     

近平滑念珠菌引起真菌性膝关节炎：病例报告及文献回顾

报告1例由近平滑念珠菌引起的膝关节炎并进行相关骨关节真菌感染的文献回顾.患者,男,53岁,有糖尿病史及左侧腘窝皮肤浅表小肿瘤切除史,因左膝关节反复疼痛7 a,逐渐加重伴跛行6个月入院.临床表现为左膝关节明显红肿,影像学发现关节腔内滑膜增生,临近胫骨上端及股骨下端有灶性骨破坏、吸收,关节腔积液查见真菌,鉴定为近平滑念珠菌.经关节镜清理术及静脉用氟康唑治疗后症状改善,随访2 a无复发,已恢复日常活动.本文结合本例患者进行了骨关节真菌感染的相关文献复习,对骨关节感染的相关因素及治疗等内容进行了回顾,其结果对临床相关医生具有重要参考价值.     

贵州地区临床分离近平滑念珠菌复合群分布及其生物膜形成相关基因研究

目的 研究贵州地区近平滑念珠菌复合群临床分布和药敏特点,检测近平滑念珠菌生物膜形成能力及其CDC28基因的表达水平。方法 收集贵州地区2所三级甲等医院送检标本中分离出的菌株,利用BACTEC^TM FX仪、Phoenix-100仪对菌种进行鉴定,通过基因测序对近平滑念珠菌复合群各分型菌株进行鉴定;采用ATB FUNGUS 3试剂盒检测菌株体外药物敏感情况;检测血源性近平滑念珠菌生物膜形成能力及利用qRT-PCR技术检测它们的CDC28基因表达水平。结果 共分离鉴定出163株近平滑念珠菌复合群,其中近平滑念珠菌Ⅰ型、Ⅱ型和Ⅲ型分别占73.0%、6.1%和20.9%;尿液和血液是分离菌株最多的标本(均为50株);血液途径感染的菌株中,Ⅰ型、Ⅱ型和Ⅲ型分别占54.0%、10.0%和36.0%;三种基因型菌株对常见抗真菌药物无明显耐药性;17株临床菌株中,16株(94.1%)具有生物膜形成能力,其中2株、4株和10株菌株生物膜形成能力强、一般、弱,CDC28在2株生物膜形成能力强以及3株生物膜形成能力一般的菌株中表达上调。结论 分离的近平滑念珠菌复合群以Ⅰ型感染为主,三种基...     

近平滑念珠菌ERG 11基因编码区的克隆及生物信息学分析

目的克隆、测序近平滑念珠菌ERG11基因的编码区序列并进行生物信息学分析。方法运用生物信息学的方法 ,通过与白念珠菌ERG11基因碱基序列同源性比对,在近平滑念珠菌基因组(www.sanger.ac.uk/sequencing/Candida/pa-rapsilosis/)中寻找可能的ERG11基因序列(CpERG11),并据此序列设计引物,经PCR扩增近平滑念珠菌标准株(ATCC22019)的ERG11基因片段,产物经电泳、纯化、克隆到质粒prG-AMAI-NotI中,转染DH10B大肠杆菌细胞,并酶切鉴定筛选阳性克隆测序分析。结果近平滑念珠菌ERG11编码区由1569个碱基组成,编码一段含522个氨基酸的多肽。近平滑念珠菌ERG11的编码区序列与白念珠菌、热带念珠菌、光滑念珠菌、酿酒酵母菌ERG11基因的同源性分别为74%、75%、65%、64%。该近平滑念珠菌ERG11的编码区为唑类药物作用靶酶基因。结论成功克隆、测序、并生物信息学分析近平滑念珠菌ERG11基因的编码区序列,为进一步的功能研究奠定基础。     

近平滑念珠菌性皮肤肉芽肿1例

<正>1临床资料患者男,59岁,"右手腕皮疹3a余,右上臂皮下结节3个月",于2014年4月就诊我院我科门诊。皮疹初起于右手腕伸侧,无明显外伤史,初起为一枚红色绿豆大丘疹,皮疹逐渐增多并融合,无明显痒痛,挤压可有黄色分泌物渗出。外院诊疗不详,无明显好转。就诊前3个月右上臂内侧出现皮下肿物,无压痛。既往体质可,高血压病史,血压控制可。查体:系统检查无特殊,右手腕伸侧见5cm×6cm大小红斑,上见多发大小不一红色斑块,表     

白色念珠菌体外诱导氟康唑耐药性的实验研究（简报）

随着广谱抗生素的普遍应用以及免疫缺陷人群的增加,机会性致病菌念珠菌感染日益增多。深部念珠菌感染已经成为重症患者死亡的重要原因,白色念珠菌(C．albicans)是其中的主要致病菌。低毒广谱的唑类抗真菌药氟康唑是既能治疗严重真菌感染又不会产生明显副作用的少数抗真菌药之一,它的广泛使用取得良好治疗效果,但也导致菌株耐药率增加而使临床治疗失败。近10年来,在治疗     

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     

Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models

Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.     

Biochemical studies carried out on different groups ofCandida parapsilosis andCandida rhagii strains by comparing some cellular and mitochondrial activities

A comparative biochemical study was performed on some strains ofCandida rhagii and on strains belonging to different subgroups ofCandida parapsilosis. Measurements of alcohol dehydrogenase activity, resistance to drugs and occurrence of an alternative pathway enabled us to confirm the classification between several subgroups within theC. parapsilosis species.     

A case of recurrent episodes of Candida parapsilosis fungemia

Candida species is the fourth most commonly isolated organism in blood stream infections in the hospital setting. Patients with candidemia frequently succumb to this infection. For those that survive an initial candidemia, an increasing number of cases of breakthrough or recurrent candidemias have been reported in the literature. We report three episodes of C. parapsilosis fungemia in a cancer patient. Molecular testing was performed and confirmed that all these episodes occurring within an eight-month period were caused by the same organism. The incidence of recurrent candidemia is likely to increase in the near future. Studies and therapeutic interventions for patients at risk for recurrent candidemias are warranted.     

Electrophoretic karyotyping and antifungal susceptibility patterns of Candida parapsilosis clinical isolates causing deep and superficial fungal infections

Forty-six isolates of Candida parapsilosis, each from a single patient, were collected from July 1993 through March 1999 at the University of Ancona Hospitals and Clinics. Twenty-eight strains were isolated from superficial lesioned sites, including skin, nails and other sources while 18 strains were isolated from blood. The isolates were typed by electrophoretickaryotyping (EK) and tested for their susceptibility to fluconazole (FLC),itraconazole (ITC), flucytosine (5-FC), and amphotericin B (AMB). Ourdata confirmed that EK is a useful technique for DNA typing of isolates ofCandida parapsilosis and showed that the source of isolation is notassociated with a given DNA type. Although strains belonging to this speciesof Candida are susceptible to the most common antifungals, including the triazoles, the degree of ITC susceptibility was dose dependent (MIC rangingfrom 0.25–0.5 μg/ml) for 98% of the isolates. This revised version was published online in June 2006 with corrections to the Cover Date.     

Elimination of the yeastCandida parapsilosis from lymphoid cells and monolayer cells in culture

Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×10⁵ cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2×10⁵/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost. This work was supported by Veterans Administration Research Funds.     

A 9-year Study Comparing Risk Factors and the Outcome of Paediatric and Adults with Nosocomial Candidaemia

Although there are numerous studies of candidaemia in adults, data on paediatrics are still limited. The aim of this study was to compare risk factors, aetiology, therapy, and the outcome of nosocomial candidaemia among paediatric and adult patients in a large Brazilian tertiary hospital (1995–2003). During this period, 78 paediatrics and 113 adults were studied. Species other than Candida albicans caused 78.2% of episodes of candidaemia in paediatrics. Compared to adults, paediatrics received more frequently broad-spectrum antibiotics, vasopressors, blood transfusions, arterial catheter, chest tube, cardiothoracic surgery, mechanical ventilation, and parenteral nutrition. Candidaemia caused by Candida parapsilosis was more common in paediatrics, as was the isolation of Candida spp. from catheters. Amphotericin B treatment was more common in paediatrics. Mortality rate was higher in adults than in paediatrics with nosocomial candidaemia. We reinforce the necessity of continuous epidemiologic surveillance to follow the dynamics of candidaemia.     

三峡水库铜绿假单胞菌分离株的耐药性与毒理评价

铜绿假单胞菌（Pseudomonas aeruginosa）是一种重要的水源性和食源性致病菌,当机体免疫力下降时,易造成感染,了解铜绿假单胞菌的耐药性和致病力,对预防铜绿假单胞菌感染及其防治有重要意义。为探究三峡水库铜绿假单胞菌分离株的耐药性及其对秀丽线虫（Caenorhabditis elegans）的致病性,从三峡水库采集水样进行细菌分离,经形态学鉴定、生理生化鉴定、16S rDNA测序和oprI特异性基因鉴定筛选出铜绿假单胞菌,研究分离菌的耐药性、耐药基因和毒力基因,并利用模式生物秀丽线虫进行毒理评价。结果显示,从三峡水库分离鉴定出14株铜绿假单胞菌。分离株对青霉素G、氨苄青霉素、头孢唑林、卡那霉素、红霉素耐药,对阿米卡星、庆大霉素、诺氟沙星、环丙沙星、美罗培南、左氧氟沙星敏感。耐药基因bla TEM、aac(6′)-Ⅱ、qacEΔ1-sull、qnrS、mexR在所有分离菌株中呈阳性;耐药基因aac(3)-Ⅰ、tetA、tetB、tetD、tetE、cat、cmlA、qnrA、qnrD在所有分离菌株中呈阴性;此外,分离菌株携带多种毒力基因。分离菌PA7、PA8、PA12使秀丽线虫的平均寿命分别下降15%、14%、16%（P<0.05）,身体弯曲频率分别下降18%、33%、23%（P<0.05）,头部摆动频率分别下降10%、13%、11%（P<0.05）,最长寿命缩短约3 d。三峡水库中铜绿假单胞菌分离株具有多重耐药性,部分菌株对秀丽线虫有致病性,可为水体铜绿假单胞菌的感染防治提供一定参考。     

Comparative studies on the glycolytic and hexose monophosphate pathways in Candida parapsilosis and Saccharomyces cerevisiae

Some enzymatic activities of the glycolytic and hexose monophosphate pathways of Candida parapsilosis, a yeast lacking alcohol dehydrogenase but able to grow on high glucose concentrations, were compared to those of Saccharomyces cerevisiae. Cells were grown either on 8% glucose or on 2% glycerol and activities measured under optimal conditions. Results were as follows: glycolytic enzymes of C. parapsilosis, except glyceraldehyde 3-phosphate dehydrogenase, exhibited an activity weaker than that of S. cerevisiae, especially when yeasts were grown on glycerol. Fructose-1,6 bisphosphatase, an enzyme implicated in gluconeogenesis and in the hexose monophosphate pathway, and known to be very sensitive to catabolite repression in S. cerevisiae, was always active in C. parapsilosis even when cells were grown on 8% glucose. However, the allosteric properties towards AMP and fructose-2,6-bisphosphate were the same in both strains. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two other enzymes of the hexose monophosphate pathway, exhibited a higher activity in C. parapsilosis than in S. cerevisiae. Regulation of two important control points of the glycolytic flux, phosphofructokinase and pyruvate kinase, was investigated. In C. parapsilosis phosphofructokinase was poorly sensitive to ATP but fructose-2,60bisphosphate completely relieved the light ATP inhibition. Pyruvate kinase did not require fructose-1,6-bisphosphate for its activity, and by this way, did not regulate the glycolytic flux. The high glyceraldehyde-3-P-dehydrogenase activity, together with the relative insensitivity of fructose-1,6-bisphosphatase to catabolite repression and the high glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities suggested that in C. parapsilosis, as in other Candida species and opposite to S. cerevisiae, the glucose degradation mainly occurred through the hexose monophosphate pathway, under both growth conditions used.Abbreviations C. parapsilosis Candida parapsilosis - S. cerevisiae Saccharomyces cerevisiae - C. utilis Candida utilis     

Candida parapsilosis fungemia in neonates: genotyping results suggest healthcare workers hands as source, and review of published studies

An outbreak of Candida parapsilosis fungemia involving 17 neonatal intensive care unit (NICU) patients was studied. There were 14 blood culture and nine colonizing isolates from other sites available. The hands of NICU healthcare workers (HCW) yielded eight isolates. Screening of the isolates by random amplified polymorphic DNA (RAPD) method showed only three profiles. Typing by restriction fragment length polymorphism (RFLP) revealed all blood isolates were RFLP subtype VII-1. Among the nine infant colonizing isolates, there were four different RFLP subtypes; four of the isolates were subtype VII-1. Seven of the eight isolates from HCW were RFLP subtype VII-1. The majority of infant colonizers were not found in the blood, suggesting a possible direct spread of the epidemic subtype VII-1 strain from HCW hands to infant blood. The source of the infant colonizing strains is unclear, but non-VII-1 strains may be largely of maternal origin and VII-1 strains from HCW. These findings reinforce prior studies that have implicated HCW hands as the source of nosocomial, including neonatal, fungemia.     

Toxicity of a heterologous leucyl-tRNA (anticodon CAG) in the pathogen Candida albicans: in vivo evidence for non-standard decoding of CUG codons

Plasmids containing derivatives of the Saccharomyces cerevisiae leucyl-tRNA (tRNA3₃^Leu) gene that vary in anticodon sequence were constructed and transformed into the pathogen Candida albicans and S. cerevisiae. C. albicans could readily be transformed with plasmids encoding leucyl-tRNA genes with the anticodons CAA and UAA (recognizing the codons UUG and UUA) and expression of the heterologous tRNA^Leu could be demonstrated by Northern RNA blotting. In contrast, no transformants were obtained if the anticodons were UAG (codons recognized CUN, UUR) and CAG (codon CUG), indicating that the insertion of leucine at CUG codons is toxic for C. albicans. All tRNA^Leu-encoding plasmids transformed S. cerevisiae with equally high efficiencies. These results provide in vivo evidence that non-standard decoding of CUG codons is essential for the viability of C. albicans.