纳豆杆菌对白假丝酵母菌的拮抗作用

目的探讨纳豆杆菌对白假丝酵母菌的拮抗作用。方法将纳豆杆菌和白假丝酵母菌混合培养24 h后,应用沙保弱平板培养基分离白假丝酵母菌,计数菌落,计算纳豆杆菌对白假丝酵母菌的拮抗率。结果纳豆杆菌对白假丝酵母菌的拮抗作用明显,拮抗率高达91.91%;纳豆杆菌肉汤培养物的除菌滤液对白假丝酵母菌也有明显的拮抗作用,拮抗率为79.05%。结论纳豆杆菌对白假丝酵母菌具有明显拮抗作用,是白假丝酵母菌的理想拮抗菌株。     

Inhibition of endothelium-dependent relaxation by Candida albicans

This study examines the effects of Candida albicans on acethylcholine-induced, endothelium-dependent relaxation of thoracic aorta of rabbits, precontracted by phenylephrine (10(-7) M). Isolated vessel rings were incubated with C. albicans, Saccharomyces cerevisiae, or their mannans, and endothelium-dependent relaxation was measured by the induction of acethylcholine. Endothelium-dependent relaxation remained unaffected after 3 hours by either C. albicans or S. cerevisiae, or their mannans. After 24 hours, however, incubation with C. albicans had completely abolished relaxation, whereas relaxation was decreased by mannan of C. albicans and continued unaffected by S. cerevisiae. In contrast, no change was registered with a 24 hours incubation of C. Albicans in a sodium nitroprusside-induced, endothelium-independent, vascular smooth muscle relaxation. Microscopical investigation of the morphological structure of vessel walls revealed penetration of C. albicans on the intimal surface after 3 hours incubation and infiltration of the yeast through the vessel wall after 24 hours. No changes in vessel morphology occurred after 3 or 24 hours with S. cerevisiae or the mannan of C. albicans. These results show the ability of C. albicans to inhibit endothelium-dependent, but not endothelium-independent, relaxation of vascular smooth muscle and may have important implications for functional damage to endothelial cells and the regulation of vessel tone and blood flow.     

Performance of Selective and Differential Media in the Primary Isolation of Yeasts from Different Biological Samples

In view of the increase in yeast infections, especially polymicrobial ones, differential culture media have acquired increasing importance. The present study evaluated the Sabouraud chloramphenicol, Biggy agar, Pagano Levin agar and CHROMagar Candida media in terms of isolation, number of yeast colony forming units per plate, and inhibition of bacteria and filamentous fungi. To this end, we used 223 biological samples, including feces, and oral, vaginal and anal mucosae from 86 patients presenting or not symptoms of fungal infections. The four media did not differ significantly in terms of detection of yeast-positive cultures. The number of colony forming units per plate ranged from zero to 2.380, with a predominance of counts of 1 to 9 colonies per plate. No significant differences were observed among the four culture media in terms of number of colonies counted, for each kind of biological material. Fifteen species belonging to the genera Candida, Saccharomyces, Cryptococcus, Trichosporon and Rhodotorula were isolated, with C. albicans being the predominant species, followed by C. parapsilosis and R. rubra. CHROMagar Candida and Biggy agar were complementary in the isolation of the different species and favored a greater recovery of polymicrobial cultures. Pagano Levin agar isolated the smallest variety of species. Sabouraud chloramphenicol agar was the least effective in terms of bacterial inhibition and favored a greater development of filamentous fungi. The results suggest that more than one culture medium should be used for an adequate primary isolation.     

SCID小鼠肠道菌群失调对深部白色念珠菌感染的影响研究

目的建立重度联合免疫缺陷（SCID）小鼠白色念珠菌感染模型,探讨肠道菌群失调与深部白色念珠菌感染的联系。方法SCID小鼠随机口服万古霉素水溶液7d,饥饿24h后给予白色念珠菌灌胃,建立小鼠白色念珠菌感染模型,观察小鼠死亡情况。荧光定量PCR检测肠道细菌总量、基质辅助激光解析电离飞行时间质谱仪鉴定肠道菌群种类,并应用扫描电镜观察肠壁黏膜组织超微结构的改变。结果应用万古霉素可致肠道菌群失调,肠道黏膜完整性受损。在万古霉素致肠道菌群失调的基础上,外源性白色念珠菌攻击可加重肠道菌群失调和肠壁黏膜损伤程度,促进深部白色念珠菌感染的发生。结论肠道菌群失衡可以导致深部白色念珠菌感染的发生,肠壁黏膜的完整性可能参与了肠道白色念珠菌播散过程。     

Active substances against trypomastigote forms of Trypanosoma cruzi and microorganisms are produced in sequence by Talaromyces flavus

The conditions for the sequential production of antibiotic activity by Talaromyces flavus were determined. The highest level of activity against Trypanosoma cruzi was obtained from the aqueous extract of the Czapeck's fermentative culture after 48 hours, with lysis of 97.58% of the trypomastigote forms of Trypanosoma cruzi (red blood cells remained normal). The antimicrobial activity was detected in the extracts of fermentative cultures from different media just after 144 hours of incubation. Maximum activities against Micrococcus luteus, Staphylococcus aureus and Candida albicans were present in chloroform, butanolic and water extracts, in this order, when Talaromyces flavus was cultivated at pH 5.0. The minimal inhibitory concentration (MIC) of extracts of Takeuchi's cultures were determined.     

慢性前列腺炎患者假丝酵母菌感染及耐药性分析

目的 了解慢性前列腺炎与假丝酵母菌感染的相关性及假丝酵母菌的耐药性。方法 采用常规沙堡平板分离360例慢性前列腺炎患者的前列腺液标本中的假丝酵母菌,疑似菌落用ATB Expression鉴定仪进行鉴定。采用ATB-Fungus真菌药敏板,对假丝酵母菌株进行药敏试验。结果 11．7％前列腺液标本（42／360）似丝酵母菌阳性,其中自假丝酵母菌23例（54．7％）,近平滑假丝酵母菌13例（30．9％）,其它6例（14．3％）。假丝酵母菌菌株对两性霉素B和制霉菌素敏感率均为100％,其次为酮康唑,敏感率为97．0％;对5-氟胞嘧啶耐药性最强,其耐药率为56．5％。结论 白假丝酵母菌和近平滑假丝酵母菌是慢性前列腺炎假丝酵母菌感染的优势菌种,假丝酵母菌株最敏感的药物是两性霉素B和制霉菌素。     

Influence of incubation conditions on cell surface hydrophobicity of Candida species fungi

Cell surface hydrophobicity (CSH) is considered to be one of several virulence factors of Candida yeast-like fungi. The aim of the study was a measurment of hydrophobic properties of Candida sp. depending on growth conditions. A total of 139 strains of Candida (80 - C. albicans and 59 - C. non-albicans) were examined. The method of salt aggregation test (SAT) was used. The strains were cultured on three different media, in two variants of incubation temperature and time. The incubation temperature and microbiological medium affected CSH of just C. albicans strains. The influence of incubation time on CSH of examined species of Candida was not occurred. There was a strong correlation between CSH and species of Candida demonstrated in the study Hydrophobic properties were more frequent and stronger among strains of C. non-albicans than C. albicans species. The results of the study indicates that CSH of Candida spp. is a dynamic feature. The ability to change surface properties may play a role in pathogenesis of candidosis.     

蜡螟抗白假丝酵母菌自噬机制的初步研究

目的探究白假丝酵母菌感染蜡螟时蜡螟的自噬相关通路蛋白的表达情况。方法用一定量的白假丝酵母菌的活化孢子感染蜡螟,经过12h,解剖蜡螟收集蜡螟细胞并裂解细胞,用真菌活性检测试剂盒检测孢子活性;解剖蜡螟取肠道组织并用PI染死细胞。取不同感染时间段的淋巴细胞,用裂解液裂解,离心取上清,用Western blot法检测上清液中的Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平。结果活化的孢子注射至蜡螟体内后,其活性受到抑制;蜡螟的肠道细胞被定位在上面的菌丝损伤并且孢子活性受到抑制。随着蜡螟感染白假丝酵母菌时间的递增,其自身的Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平在不断增高且在感染24h最高。结论白假丝酵母菌感染蜡螟后,蜡螟的淋巴细胞和肠道细胞通过升高Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平发挥杀伤孢子的作用。     

大蒜素对白念珠菌形态转换的影响研究

目的探讨大蒜素对白念珠菌形态转换的影响及其作用机制。方法倒置显微镜观察白念珠菌菌丝形成的体外动力学过程;采用CLSI-M27-A3微量液基稀释法检测大蒜素对白念珠菌的最小抑菌浓度(minimum inhibitory concentration,MIC);倒置显微镜观察不同浓度大蒜素对白念珠菌在Spider液体培养基中菌丝形成的影响;qRT-PCR法检测在不同浓度大蒜素作用下白念珠菌菌丝相关基因HWP1、ALS1、EFG1、PDE2表达水平的变化。结果白念珠菌在Spider液体培养基中6 h时出现较长菌丝,24 h后镜下可见大量念珠菌菌丝包裹酵母细胞,紧密交错;大蒜素对白念珠菌的MIC值为25μg/mL;倒置显微镜观察(25~100)μg/mL浓度的大蒜素能明显抑制Spider液体培养基中白念珠菌菌丝的生长;qRT-PCR结果显示,在(25~100)μg/mL浓度的大蒜素作用下,白念珠菌菌丝相关基因表达下调。结论大蒜素能有效抑制白念珠菌的形态转换,其作用机制可能与调节菌丝形成相关基因的表达水平有关。     

Etiologic agents of fungemia in hospitalized patients]

The aim of performed examinations was the analysis of fungi as etiological agents of blood infections in patients hospitalized in surgical wards, internal medicine wards and intensive care units of the Medical Academy Central Clinical Hospital in Warsaw. Blood samples from patients hospitalized in 1997 were examined. Peripheral blood samples were incubated in BacT/Alert system (Organon Teknika, USA). Positive blood samples were inoculated on Sabouraud medium with chloramphenicol (bioMerieux, France or Oxoid, England). The time of cultivation was from 48 hours to 7 days at 30 degrees C. Fungal strains were identified by standard mycological procedures with the use of chromogenic medium BBL CHROMagar Candida (Becton Dickinson, USA) and biochemical test ID 32 C (bioMerieux, France). Susceptibility of strains to antifungal agents was determined by ATB FUNGUS method (bioMerieux, France). The total number of positive blood cultures in 1997 was 1380. Forty-two fungal strains were isolated from blood samples (3%). Strains belonged to the following species: C. albicans (17 isolates), C. parapsilosis (15), C. glabrata (3), melibiosica (2), C. pelliculosa (2), C. guilliermondii (1), C. tropicalis (1) and T. beigelii (1). Among fungi cultured from patients hospitalized in operative wards dominated C. parapsilosis (11) and C. albicans (10) strains, whereas from patients hospitalized in conservative wards most often C. albicans (6) strains were isolated. Candida strains were mostly susceptible to antifungal agents tested. It was interesting to culture Trichosporon beigelii (T. cutaneum) strain as an etiological agent of fungemia. This strain was multidrug-resistant.     

卡泊芬净、米卡芬净对念珠菌体外药物敏感性的动态研究

目的 动态研究卡泊芬净、米卡芬净体外对念珠菌的药物敏感性.方法 参照CLSI公布的M-27A方案微量液体稀释法分别测定卡泊芬净、米卡芬净、氟康唑对85株念珠菌的体外敏感性,并连续7d观测结果.结果 48 h卡泊芬净对白念珠菌、光滑念珠菌及其他念珠菌MIC50、MIC90中位数分别为0.030μg/mL、0.030 μg/mL,0.060μg/mL、0.125 μg/mL,0.125 μg/mL、0.500 μg/mL.48 h米卡芬净对白念珠菌、光滑念珠菌及其他念珠菌MIC50、MIC90中位数分别为0.030 μg/mL、0.030 μg/mL,0.060 μg/mL、0.060 μg/mL,0.250 μg/mL、0.500 μg/mL.48 h氟康唑对白念珠菌、光滑念珠菌及其他念珠菌MIC80、MIC100中位数分别为2μg/mL、128 μg/mL,64 μg/mL、128 μg/mL,2μg/mL、32μg／mL.85株念珠菌中未见对3种药物同时耐药的菌株.卡泊芬净组白念珠菌MIC50、MIC90 24 h后不再升高;光滑念珠菌MIC50 72 h后不再升高,MIC90 120 h后不再升高;其他念珠菌组MIC50 168 h、MIC90 96 h后不再升高.米卡芬净组白念珠菌、光滑念珠菌MIC50、MIC90 24 h后不再升高;其他念珠菌MIC50、MIC90在72 h后不再升高.结论卡泊芬净、米卡芬净对念珠菌属有较好的抗菌作用,其中对白念珠菌、光滑念珠菌作用更强,且MICs随着作用时间延长而升高并存在药物特异性和念珠菌种属特异性.     

致病真菌对甲侵袭性的体外初步研究

目的观察不同致病真菌在体外对甲板的侵袭能力。方法将分离自甲真菌病患者的致病真菌包括白念珠菌、红色毛癣菌和短帚霉,接种到沙堡培养基中,同时将灭菌甲板埋植入接种处。第28d时,取出甲板,进行病理切片,观察真菌对甲板的侵袭能力。结果病理显示接种于红色毛癣菌及短帚霉的甲板内可见菌丝生长,而接种于白念珠菌的甲板内无菌丝生长。结论皮肤癣菌和霉菌可以在甲板内侵袭生长,而白念珠菌对甲板无侵袭能力。     

地衣芽胞杆菌对白色念珠菌等的拮抗作用

目的了解地衣芽胞杆菌在试管内与阴道正常菌群共生关系的情况。方法将地衣芽胞杆菌菌液分别与葡萄球菌、大肠埃希菌、白色念珠菌、德氏乳杆菌混合培养,定量计数各菌在不同时间内单独培养和混合培养时各菌的活菌数。结果地衣芽胞杆菌生长不受金黄色葡萄球菌、白色念珠菌和大肠埃希菌的影响,金黄色葡萄球菌和白色念珠菌在有地衣芽胞杆菌存在的情况下,其生长受到明显的抑制（P〈0.05）;乳杆菌在12-48 h内,有显著的抑制地衣芽胞杆菌生长的作用,而乳杆菌的生长不受地衣芽胞杆菌的存在与否而正常生长。结论地衣芽胞杆菌对金黄色葡萄球菌及白色念珠菌在体外具有明显的拮抗作用,地衣芽胞杆菌对大肠埃希菌、乳杆菌无明显的体外拮抗作用。     

In vitro activity of amphotericin B and anidulafungin against Candida spp. biofilms

Invasive infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. A large number of manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces. Candida spp. biofilms are recalcitrant to treatment with conventional antifungal therapies. The aim of this study was dual 1) to determine the prevalence of biofilm producers among clinical isolates from catheter (16 C. albicans ) and blood culture (2 C. albicans and 30 C. tropicalis), and 2) to determine the activity of amphotericin B and anidulafungin against C. albicans and C. tropicalis biofilms of 24 and 48 hours of maturation. Biofilms were developed using a 96-well microtitre plate model and production and activity of antifungal agents against biofilms were determined by the tetrazolium (XTT) reduction assay. Of catheter and blood isolates, 62.5 and 56.25%, respectively, produced biofilms. By species, 68.42% of C. albicans and 53.33% of C. tropicalis were biofilm producers. C. albicans biofilms showed more resistance to amphotericin B and anidulafungin than their planktonic counterparts. Complete killing of biofilms was never achieved, even at the highest concentrations of the drugs tested. Anidulafungin displayed more activity than amphotericin B against C. albicans biofilms of 24 hours of maturation (GM MIC 0.354 vs. 0.686 microg/ml), but against C. tropicalis biofilms amphotericin B was more active (GM MIC 11.285 vs. 0.476 microg/ml). In contrast, against biofilms with 48 hours maturation, amphotericin B was more active against both species.     

Amphotericin B-metronidazole combination against Candida spp

Oropharyngeal candidiasis (OPC) remains a common opportunistic infection in HIV-infected patients. Candida albicans is the most frequent causative agent of OPC. However, non-albicans spp. are being increasingly isolated. Candidal cell wall proteins and mannoproteins play important roles in the biology and patogenesis of candidiasis. In the present study, we have analyzed the proteinaceous components associated with cell wall extracts from C. albicans, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii and Candida rugosa obtained from HIV-infected patients with recurrent OPC. Cell wall proteinaceous components were extracted with beta-mercaptoethanol and analyzed using electrophoresis, immunoblotting (with antisera generated against C. albicans cell wall components, and with serum samples and oral saline rinses from patients with OPC), and lectin-blotting (concanavalin A) techniques. Numerous molecular species were solubilized from the various isolates. Major qualitative and quantitative differences in the polypeptidic and antigenic profiles associated with the cell wall extracts from the different Candida spp. were discernible. Some of the antibody preparations generated against C. albicans cell wall components were able to recognize homologous materials present in the extracts from non-albicans spp. Information on cell wall antigens of Candida species may be important in the therapy and prevention of HIV-related OPC.     

Candida dubliniensis identification in Brazilian yeast stock collection

We investigated the presence of Candida dubliniensis among isolates previously identified as Candida albicans and maintained in a yeast stock collection from 1994 to 2000. All isolates were serotyped and further evaluated for antifungal susceptibility profile. After doing a screening test for C. dubliniensis isolates based on the capability of colonies to grow at 42 C, its final identification was obtained by randomly amplified polymorphic DNA (RAPD) analysis using three different primers. A total of 46 out of 548 screened isolates did not exhibit growth at 42 C and were further genotyped by RAPD. Eleven isolates were identified as C. dubliniensis with RAPD analysis. Regarding serotypes, 81.5% of C. albicans and all C. dubliniensis isolates belonged to serotype A. Of note, 9 out of 11 C. dubliniensis isolates were obtained from patients with acquired immunodeficiency syndrome (Aids) and all of them were susceptible to azoles and amphotericin B. We found 17 (3%) C. albicans isolates that were dose-dependent susceptibility or resistant to azoles. In conclusion, we found a low rate of C. dubliniensis isolates among stock cultures of yeasts previously identified as C. albicans. Most of these isolates were recovered from oral samples of Aids patients and exhibited high susceptibility to amphotericin B and azoles. C. albicans serotype A susceptible to all antifungal drugs is the major phenotype found in our stock culture.     

Vascular clearance of blastospore and pseudomycelial phase Candida albicans.

Blastospore phase and pseudomycelial phase Candida albicans were infused at a constant rate into the proximal aorta of rabbits and simultaneous quantitative blood cultures were obtained from the abdominal aorta and inferior vena cava. Arteriovenous differences were greater with pseudomycelial phase C. albicans at all concentrations tested. Rates of clearance of C. albicans as blastospores or with pseudomycelia yielded mean T one-half values of 22 seconds and 17 seconds, respectively. Similarly, hepatic clearance of pseudomycelial phase C. albicans was more efficient. Prior immunization with heat-killed Candida albicans had no effect on the vascular clearance of either pseudomycelial phase or blastospore phase C. albicans.     

8种致病真菌在5种材料上的存活时间研究

目的了解实验所用常见致病真菌能否在常用医用原材料上生存及生存时间。方法选择8种常见致病真菌,活化后将它们分别接种在5种常用的医用原材料上,接种后及以后的每24h将接种有真菌的医用材料放入巯基乙酸盐液体培养基中并观察能否生存,记录存活时间。结果红色毛癣菌和须癣毛癣菌在棉布、纸板、铁皮、玻璃表面存活25～28d,在地板胶表面存活14～15d;新生隐球菌在棉布、纸板、铁皮表面存活24～27d,在玻璃和地板胶表面存活14～18d;茄病镰刀菌在上述5种材料上存活22～27d;石膏样小孢子菌在棉布、纸板、铁皮和玻璃表面存活20～27d,在地板胶表面存活8d;申克孢子丝菌在棉布、纸板、玻璃和地板胶表面存活17～24d,在铁皮表面存活10d;白念珠菌在棉布、纸板、铁皮和地板胶表面存活15～20d,在玻璃表面存活4d;犬小孢子菌在棉布、纸板、铁皮、玻璃和地板胶上存活时间分别为10d、9d、3d、2d和1d。结论实验所用各种真菌在医用原材料上都能存活,其时间长短不仅与菌种有关,还与所存在的材料有关。真菌在吸水性强、表面粗糙的材料(棉布、纸板)上的平均存活时间长于在吸水性差、表面光滑的材料(玻璃、铁皮和地板胶)。     

氨基酸对白念珠菌形态学影响的研究

目的初步探讨单个氨基酸对白念珠菌形态学的影响。方法用0．67％的酵母氮源基础培养基和2％葡萄糖配制成SD合成培养基,37％恒温摇床培养,研究单个天然氨基酸对白念珠菌形态学的影响,并分别通过不添加碳源和厌氧条件下培养观察对精氨酸诱导的菌丝的影响。结果在含10mmol／L的L－精氨酸的SD液体培养基中,可见大量的菌丝。在含10mmol／L的L一半胱氨酸、L．苏氨酸、L－缬氨酸和L－色氨酸的sD液体培养基中,可见典型的酵母细胞,未见菌丝。在含10mmol／L的其他单个氨基酸的SD液体培养基中可见混合的酵母和菌丝结构。在不含氨基酸或含各种天然氨基酸的SD固体培养基上,白念珠菌的菌落均光滑。但在含10mmol／L的L-精氨酸固体培养基上,光滑的菌落周围可见小的突起,镜下可见菌丝。无氧条件下,无论有无碳源,含精氨酸的SD培养液中白念珠菌只能形成酵母细胞,生长部分受到抑制。结论精氨酸可以诱导白念珠菌菌丝形成,厌氧条件下精氨酸不能诱导白念珠菌菌丝形成。     

Methods for DNA extraction from Candida albicans

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.