Inhibition of hyphal development and kill of Candida albicans blastospores by noxythiolin in vitro

The cidal activity of the antimicrobial agent, noxythiolin, was investigated against a laboratory strain and a fresh isolate of Candida albicans. The order of resistance to noxythiolin was hyphal form (isolate) ≥ 25°C-grown blastospores (isolate) ≥ 37°C-grown blastospores (isolate) > laboratory strain blastospores. Noxythiolin activity was superior to that of 'equivalent' formaldehyde concentrations. Mycelial transformation in C. albicans was examined by light and scanning electron microscopy and measured in terms of percentage germination and hyphal extension. Noxythiolin, 2·5%, in contact for 30 min prevented germination of the blastospore population whereas the decomposition products, formaldehyde and N-methylthiourea, showed no appreciable effect in the expected concentrations. The implications of these results are discussed in relation to the observed clinical efficacy of noxythiolin.     

Reduced adherence and inhibition of hyphal development of Candida albicans by the antimicrobial agent polynoxylin

Polynoxylin (Anaflex R) was investigated for antimicrobial activities ancillary to its known cidal and static effects. Significant reductions in adherence of Candida albicans blastospores (oral strain) to human buccal epithelial cells were observed following polynoxylin treatment. The anti-adherence activity was concentration and time-dependent. Treatment of the epithelial cells did not result in significant reductions in adherence. Polynoxylin was also shown to inhibit the germination and hyphal development of C. albicans.     

Candida dubliniensis and Candida albicans display surface variations consistent with observed intergeneric coaggregation

Adherence of yeasts to other microorganisms and epithelial cell surfaces is important in their colonization. Comparative studies based on the coaggregation of Candida dubliniensis versus Candida albicans with Fusobacterium nucleatum and other oral bacteria suggested differences in the surfaces of these yeasts. Transmission electron microscopy was used to test the hypothesis that there are morphologic variations in the cell surface of these two species. C. dubliniensis type strain CD36 and C. albicans ATCC 18804 were grown on Sabouraud's dextrose agar at various growth temperatures. In some experiments suspensions of yeast cells were treated with dithiothreitol. Fixation for transmission electron microscopy was accomplished using dimethylsulfoxide and alcian blue added to 3% paraformaldehyde and 1% glutaraldahyde in cacodylate buffer. The cell wall of both species was predominantly electron lucent and was visibly differentiated into several layers. A thin electron dense outer layer was seen with clearly visible fibrillar structures, closely associated to the cytoplasmic membrane. The length of the fibrils of the C. albicans cells grown at 37 degrees C was approximately two times greater than those of the cells grown at 25 degrees C. The fibrils of the 37 degrees C-grown cells were thin, distinct and tightly packed whereas those of the 25 degrees C-grown cells appeared blunt, loosely spaced and aggregated. C. dubliniensis demonstrated short, blunt fibrils appearing similar to those of the 25 degrees C-grown C. albicans cells. C. dubliniensis showed no difference in the density, length and arrangement of fibrils between the 25 degrees C and 37 degrees C growth temperatures. The shortest and most aggregated fibrils seen were of the 45 degrees C-grown C. albicans cells. Dithiothreitoltreated 37 degrees C-grown C. albicans cells revealed a distorted and partially destroyed fibrillar layer. In this investigation C. dubliniensis, unlike C. albicans, displayed an outer fibrillar layer that did not vary with variations in growth temperature. In addition, the fibrils on the C. dubliniensis cells were similar to those of the 25 degrees C-grown C. albicans in that they were considerably shorter and less dense than those of the 37 degrees C-grown C. albicans cells. It can be postulated, that C. dubliniensis exhibits constant cell surface characteristics consistent with hydrophobicity and that this property may give this species an ecological advantage. Therefore, C. dubliniensis may compete well in oral environments via enhanced attachment to oral microbes and other surfaces, perhaps even more efficiently than C. albicans.     

Decrease in adherence of bacteria and yeasts to human mucosal epithelial cells by noxythiolin in vitro

Adherence of buccal and vaginal isolates of Candida albicans to buccal epithelial cells and the adherence of urine isolates of Escherichia coli and Staphylococcus saprophyticus to uroepithelial cells was quantified by light microscopy. The antimicrobial agent noxythiolin reduced the adherence of these micro-organisms in both exponential and stationary growth phases. Adherence of both the blastospore and pseudohyphal forms of C. albicans was reduced. Treatment of epithelial cells and/or micro-organisms with noxythiolin resulted in decreased adherence. No anti-adherence effect was observed with formaldehyde and N-methylthiourea, the degradative products of noxythiolin.     

An isolated Candida albicans TL3 capable of degrading phenol at large concentration

An isolated yeast strain was grown aerobically on phenol as a sole carbon source up to 24 mM; the rate of degradation of phenol at 30 degrees C was greater than other microorganisms at the comparable phenol concentrations. This microorganism was further identified and is designated Candida albicans TL3. The catabolic activity of C. albicans TL3 for degradation of phenol was evaluated with the K(s) and V(max) values of 1.7 +/- 0.1 mM and 0.66 +/- 0.02 micromol/min/mg of protein, respectively. With application of enzymatic, chromatographic and mass-spectrometric analyses, we confirmed that catechol and cis,cis-muconic acid were produced during the biodegradation of phenol performed by C. albicans TL3, indicating the occurrence of an ortho-fission pathway. The maximum activity of phenol hydroxylase and catechol-1,2-dioxygenase were induced when this strain grew in phenol culture media at 22 mM and 10 mM, respectively. In addition to phenol, C. albicans TL3 was effective in degrading formaldehyde, which is another major pollutant in waste water from a factory producing phenolic resin. The promising result from the bio-treatment of such factory effluent makes Candida albicans TL3 be a potentially useful strain for industrial application.     

Noxythiolln-resistant organisms in a district general hospital.

Twelve strains of Pseudomonas aeruginosa, three strains of Klebsiella aerogenes, and two strains of Escherichia coli were found to be resistant to noxythiolin. Some of the pseudomonads were isolated from patients in the same ward, not all of whom were on noxythiolin treatment. The strains from these patients were indistinguishable from each other on phage typing, which suggested cross-contamination. No Gram-positive organism was found to be resistant to noxythiolin. Noxythiolin should not be used before a disc diffusion sensitivity test has been performed to determine whether the organisms are sensitive to it. This is particularly important when pseudomonads are the offending organisms.     

Opsonic effect of C-reactive protein on phagocytosis and intracellular killing of virulent and attenuated strains of Candida albicans by human neutrophils.

In the presence of autologous complement, maximal phagocytosis of Candida albicans blastospores of both a virulent and attenuated strain by human neutrophils in a monolayer assay was achieved after 30 min. The proportion of phagocytes containing intracellular blastospores was 33-36% with an average of 1.5 blastospores per phagocyte. In contrast to the attenuated strain of C. albicans, the virulent strain resisted opsonization by C-reactive protein (CRP) and of those blastospores ingested, only 8% were killed. These findings support the concept that CRP may play a protective role in candidosis independent of complement. The fate of strains of different virulence may be a result of differences in CRP receptors or killing mechanisms.     

Role of blastospores in protecting Aspergillus parasiticus NRRL 3240 from high levels of aflatoxins.

The role of blastospores in the protection of Aspergillus parasiticus from high levels of aflatoxins was studied. The strain protects itself from aflatoxicity by forming thick-walled blastospores. The formation of blastospores was not observed under conditions of reduced aflatoxin formation, e.g., under zinc and asparagine deficiencies. The germination of blastospores coincided with an increase in the specific activity of glutamate dehydrogenase (NADP) and a simultaneous decrease in the specific aflatoxin production.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

A comparison of phospholipase activity, cellular adherence and pathogenicity of yeasts

Phospholipase A and lysophospholipase activities were measured in the culture fluid and in the blastospores of Candida albicans. When phospholipase activity was measured in six yeasts (four strains of C. albicans and a single strain each of Candida parapsilosis and Saccharomyces cerevisiae) a correlation was found between this activity and two potential parameters of pathogenicity. The C. albicans isolates which adhered most strongly to buccal epithelial cells and were most pathogenic in mice had the highest phospholipase activities. Non-pathogenic yeasts, including C. albicans isolates which did not adhere and did not kill mice, had lower phospholipase activities.     

重要虫生真菌莱氏野村菌芽生孢子的形态发生

经对比,萨氏麦芽糖-酵母浸粉培养液(SMY)较适合制备莱氏野村菌Nomuraea rileyi芽生孢子。以菌株Nr09接种该培养基,在130r/min、25℃全光照下震荡培养,观察芽生孢子在不同时期发育的形态变化。结果表明,分生孢子萌发产生芽管;24h萌发率为42%,36h时达84%。芽管迅速伸长形成菌丝。在48h前芽生孢子通过菌丝顶端及分枝末端缢缩的方式形成,后期则主要通过芽生孢子继续出芽的方式大量形成,78h芽生孢子数量达到最大值。芽生孢子的形成方式可为一端、两端或多端芽殖。芽生孢子的形成过程可分为5个阶段:(I)分生孢子膨大期;(II)芽管萌发期;(III)芽管延长期;(IV)芽生孢子形成初期;(V)芽生孢子指数生长期。30h后培养液中有少量草酸钙结晶出现并逐渐增多。到84h时有31%的芽生孢子细胞内液泡聚集增大,表明芽生孢子已开始进入衰老阶段。使用指数生长期制备的Nr09芽生孢子进行几丁质酶基因转化,转化效率达79个转化子/μgDNA。     

Rapid small scale preparation of bacterial genomic DNA, suitable for cloning and hybridization analysis

The uptake of noxythiolin by a urinary isolate of Escherichia coli was examined initially at 37°C but the adsorption isotherm was complicated by the concomitant degradation of the compound. When drug adsorption was investigated at 4°C, to reduce the degradation rate of the compound, it was observed that noxythiolin was taken up by the urinary isolate in a linear fashion. The resulting adsorption patterns are discussed in relation to their possible classification. The implications of this uptake are considered with respect to the antimicrobial activity of noxythiolin.     

防治西花蓟马的病原真菌-白僵菌微菌核的液体培养

微菌核是许多植物病原真菌产生的越冬结构,目前还没有关于白僵菌微菌核的报道。用含不同氮源的介质培养出了2株白僵菌菌株,RSB和SZ21的微菌核。在液体培养条件下,2株菌株不仅能产生典型的芽孢和菌丝,还能产生微菌核。在以玉米粉(RSB和SZ21菌株分别为6.5×102和6.3×102mL-1)为氮源的介质中产生的微菌核浓度高于以大豆粉(RSB和SZ21菌株分别为2.5×102和2.2×102mL-1)为氮源的介质。菌株SZ21产生的芽孢浓度(在以大豆粉和玉米粉为氮源的介质中,分别为17.9×105和7.4×105L-1)高于RSB菌株的芽孢浓度(在以大豆粉和玉米粉为氮源的介质中,分别为14.8×105和6.2×105L-1)。含有硅藻土的微菌核制剂能在真空干燥(<5%湿度)后存活下来,并且对于活力没有显著影响。干燥后的微菌核颗粒在水洋菜培养基上复水和培养后,能够萌发菌丝和产生高浓度的分生孢子。将微菌核颗粒预混到土壤中能引起西花蓟马Frankliniella occidentalis(Pergande)土栖阶段的高死亡率。这是首次报道白僵菌的微菌核的培养,并为用虫生真菌防治土栖昆虫提供了一个新的方法。     

Viability and virulence of blastospores of Metarhizium anisopliae (Metch.) Sorokin after storage in various liquids at different temperatures

Blastospores of three strains of Metarhizium anisopliae were stored in 18 liquids at 4°C, 20°C and 35°C for 18 weeks, 12 weeks or 9 days respectively. Viability was quantified by determination of their germination. In bioassays the virulence of stored blastospores was studied using adults and third instars of Locusta migratoria migratorioides (R. & F.) and compared to those of freshly produced blastospores and conidia. Generally, there was great variability in the viability of blastospores, depending on the fungal strain and the liquids used. Blastospores survived best at 4°C in 10% hydroxyethyl starch; for example, germination of M. anisopliae strain 97 still amounted to more than 80% after storage for 18 weeks. Other suitable liquids were deionized water, 25% Ringer's solution and 1% sodium alginate. The viability of blastospores stored at 20°C was considerably shorter than at 4°C. During storage for 12 weeks at 20°C the best protective liquids for M. anisopliae strain 97 were 25% Ringer's solution (43% germination), deionized water (23%) and 10% hydroxyethyl starch (23%). At 35°C, 45% of M. anisopliae strain 97 blastospores still germinated after storage for 7 days in 25% glycerol. The bioassays revealed that the virulence of blastospores after storage was comparable to that of fresh ones and even better than that of fresh conidia. In general, the LT₅₀ was about 4–6 days at an alternating day/night temperature of 28/20°C.     

The effects of amphotericin B on the interaction of Candida albicans with fibroblast cultures

Sublethal amounts of amphotericin B inhibited the interaction of Candida albicans with cultured fibroblasts. Different C. albicans clinical isolates exhibited varying degrees of sensitivity to the drug, but those isolates that were the most infective in control cultures appeared to be the most resistant to amphotericin B mediated infection inhibition. Although amphotericin B inhibited germ tube formation at the sublethal concentration of 0.3 microgram/mL, lower concentrations inhibited infection without preventing germination. The extent of this latter activity varied with the isolate and amphotericin B concentration and appeared to be related to sublethal effects on germinated yeasts. While amphotericin B effectively prevented new fibroblast infection, it did not dissociate those yeasts which had established an infection before its addition.     

Cell wall composition and protoplast regeneration in Candida albicans

The transition of blastospores to the mycelial phase in Candida albicans was induced after the blastospores were kept at 4 degrees C for several hours and then transferred to a fresh medium prewarmed at 37 degrees C. Glucan was the most abundant polymer in the wall in the two morphogenetic forms but the amount of chitin was higher in the mycelial form than in blastospores. Efficient protoplasting required reducing agents and proteases together with beta-glucanases (zymolyase). Protein synthesis in regenerating protoplasts was initiated after about 30 min. Chitin synthetase, initially very low, was incorporated in important amounts into cell membranes mainly in a zymogenic state. After a few hours chitin was the most abundant polymer found in the aberrant wall of the regenerating protoplast.     

Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects

Microsclerotia (MS), overwintering structures produced by many plant pathogenic fungi, have not been described for Metarhizium anisopliae. Three strains of M. anisopliae – F52, TM109, and MA1200 – formed MS in shake flask cultures using media with varying carbon concentrations and carbon-to-nitrogen (C:N) ratios. Under the conditions of this study, all strains produced MS, compact hyphal aggregates that become pigmented with culture age, in addition to more typical blastospores and mycelia. While all strains formed desiccation tolerant MS, highest concentrations (2.7–2.9 × 10⁸ L⁻¹ liquid medium) were produced in rich media with C:N ratios of 30:1 and 50:1 by strain F52. All three strains of M. anisopliae produced similar biomass concentrations when media and growth time were compared. Strain MA1200 produced higher concentrations of blastospores than the other two strains of M. anisopliae with highest blastospore concentrations (1.6 and 4.2 × 10⁸ blastospores ml⁻¹ on days 4 and 8, respectively) in media with the highest carbon and nitrogen concentrations. Microsclerotial preparations of M. anisopliae containing diatomaceous earth survived air-drying (to <5 % moisture) with no significant loss in viability. Rehydration and incubation of air-dried MS granules on water agar plates resulted in hyphal germination and sporogenic germination to produce high concentrations of conidia. Bioassays using soil-incorporated, air-dried MS preparations resulted in significant infection and mortality in larvae of the sugar beet root maggot, Tetanops myopaeformis. This is the first report of the production of sclerotial bodies by M. anisopliae and provides a novel approach for the control of soil-dwelling insects with this entomopathogenic fungus.     

Fungicidal activity of Candida albicans-induced murine lymphokine-activated killer cells against C. albicans hyphae in vitro.

Multiple intraperitoneal injections of inactivated Candida albicans cells resulted in the generation of cytotoxic peritoneal cells with phenotypical and functional properties similar to in vitro-generated lymphokine-activated killer (LAK) cells. Using an in vitro [3H]glucose uptake assay, C. albicans-induced LAK-like (CA-LAK) cells exhibited high levels of anti-hyphal activity, the effects being effector to target cell (E:T) ratio- and time-dependent. Maximal levels of anti-C. albicans activity (approximately 60%) were observed after 4 h and at E:T greater than or equal to 300:1. Similar patterns of anti-C. albicans activity were exerted by in vivo-activated natural killer (NK) cells, in vitro interleukin-2- (IL-2) generated LAK cells and polymorphonuclear cells. The anti-hyphal activity of CA-LAK cells was enriched by separation on a Percoll gradient, F2 and F3 fractions retaining most of the activity. Experiments using immunodepressed animals demonstrated that the in vivo lethality of the C. albicans hyphal form is significantly affected by in vitro pre-exposure to CA-LAK cells. While control mice receiving C. albicans alone had a median survival time of 2 d, mice receiving C. albicans pre-exposed to CA-LAK cells (E:T = 300:1) had a median survival time of 15 d. Overall, the susceptibility of the C. albicans hyphal form to CA-LAK cells suggests that C. albicans-induced effectors might play a significant role as a second-line defence mechanism against the C. albicans hyphal form.     

Methylotrophic extremophilic yeast Trichosporon sp.: a soil-derived isolate with potential applications in environmental biotechnology

A yeast isolate revealing unique enzymatic activities and substrate-dependent polymorphism was obtained from autochthonous microflora of soil heavily polluted with oily slurries. By means of standard yeast identification procedures the strain was identified as Trichosporon cutaneum. Further molecular PCR product analyses of ribosomal DNA confirmed the identity of the isolate with the genus Trichosporon. As it grew on methanol as a sole carbon source, the strain appeared to be methylotrophic. Furthermore, it was also able to utilize formaldehyde. A multi-substrate growth potential was shown with several other carbon sources: glucose, glycerol, ethanol as well as petroleum derivatives and phenol. Optimum growth temperature was determined at 25 degrees C, and strong inhibition of growth at 37 degrees C together with the original soil habitat indicated lack of pathogenicity in warm-blooded animals and humans. The unusually high tolerance to xenobiotics such as diesel oil (>30 g/l), methanol (50 g/l), phenol (2 g/l) and formaldehyde (7.5 g/l) proved that the isolate was an extremophilic organism. With high-density cultures, formaldehyde was totally removed at initial concentrations up to 7.5 g/l within 24 h, which is the highest biodegradation capability ever reported. Partial biodegradation of methanol (13 g/l) and diesel fuel (20 g/l) was also observed. Enzymatic studies revealed atypical methylotrophic pathway reactions, lacking alcohol oxidase, as compared with the conventional methylotroph Hansenula polymorpha. However, the activities of glutathione-dependent formaldehyde dehydrogenase, formaldehyde reductase, formate dehydrogenase and unspecific aldehyde dehydrogenase(s) were present. An additional glutathione-dependent aldehyde dehydrogenase activity was also detected. Metabolic and biochemical characteristics of the isolated yeast open up new possibilities for environmental biotechnology. Some potential applications in soil bioremediation and wastewater decontamination are discussed.     

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.