哈尔滨地区假丝酵母菌DNA异质性及药物敏感性分析

研究假丝酵母菌的DNA异质性及药物敏感性,为预防和监控院内假丝酵母菌感染奠定基础。将临床分离的假丝酵母菌菌株,用科玛嘉显色培养基鉴定菌种,经纸片扩散法进行药敏试验,应用随机扩增多态性DNA（RAPD）技术对这些菌株进行基因分型。结果显示：93株假丝酵母菌中白假丝酵母菌68株,非白假丝酵母菌25株,所有菌株对制霉菌素,两性霉素B两种药物的敏感率最高（100％）,酮康唑其次（70．9％）,氟康唑的敏感率最低（50．5％）,引物1和引物2将来源不同的68株白假丝酵母菌分别分成4型（A1、B1、C1、D1）和6型（A2、B2、C2、D2、E2、F2）。哈尔滨地区的假丝酵母菌感染以白假丝酵母菌为主,且主要为A1、B1型（引物1）或A2、B2型（引物2）;基因型与药敏谱无明显相关性。     

A study of the use of rapid methods for preservative efficacy testing of pharmaceuticals and cosmetics

Three rapid microbiological methods, impedance, the direct epifluorescence technique (DEFT-MEM) and ATP bioluminescence (ATP-B) were evaluated for their applicability to preservative efficacy testing (PET) of pharmaceuticals and cosmetics. A good correlation between rapid method response and total colony counts was obtained for untreated suspensions of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans with all three methods but, for Aspergillus niger , with impedance only. For chlorhexidine-treated suspensions of Staph. aureus and C. albicans , a good dose—response curve was obtained with impedance, but ATP-B and DEFT-MEM methods underestimated the kill by the order of 1–6 logs. From the results of this study it is concluded that impedance offers an alternative method to colony counting methods for PET but, at their present level of method development, neither DEFT-MEM nor ATP-B can be considered as satisfactory.     

Anticandidal properties of N-acylpeptides containing an inhibitor of glucosamine-6-phosphate synthase

A series of N-acyl peptides 1-9, containing an inhibitor of glucosamine-6-phosphate synthase have been synthesised and tested against Candida strains. N-Acylated peptides inhibit glucosamine-6-phosphate synthase in cell free extracts from Candida albicans. Antifungal activities of the tested compounds correlated with their lipophilic properties. Peptides acylated with decanoic acid were found to be the most potent in the series. N-decanoylpeptides also showed activity against Candida albicans Gu5 resistant mutant with Cdr1 and Cdr2 drug extrusion proteins that causes MDR by an active efflux mechanism.     

Involvement of aspirin-sensitive oxylipins in vulvovaginal candidiasis

3(R)-Hydroxyoxylipins are produced via an aspirin-sensitive pathway in Candida albicans, an abundant pathogen in vulvovaginal candidiasis. In the present study, we have investigated the effect of aspirin on vaginal isolates of C. albicans from patients with recurrent candidiasis. Aspirin alone and with clotrimazole, a commonly used drug, strongly suppressed growth of C. albicans. 3(R)-Hydroxyoxylipins, which were selectively located in hyphae and other filamentous structures, but not in free blastospores, were almost totally suppressed by aspirin. Moreover, C. albicans stimulated prostaglandin E(2) (PGE(2)) production in HeLa cells. PGE(2) is a stimulus for germ tube formation in C. albicans. We conclude therefore that the administration of aspirin should be beneficial in the treatment of vulvovaginal candidiasis by dual ways: (i) by inhibition of 3(R)-hydroxyoxylipin formation, and (ii) by inhibition of PGE(2) formation in the infected host tissue.     

Investigation of the anti-fungal activity of coptisine on Candida albicans growth by microcalorimetry combined with principal component analysis

Aims:  This study investigated the anti-fungal activity of coptisine on Candida albicans growth.
Methods and Results:  The metabolic power-time curves of Candida albicans growth at 37°C affected by coptisine were measured by microcalorimetry using an LKB-2277 Bioactivity Monitor with stop-flow mode. Then, the diameter of inhibitory zones in the agar layer was observed using agar cup method, and the minimal inhibitory concentration (MIC) of coptisine on Candida albicans growth was determined by serial dilution method. From the principal component analysis on nine quantitative parameters obtained from the power-time curves, we could easily evaluate the anti-fungal activity of coptisine by analysing the change of values of the main two parameters, growth rate constant k and maximum power output in the log phase P _{m, log}. The results showed that coptisine had strong anti-fungal activity: at a low concentration (45  μ g ml⁻¹) began to inhibit the growth of Candida albicans and at a high concentration (500  μ g ml⁻¹) completely inhibited Candida albicans growth. Coptisine gave big inhibitory zones with diameters between 11 and 43 mm within test range, and the MIC of it was 1000  μ g ml⁻¹.
Conclusions:  Coptisine had strong anti-fungal activity on Candida albicans growth. The method of microcalorimetry applied for the assay of anti-fungal activity of coptisine was quantitative, sensitive and simple.
Significance and Impact of the Study:  This work will provide useful information for the development of chemical biology policy in the use of anti-microbials in food and drug production.     

CpG oligodeoxynucleotides protect mice from lethal challenge with Candida albicans via a pathway involving tumor necrosis factor-alpha-dependent interleukin-12 induction

In this study, we have attempted to determine whether the systemic administration of CpG oligodeoxynucleotide (CpG-ODN) 1826 would protect mice against systemic lethal Candida albicans infection. CpG-ODNs were found completely to protect mice from death and also reduced the growth of C. albicans in the kidneys. The administration of CpG-ODNs resulted in early interleukin (IL)-12 mRNA expression in the kidneys and an increase in serum IL-12 levels. The protective activity of CpG-ODN was abolished in IL-12-deficient (IL-12-/-) mice, thereby indicating the IL-12-dependency inherent to the effects of CpG-ODN. The protective effect of CpG-ODN was not associated with the activity of NF-kappaB. Interestingly, in tumor necrosis factor (TNF)-alpha-deficient (TNF-/-) mice CpG-ODN neither exerted protective effects nor induced IL-12 expression. These data indicate that CpG-ODN protects animals against lethal C. albicans challenge via a pathway that involves the TNF-alpha-dependent induction of IL-12.     

Cdc28 provides a molecular link between Hsp90, morphogenesis, and cell cycle progression in Candida albicans

The trimorphic fungus Candida albicans is the leading cause of systemic candidiasis, a disease with poor prognosis affecting immunocompromised individuals. The capacity of C. albicans to transition between morphological states is a key determinant of its ability to cause life-threatening infection. Recently the molecular chaperone heat shock protein 90 (Hsp90) was implicated as a major regulator of temperature-dependent C. albicans morphogenesis; compromising Hsp90 function induces filamentation and relieves repression of Ras1-protein kinase A (PKA) signaling, although the mechanism involved remains unknown. Here we demonstrate that filaments generated by compromise of Hsp90 function are neither pseudohyphae nor hyphae but closely resemble filaments formed in response to cell cycle arrest. Closer examination revealed that these filaments exhibit a delay in mitotic exit mediated by the checkpoint protein Bub2. Furthermore, Hsp90 inhibition also led to a distinct morphology with defects in cytokinesis. We found that the cyclin-dependent kinase Cdc28 was destabilized in response to depletion of Hsp90 and that Cdc28 physically interacts with Hsp90, implicating this major cell cycle regulator as a novel Hsp90 client protein in C. albicans. Taken together, our results suggest that Hsp90 is instrumental in the regulation of cell division during yeast-form growth in C. albicans and exerts its major effects during late cell cycle events.     

Survival of Candida albicans in tropical marine and fresh waters.

A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard.     

Survival of Candida albicans in tropical marine and fresh waters

A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard.     

2-Hydroxyphenacyl azoles and related azolium derivatives as antifungal agents

2-Hydroxyphenacyl azole and 2-hydroxyphenacyl azolium compounds have been described as a new class of azole antifungals. Most target compounds showed significant in vitro antifungal activities against tested fungi (Candida albicans, Saccharomyces cerevisiae, Aspergillus niger, and Microsporum gypseum) with low MICs values included in the range of 0.25-32 microg/mL comparable to reference drug fluconazole. The most active compounds were also assessed for their cytotoxicity using MTT colorimetric assay on normal mouse fibroblast (NIH/3T3) cells. The results of antifungal activity and toxicity tests indicated that these compounds display antifungal activity at non-cytotoxic concentrations.     

A Candida albicans mutant impaired in the utilization of N-acetylglucosamine

Indicator plates containing eosin, methylene blue, glucosamine and proline were used to select mutants of Candida albicans impaired in the utilization of glucosamine. One such mutant, strain hOG298, grew on glucosamine at a slower rate than the parent and was severely impaired in growth on N-acetylglucosamine. The mutant was unable to express the first three steps in the N-acetylglucosamine pathway: viz the permease, N-acetylglucosamine kinase and N-acetylglucosamine-6-phosphate deacetylase. Glucosamine-6-phosphate deaminase was, however, induced by N-acetylglucosamine. The mutant still possessed a constitutive uptake system and kinase activity for glucosamine but glucosamine neither increased the glucosamine kinase activity nor induced N-acetylglucosamine kinase. These findings accounted for the decreased growth rate on glucosamine. The parent strain formed germ-tubes in N-acetylglucosamine or 4% (v/v) serum but the mutant formed germ-tubes only in serum.     

Discovery of uracil-based histone deacetylase inhibitors able to reduce acquired antifungal resistance and trailing growth in Candida albicans

Among fungal pathogens such as Candida albicans, acquired drug resistance has not been associated with plasmids or other transferable elements, but it is thought to involve primarily mutations and genetic or epigenetic phenomena. This prompted us to test some histone deacetylase inhibitors (HDACi) from our library, in combination with fluconazole, against C. albicans strains in vitro. Among the tested compounds, the two chloro-containing uracil-hydroxamates 1c and 1d showed a strong reduction of the MIC values on Candida strains that show the trailing growth effect. In this assay, 1c,d were more potent than SAHA, a well-known HDAC inhibitor, in reducing the Candida growth. More interestingly, 1c,d as well as SAHA were able to inhibit the fluconazole-induced resistance induction in Candida cultures.     

Production of anti-Candida antibodies in mice with gut colonization of Candida albicans

BACKGROUND: Production of antibodies that are specific for allergens is an important pathological process in inflammatory allergic diseases. These contain the antibodies against antigens of Candida albicans, one of the normal microbial flora in an intestinal tract. We studied the effects of the prednisolone administration on the production of anti-Candida antibodies in the gastrointestinally C. albicans-colonized mice. METHODS AND MATERIALS: BALB/c mice, treated with antibacterial antibiotics to decontaminate indigenous intestinal bacterial flora, were inoculated intragastrically with C. albicans. The mice, in which C. albicans grows intestinally, were administered prednisolone to induce temporary immunosuppression. The Candida growth in their intestinal tract and their antibody response to Candida were examined. RESULTS: Antibiotic treatment allowed establishment of C. albicans gastrointestinal colonization, but did not cause subsequent systemic dissemination of C. albicans in all the animals. When these animals received an additional treatment with prednisolone, they showed a significantly higher population of C. albicans in their feces than those of animals treated with antibiotics alone, and the organisms were recovered even from their kidney. This systemic dissemination by C. albicans appeared to be temporal, because all the mice survived without any symptoms for more than 2 months. Examination of the serum titers of total immunoglobulin (Ig)E antibodies and specific IgE and IgG antibodies against Candida antigens demonstrated that titers of total IgE increased, partially by day 14 and clearly at day 27, in prednisolone-treated Candida-colonized mice. Without prednisolone treatment, an increment of the serum titer was scarcely observed. By day 27, corresponding to the increase of total IgE, the anti-Candida IgE and IgG titer increased in mice of the prednisolone-treated group. CONCLUSION: Administration of prednisolone to Candida-colonized mice can induce production of the IgG, IgE antibodies against Candida antigens, perhaps through temporal systemic dissemination of Candida from the intestinal tract.     

In Vitro Inhibition of Oral Candida albicans by Chicken Egg Yolk Antibody (IgY)

This study was conducted to measure Candida albicans-specific chicken egg yolk antibody (IgY) inhibition of fluconazole-sensitive and resistant strains of C. albicans in order to assess potential use in the prevention and treatment of oral candidiasis. In this study, laying hens were immunized, and IgY was extracted by water dilution. The Minimal Inhibitory Concentrations (MICs) of IgY for inhibiting C. albicans growth were determined using the broth microdilution method from the CLSI M27-A2 protocol. Fluconazole (FLC) was used as the control. The results were analyzed with the chi(2) test. The anti-Candida titer of anti-C. albicans IgY was 1:12,000. The concentration of the IgY extract that effectively inhibited the growth of C. albicans was between 1.25 g/l and 5.0 g/l, and the efficacy rate was 82.98% during the observed 24-48 h time period. No correlation was recorded between the drug resistance of FLC and growth inhibition by IgY. It was concluded that anti-C. albicans IgY inhibited the growth of C. albicans in vitro and there was no correlation between the drug resistance of FLC and the growth inhibition by IgY (P > 0.99).     

白假丝酵母菌耐药与抗氰呼吸的相关性研究

目的探讨白假丝酵母菌的耐药情况及其与抗氰呼吸的相关性。方法用真菌药敏测定试剂盒测定从临床分离出来的37株白假丝酵母菌的耐药性,并从中选出5株耐药菌和5株敏感菌进行抗氰呼吸的研究。结果白假丝酵母菌对益康唑的耐药率最高,达54.1%,耐药白假丝酵母菌的抗氰呼吸速率均值为(17.56±6.75)nmol/(min.A620),敏感白假丝酵母菌的抗氰呼吸速率均值为(7.99±5.80)nmol/(min.A620),耐药白假丝酵母菌的抗氰呼吸速率明显升高,且耐药菌株抗氰呼吸速率占总呼吸的比例明显高于敏感菌株(P0.05),差异具有显著性。结论兰州市区白假丝酵母菌对益康唑耐药性较高,且白假丝酵母菌的耐药与抗氰呼吸途径相关。     

pH及氧气对白念珠菌菌丝形成的影响

目的探讨pH值和氧气对白念珠菌菌丝形成的影响。方法通过调节Muller—Hinton液体培养基的pH值和去除培养基中的氧气来观察白念珠菌的生长曲线、倍增时间和菌丝形成率的变化。结果在无氧气的液体培养基中,白念珠菌生长缓慢,不能产生菌丝结构,只有酵母细胞形成。生长曲线的延缓期内各组没有明显差异,而在生长的对数期pH3和pH4的条件下念珠菌生长速度明显慢于pH5、pH6、pH7、pH8和pH9。菌丝形成率在pH3、pH4和pH5条件下〈20％,而在pH6、pH7、pH8和pH9条件下可高达70％。结论厌氧条件抑制白念珠菌的菌丝形成,只形成酵母细胞。白念珠菌在pH3—9的范围内均能生长,偏酸性环境有利于白念珠菌酵母形成,偏碱性的环境有助于菌丝的形成。     

PIC-BE诱导K562/ADM细胞凋亡及逆转其MDR的研究

β榄香烯吗素（ＰＩＣ－ＢＥ）是抗癌新药β榄香烯的水溶性衍生物．采用人红白血病的多药耐药性（ＭＤＲ）细胞株Ｋ５６２／ＡＤＭ作为实验模型,观察ＰＩＣ－ＢＥ对Ｋ５６２／ＡＤＭ细胞的生长抑制和凋亡诱导作用,并进而研究其对该细胞ＭＤＲ的可能影响．结果显示：（１）Ｋ５６２／ＡＤＭ细胞对ＡＤＭ具有明显的抗性,与Ｋ５６２细胞相比,抗性倍数约为４０倍,而两者对ＰＩＣ－ＢＥ的ＩＣ５０接近,无显著差异;（２）ＰＩＣ－ＢＥ（１０．０～３０．０μｇ／ｍｌ）对Ｋ５６２／ＡＤＭ细胞具有明显的生长抑制和凋亡诱导作用,两种作用的强度在一定的范围内均具药物浓度和作用时间依赖性;（３）低毒剂量ＰＩＣ－ＢＥ（１０．０μｇ／ｍｌ）与ＡＤＭ（４．０μｇ／ｍｌ）联合应用,可显著增强ＡＤＭ对该细胞的生长抑制和凋亡诱导作用,升高细胞内ＡＤＭ的浓度,降低该细胞对ＡＤＭ的ＩＣ５０,使该细胞对ＡＤＭ的抗性有数倍逆转．上述结果提示,ＰＩＣ－ＢＥ不仅是一种有效的广谱抗肿瘤剂,而且也是一种有效的ＭＤＲ逆转剂     

Environmental dimensionality controls the interaction of phagocytes with the pathogenic fungi Aspergillus fumigatus and Candida albicans

The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why "delocalized" Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals.