Selective inhibition of 14 alpha-desmethyl sterol synthesis in Candida albicans by terconazole, a new triazole antimycotic

Terconazole, a new broad spectrum antimycotic triazole derivative, has been shown to have potent activity against Candida albicans in vitro and to be effective in animal models of yeast infections. The present study explored a possible mechanism of anticandidal activity of terconazole. The compound inhibited production of 14 alpha-desmethyl sterols (e.g. ergosterol) in C. albicans at concentrations (IC50 = 3-6 x 10(-9) M) lower than those inhibiting the in-vitro growth of the yeast. There was concomitant accumulation of methylated sterols, (e.g. lanosterol), which are considered detrimental to normal yeast cell membrane function. Terconazole stimulated incorporation of 14C-acetate into triglycerides, but had no other effect on C. albicans lipid metabolism. At concentrations greater than or equal to 10(-6)M terconazole inhibited the oxidation of 14C-acetate into 14CO2 in C. albicans although the mechanism for this effect remains unclear. These data indicate that terconazole is a specific inhibitor of yeast C-14 desmethyl sterol production in C. albicans. Furthermore, terconazole reduced cytochrome P-450 levels in yeast microsomes at concentrations 10,000-fold below those at which it showed effects on rabbit liver microsomes. These data indicate a species specificity for the biochemical actions of terconazole. The C-14 alpha-desmethylase system in yeast cell membranes is cytochrome P-450 associated. Thus, terconazole, was a potent inhibitor of C-14 desmethyl sterol synthesis. This effect could contribute to the anticandidal activity of the drug.     

Terconazole - a new broad-spectrum antifungal

Terconazole, a new triazole ketal, is found to be highly active in vitro on a wide range of yeasts and mycelium-forming fungi. The in vitro activity depends largely on the medium used. In vitro it is a potent antifungal agent in preventing the morphogenetic transformation of the yeast into the (pseudo-)mycelium form of Candida albicans. In vivo terconazole is highly active in topical treatment of various experimental models of dermatophytosis and candidosis. It also possesses moderate oral broad-spectrum activity. No side effects were observed.     

Susceptibility of clinical isolates of Candida spp. to terconazole and other azole antifungal agents

Terconazole is a triazole ketal derivative with potent, broad-spectrum antifungal activity. We investigated the in vitro activity of terconazole, miconazole, and clotrimazole, against 94 clinical isolates of Candida spp.: C. albicans (n = 68), C. tropicalis (n = 18), and C. parapsilosis (n = 8). In vitro susceptibility testing was performed using a broth microdilution method. The minimal inhibitory concentrations of terconazole were less than those of miconazole against C. albicans and C. parapsilosis but higher against C. tropicalis. Terconazole was more active than clotrimazole against C. parapsilosis and less active against C. albicans and C. tropicalis. Terconazole inhibited the uptake of 14C-labeled glucose, leucine, and hypoxanthine into C. albicans and caused the rapid release of intracellular K+. Based on these studies, terconazole has promising anticandidal activity and warrants further in vitro and in vivo investigation.     

Effects of temperature on anti-Candida activities of antifungal antibiotics.

The relative growth (percentage of growth relative to control growth) of 767 Candida isolates representing five species was measured in microcultures at 25 and 37 degrees C. In the presence of 10(-4) M flucytosine, the distribution of relative yeast growth data indicated that Candida albicans isolates were less susceptible at 25 degrees C than at 37 degrees C, while the opposite was found with 4 x 10(-5) M amorolfine for most of the isolates tested. Repetition of the experiments at four different temperatures with 99 C. albicans isolates and five antifungal agents confirmed a direct relationship between growth inhibition and increasing temperature from 25 to 40 degrees C with amphotericin B, flucytosine, and terconazole; a strong inverse relationship between inhibition and temperature with amorolfine; and a weak inverse relationship with terbinafine. However, these relationships were not always noted with other Candida spp.: in particular, the growth of C. glabrata and C. parapsilosis isolates tended to be greater at 37 degrees C than at 25 degrees C in the presence of the azole-derivative antifungal agents itraconazole and terconazole. These findings stress the species-specific individuality of yeast susceptibility to azole antifungal agents. The results with C. albicans and amorolfine and terbinafine accord with their known in vivo efficacy in mycoses involving low-temperature superficial sites and poor activity against mycoses involving deep body sites. The data also reinforce the need for control of experimental variables such as temperature in the design of standardized yeast susceptibility tests.     

Effects of terconazole and other azole antifungal agents on the sterol and carbohydrate composition of Candida albicans

The effects of terconazole, a triazole antifungal, on the sterol and carbohydrate composition of Candida albicans was compared with that of three imidazoles: clotrimazole, miconazole, and butoconazole. Exposure of C. albicans to terconazole resulted in a profound depletion of ergosterol with a corresponding increase in lanosterol content versus control cells. Carbohydrate analysis revealed a significant (245%) increase in chitin and a minimal effect on glucan and mannan in terconazole-treated cells. Similar effects on sterol and carbohydrate composition were observed with clotrimazole and miconazole. Butoconazole had a similar effect on sterol composition but had no effect on carbohydrate composition. The decreased ergosterol and increased lanosterol content is consistent with 14 alpha-demethylase inhibition by terconazole and the other azoles. The increase in cell wall chitin is most likely due to deregulation of chitin synthesis secondary to ergosterol depletion in the cell membrane. Because both chitin and ergosterol are critical components of the fungal cell, perturbation of the production and localization of these components by terconazole is likely to contribute to the selective toxicity of this compound for C. albicans and other fungi.     

Effect of an arginine-glycine-aspartic acid-containing peptide on hematogenous candidal infections in rabbits.

The adherence of Candida albicans yeast cells to the subendothelial extracellular matrix, fibronectin, laminin, and type I and IV collagen was tested. Fibronectin (10(-7) M) and a peptide, PepTite-2000 (Telios Pharmaceuticals Inc., San Diego, Calif.), containing the sequence arginine-glycine-aspartic acid (RGD) inhibited Candida adherence to these targets by greater than 90%. When C. albicans was perfused over ex vivo rabbit aortic endothelium, there was no significant difference in the amount of adherence in the presence or absence of the RGD-containing peptide. However, the RGD-containing peptide reduced the number of Candida organisms present in liver, brain, heart, and kidneys (P less than 0.05) of rabbits 4 h after intravenous inoculation of 5 x 10(7) C. albicans yeast cells. The peptide also reduced the number of macroscopic Candida abscesses in the kidneys of rabbits 72 h after intravenous inoculation of 10(7) C. albicans yeast cells (P less than 0.05). Inhibition of Candida adherence in vitro and in vivo may occur because the peptide blocks a fungal receptor that is necessary for adherence.     

Growth phase in relation to ketoconazole and miconazole susceptibilities of Candida albicans

The antifungal imidazoles miconazole and ketoconazole inhibit synthesis of essential cell membrane components. Furthermore, miconazole can exert direct physicochemical cell membrane damage at relatively high levels, but ketoconazole cannot. Experiments were designed to explain our previous observation that concentrations of miconazole capable of causing direct membrane damage were no more active against Candida albicans than equimolar levels of ketoconazole. When stationary-phase cells were inoculated into medium containing either drug at 3.8 X 10(-5) M, fungistatic effects were indistinguishable. If, however, such cultures were incubated 3 h before drug addition, differences were remarkable. After 3 h, miconazole caused a 99% reduction in CFU per milliliter within 20 min, but ketoconazole again was only fungistatic. The immediate onset, rapidity, and magnitude of the miconazole effect were indicative of direct lethal cell damage. Miconazole concentrations as low as 1.0 X 10(-5) M were similarly active. It was concluded that C. albicans undergoes phenotypic changes during the growth cycle that coincidentally confer susceptibility or resistance to the lethal direct membrane damage effect of miconazole. The fungistatic or metabolic effects of ketoconazole or low-level miconazole appeared to be independent of growth phase.     

In vitro activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole compared with those of amphotericin B and fluconazole against clinical isolates of Candida spp. and fluconazole-resistant Candida albicans

OBJECTIVES: The aim of this study was to investigate the in vitro antifungal activity of an isothiosemicarbazone cyclic analogue against isolates of Candida spp. including fluconazole-resistant Candida albicans. METHODS: We investigated the activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole (EM-01D2) against 114 clinical isolates of Candida spp., representing five different species, by microdilution, according to the NCCLS method 27-A. The activity against C. albicans biofilms was also investigated. Toxicity in vitro was evaluated by MTT reduction assay. RESULTS: EM-01D2 demonstrated low toxicity, broad spectrum, fungicidal activity and was active against C. albicans and Candida krusei at concentrations lower than those shown by amphotericin B and fluconazole (P < 0.05). It maintained potent in vitro activity against fluconazole-resistant C. albicans isolates. Fungicidal activity occurred at concentrations 1-2 doubling dilutions greater than the corresponding MICs, and time-kill analysis indicated that a 99.9% loss of C. albicans viability occurred after 6 h of incubation in the presence of EM-01D2 at concentrations equal to four times the MIC. EM-01D2 was also active in inhibiting the growth of C. albicans ATCC 10231 biofilms, even though such inhibition occurred at concentrations higher than the MICs determined under planktonic growth conditions. However, when C. albicans biofilms were pre-exposed to subinhibitory concentrations of EM-01D2, a reduction of MIC50 of amphotericin B was observed. CONCLUSIONS: Based on these results, EM-01D2 could represent a template for the development of novel fungicidal agents.     

Regulated overexpression of CDR1 in Candida albicans confers multidrug resistance

OBJECTIVES: Information on the function of Candida albicans ATP-binding cassette (ABC) membrane transporter Cdr1p has come from studying the effect of gene inactivation in C. albicans and from heterologous Cdr1p expression in the yeast Saccharomyces cerevisiae. These approaches, however, give only an indirect indication of Cdr1p function in C. albicans itself. The objective of this study was to determine Cdr1p function in C. albicans by induced overexpression of Cdr1p in a C. albicans CDR1-deleted strain. METHODS: The C. albicans CDR1 open reading frame was fused to the C. albicans HEX1 promoter and used to complement a CDR1-null mutant to create strain FL3. The effect of inducing the FL3 HEX1 promoter, by growth on medium containing N-acetylglucosamine (GlcNAc) as the carbon source, on CDR1 expression and drug susceptibility was determined. RESULTS: C. albicans FL3 cells grown on medium containing GlcNAc overexpressed CDR1 mRNA and a 170 kDa plasma membrane protein that reacted with anti-Cdr1p antibodies. Overexpression of Cdr1p in C. albicans FL3 conferred resistance to structurally unrelated chemicals such as terbinafine, brefeldin A, cerulenin and nigericin as well as to azole antifungal agents, but not resistance to polyene antibiotics. FK506, ascomycin and ciclosporin A chemosensitized FL3 to fluconazole. FL3 cells grown on GlcNAc effluxed 5.3 times as much Cdr1p substrate rhodamine 6G, over a 10 min period, as FL3 cells grown on glucose, and this rhodamine 6G efflux was inhibited by including fluconazole in the assay. CONCLUSION: This study provides the first direct demonstration of Cdr1p pump activity in C. albicans.     

Proposed quality control ranges for 2-microg anidulafungin disk diffusion testing and ability of the method to detect strains with elevated echinocandin MIC values

Anidulafungin, a newer echinocandin with potent antifungal activity, was tested in an 8-laboratory M23-style quality control (QC) study to determine disk diffusion (DD) method ranges. Two lots of 2-microg disks (MAST and BD) and 3 lots of Mueller-Hinton agar (with 0.5 microg/mL methylene blue + 2% glucose) were used against 4 QC strains. Proposed ranges are Candida albicans ATCC 90028 at 24 to 39 mm (95.0% in range), Candida krusei ATCC 6258 at 20 to 35 mm (94.7%), Candida parapsilosis ATCC 22019 at 15 to 27 mm (95.5%), and Candida tropicalis ATCC 750 at 21 to 38 mm (94.6%); however, the ranges were very wide (13-18 mm), indicating between observer interpretive or reagent variation and compromised reproducibility. Challenge strains with elevated echinocandin MIC values (> or = 4 microg/mL, nonsusceptible) were tested (DD and MIC) and compared with isolates with reproducible anidulafungin MIC results at 2 microg/mL (susceptible). Discrimination between these nonsusceptible and susceptible (MIC, < or = 2 microg/mL) yeast populations was very acceptable, for example, 97.6% categoric agreement by DD method.     

Effect of matrix metalloprotease inhibitors on the 95 kDa metallopeptidase of Candida albicans

A 95 kDa metallopeptidase of Candida albicans could be involved in the process of dissemination of the yeast. Matrix metalloproteases (MMPs) are also responsible for collagen breakdown in inflammatory and malignant processes. We tested six compounds on the C. albicans enzyme. Doxycycline, gentamicin, cefalothin, galardin, and elaidic and oleic acids are known for their capacity to inhibit some MMPs. Amongst these agents, only oleic acid was able to markedly inhibit the purified metallopeptidase at very low concentrations. Moreover, this fatty acid inhibited the secretion of the enzyme in the culture medium without altering the yeast viability.     

不同浓度5-盐酸氨酮戊酸光动力疗法对白色念珠菌耐药菌株的体外抑菌作用

目的探索不同浓度5-盐酸氨酮戊酸光动力疗法对白色念珠菌耐药菌株的体外抑菌作用,为临床解决白色念珠菌耐药问题提供理论依据。方法先用酵母样真菌药敏试剂盒(微量稀释法)确定白色念珠菌耐药菌株的药物敏感性,然后利用光动力疗法对其进行体外抑菌试验,分别利用XTT法和菌落计数平板法检测抑菌和灭菌效果,最后利用透射电子显微镜观察光动力疗法处理后的白色念珠菌微观形态。结果白色念珠菌耐药菌株对氟康唑(FCA)、伊曲康唑(ITR)和伏立康唑(VRC)耐药。XTT法检测得出,随着ALA治疗浓度升高,白色念珠菌的活力逐渐降低,在0.05~0.1 mol/L浓度段活力下降最快,当ALA治疗浓度达到0.15 mol/L时,白色念珠菌的活力基本消失。菌落计数平板法显示,浓度为2.25 mol/L的ALA能彻底使1×10~5CFU/mL的白色念珠菌死亡。白色念珠菌耐药菌株经过1 mol/L的ALA光动力治疗后,透射电子显微镜下显示细胞壁破坏,细胞膜扭曲,胞质未见凝结,细胞核较完整。结论体外光动力疗法对白色念珠菌耐药菌株有明显的抑菌作用,不同浓度ALA的抑菌和灭菌效果不同,光动力疗法可试用于临床白色念珠菌耐药病例。     

Commercial systems for fluconazole susceptibility testing of yeasts: comparison with the broth microdilution method

Fluconazole susceptibility was tested in 100 clinical yeast isolates (65 Candida albicans, 13 C. glabrata, 8 C. tropicalis, 7 C. parapsilosis, 3 Saccharomyces cerevisiae, 1 each of C. krusei, C. lusitaniae, Cryptococcus neoformans, Rhodotorula glutinis) and two control strains (Candida krusei ATCC 6258, C. parapsilosis ATCC 22019) using broth microdilution (reference method), disk diffusion, Etest strips, Sensititre YeastOne, Candifast, Integral System Yeasts. Using M27-A breakpoints, isolates were classified as susceptible (81%), susceptible-dose dependent or Resistant with broth dilution. Rates of concordance with the reference method were good for Sensititre YeastOne, Etest and disc-diffusion (81.2%-94.7%) but very low for the Candifast (3.1%) and Integral System (16.6%), which classified most susceptible isolates as resistant. Lack of standardisation (inoculum, medium composition) and non-objective interpretation schemes may be the cause of their poor performance. Sensititre YeastOne, Etest and disc-diffusion are potentially useful for fluconazole antifungal susceptibility testing of yeasts in clinical laboratories.     

Sodium hypochlorite decontamination of split-thickness cadaveric skin infected with bacteria and yeast with subsequent isolation and growth of basal cells to confluency in tissue culture.

The ability of sodium hypochlorite to decontaminate skin while leaving sufficient epidermal cell viability for growth in tissue culture was investigated with an in vitro system. Split-thickness cadaveric skin was infected with Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans and subsequently treated with various concentrations of sodium hypochlorite for various time intervals. Exposure to a 0.5% solution of sodium hypochlorite for 6 min effectively decontaminated the skin while leaving 66% of the basal cells viable. The basal cells were subsequently grown to confluency in tissue culture. This study demonstrates that microbial colonization of skin can be eliminated by exposure to dilute hypochlorite. This procedure, while decontaminating the skin, leaves sufficient viability of epidermal cells for subsequent growth and expansion in tissue culture, elements essential for grafting over wounds.     

Initial results from a longitudinal international surveillance programme for anidulafungin (2003)

OBJECTIVES: This longitudinal study evaluated the in vitro activity of anidulafungin against 880 clinical yeast isolates and 68 mould isolates from 64 medical centres in North America, Latin America and Europe. METHODS: MICs of anidulafungin, amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, ketoconazole and voriconazole were determined using reference method (M27-A2) guidelines. The M38-A reference method was used for the filamentous fungi, including determination of minimum effective concentrations (MECs) of anidulafungin. RESULTS: Anidulafungin was more active when compared with fluconazole and itraconazole for Candida albicans (MIC(90), 0.06 mg/L), Candida tropicalis (MIC(90), 0.06 mg/L), Candida glabrata (MIC(90), 0.12 mg/L), Candida krusei (MIC(90), 0.06 mg/L) and Candida lusitaniae (MIC(90), 1 mg/L) as well as the less-often encountered yeast species. Anidulafungin was less active against Candida parapsilosis, Candida guilliermondii and Candida famata (MIC(50), 1-2 mg/L). Anidulafungin also exhibited excellent activity against all Aspergillus spp. (MEC(90), < or =0.03 mg/L). Anidulafungin was also evaluated comparing two end point reading criteria and two incubation intervals. Data indicate that longer incubation periods do not significantly influence overall MIC ranges. These international surveillance results for anidulafungin confirm the activity observed in studies of smaller numbers of isolates.     

Effect of fluconazole on viability of Candida albicans over extended periods of time.

The treatment of chronic mycoses may expose the infecting organisms to antimicrobial agents for extended periods of time. It is possible that an azole antifungal drug such as fluconazole, with primarily fungistatic activity in standard in vitro susceptibility tests, might be able to damage the fungal cells and reduce their viability over prolonged incubations under nonproliferating conditions. To test this possibility, Candida albicans yeast cells were exposed to various concentrations of fluconazole in RPMI 1640 tissue culture medium for 4 h at 37 degrees C, washed free of the drug, and then incubated at 37 degrees C for a 28-day period; enumeration of the remaining CFU at various times during this period revealed no increased loss of viability for the fluconazole-exposed organisms. However, when fluconazole was added to the organisms maintained in distilled water (with or without pretreatment with the drug), a marked reduction of viability was found. At 14 days of incubation with two strains of C. albicans, negative cultures were found for 7 of 10 and 10 of 11 samples, respectively, containing 1.0 microgram of fluconazole per ml versus 0 of 10 and 1 of 11 control samples (P of < 0.01 and 0.001, respectively). The effect of fluconazole on fungal viability under these conditions became noticeable at approximately 7 days and was greater when the samples were incubated at 37 degrees C rather than 25 degrees C. These findings suggest that fluconazole may have fungicidal effects on fungal cells during prolonged exposures under conditions in which the organisms are prevented from proliferating by lack of nutrients.     

In vitro studies of a new antifungal triazole, D0870, against Candida albicans, Cryptococcus neoformans, and other pathogenic yeasts.

We investigated the effects of various assay conditions on the activity of D0870 against seven species of fungi in the broth macrodilution testing procedure proposed by the National Committee for Clinical Laboratory Standards (NCCLS). Multivariate analysis demonstrated that endpoint definition, starting inoculum size, medium composition, type of buffer, and length of incubation, but not pH or temperature, had significant effects on results. Increasing the inoculum from 10(2) to 10(5) yeast cells/ml raised the MICs for all isolates up to > 75,000 fold. This effect was greatest when endpoints corresponded to a 90% reduction in visually determined turbidity (MIC90), was less prominent with an 80% inhibition visual endpoint (MIC80), and was nearly absent with a 50% endpoint measured by a spectrophotometer (IC1/2). Differences due to medium composition were attributable to antibiotic medium 3 with RPMI and yeast nitrogen base media performing nearly identically. Under standardized conditions as specified in NCCLS document M27-P (Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Proposed Standard, 1992), 79 strains (5 to 25 strains for each species) demonstrated median MIC80s of 0.0037 and 0.0075 microgram/ml for Candida albicans and Cryptococcus neoformans, respectively. In contrast, Candida krusei and Torulopsis glabrata had a median MIC80 of 1.0 microgram/ml. Our studies indicate that the pathogenic yeasts C. albicans and C. neoformans are more susceptible to D0870 than other pathogenic yeasts.     

白假丝酵母菌不同菌株荚膜厚度对动物致病性影响的对比

背景：作者前期实验发现多株白假丝酵母菌菌株具有荚膜结构，荚膜与该菌的毒力是否有关？ 目的：观察白假丝酵母菌标准菌株与临床分离菌株对动物的致病差异，验证荚膜是否是该菌的毒力因子，并分析不同菌株对动物致病性与荚膜厚度的关系。 设计：随机对照动物实验。 单位：赣南医学院病原生物学教研室。 材料：实验于2005—05／06在赣南医学院科研中心完成。选用BALB／c小鼠120只，健康成年家兔72只。白假丝酵母菌：中央标准菌株（菌号：CCCMC1a），国际标准菌株（菌号：ATCC 14053），分离培养的4株临床菌株暂行自编菌号为C1—1，C1—2，C1—3，C1-4。 方法：将实验动物按随机数字表法分为6组，即CCCMC1a，ATCC14053，C1—1，C1-2，C1—3，C1-4组，每组小鼠20只，家兔12只。将实验菌种沙保氏琼脂36h培养物用生理盐水配成菌悬液，家兔耳缘静脉注射，1．5mL/只；BALB/c小鼠腹腔注射，0．5mL/只。注射后6h开始观察动物的反应，记录动物发病时间、死亡时间及死亡率。 主要观察指标：①家兔肾组织印片及小鼠腹腔液涂片观察。②Hiss荚膜染色镜检，显微测微计测量荚膜层厚度（n=40），比较各菌株荚膜层厚度均值。 结果：①与C1-1，C1-3，CCCMC1a，ATCC 14053相比，C1—2和C1—4对动物具有很强的致病性。②标准菌株和临床分离菌株在家兔及小白鼠体内均可形成荚膜，荚膜层的厚度可因菌株和动物种属不同而存在差异（P〈0．05—0．01）。兔肾感染灶的菌细胞较小鼠腹腔内菌细胞的荚膜宽厚；C1—1，C1-2，C1—3，C1-4组兔肾感染灶的菌细胞及鼠腹腔内菌细胞的荚膜分别比同种属CCCMC1a，ATCC 14053组宽厚，其中C1-2，C1—4组兔肾感染灶的菌细胞及鼠腹腔内菌细胞的荚膜最为宽厚。结论：白假丝酵母菌C1-2和C1-4菌株对动物具有很强的致病性，它们的荚膜比其他菌株更宽厚。初步验证了荚膜可能是白假丝酵母菌的毒力因子，荚膜的宽厚与动物致病性有着密切的联系。     

The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro

Caspofungin acetate is an antifungal antibiotic that inhibits synthesis of 1,3-beta-D-glucan, an essential component of the fungal cell wall. While caspofungin causes cell death in yeasts and dimorphic fungi such as Candida albicans, its effect on Aspergillus fumigatus is less well understood. We used the fluorescent dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC), which stain live and dead cells, respectively, to further characterize the antifungal activity of caspofungin. For comparison, compounds whose mode of action was either fungistatic (fluconazole, itraconazole) or fungicidal (amphotericin B) were also evaluated. A correlation between caspofungin-induced loss of viability, decreased CFDA staining, and increased DiBAC staining was established first with C. albicans. For A. fumigatus, caspofungin caused similar dye-staining changes, which were quantified by fluorimetric analysis of stained hyphae grown in a medium that promoted dispersed growth. The minimum concentration of caspofungin required to produce these changes also decreased the level of growth-dependent reduction of the indicator dye Alamar Blue. We observed a differential effect of caspofungin as a function of cell position: 88% of apical cells and 61% of subapical branching cells failed to stain with the viable dye CFDA, but only 24% of subapical cells were unstained. Complementary results were seen with germlings from DiBAC-stained, caspofungin-treated cultures. Extended incubation of A. fumigatus with a single dose of caspofungin affected the same proportion of apical and subapical branching cells for up to 72 h. The dye-staining patterns illustrate that the cells at the active centers for new cell wall synthesis within A. fumigatus hyphae are killed when they are exposed to caspofungin.     

Morphological Changes in Yeasts as a Result of the Action of 5-Fluorocytosine

The mode of action of 5-fluorocytosine on Candida albicans and Saccharomyces cerevisiae was studied with special reference to morphology and ultrastructure. Shortly after contact of yeast cells with 10 mug of 5-fluorocytosine per ml, marked enlargement of the cells occurred. The initial increase in cell size was almost the same with both yeast strains. As incubation proceeded, further increase in cell volume continued with C. albicans, whereas no further change in cell volume was observed with S. cerevisiae. An electron microscope examination of C. albicans cells after exposure to the drug revealed characteristic changes, consisting of an enlarged nucleus and a thin cell wall. The morphological changes were exactly comparable to those reported with inhibitors of deoxyribonucleic acid synthesis in mammalian cells and some bacteria.