紫草素对白色念珠菌的抑制作用机制

【目的】研究紫草素抑制白色念珠菌的作用机制。【方法】通过微量稀释法测定紫草素对白色念珠菌的最低抑菌浓度(MIC)和最低杀菌浓度(MFC);紫外分光光度法测定紫草素对白色念珠菌细胞膜渗透性的影响;扫描电镜观察紫草素对菌体形态的影响;激光共聚焦显微镜测定紫草素对白色念珠菌细胞内钙离子浓度的影响;卵黄平板培养基法检测紫草素对白色念珠菌的细胞膜磷脂酶活性的影响;RT-PCR检测紫草素对白色念珠菌PLB1和PLB2基因表达量的影响。【结果】紫草素对白色念珠菌有较强的抑制作用,其对白色念珠菌的MIC和MFC分别为16μg/m L和32μg/mL。紫草素能破坏白色念珠菌细胞膜的完整性,使细胞膜的通透性增加,导致细胞内DNA和RNA等大分子物质的泄漏和细胞内钙离子的流失。其中MIC的紫草素作用菌体16 h后,上清液中的DNA和RNA等大分子含量与对照组相比增加了117.32%(P0.01);细胞内的[Ca~(2+)]降低了72.02%(P0.01)。扫描电镜结果也证明了紫草素对白色念珠菌细胞膜的破坏作用。紫草素也能抑制白色念珠菌分泌磷脂酶,且呈浓度剂量依赖。其中,与对照组相比,MIC的紫草素能使白色念珠菌分泌磷脂酶的量下降56.3%(P0.01)。RT-PCR结果显示,紫草素能抑制编码磷脂酶B的基因PLB1和PLB2的表达量,其中1/2 MIC的紫草素作用白色念珠菌16 h后,与对照组相比,PLB1和PLB2基因的相对表达量分别降低了56.4%和61.4%(P0.01)。【结论】紫草素对白色念珠菌有较强的抑杀作用,其作用机制是通过破坏白色念珠菌细胞膜的完整性,增加菌体细胞膜的通透性,导致细胞内DNA和RNA等大分子的泄漏和细胞内[Ca~(2+)]的流失,最终引起菌体的死亡。而紫草素对白色念珠菌磷脂酶分泌的抑制作用,致使其不能及时维护和修复由紫草素造成的细胞膜的破坏和损伤,也是导致菌体死亡的原因。     

乳酸杆菌发酵滤液对人宫颈癌细胞株Hela细胞的细胞周期和细胞凋亡的影响

目的观察乳酸杆菌DM9811发酵滤液对宫颈癌细胞株Hela细胞的细胞周期和细胞凋亡的影响,探索乳酸杆菌发酵滤液对宫颈癌细胞作用的可能机制。方法用光镜、电镜和流式细胞仪分析不同浓度乳酸杆菌DM9811发酵滤液对Hela细胞凋亡的诱导效果;用流式细胞仪分析不同浓度乳酸杆菌DM9811发酵滤液对Hela细胞细胞周期的影响。结果（1）乳酸杆菌DM9811发酵滤液可诱导宫颈癌Hela细胞凋亡。形态学观察处理后的Hela细胞,可见细胞变形,细胞皱缩,体积变小,细胞间隙增大,细胞核固缩。流式细胞仪分析,1%、2%的乳酸杆菌DM9811发酵滤液在48、72h可诱导Hela细胞凋亡;5%的乳酸杆菌发酵滤液在24、48和72h均可诱导Hela细胞凋亡。（2）乳酸杆菌DM9811发酵滤液阻滞宫颈癌Hela细胞于S期,不同浓度的乳酸杆菌发酵滤液作用24、48和72h均可使S期细胞比阴性对照组增多。结论乳酸杆菌DM9811发酵滤液可诱导部分Hela细胞凋亡,其对Hela细胞的生长抑制作用可能通过S期阻滞实现。     

和厚朴酚通过ROS的积累和破坏细胞膜杀死白色念珠菌

[目的]研究在体外情况下和厚朴酚对白色念珠菌的抑制作用及其可能机制。[方法]采用微量稀释法测定和厚朴酚对白色念珠菌的最低抑菌浓度（MIC₈₀）和最低杀菌浓度（MFC）;用透射电镜观察不同浓度和厚朴酚对白色念珠菌超微结构的影响;采用Annexin V-FITC/PI染色法分析不同浓度和厚朴酚对白色念珠菌细胞凋亡的影响;用DCFH-DA染色法测定不同浓度和厚朴酚对白色念珠菌细胞内活性氧积累的影响;用JC-1染色法分析不同浓度和厚朴酚对白色念珠菌线粒体膜电位的影响;用碘化丙啶染色、考马斯亮蓝G-250染色检测和厚朴酚对白色念珠菌细胞膜通透性的影响;通过测定加入麦角甾醇后,和厚朴酚对白色念珠菌的抑制作用的变化,检测和厚朴酚对白色念珠菌细胞膜的影响。[结果]和厚朴酚对白色念珠菌具有很强的抑制作用,MIC和MFC分别为16 μg/mL和32 μg/mL。对白色念珠菌细胞壁、细胞膜和胞浆均有明显的影响。和厚朴酚是通过增加活性氧的产生和破坏线粒体功能来诱导白念珠菌的细胞凋亡和坏死。它也影响细胞膜的通透性,这可能和细胞壁的破坏和与麦角固醇的结合有关。[结论]和厚朴酚通过产生活性氧并伴随着一系列的细胞损伤这种复杂的机制从而对白色念珠菌产生抑制作用,使和厚朴酚成为一种潜在的抗真菌药物。     

苦楝素联合BIT对白色念珠菌的协同抑菌效果研究

探讨苦楝素与BIT分别单独及二者联合使用对白色念珠菌的浮游菌生长及其生物膜形成的影响。以预加菌液倾注平板打孔抑菌实验法测定苦楝素、BIT及二者联合使用对白色念珠菌菌体生长的抑制效果,96孔板微量稀释法培养该菌浮游菌及其生物膜,分别测定浮游菌及其生物膜的吸光度并进行比较分析,判断最小抑菌浓度(MIC),计算二者联合时的抑菌指数(FIC)。平板打孔抑菌实验结果显示,其抑菌效果依次为:二者联合BIT苦楝素;96孔微量稀释法结果显示:二者联合的FIC系数为0.75;二者联合抑制该浮游菌生长比单用时药效增加了20%～50%,抑制其生物膜的形成比单用时药效增加了33%～50%。苦楝素与BIT联合使用具有对白色念珠菌生长的复配增效抑制作用,不但有效抑制该菌浮游菌生长,也能抑制其生物膜的形成。     

白色念珠菌形态转换的调控

白色念珠菌 (Ｃａｎｄｉｄａａｌｂｉｃａｎｓ)是一种常见的致病真菌 ,属于半知菌亚门芽孢菌纲隐球酵母科念珠菌属。在健康人体粘膜表面 ,如肠道、口腔 ,白色念珠菌通常不会引起疾病。但在免疫系统受到损害或抑制的人体中 ,比如化疗病人、器官移植病人或是艾滋病人中 ,白色念珠菌能够引起严重的白色念珠菌病 ,危及生命。许多因素可能与白色念珠菌的致病性有关 ,如菌体对寄主细胞的粘附能力、白色念珠菌产生的分泌性蛋白酶、菌落形态的转换以及细胞形态的转换等[1] 。1 .影响白色念珠菌形态转换的因素白色念珠菌能够以三种形态生长 ,即菌体形…     

中药野菊花和山楂核提取液对 宫颈常见病原体抑菌效果的比较

目的了解中药野菊花和山楂核提取液对宫颈常见病原体大肠埃希菌、金黄色葡萄球菌、白色念珠菌的抑菌效果,比较野菊花和山楂核提取液抑菌作用的效果。方法采用二倍稀释法药敏试验测定抑菌浓度(MIC)。结果野菊花、山楂核提取液对大肠埃希菌、金黄色葡萄球菌、白色念珠菌有不同程度的抑菌效果,三种菌的总抑菌效果差异具有统计学意义(χ2=21.781,P=0.00000.05),四种浓度药物的实验,两两比较后得出山楂核提取液的抑菌效果更好,抑菌率明显高于野菊花组,差异有统计学意义(χ2=8.6660,P=0.00300.05)。结论野菊花、山楂核提取液对大肠埃希菌、金黄色葡萄球菌、白色念珠菌有不同程度的抑菌效果,山楂核提取液的抑菌效果更好。     

妇肤康喷雾剂对阴道常见微生物增殖影响的体外研究

目的体外了解妇肤康喷雾剂溶液对阴道常见的益生乳杆菌和致病菌的影响,为阴道感染疾病提供药物应用的理论依据,指导临床用药。方法对从中国健康妇女阴道分离的乳杆菌、ATCC阴道来源的加德纳菌和白色念珠菌进行体外培养,以中药制剂妇肤康喷雾剂作为培养基,通过绘制生长曲线,评价妇肤康溶液对阴道常见微生物的影响。结果妇肤康实验组中,乳杆菌和白色念珠菌生长曲线随着时间延长轻微下降。加德纳菌在15h后可完全杀灭。结论体外培养时,妇肤康溶液轻微抑制乳杆菌生长,其能够抑制白色念珠菌生长,可以杀灭加德纳菌。     

黄酮和甾体类化合物的抗真菌活性

用琼脂扩散法对从药用植物中分离到的12个黄酮和5个甾体化合物进行了抗真菌活性实验,其中黄芩甙元(baicalein)、funkioside C和胡萝卜甙(daucosterol)对尖孢镰刀菌(Fusarium oxysporum)和白色念珠菌(Candida albicans)的生长均有抑制作用,对尖孢镰刀菌的最低抑菌浓度(MIC)分别为0.112、0.217和0.108g/L,对白色念珠菌的MIC分别为0.264、0.310和0.078g/L。对其构效关系进行了初步讨论。     

都柏林念珠菌的鉴定及与白念珠菌3种表型鉴别方法的研究

目的 从临床分离的念珠菌中进一步鉴定都柏林念珠菌,并评价3种表型鉴别白念珠菌和都柏林念珠菌的方法.方法 对17株临床分离并初步鉴定的白念珠菌和1株ATCC白念珠菌标准株,采用PHR1同源序列PCR法检测,鉴定出其中的都柏林念珠菌;分别采用45℃生长试验、YEPD(1%酵母浸膏,2%蛋白胨,2%葡萄糖)液基39℃芽管生成试验、Staib琼脂(鸟食琼脂)厚壁孢子形成试验对两种菌的表型特点进行比较.结果 17株临床分离的白念珠菌中有3株鉴定为都柏林念珠菌;45℃时,两种菌在改良沙堡弱琼脂上均无明显生长,YEPD液基中仅有1株白念珠菌生长良好;YEPD液基39℃培养2种菌均无芽管生成;Staib琼脂培养72h,3株都柏林念珠菌中有2株可形成厚壁孢子,而白念珠菌则无,与PHR1同源序列检测结果基本一致.结论 PHR1同源序列检测是鉴别都柏林念珠菌与白念珠菌的可靠方法,Staib琼脂厚壁孢子形成试验有助于鉴别两菌,45℃生长试验和YEPD液基39℃芽管生成试验则不能有效鉴别两菌.     

乳杆菌DM9811代谢产物脂肪酸组分对义齿性口炎患者口腔中白假丝酵母菌的抑制

目的从调整口腔菌群平衡的角度出发,观察乳杆菌DM9811代谢产物脂肪酸组分对义齿性口炎患者口腔菌群的影响。方法（1）体外试验：配制不同浓度乳杆菌DM9811代谢产物脂肪酸组分的沙包液体培养基,分别接种10^8CFU／ml白假丝酵母菌菌液,用CFU活菌记数法计数。（2）临床试验：符合义齿性口炎病例40例,随机分为试验组20例和对照组20例（阴性对照组和阳性对照组各10例）。分别给予乳杆菌DM9811代谢产物脂肪酸组分、纯净水、洗必泰含漱液。观察菌群变化。结果（1）体外试验：乳杆菌DM9811代谢产物脂肪酸组分在浓度为1．6、0．8和0．4mg／ml时对白假丝酵母菌的抑制作用分别为100％、98．11％和62．72％。（2）临床试验：试验组在治疗后口腔黏膜白假丝酵母菌数量明显降低,口腔链球菌数量升高,较治疗前差异均有显著性（P〈0．01）;停药后第21天较治疗7d差异无显著性（P〉0.05）。结论乳杆菌DM9811代谢产物脂肪酸组分对口腔白假丝酵母菌有明显的抑杀作用;对口腔链球菌有促进作用,可能有调整口腔菌群平衡的作用。     

A micromethod for the quantitative determination of the viability of Candida albicans hyphae

A micromethod for the quantitative determination of the viability of Candida albicans hypae was devised which takes advantage of the dimorphic nature of C. albicans which grows exclusively in the yeast form when incubated aerobically on Sabouraud dextrose agar at 30°C. When tested by thisd method, all viable, C. albicans hyphae were recognized as microcolonies consisting of one hypha surrounded by several yeast form progeny. In contrast to this, no yeast form progeny emerged from nonviable hypae. By counting appropriate total numbers (200–400) of microcolony-forming hypae and infertile hyphae, it was possible to determine the ratio of viable to nonviable cells in a given hyphal suspension. This micromethod may be used for quantitative assessment of the candidacidal effects of various antimycotic agents or phagocytes C. albicans hyphae whose viability could not have been determined by the conventional plating technique because of the species' high propensity to clump.     

Bioconversion of Ferulic Acid to 4-Vinylguaiacol and 4-Ethylguaiacol and of 4-Vinylguaiacol to 4-Ethylguaiacol by Halotolerant Yeasts Belonging to the Genus Candida

In order to examine the genesis of the characteristic flavors of soy sauce and miso, seven novel halotolerant yeast strains of two types, which showed convertibility of ferulic acid (FA) to 4-vinylguaiacol (4-VG) and to 4-ethylguaiacol (4-EG), were isolated from miso-koji and miso pastes. Two of these strains were identified as Candida guilliermondii (anamorph of Pichia guilliermondii), and Candida fermentati (anamorph of Pichia caribbica), based on sequence analyses of a partial 26S ribosomal RNA gene and the region of internal transcribed spacers 1 and 2, and the 5.8S ribosomal RNA gene. Moreover, we also found three Candida etchellsii strains which showed convertibility of FA to 4-VG, but not to 4-EG, and two atypical strains of Candida versatilis which showed no convertibility of FA to 4-VG, but did show convertibility of 4-VG to 4-EG from soy sauce mashes. The bioconversion pathway from FA to 4-EG via 4-VG in halotolerant yeasts and bacteria is discussed.     

Extreme diversification driven by parallel events of massive loss of heterozygosity in the hybrid lineage of Candida albicans

Candida albicans is the most commonly reported species causing candidiasis. The taxonomic classification of C. albicans and related lineages is controversial, with Candida africana (syn. C. albicans var. africana) and Candida stellatoidea (syn. C. albicans var. stellatoidea) being considered different species or C. albicans varieties depending on the authors. Moreover, recent genomic analyses have suggested a shared hybrid origin of C. albicans and C. africana, but the potential parental lineages remain unidentified. Although the genomes of C. albicans and C. africana have been extensively studied, the genome of C. stellatoidea has not been sequenced so far. In order to get a better understanding of the evolution of the C. albicans clade, and to assess whether C. stellatoidea could represent one of the unknown C. albicans parental lineages, we sequenced C. stellatoidea type strain (CBS 1905). This genome was compared to that of C. albicans and of the closely related lineage C. africana. Our results show that, similarly to C. africana, C. stellatoidea descends from the same hybrid ancestor as other C. albicans strains and that it has undergone a parallel massive loss of heterozygosity.     

Candida riodocensis and Candida cellae, two new yeast species from the Starmerella clade associated with solitary bees in the Atlantic rain forest of Brazil

Two new ascomycetous yeast species belonging to the Starmerella clade were discovered in nests of two solitary bee species in the Atlantic rain forest of Brazil. Candida riodocensis was isolated from pollen-nectar provisions, larvae and fecal pellets of nests of Megachile sp., and Candida cellae was found in pollen-nectar provisions of Centris tarsata. Analysis of the sequences of the D1/D2 large-subunit ribosomal DNA showed that C. riodocensis is phylogenetically related to C. batistae, and the closest relative of C. cellae is C. etchellsii. The type strains are C. riodocensis UFMG-MG02 (=CBS 10087(T) = NRRL Y-27859(T)) and C. cellae UFMG-PC04 (=CBS 10086(T) = NRRL Y-27860(T)).     

Effect of the incubation conditions on the production of patulin by Penicillium griseofulvum isolated from wheat

A total of 290 Candida isolates from patients were investigated for in vitro proteinase production. Overall, sixty percent of these strains were found to be proteinase producers. Of the C. albicans strains, 81.4% of the significant isolates in contrast to 19.7% of nonsignificant isolates were proteinase producers, the difference being statistically significant (P<0.001). Amongst the different Candida species, the proteinase production was found not only in Candida albicans, but also in C. tropicalis, C. parapsilosis and C. glabrata. Thus this in vitro method of demonstration of proteinase may be a good adjunct to smear and culture examination in identifying pathogenic Candida species from anatomical sites where they can also be present as commensals.     

Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

Biotypes of Candida albicans using the API 20C system

Abstract A total of 130 isolates of Candida albicans obtained from oral, vaginal and skin sites, were biotyped using the API 20C sugar assimilation system. One major biotype accounted for 75% of the isolates, while 12 minor biotypes accounted for the rest. The API 20C system may therefore be useful in supplementing other biotyping schemes of C. albicans available at present.     

Purification and germination of Candida albicans and Candida dubliniensis chlamydospores cultured in liquid media

Candida albicans and Candida dubliniensis are the only Candida sp. that have been observed to produce chlamydospores. The function of these large, thick-walled cells is currently unknown. In this report, we describe the production and purification of chlamydospores from these species in defined liquid media. Staining with the fluorescent dye FUN-1 indicated that chlamydospores are metabolically active cells, but that metabolic activity is undetectable in chlamydospores that are >30 days old. However, 5–15-day-old chlamydospores could be induced to produce daughter chlamydospores, blastospores, pseudohyphae and true hyphae depending on the incubation conditions used. Chlamydospores that were preinduced to germinate were also observed to escape from murine macrophages following phagocytosis, suggesting that these structures may be viable in vivo . Mycelium-attached and purified chlamydospores rapidly lost their viability in water and when subjected to dry stress, suggesting that they are unlikely to act as long-term storage structures. Instead, our data suggest that chlamydospores represent an alternative specialized form of growth by C. albicans and C. dubliniensis .