焦性没食子酸联合唑类药物对念珠菌的抑菌作用

目的了解光滑念珠菌临床菌株的药敏情况以及中药单体焦性没食子酸联合唑类药物对念珠菌的抑菌作用。方法收集临床分离的光滑念珠菌116株、白念珠菌49株、热带念珠菌42株、克柔念珠菌4株和近平滑念珠菌13株,采用ATB FUNGUS3药敏试条检测光滑念珠菌的药敏情况;同时采用棋盘肉汤稀释法检测焦性没食子酸联合唑类药物对念珠菌的抑菌情况。结果116株光滑念珠菌中,14.66%(17株)的菌株对氟康唑耐药,22.41%(26株)对伊曲康唑表现为非野生型和81.03%(94株)对伏立康唑表现为非野生型。焦性没食子酸对5种念珠菌的抑菌情况,46.55%光滑念珠菌的MIC值为64μg/mL;34.69%白念珠菌的MIC值为64μg/mL;59.52%热带念珠菌的MIC值为64μg/mL;25%克柔念珠菌的MIC值为128μg/mL;46.15%近平滑念珠菌的MIC值为128μg/mL。唑类药物与焦性没食子酸联合用药时,100%、99.14%、99.14%的光滑念珠菌分别对氟康唑、伊曲康唑和伏立康唑表现为协同作用或相加作用,差异无统计学意义(P>0.05);而白念珠菌、热带念珠菌、克柔念珠菌和近平滑念珠菌均表现为无关作用和拮抗作用。与单独用药相比,联合用药时81.03%、68.1%、77.59%的光滑念珠菌分别对氟康唑、伊曲康唑、伏立康唑的MIC值降低2~3个浓度梯度,且耐药组与非耐药组之间差异具有统计学意义(P<0.05)。结论光滑念珠菌对唑类药物具有耐药性,伏立康唑非野生型菌株所占比例最高。焦性没食子酸单独用药时,5组念珠菌中对光滑念珠菌的抑菌效果最好,且敏感组比耐药组的抑菌效果更加显著。焦性没食子酸联合唑类药物显著降低了光滑念珠菌唑类药物的MIC值,且耐药组比非耐药组的效果更加显著,为中西医结合治疗临床光滑念珠菌感染提供实验依据。     

Antifungal activity and mode of action of silver nano-particles on Candida albicans

In this study, the antifungal effects of silver nano-particles (nano-Ag) and their mode of action were investigated. Nano-Ag showed antifungal effects on fungi tested with low hemolytic effects against human erythrocytes. To elucidate the antifungal mode of action of nano-Ag, flow cytometry analysis, a glucose-release test, transmission electron microscopy (TEM) and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH), as a plasma membrane probe, were performed with Candida albicans. The results suggest nano-Ag may exert an antifungal activity by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity. The present study indicates nano-Ag has considerable antifungal activity, deserving further investigation for clinical applications. K.-J. Kim and W. S. Sung contributed equally to this work and should be considered co-first authors.     

Essential Oils,Silver Nanoparticles and Propolis as Alternative Agents Against Fluconazole Resistant Candida albicans,Candida glabrata and Candida krusei Clinical Isolates

Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO₂ against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.     

Antifungal Activity of Geldanamycin Alone or in Combination with Fluconazole Against Candida species

A standardized broth microdilution method was used to test the antifungal activity of geldanamycin (GA), an inhibitor of heat shock protein 90 (Hsp90), alone or in combination with the antifungal agent fluconazole (FLC) against 32 clinical isolates of Candida spp. In addition, a disk diffusion test was also used to evaluate the antifungal effect of these two drugs against Candida spp. by measuring the inhibition zone diameters. We found that the range of minimal inhibitory concentrations (MICs) for GA alone against Candida spp. was 3.2–12.8 mg/L and the geometric mean of MICs was 6.54 mg/L. In addition, the combination of GA with FLC showed synergistic effects in vitro against 2 FLC-susceptible and 6 FLC-resistant isolates of C. albicans. As for the other isolates, indifference but no antagonism was observed. In the disk diffusion assay, the diameter of inhibition zones for FLC combined with GA against FLC-resistant C. albicans isolates was 30 mm, while no inhibition was observed with FLC alone. These results demonstrate that GA possesses antifungal activity against Candida spp., and the combination of GA with FLC shows in vitro synergistic activity against some C. albicans isolates, especially those resistant to FLC.     

Antifungal activities of origanum oil against Candida albicans

The antimicrobial properties of volatile aromatic oils from medicinal as well as other edible plants has been recognized since antiquity. Origanum oil, which is used as a food flavoring agent, possesses a broad spectrum of in vitro antimicrobial activities attributed to the high content of phenolic derivatives such as carvacrol and thymol. In the present study, antifungal properties of origanum oil were examined both in vitro and in vivo. Using Candida albicans in broth cultures and a micro dilution method, comparative efficacy of origanum oil, carvacrol, nystatin and amphotericin B were examined in vitro. Origanum oil at 0.25 mg/ml was found to completely inhibit the growth of C. albicans in culture. Growth inhibitions of 75% and >50% were observed at 0.125 mg/ml and 0.0625 mg/ml level, respectively. In addition, both the germination and the mycelial growth of C. albicans were found to be inhibited by origanum oil and carvacrol in a dose-dependent manner. Furthermore, the therapeutic efficacy of origanum oil was examined in an experimental murine systemic candidiasis model. Groups of mice (n = 6) infected with C. albicans (5 × LD₅₀) were fed varying amounts of origanum oil in a final vol. of 0.1 ml of olive oil (vehicle). The daily administration of 8.6 mg of origanum oil in 100 l of olive oil/kg body weight for 30 days resulted in 80% survivability, with no renal burden of C. albicans as opposed to the group of mice fed olive oil alone, who died within 10 days. Similar results were obtained with carvacrol. However, mice fed origanum oil exhibited cosmetically better clinical appearance compared to those cured with carvacrol. The results from our study encourage examination of the efficacy of origanum oil in other forms of systemic and superficial fungal infections and exploration of its broad spectrum effect against other pathogenic manifestations including malignancy.     

Antifungal evaluation of traditional herbal monomers and their potential for inducing cell wall remodeling in Candida albicans and Candida auris

Abstract

Traditional herbal monomers (THMs) are widely distributed in many traditional Chinese formulas (TCFs) and decoctions (TCDs) and are frequently used for the prevention and treatment of fungal infections. The antifungal activities of five common THMs, including sodium houttuyfonate (SH), berberine (BER), palmatine (PAL), jatrorrhizine (JAT) and cinnamaldehyde (CIN), and their potential for inducing cell wall remodeling (CWR), were evaluated against Candida albicans SC5314 and Candida auris 12372. SH/CIN plus BER/PAL/JAT showed synergistic antifungal activity against both Candida isolates. Furthermore, SH-associated combinations (SH plus BER/PAL/JAT) induced stronger exposure of β-glucan and chitin than their counterparts, while CIN triggered more marked exposure compared with CIN-associated combinations (CIN plus BER/PAL/JAT). Collectively, this study demonstrated the anti-Candida effect and the CWR induction potential of the five THMs and their associated combinations, providing a possibility of their in vivo application against fungal-associated infections.     

家蝇抗菌肽AMP-17对白色念珠菌菌丝的抑制作用

【背景】AMP-17是从微生物诱导的家蝇转录组数据库筛选到的一条特异性高表达基因,采用原核表达体系获得其重组蛋白并证实了具有显著的抗菌效果,特别是对白色念珠菌具有较强的抗菌活性。【目的】研究抗菌肽AMP-17对白色念珠菌菌丝的抑制作用。【方法】采用微量液体稀释法测定AMP-17对11株白色念珠菌的最小抑菌浓度(minimal inhibitory concentration,MIC);根据对AMP-17的敏感程度选取3株绘制生长曲线;通过光学显微镜观察并计数经AMP-17作用后白色念珠菌芽生孢子生成率及芽管形成率;倒置荧光显微镜观察白色念珠菌酵母相向菌丝相转化及以菌丝相为起点AMP-17促进菌丝相转化为酵母相的情况。【结果】AMP-17对支气管肺泡灌洗液分离株16105的MIC为10μg/mL,对粪便分离株16214的MIC为40μg/mL,对其余9株白色念珠菌的MIC均为20μg/mL;白色念珠菌经不同浓度的AMP-17作用后,各时间点的芽生孢子生成率均显著低于对照组,尤其是40μg/mL的AMP-17组,芽生孢子生成率仅15%,显著低于阳性药物氟康唑;各实验组芽管形成率显著低于对照...     

Requirement for nonprotonated drug molecules in the direct lethal action of miconazole against Candida albicans

At 10^–5 M, miconazole (MCZ) can exert a direct physicochemical cell-damaging lethal action against logarithmic phase yeasts of Candida albicans. The imidazole moiety of MCZ has a pKa 6.5. Thus, in media of pH >6.5 most drug molecules are nonprotonated (MCZ). Conversely, at pH < 6.5 the majority are protonated and carry a positive charge (MCZH⁺). Our earlier work suggesting that MCZ is required for direct lethal action was tested further. In support of such a requirement, we established a minimal lethal concentration of MCZ (i.e. 5×10^–6 M) that was relatively independent of pH, MCZ concentration, and MCZMCZH⁺ ratio.     

Effect of calcium ion uptake on Candida albicans morphology

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.     

氯己定联合联苯苄唑对念珠菌的协同抗菌活性

本文用两种方法评价了联苯苄唑与氯己定联用对临床分离念珠菌的抑菌活性。微量液基稀释法显示,氯己定单独作用于念珠菌时,对念珠菌没有明显的抑制作用,最低抑菌浓度均大于16μg/mL;与联苯苄唑联合应用时,对17株念珠菌均表现为协同抑菌作用。纸片扩散法也观察到,含氯己定和联苯苄唑的纸片对4种念珠菌均能产生直径大于单药纸片的抑菌圈。结果显示,联苯苄唑联合低浓度氯己定对多种念珠菌具有协同抑菌作用。     

Antifungal activity of Rubus chingii extract combined with fluconazole against fluconazole‐resistant Candida albicans

This study aimed to investigate the antifungal activity of Rubus chingii extract in combination with fluconazole (FLC) against FLC‐resistant Candida albicans 100 in vitro. A R. chingii extract and FLC‐resistant C. albicans fungus suspension were prepared. The minimum inhibitory concentration and fractional inhibitory concentration index of R. chingii extract combined with FLC against C. albicans were determined, after which growth curves for C. albicans treated with R. chingii extract, FLC alone and a combination of these preparations were constructed. Additionally, the mechanisms of drug combination against C. albicans were explored by flow cytometry, gas chromatographic mass spectrometry and drug efflux pump function detection. R. chingii extract combined with FLC showed significant synergy. Flow cytometry suggested that C. albicans cells mainly arrest in G1 and S phases when they have been treated with the drug combination. The drug combination resulted in a marked decrease in the ergosterol content of the cell membrane. Additionally, efflux of Rhodamine 6G decreased with increasing concentrations of R. chingii extract. R. chingii extract combined with FLC has antifungal activity against FLC‐resistant C. albicans.     

中药水提物对白念珠菌生物膜抑制作用的研究

目的研究中药水提物对体外白念珠菌生物膜的影响。方法体外构建白念珠菌生物膜,微量稀释法测定中药提取物对白念珠菌浮游菌最低抑菌浓度（M IC）,XTT减低法评价中药提取物对白念珠菌生物膜SM IC（SM IC80,SM IC50）及对白念珠菌黏附能力的影响。结果野菊花、土槿皮和决明子SM IC80为125 mg/m l,其余中药SM IC80为250或500 mg/m l;地肤子SM IC50为7.81 mg/m l,川芎、苏梗、藿香和决明子SM IC50为15.63 mg/m l,其他中药SM IC50在62.5~250 mg/m l。10味中药在100 mg/m l时均能显著抑制白念珠菌细胞的黏附,10 mg/m l时地肤子、苏梗和苦参对黏附仍具抑制作用。结论该10味中药水提物对体外白念珠菌生物膜有较强抑制效应。     

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 .     

Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans

Human cathelicidin LL37 and its fragments LL13–37 and LL17–32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13–37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13–37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13–37. Bimane-labeled LL13–37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13–37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.     

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。     

Susceptibilities of Candida albicans Mouth Isolates to Antifungal Agents, Essentials Oils and Mouth Rinses

Forty Candida albicans strains isolated from patient's mouth with fixed orthodontic appliances were analyzed to their susceptibilities to antifungal agents, mouth rinses and essential oils. Susceptibility to fluconazole, econazole, miconazole and ketoconazole, amphotericin B and nystatin was assessed by the disk diffusion (DD) method based on the Clinical and Laboratory Standards Institute M44-A protocol, and by Etest (fluconazole and amphotericin B). The susceptibilities to mouth rinses and essential oils were also determined by the DD technique. All isolates tested were susceptible (S) to amphotericin B, nystatin and fluconazole. The overall concordance between the DD and the Etest was 100% for amphotericin and fluconazole. One isolate was resistant to econazole (2.5%) and the other to ketoconazole (2.5%). Econazole and ketoconazole had the highest percentages of susceptible dose dependent (SDD), 55 and 95%, respectively. Regarding to the susceptibility isolates profile, seven phenotypes were detected, and the 3 more represented (90% of the isolates) of them were SDD to one, two or three azoles. The study of mouth rinses showed a high variability of efficacy against C. albicans. The results showed that the isolates susceptibility to essential oils differed (P < 0.05). The profile activity was: cinnamon > laurel > mint > eucalyptus > rosemary > lemon > myrrh > tangerine. The main finding was that the susceptibility to cinnamon and laurel varied among the three more representative antifungal phenotypes (P < 0.05). The susceptibility of econazole-SDD isolates to cinnamon and lemon was higher than those of the econazole-S yeasts (P < 0.05). In contrast, econazole-SDD isolates were less affected by laurel than econazole-S counterparts (P < 0.05).     

Candida albicans biofilm formation is associated with increased anti-oxidative capacities

Candida albicans is a common, opportunistic, human fungal pathogen that causes a variety of mucosal and systemic afflictions. It exists in nature both in the biofilm or the sessile phase, as well as in the free-floating or the planktonic phase. Candida biofilms, in particular, display unique characteristics that confer survival advantages over their planktonic counterparts, such as their recalcitrance to common antifungals. The mechanisms underlying Candida biofilm formation and their attributes are poorly understood. In this study, we used a 2-DE-based approach to characterize the protein markers that are differentially expressed in Candida biofilms in comparison to their planktonic counterparts. Using tandem mass spectrometric analysis, we have identified a significant number of proteins including alkyl hydroperoxide reductase, thioredoxin peroxidase, and thioredoxin involved in oxidative stress defenses that are upregulated in the biofilm phase. These proteomic findings were further confirmed by real-time PCR and lucigenin-based chemiluminescence assays. In addition, we demonstrate that a drug target for the new antifungal agent echinocandin, is abundantly expressed and significantly upregulated in Candida biofilms. Taken together, these data imply that the biofilm mode, Candida, compared with their planktonic counterparts, exhibits traits that can sustain oxidative stress (anti-oxidants), and thereby exert resistance to commonly used antifungals.     

肉桂醛对根管内白色念珠菌生物膜作用的体外研究

目的研究肉桂醛对体外白色念珠菌生物膜的影响。方法采用琼脂扩散法进行肉桂醛和洗必泰对白色念珠菌敏感性的比较;MTT法评价肉桂醛对白色念珠菌生物膜及细胞黏附的影响。结果 2 048μg/mL肉桂醛与2%洗必泰抑菌环直径比较差异无统计学意义(P>0.05);4 096μg/mL肉桂醛对白色念珠菌生物膜的抑菌率达93.02%;不同浓度肉桂醛对60、90和120 min的白色念珠菌细胞粘附都具有抑制作用。结论肉桂醛对体外白色念珠菌生物膜有较明显的抑制作用。     

Molecular Analysis and Susceptibility Profiling of Candida albicans Isolates from Immunocompromised Patients in South India

The genetic diversity and in vitro antifungal susceptibility profiles of 55 Candida albicans from immunocompromised patients were studied. PCR based analysis of the transposable intron in the 25S rDNA revealed 39 genotype A, 4 genotype B and 12 genotype C isolates. Serotype analysis categorized 52 isolates as serotype A and 3 as serotype B. All strains were susceptible to micafungin, 5-flucytosine and miconazole, whereas resistance against amphotericin B (3.6%), fluconazole (3.6%), itraconazole (7.3%) and voriconazole (5.5%) was observed. No association was seen between antifungal resistance and genotype/serotype status.     

土槿乙酸对白假丝酵母菌生物膜的抑制作用

目的 探讨土槿乙酸（pseudolaric acid B,PAB）对体外白假丝酵母菌生物膜的影响。方法 甲基四氮盐（XTT）法检测不同浓度PAB和AMB（两性霉素B）对白假丝酵母菌生物膜的抑制作用。血清芽管试验检测不同浓度PAB对芽管生成的影响。结果 PAB对白假丝酵母菌生物膜的SMIC50（抑制生物膜50%的药物浓度）为256~512 μg/mL;1024和512 μg/mL PAB对早期2 h生物膜的抑制率分别为（99.5±0.28）%和（97.1±0.38）%;512 μg/mL PAB对早期（2 h）、中期（8 h）及成熟期（24 h）生物膜的抑制率分别为（97.1±0.38）%、（90.4±0.32）%和（80.1±0.67）%;不同浓度PAB的血清芽管试验显示,64 μg/mL PAB可以完全抑制白假丝酵母菌的出芽生长,16 μg/mL PAB可以抑制83.5%的白假丝酵母菌出芽生长。结论 PAB对体外白假丝酵母菌生物膜有抑制作用,对白假丝酵母菌的出芽生长过程抑制作用显著。