芍药苷对白色念珠菌生物膜的作用

背景：研究证实中药赤芍有效成分对白色念珠菌有较好的抑制作用,但其单体芍药苷对白色念珠菌生物膜是否有抑制作用未见报道。  目的：观察芍药苷对体外白色念珠菌生物膜的影响。 方法：用RPMI-1640分别按2倍稀释法制备5个浓度梯度(4,2,1,0.5,0.25 g/L)的芍药苷溶液。用RPMI-1640稀释洗必泰为5个浓度梯度(2%,1%,0.5%,0.25%,0.125%)。采用琼脂扩散法检测不同浓度梯度芍药苷或洗必泰对白色念珠菌的抑菌直径。MTT法检测不同浓度洗必泰或芍药苷对白色念珠菌细胞黏附的作用,以及对白色念珠菌生物膜的抑制作用,并且利用激光共聚焦扫描显微镜和死菌活菌荧光染色技术相结合方法观察常态及药物作用下的白色念珠菌生物膜。 结果与结论：洗必泰与芍药苷均有抑菌能力,抑菌环直径与药物浓度呈正相关;除2 g/L芍药苷组与1%,2%洗必泰组抑菌环直径无差异外,其余组间两两比较差异均有显著性意义。不同质量浓度芍药苷对白色念珠菌的细胞黏附都具有抑制作用,对白色念珠菌生物膜也具有抑制作用,且抑制率与药物质量浓度呈正相关。观察48 h时常态生物膜结构中大部分是活菌,有少量死菌存在;随芍药苷质量浓度改变白色念珠菌生物膜中死菌比例不断增高,其抑菌活性相对弱于洗必泰。表明芍药苷对体外白色念珠菌生物膜有较明显的抑制作用。|中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

不同pH值纳米羟基磷灰石复合根充糊剂的体外抑菌性

背景：目前临床应用的根管充填糊剂抗菌作用有限,存在组织刺激性强、成形困难、根管封闭性较差及易降解等缺点。 目的：比较不同pH值纳米羟基磷灰石复合根充糊剂对感染根管常见菌的体外抑菌作用。 方法：建立体外根管粪肠球菌、白色念球菌感染模型,评价pH 8纳米羟基磷灰石糊剂、pH 9纳米羟基磷灰石糊剂、pH 10纳米羟基磷灰石糊剂在离体牙根管内的抗菌作用,并以氧化锌丁香油做对照。 结果与结论：所有根充糊剂均具有抑菌作用。各pH值纳米羟基磷灰石糊剂对粪肠球菌抑制作用实验组优于氧化锌丁香油组(P < 0.05),不同pH值纳米羟基磷灰石组组间比较差异无显著性意义(P > 0.05)。而对白色念珠菌抑制作用各pH值纳米羟基磷灰石组不及氧化锌丁香油组(P < 0.05),各pH值纳米羟基磷灰石组组间比较,pH10纳米羟基磷灰石组>pH 9纳米羟基磷灰石组>pH 8纳米羟基磷灰石组(P < 0.05)。结果表明,感染根管模型中,纳米羟基磷灰石根充糊剂对粪肠球菌的抑制作用好于氧化锌丁香油(P < 0.05),而对白色念珠菌的抑制作用不及氧化锌丁香油(P < 0.05)。pH=10纳米羟基磷灰石复合根充糊剂的抑菌作用最佳。     

体内外光滑假丝酵母菌氟康唑耐药机制的比较研究

目的 研究比较临床分离和体外诱导的耐氟康唑光滑假丝酵母菌的耐药机制.方法 选取临床分离的4对氟康唑敏感-耐药配对的光滑假丝酵母菌,其中4株敏感株在体外氟康唑的作用下均被诱导为耐药株.利用罗丹明6G试验比较敏感株与两种耐药株的外排泵作用,实时荧光定量RT-PCR检测外排相关基因CDR1、CDR2、SNQ2和ERG11的表达.同时,对PDR1基因进行PCR扩增和测序,对比分析临床耐药株和体外诱导耐药株的突变位点.结果 临床耐药株和体外诱导耐药株外排泵的功能均明显强于敏感株；两者CDR1表达均显著升高,而CDR2和SNQ2则无明显变化；ERG11在敏感与耐药菌株之间的表达水平也无显著差别；两种耐药株的PDR1均发现错义突变位点,其中P927S、L543P及S947L突变尚未被报道过.结论 光滑假丝酵母菌在体内外氟康唑作用下PDR1均产生突变,引起外排相关基因,尤其是CDR1的表达增加从而增强了外排泵的作用导致耐药.     

纳米羟基磷灰石复合根充糊剂的抑菌性及微渗漏

背景：不同pH值形成纳米羟基磷灰石材料的纳米纯度不同,所形成的根充糊剂是否对抑菌性、根管封闭及根尖组织相容性产生影响尚无报道。 目的：比较不同pH值纳米羟基磷灰石根管充填材料、纳米羟基磷灰石复合材料和传统根管充填材料的抑菌性及与根管的密合性。 方法：采用抑菌环实验,研究pH值为8.0,9.0,10.0的纳米羟基磷灰石复合根充糊剂,Vitapex糊剂和AH-Plus糊剂及对以上各组根充糊剂添加奥硝唑以后的5组实验样本(共10组)对粪肠球菌、白色念球菌、金黄色葡萄球菌的抑菌性能;此外建立微渗漏模型,通过葡萄糖氧化酶比色测定法测量pH值为8.0,9.0的纳米羟基磷灰石复合根充糊剂,pH值为10的纳米羟基磷灰石复合根充糊剂+奥硝唑,氧化锌丁香油,Vitapex糊剂和AH-Plus糊剂与根管的密合性。 结果与结论：不同pH值的纳米羟基磷灰石本身无抑菌性,加入奥硝唑后有抑菌性,对粪肠球菌抑制性最高,其次是金黄色葡萄球菌,最后是白色念珠株菌。Vitapex仅对金黄色葡萄球菌有抑制作用。AH-Plus本身有抑菌性,加入奥硝唑后抑菌性降低。不同pH值纳米羟基磷灰石糊剂的根管封闭性优于氧化锌丁香油糊剂、Vitapex糊剂,但次于AH-Plus糊剂。将奥硝唑加入纳米羟基磷灰石糊剂对根管封闭性短期无影响。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

纳米氧化锌对白色念珠菌的抗菌作用

背景：纳米氧化锌作为无机抗菌剂,与有机杀菌剂的抗菌机制明显不同。 目的：探讨纳米氧化锌对白色念珠菌等真菌的抗菌作用。 方法：分别配制成100,75,50,25,10 g/L的纳米氧化锌与普通氧化锌悬浊液,以生理盐水作为阴性对照,采用KB纸片扩散法将30 μL不同浓度悬浊液分别作用于白色念珠菌,将培养平板上置于37 ℃恒温箱培养24 h观察并测量抑菌圈大小。 结果与讨论：随着纳米氧化锌浓度的提高,对白色念珠菌的抑制作用逐渐增强,抑菌圈直径逐渐增大。普通氧化锌的无明显抑菌作用。证实纳米氧化锌能够抑制白色念珠菌等真菌的生长。     

VVC患者临床分离白念珠菌Cap1、Mrr1和MDR1在唑类药物交叉耐药中的作用

目的探讨白念珠菌锌簇转录因子Cap1、Mrr1基因突变／高表达及外排泵基因MDR1的mRNA表达水平在唑类抗真菌药物氟康唑（FCA）、伊曲康唑（ITR）、伏立康唑（VRC）交叉耐药中的作用。方法收集山西医科大学第二医院皮肤性病科门诊疑似外阴阴道念珠菌病（VVC）患者阴道分泌物标本,进行真菌直接镜检、培养、分离鉴定和体外药物敏感性试验,获得试验用白念珠菌耐药和敏感菌株共68株。采用PCR方法扩增68株白念珠菌临床分离菌株Cap1、Mrr1基因,并进行双向测序,应用Blast软件,将测序结果与GenBank中已发表的序列进行比对,分析基因突变情况,实时荧光定量PCR检测锌簇转录因子Cap1、Mrr1和外排泵基因MDR1的mRNA表达水平。结果Cap1测序中1株ITR、VRC交叉耐药菌株出现P311S错义突变。Mrr1测序中1株FCA、ITR、VRC交叉耐药菌株出现5个错义突变（T917M,I＇923I,E1020Q,F1032L,S1037L）;FCA、ITR、VRC交叉耐药菌株组中Cap1／Mrr1／MDR1的mRNA表达量分别高于敏感菌株组;CA、ITR、VRC交叉耐药菌株组Cap1和MDR1（r=0．173,P=0．571）,Mrr1和MDR1（r=-0．091,P=0．766）,Cap1和Mrr1基因（r=0．025,P=0．936）的mRNA表达量不相关（均P〉0．05）。结论Cap1和Mrr1的突变可能和唑类药物交叉耐药有关。     

夫西地酸钠联合利福平对表皮葡萄球菌体外培养生物膜的作用

背景：表皮葡萄球菌是材料植入后感染的主要病原体,致病机制是在材料表面形成生物膜,生物膜形成后,单一用药抑制其细菌生长的效果较差。 目的：建立表皮葡萄球菌生物膜体外模型,观察夫西地酸钠联合利福平对表皮葡萄球菌体外培养生物膜的作用。 方法：体外培养生物膜,微量肉汤稀释法检测最低抑菌浓度,将低于最低抑菌浓度的夫西地酸钠和利福平单独或联合使用作用于表皮葡萄球菌早期与成熟期生物膜,观察其对表皮葡萄球菌早期和成熟生物膜的影响。 结果与结论：夫西地酸钠和利福平均可抑制表皮葡萄球菌的黏附和生物膜的形成,对早期及成熟期生物膜内细菌有杀菌效应,二者联合应用后疗效明显优于单独应用(P < 0.05)。提示夫西地酸钠联合利福平可明显抑制抑制表皮葡萄球菌的黏附和生物膜的形成,对早期及成熟期表皮葡萄球菌-生物膜有破坏作用,具有明显杀菌活性,是治疗细菌生物被膜相关感染的良好选择。     

根管充填体外模型构建中X射线牙片的作用

背景：X射线检查在根管治疗中的局限性逐渐被发现,然而相关病例的跟踪研究和有效体外模型的建立报道较少。 目的：评价X射线牙片在根管治疗中的局限性。 方法：通过对1例1年前认为需要拔除的患牙进行常规根管治疗,同时构建体外模型观察根管充填情况,分析X射线牙片在根管治疗前诊断和根管充填过程中的价值。 结果与结论：患者原需要拔除的患牙得以保留,体外模型显示不同角度观察的根管充填情况差异较大。结果提示,X射线牙片不能很好反映出根尖周的病变和根管充填情况。     

粉防己碱抑制耐氟康唑热带念珠菌外排泵机制的研究

目的探讨粉防己碱抑制耐氟康唑热带念珠菌外排泵的机制。方法通过MTT法确定24h粉防己碱对耐氟康唑热带念珠菌的无细胞毒性剂量,并以此剂量处理5株耐氟康唑热带念珠菌。粉防己碱组（20mg／L）及对照组（PBS）加入不同浓度的氟康唑（10～50mg/L）处理48h以测定MIC值。半定量PCR分析粉防己碱（20mg/L）作用24h前后,耐氟康唑热带念珠菌外排泵基因CDR1和MDR1转录水平的变化;流式细胞术检测粉防己碱（20mg／L）作用24h后对耐氟康唑热带念珠菌罗丹明123的蓄积能力的影响。结果粉防己碱对耐氟康唑热带念珠菌的最大无细胞毒性剂量为20mg／L。以此浓度处理5株耐氟康唑热带念珠菌,与对照组相比,MIC的范围由64（64～128）mg/L减少到32（24-64）mg/L（P〈0．05）。粉防己碱可以将耐氟康唑热带念珠菌CDR1和MDR1基因mRNA水平的表达由0．905±0．026及b．649±0．028分别下调至0．715±0．131和0．342±0．068（均P〈0．05）。粉防己碱作用后的耐氟康唑热带念珠菌对罗丹明123的蓄积量提升至（37．7±3．5）％,高于对照组的（16．3±2．2）％（P〈0．05）。结论粉防己碱可以减少耐氟康唑热带念珠菌外排蛋白的表达,增加细胞内药物的蓄积,从而抑制氟康唑的耐药作用。     

Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols

Candida albicans biofilms are formed through three distinct developmental phases and are associated with high fluconazole (FLU) resistance. In the present study, we used a set of isogenic Candida strains lacking one or more of the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p to determine their role in FLU resistance of biofilms. Additionally, variation in sterol profile as a possible mechanism of drug resistance was investigated. Our results indicate that parent and mutant strains formed similar biofilms. However, biofilms formed by double and triple mutants were more susceptible to FLU at 6 h (MIC = 64 and 16 microg/ml, respectively) than the wild-type strain (MIC > 256 microg/ml). At later time points (12 and 48 h), all the strains became resistant to this azole (MIC > or = 256 microg/ml), indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation. Northern blot analyses revealed that Candida biofilms expressed CDR and MDR1 genes in all the developmental phases, while planktonic cells expressed these genes only at the 12- and 48-h time points. Functionality of efflux pumps was assayed by rhodamine (Rh123) efflux assays, which revealed significant differences in Rh123 retention between biofilm and planktonic cells at the early phase (P = 0.0006) but not at later stages (12 and 48 h). Sterol analyses showed that ergosterol levels were significantly decreased (P < 0.001) at intermediate and mature phases, compared to those in early-phase biofilms. These studies suggest that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms.     

An update on antifungal targets and mechanisms of resistance in Candida albicans.

Much progress has been made in the last decade in identifying genes responsible for antifungal resistance in Candida albicans. Attention has focused on five major C. albicans genes: ABC transporter genes CDR1 and CDR2, major facilitator efflux gene MDR1, and ergosterol biosynthesis genes ERG11 and ERG3. Resistance involves mutations in 14C-lanosterol demethylase, targeted by fluconazole (FLZ) and encoded by ERG11, and mutations that up-regulate efflux genes that probably efflux the antifungals. Mutations that affect ERG3 mutations have been understudied as mechanism resistance among clinical isolates. In vitro resistance in clinical isolates typically involves step-wise mutations affecting more than one of these genes, and often unidentified genes. Different approaches are needed to identify these other genes. Very little is understood about reversible adaptive resistance of C. albicans despite its potential clinical significance; most clinical failures to control infections other than oropharyngeal candidiasis (OPC) occur with in vitro susceptible strains. Tolerance of C. albicans to azoles has been attributed to the calcineurin stress-response pathway, offering new potential targets for next generation antifungals. Recent studies have identified genes that regulate CDR1 or ERG genes. The focus of this review is C. albicans, although information on Saccharomyces cerevisiae or Candida glabrata is provided in areas in where Candida research is underdeveloped. With the completion of the C. albicans genomic sequence, and new methods for high throughput gene overexpression and disruption, rapid progress towards understanding the regulation of resistance, novel resistance mechanisms, and adaptive resistance is expected in the near future.     

Tyrosol和Farnesol对白念珠菌生物被膜形成作用初探

目的 研究密度感应分子(quorum sensing molecule)tyrosol(对羟基苯乙醇)和farnesol(法呢醇)对白念珠菌生物被膜形成的调控作用.方法 在tyrosol和farnesol干预下构建白念珠菌临床株和标准株生物被膜,在倒置显微镜下观察细胞形态,应用RT-PCR技术检测密度感应分子对白念珠菌HTA1和EFG1基因表达的调控作用,并采用MTT法观察密度感应分子对细胞活性的影响.结果 tyrosol对白念珠菌生物被膜的菌丝发生和细胞活性无明显促进作用,也无法中和farnesol对菌丝发生和细胞活性的抑制作用.tyrosol使白念珠菌生物被膜内细胞HTA1的表达增强,对EFG1的表达并无明显影响;tyrosol不能改变famesol对HTA1和EFG1表达的抑制作用.结论 tyrosol能在一定程度恢复口腔白念珠菌生物被膜内细胞的活跃状态,但当tyrosol与famesol同时存在时,tyrosol的作用被后者的抑制效应所掩盖,细胞对farnesol更敏感.     

Phenotypic and genotypic evaluation of fluconazole resistance in vaginal Candida strains isolated from HIV-infected women from Brazil.

We conducted a study to determine the antifungal susceptibility of vaginal Candida isolates from HIV-infected Brazilian women. Among 127 women enrolled, positive cultures for yeast were obtained from 31 of 38 (81%) women with symptomatic vulvovaginitis, and from 41 of 89 (46%) asymptomatic women. Susceptibility testing demonstrated 11 of the 72 isolates had either resistance or dose-dependent susceptibility to azole drugs, including four Candida albicans strains. Expression of the MDR1, CDR1, CDR2 and ERG11 genes was evaluated in all of the C. albicans isolates, and all four of the strains with reduced susceptibility to fluconazole had increased expression of CDR1 as compared to the fluconazole-sensitive strains. No increased expression of the other genes was identified. This large survey of Candida isolates from HIV-infected women from Brazil demonstrates that reduced susceptibility to azoles occurs at a low frequency among vaginal yeast isolates, and when present in C. albicans, azole resistance is associated with increased expression of CDR1.     

Alcohol dehydrogenase restricts the ability of the pathogen Candida albicans to form a biofilm on catheter surfaces through an ethanol-based mechanism

Candida biofilms formed on indwelling medical devices are increasingly associated with severe infections. In this study, we used proteomics and Western and Northern blotting analyses to demonstrate that alcohol dehydrogenase (ADH) is downregulated in Candida biofilms. Disruption of ADH1 significantly (P = 0.0046) enhanced the ability of Candida albicans to form biofilm. Confocal scanning laser microscopy showed that the adh1 mutant formed thicker biofilm than the parent strain (210 microm and 140 microm, respectively). These observations were extended to an engineered human oral mucosa and an in vivo rat model of catheter-associated biofilm. Inhibition of Candida ADH enzyme using disulfiram and 4-methylpyrazole resulted in thicker biofilm (P < 0.05). Moreover, biofilms formed by the adh1 mutant strain produced significantly smaller amounts of ethanol, but larger amounts of acetaldehyde, than biofilms formed by the parent and revertant strains (P < 0.0001), demonstrating that the effect of Adh1p on biofilm formation is mediated by its enzymatic activity. Furthermore, we found that 10% ethanol significantly inhibited biofilm formation in vitro, with complete inhibition of biofilm formation at ethanol concentrations of >/=20%. Similarly, using a clinically relevant rabbit model of catheter-associated biofilm, we found that ethanol treatment inhibited biofilm formation by C. albicans in vivo (P < 0.05) but not by Staphylococcus spp. (P > 0.05), indicating that ethanol specifically inhibits Candida biofilm formation. Taken together, our studies revealed that Adh1p contributes to the ability of C. albicans to form biofilms in vitro and in vivo and that the protein restricts biofilm formation through an ethanol-dependent mechanism. These results are clinically relevant and may suggest novel antibiofilm treatment strategies.     

In vitro activity of caspofungin (MK-0991) against Candida albicans clinical isolates displaying different mechanisms of azole resistance

Caspofungin inhibits the synthesis of 1,3-beta-D-glucan, a key step in fungal cell wall biosynthesis. Here we report on its potent in vitro activity (MIC at which 90% of the isolates tested are inhibited = 1 microg per ml of RPMI medium) against 32 Candida albicans fluconazole-susceptible and -resistant clinical isolates irrespective of the underlying resistance mechanism (alterations in ERG11 and/or upregulation of MDR and CDR genes encoding efflux pumps) and provide further evidence that caspofungin is not a substrate for multidrug transporters.     

Comparison of Biofilms Formed by Candida albicans and Candida parapsilosis on Bioprosthetic Surfaces

Little is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by different Candida species, particularly Candida albicans and C. parapsilosis, was evaluated by using a clinically relevant model of Candida biofilm on medical devices. Candida biofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes. Although minimal variations in biofilm production among invasive C. albicans isolates were seen, significant differences between invasive and noninvasive isolates (P < 0.001) were noted. C. albicans isolates produced more biofilm than C. parapsilosis, C. glabrata, and C. tropicalis isolates, as determined by DW assays (P was <0.001 for all comparisons) and microscopy. Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates. On microscopy, C. albicans biofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae. In contrast, C. parapsilosis biofilms had less volume than C. albicans biofilms and were comprised exclusively of clumped blastospores. Unlike planktonically grown cells, Candida biofilms rapidly (within 6 h) developed fluconazole resistance (MIC, >128 microg/ml). Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active. In conclusion, our data show that C. albicans produces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C. parapsilosis.     

The effect of biomaterials and antifungals on biofilm formation by Candida species: a review

Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis are able to form biofilms on virtually any biomaterial implanted in a human host. Biofilms are a primary cause of mortality in immunocompromised and hospitalized patients, as they cause recurrent and invasive candidiasis, which is difficult to eradicate. This is due to the fact that the biofilm cells show high resistance to antifungal treatments and the host defense mechanisms, and exhibit an excellent ability to adhere to biomaterials. Elucidation of the mechanisms of antifungal resistance in Candida biofilms is of unquestionable importance; therefore, this review analyzes both the chemical composition of biomaterials used to fabricate the medical devices, as well as the Candida genes and proteins that confer drug resistance.