Evaluation of antifungal volatile compounds on the basis of the elongation rate of a single hypha

A novel method is proposed for the evaluation of the activity of an antifungal agent administered as a gas. This system is composed of a batch-flow type reaction vessel, a gas flow system, and a microscopic observation system. The agar plate was prepared on the ceiling of the reaction vessel, and the mycelium of a fungus (Aspergillus niger or Rhizoctonia solani) was inoculated onto it. After preincubation at 25 degrees C for 24 h, the reaction vessel was connected to the gas flow system. An appropriate hypha was selected, and its elongation rate was measured. Then a sample holder containing an antifungal compound was inserted into the reaction vessel from the side hole to saturate the atmosphere inside with its vapor. The retardation or inhibition of the hypha elongation was observed on a television monitor and recorded on a video tape recorder. The antifungal compound was then removed, and the reaction vessel was flushed with air. If the hypha lived, it began to elongate again. By this method, antifungal activity of seven odor compounds could be evaluated quantitatively within several hours.     

Automatic antifungal activity analyzing system on the basis of dynamic growth process of a single hypha

A system for the evaluation of antifungal activity of volatile compounds has been developed that is based on dynamic growth of a single hypha. The newly developed system is composed of a reaction vessel under a microscope, automatic stage, charge coupled device (CCD) camera, TV monitor, video tape recorder (VTR), and a microcomputer. A fungus was inoculated in the reaction vessel containing agar medium and then was treated with an antifungal reagent in the gas phase either in batch or flow reaction manner. The apex of a growing hypha displayed on a TV monitor was followed automatically. From the ratio of the growth rate under exposure of a reagent (U_EXPO) to the growth rate before the exposure (U_PRE), the antifungal activity was expressed quantitatively.     

Automatic evaluation of antifungal volatile compounds on the basis of the dynamic growth process of a single hypha

An automatic analysing system was developed and employed for the evaluation of antifungal activity of volatile compounds in the gas phase. Aspergillus niger was inoculated on agar medium in the reaction vessel. The reaction vessel was incubated at 28° C for 24 h and then a volatile compound was introduced into the vessel either in a batch or flow manner. The antifungal activity of the respective compounds estimated in situ was expressed by the dynamic response parameters of a single hypha. All volatiles tested in the present system inhibited hyphal growth, except linalyl acetate: Limone and geraniol were the most inhibitory. In contrast, linalyl acetate promoted hyphal growth. By definition of the parameters, the fungicidal and fungistatic effects could be distinguished. Correspondence to: H. Matsuoka     

Evaluation of antifungal activity of antimycotics by automatic analyzing system

Antifungal activity of several antimycotics has been evaluated using an automatic analyzing system (AAS), which is composed of a specially designed reaction vessel, microscopic observation system, image analyzing system, and computer program for automatic tracing of hypha growth. The agar plate was prepared on the ceiling of the reaction vessel, and spore mass of a fungus (Aspergillus niger) was inoculated onto it. After the preincubation at 28 °C for 24 h the reaction vessel was set on a microscope stage and connected to the liquid flow system. An appropriate hypha was selected for the measurement of growth process during the following steps: first contact with saline for 30 min for the adaptation, the second contact with same saline for 30 min, contact with saline containing an antimycotic substance for 60 min, and contact with flushing saline for 60 min. During a sequence of these steps, the apical tip of a growing hypha displayed on a TV monitor was followed automatically. The dynamic response of hypha to an agent was analyzed by several parameters. Morphological changes of the hypha caused by respective agents were recorded on VTR for further analysis. By using this system, the antifungal activity of antimycotics could be quantitatively determined within several hours.     

Transmission of the eect of an antifungal agent within a single hypha

A micro-compartment culture method was devised in which a single hypha of Rhizopus stolonifer growing on an agar section traversed an antifungal non-diffusible barrier to another agar section; thus the local environment of the distal or proximal part of the hypha could be controlled independently. The responses in terms of hyphal extension of the test fungus to local application of amphotericin B in this culture system were estimated by using an automatic analysing system. After hyphae had traversed the barrier, distal application of amphotericin B caused no appreciable effect on the proximal hyphae. In contrast, proximal application of amphotericin B caused inhibition of the extension of distal hyphae. The reversal of polarized cytoplasmic streaming also occured during the inhibition of distal hyphal extension. The extents of inhibition of the distal hyphal extension and the cytoplasmic streaming were dependent upon the hyphal distance between the amphotericin B application site and the hyphal tip. These results show that the effect of an antifungal agent on a hypha depends on the region of the hypha exposed. Cytoplasmic streaming may play key role in the transmission of antifungal effects within a single hypha.     

Mannan changes induced by 3-methyl-5-aminoisoxazole-4-thiocyanate,a new azole derivative,on Epidermophyton floccosum

The antifungal activity of 3-methyl-5-aminoisoxazole-4-thiocyanate, a new azole derivative, was studied on the dermatophyte Epidermophyton floccosum. The compound strongly inhibited the in vitro growth of two different strains of the fungus and even induced profound morphogenetic anomalies. Optical and electron microscopy showed that such treatment targets the endomembrane system, particularly the plasmalemma, causing abnormal extrusion of the wall mannans. This results in improper arrangement of the different parietal materials; the walls are thus weak and subject to subapical rupture which terminates cell growth and elongation of the hypha. The morphological results and the preliminary biochemical data on fungal sterols suggest that this compound employs an action mechanism similar to that of other azoles used in therapy.     

Influence of antifungal pre-treatment in preparing test mycelium on MIC values of several antifungal agents againstAspergillus niger

Antifungal activity of two imidazoles (miconazole and ketoconazole) and one polyene (amphotericin B) was evaluated using an automatic growth analysis system. Spores ofAspergillus niger were inoculated on the polylysine-coated glass bottom of a culture vessel. A colony formed in liquid medium was exposed to an antifungal agent and subsequently washed. Based on the dynamic growth rate of a test hypha selected from the colony in response to the antifungal agent, the minimum inhibitory concentration (MIC) was evaluated. The influence of time of reading (1, 2 and 3 h after washing) on the MIC determined was investigated. MICs for test hyphae subjected to antifungal pre-treatment were compared with those for hyphae without pre-treatment. Hyphae pre-treated with an antifungal agent for 1 h were found to become adapted and tolerant to that antifungal agent. Hyphae exposed and adapted to an imidazole obtained tolerance to amphotericin B as well as to the other imidazole.     

Determination of anti-Aspergillus activity of antifungal agents based on the dynamic growth rate of a single hypha

The dynamic growth rate of a single hypha of Aspergillus niger was analysed using an automatic system. A colony of A. niger was in contact with saline, saline containing an antifungal agent, and flushing saline, in sequence. The growth rate of a test hypha selected arbitrarily from the colony responded dynamically to the antifungal agent. The minimum concentration that caused the complete inhibition of hyphal growth was defined as the minimum inhibitory concentration (MIC). The MIC values obtained were compared with those determined by conventional methods based on increasing rate of colony diameter or dry matter weight.     

Synthesis and antifungal activity of novel s-substituted 6-fluoro-4-alkyl(aryl)thioquinazoline derivatives

6-Fluoro-4-quinazolinol is prepared by the cyclization reaction of 2-amino-5-fluorobenzoic acid and formamide. The resulting thiol obtained by treatment of hydroxyl group with phosphorus (V) sulfide is converted under phase transfer condition to 4-substituted 4-alkylthio-6-fluoroquinazoline derivatives by reaction with halide. The structures of the compounds are confirmed by elemental analysis, IR, and (1)H NMR. Title compounds 3a, 3g, and 3h are found to possess good antifungal activities. Using the mycelial growth rate method in the laboratory, the mechanism of action of 3g against Fusarium oxysporum in vitro is studied. The results indicate that 3a, 3g, and 3h have high inhibitory effect on the growth of most of the fungi with EC(50) values ranging from 8.3 to 64.2 microg/mL. After treating F. oxysporum with compound 3g at 100 microg/mL, only 6.5% of its spore bourgeoned. The permeability of the cell membrane increases along with the malformation of the hypha and condensation of its endosome. After treatment with compound 3g at 100 microg/mL within 12h, the mycelial reducing sugar, D-GlcNAc, content and chitinase activity decline, but the soluble protein content shows no obvious change.     

Key differences between lateral and apical branching in hyphae of Neurospora crassa

We examined in fine detail growth kinetics and intracellular events during lateral and apical branching in hyphae of Neurospora crassa. By high-resolution video-enhanced light microscopy, we found remarkable differences in the events preceding lateral vs apical branching. While apical branching involved a significant disturbance in the apical growth of the parental hypha, lateral branching occurred without any detectable alterations in the growth of the parental hypha. Prior to the emergence of a lateral branch, an incipient Spitzenk?rper was formed about 12-29 microm behind the apex. Lateral branch formation did not interfere with the elongation rate of the primary hypha, the shape of its apex or the behavior of its Spitzenk?rper. In sharp contrast, apical branching was preceded by marked changes in physiology and morphology of the parental hypha and by a sharp drop in elongation rate. The sequence involved a cytoplasmic contraction, followed by a retraction, dislocation, and disappearance of the Spitzenk?rper; hyphal elongation decreased sharply and a transient phase of isotropic growth caused the hyphal apex to round up. Growth resumed with the formation of two or more apical branches, each one with a Spitzenk?rper formed by gradual condensation of phase-dark material (vesicles) around an invisible nucleation site. The observed dissimilarities between lateral and apical branching suggest that these morphogenetic pathways are triggered differently. Whereas apical branching may be traced to a sudden discrete disruption in cytoplasmic organization (cytoplasmic contraction), the trigger of lateral branching probably stems from the subapical accumulation of wall precursors (presumably vesicles) reaching a critical concentration.     

CO2 angiography: studies of gas filling of vessels and assessing the factors of influence on the injection process with a cardiovascular model]

Following the advent of digital subtraction angiography (DSA), carbon dioxide gas has become a useful contrast agent for arterial angiography. Former manual injection methods had precluded accurate dosing and reproducibility. An original gas injector was therefore developed and tested in a circulatory system model. This permitted an accurate evaluation of vascular gas-filling. it also proved possible to measure the injection parameters taking different influencing factors into account. It was shown that vessels up to 10 mm in diameter are virtually completely filled with gas. In vessels larger than 10 mm in diameter, a residual fluid level remains, which in turn reduces the possibility for complete vessel imaging. The injection flow is dependent primarily on the vascular circulation rate, vessel diameter and the inclination of the vessel. With respect to other factors examined, neither catheter size, number of holes nor the direction of the injection have any relevant influence.     

Measuring kLa with randomly pulsed dynamic method

This reports on the determination of the overall oxygen transfer coefficient in a mechanically agitated vessel using a randomly pulsed dynamic method. This method consists in exciting the system by randomly switching the inlet gas stream with air or nitrogen with an identical volumetric flow rate. A pseudo-random binary sequence was used. This procedure is routinely used in process control for the identification of system's transfer function. The pulsed dynamic method gives good reliability (as compared with the traditional gassing-out method) and reproducibility in water. However, further improvement is needed before it can be used to monitor on-line the k(L)a during a fermentation.     

水霉拮抗菌的筛选及其拮抗活性物质稳定性初步研究

【目的】从海底沉积物中分离、筛选水霉拮抗放线菌菌株,鉴定目标菌株及其无菌发酵液对水霉生长的抑制效果,并初步分析拮抗活性物质的稳定性。【方法】用稀释涂布法从采集的海底沉积物中分离得到海洋放线菌,以水霉为靶菌,通过平板对峙法在PDA平板上筛选出对水霉有拮抗作用的菌株;利用其发酵液对水霉菌丝和孢子进行初步拮抗效果研究;通过16S rRNA基因序列分析对目标菌株的种属进行初步鉴定。【结果】从分离到的数十株海洋放线菌中筛选到5株水霉拮抗菌,其中拮抗效果最强的为S26菌株,16S rRNA基因序列分析结果显示其为链霉菌,并与紫色链霉菌具有较近的亲缘关系;S26马铃薯葡萄糖液体培养基发酵液在平板抑菌圈实验中,对水霉孢子萌发的抑菌圈直径达32.00 mm±0.81 mm,其5倍浓缩无菌发酵液对水霉菌丝的抑菌圈直径达39.75 mm±0.50 mm;5倍浓缩无菌发酵液抑菌活性的3.125%即能完全抑制水霉孢子的萌发;5倍浓缩液对温度具有较强耐受性,经100 °C高温30 min处理后平板抑菌圈直径为25.50 mm±0.58 mm;经不同pH值处理12 h后,pH 5.0–9.0之间仍保持较好的拮抗活性;在37 °C下蛋白酶处理2 h后实验组与对照组存在显著性差异,但平板抑菌圈直径仍可达33.25 mm以上,推测拮抗物质活性成分由多肽和非多肽类代谢物共同组成。【结论】海洋链霉菌株S26产生的活性物质对病原水霉真菌有较强的抑制作用,并对外界环境变化有较强的适应能力,因而在水霉病的生物防治中具有潜在的应用价值。研究结果同时也显示海洋链霉菌在水产病害生物防治应用领域有较好的发展前景和更广阔的挖掘空间。     

Model for Branch Initiation in Aspergillus nidulans Based on Measurements of Growth Parameters

The rate of hyphal elongation and the number of branches per hypha were measured on short sporelings of Aspergillus nidulans growing at different rates. The rate of elongation was proportional to total length in unbranched and branched hyphae. At each growth rate, the number of branches per hypha increased with increasing length and gave approximately straight-line graphs when plotted against length. The average number of branches per unit of hyphal length was quite different for the various growth rates and increased in direct proportion to the growth rate. The results are interpreted to mean that (i) growing tips have a maximum rate at which they can elongate and which is reached at hyphal lengths characteristic of the particular growth rate and (ii) a new branch is formed when the capacity of the hypha to elongate exceeds that of the existing tips.     

丛生丝孢酵母菌的生物学特性观察

探讨了丛生丝孢酵母菌的生物学鉴定特性。采用真菌培养法观察丛生丝孢酵母菌的培养特性、同化试验;耐受试验测定对放线菌酮的耐受性;体外双相形态转换试验确定霉菌相及酵母相;微量稀释法进行抗真菌药物敏感试验;温度耐受性试验、干燥试验、紫外线照射试验测定其抵抗力;小鼠腹腔接种法观察其侵袭力。本菌为双相型真菌,经分离培养该菌呈现真菌所特有的菌落特征,在沙氏肉汤中呈菌膜和沉淀生长;玉米粉琼脂小培养,在镜下可见到大小不等圆形和卵圆形孢子。对10种糖类同化试验阳性,不分解尿素,还原硝酸盐。对氟康唑等4种抗真菌药均敏感;对温度、干燥、紫外线有一定耐受力;侵袭力较弱,组织内酵母相呈细胞内感染。结论观察丛生丝孢酵母菌的生物学鉴定特性,对丛生丝孢酵母菌的实验室鉴定奠定基础和临床诊断提供依据。     

In vivo inhibitory effect on the biofilm formation of Candida albicans by liverwort derived riccardin D

Riccardin D, a macrocyclic bisbibenzyl isolated from Chinese liverwort Dumortiera hirsute, has been proved to have inhibitory effect on biofilms formation of Candida albicans in in vitro study. Our present study aims to investigate the in vivo effect and mechanisms of riccardin D against C. albicans biofilms when used alone or in combination with clinical using antifungal agent fluconazole. XTT reduction assay revealed riccardin D had both prophylactic and therapeutic effect against C. albicans biofilms formation in a dose-dependent manner when using a central venous catheter related infective animal model. Scanning electron microscope and laser confocal scanning microscope showed that the morphology of biofilms was altered remarkably after riccardin D treatment, especially hypha growth inhibition. To uncover the underlying molecular mechanisms, quantitative real-time RT-PCR was performed to observe the variation of related genes. The downregulation of hypha-specific genes such as ALS1, ALS3, ECE1, EFG1, HWP1 and CDC35 following riccardin D treatment suggested riccardin D inhibited the Ras-cAMP-Efg pathway to retard the hypha formation, then leading to the defect of biofilms maturation. Moreover, riccardin D displayed an increased antifungal activity when administered in combination with fluconazole. Our study provides a potential clinical application to eliminate the biofilms of relevant pathogens.     

A novel triterpenoid 16-hydroxy betulinic acid isolated from Mikania cordata attributes multi-faced pharmacological activities

The aerial parts of extensively used ethnomedicinal plant Mikania cordata (Burm. f.) Robinson growing wild in Bangladesh were investigated to isolate and characterize compounds responsible for the bioactivities of the plant. In the present study, a new derivatives of betulinic acid, 16-hydroxy betulinic acid [3β,16-dihydroxy-lup-20(29)-en-28-oic] was isolated and the structure of the compound was determined by NMR spectroscopic means and comparing with available literature data. The isolated compound was then investigated for different pharmacological activities including antibacterial, antifungal, analgesic, anti-inflammatory and antipyretic potential employing different methods. The compound showed potent antibacterial activity with inhibition zone of diameter ranging from 12.0 to 17.5?mm and antifungal activity with mycelial growth inhibition ranging from 37.6 to 54.5%. The MIC values for antibacterial and antifungal activities ranged from 31.5–125 and 250–1000?μg/mL respectively. The compound (50 and 100?mg/kg body weight) showed potent peripheral and central analgesic activity with 55.19% and 41% of writhing inhibition at 90?min after administration of the compound and the highest 55.98%, 79.18% elongation of reaction time, respectively. In anti-inflammatory activity screening, the compound (100?mg/kg b.w.) revealed the highest 77.08% edema inhibition at 4?h after administration of carrageenan. In antipyretic assay, 16-hydroxy betulinic acid displayed a strong antipyretic effect in yeast-induced rats. From the present study it is apparent that 16-hydroxy betulinic acid might play vital role to establish M. cordata as ethnomedicinal plant to treat wound, cuts and fever.     

Synthesis,Antifungal Activity,and Molecular Simulation Study of L–Carvone-Derived 1,3,4-Oxadiazole-Thioether Compounds

To discover potent antifungal molecules with new and distinctive structures, 20 novel L-carvone-derived 1,3,4-oxadiazole-thioether compounds 5 a – 5 t were synthesized through multi-step reaction of L-carvone, and their structures were confirmed by FT-IR, ¹H-NMR, ¹³C-NMR, and HR-MS. The antifungal activities of compounds 5 a – 5 t were preliminarily tested by in vitro method, and the results indicated that all of the title compounds displayed certain antifungal activities against the eight tested plant fungi, especially for P. piricola. Among them, compound 5 i (R=p-F) with the most significant antifungal activity deserved further study for discovering and developing novel natural product-based antifungal agents. Moreover, two molecular simulation technologies were employed for the investigation of their structure–activity relationships (SARs). Firstly, a reasonable and effective 3D-QSAR model was established by the comparative molecular field (CoMFA) method, and the relationship of the substituents linked with the benzene rings and the inhibitory activities of the title compounds against P. piricola was elucidated. Then, the binding mode of compound 5 i (R=p-F) and its potential biological target (CYP51) was simulated by molecular docking, and it was found that compound 5 i could readily bind with CYP51 in the active site, and the ligand-receptor interactions involved three hydrogen bonds and several hydrophobic effects.     

Evaluation of <Emphasis Type="Italic">Fusarium solani</Emphasis> Hyphae and Conidia Susceptibility to Amphotericin B and Itraconazole: Study of a Clinical Case

Fusarium species are hyaline moulds belonging to the hyalohyphomycosis group that are usually found in the soil and plants. This organism has emerged as a cause of disseminated invasive disease. The correlation between in vitro value and clinical efficacy is low and many patients remain unresponsive to treatment despite in vitro susceptibility. We determined growth control for Fusarium solani using the BioCell-Tracer system that measures the growth rate of a single fungal hypha, and the effect of different concentrations of amphotericin B and itraconazole. The MIC for these two drugs was also determined by a broth microdilution technique, using RPMI 1640. Different MICs for amphotericin B were obtained by the two different methods. This paper describes a case of infection due to Fusarium solani in an allogeneic bone marrow transplanted patient, the microbiological diagnostic, antifungal susceptibility tests for conidia and hypha and clinical correlation.     

Studies on the synergistic effect of amphotericin B and 5-fluorocytosine on the growth rate of single hyphae ofAspergillus fumigatus by a biocell-tracer system

The biocell-tracer system is a microscopical system to measure the growth rate of a single fungal hypha. The synergistic effect of amphotericin B (AMPH) and 5-fluorocytosine (5-FC) on the growth of hyphae ofAspergillus fumigatus was studied by using this system. Although neither 2µg/ml of AMPH nor 250µg/ml of 5-FC alone showed any effect on the hyphal growth, their combination at these concentrations showed a distinct inhibitory activity. The biocell-tracer system is useful for antifungal activity testing in filamentous fungi.