Regional Differences in Antifungal Susceptibility of the Prevalent Dermatophyte Trichophyton rubrum

Mycopathologia - In vitro susceptibility testing for Trichophyton rubrum has shown resistance to terbinafine, azoles and amorolfine, locally, but epidemiological cutoffs are not available. In order...     

Antifungal activity of 5 new synthetic compounds vs. Trichophyton rubrum and Epidermophyton floccosum

The antifungal activity of five new synthetic compounds was evaluated on two dermatophytes: Epidermophyton floccosum and Trichophyton rubrum. The data showed that the imidazo-pyrazole and pyrazolo-thiazoles were not particularly effective, while the two pyrazole-thiocyanates proved highly active on both fungi. The most active 5-amino-3-methyl-1-phenylpyrazolo-4-thiocyanate was chosen to perform SEM and TEM morphological studies on both fungi. Both SEM and TEM observations revealed interesting alterations on the two dermatophytes, particularly involving the endomembrane system. This revised version was published online in June 2006 with corrections to the Cover Date.     

红色毛癣菌的生物学特性研究

目的 观察红色毛癣菌在不同温度、不同培养基上的生长和产孢情况,并对其进行分子生物学鉴定.方法 ①大培养:采用沙堡葡萄糖琼脂(SDA)和马铃薯葡萄糖琼脂(PDA)平皿,27℃、35℃黑暗培养,测量菌落直径,绘成生长曲线.②小培养(钢圈法):采用SDA、PDA、溴甲酚紫乳固体葡萄糖琼脂(BCP-MSG)、乳蜜琼脂(M)和复合维生素B(VitB)培养基,27℃、30℃黑暗培养,观察镜下菌丝生长、孢子产生情况.③进行rDNA 18S和ITS序列测定.结果 在SDA,PDA上,27℃条件下菌落生长速度较35℃快;在5种培养基上,SDA、PDA产孢较快较多,复合维生素B培养基产孢较慢,但产生大分生孢子较多.30℃产孢更丰富.对部分菌株rDNA ITS、18S PCR扩增产物纯化后直接测序,结果在GenBank中比对、分析,相似度为98%～100%,均鉴定为红色毛癣菌.结论 SDA、PDA均为鉴定和分离红色毛癣菌的合适培养基.5种培养基均可用来刺激红色毛癣菌产孢,其中SDA、PDA产孢较早、较丰富.红色毛癣菌rDNA 18S和ITS序列测定是一种快速准确的红色毛癣菌分子生物学鉴定方法.     

Factors Affecting the Antigenicity of Trichophyton rubrum

Nitrogen determinations, performed upon the mycelia of Trichophyton rubrum, indicated that both the total nitrogen to mycelial weight ratio and the protein nitrogen to mycelial weight ratio decreased as the age of the mycelia increased. An increase in nitrogen concentration in the medium produced an increase in the total nitrogen to mycelia weight ratio, but did not necessarily increase the protein nitrogen to mycelial weight ratio. The optimal nitrogen source concentration which produced the highest protein nitrogen to mycelium ratio was found to be considerably less than that recommended in most standard Sabouraud medium formulations. Antisera to antigen preparations, grown on low concentrations of Multipeptone, produced more lines in the gel diffusion reaction than did antisera to antigens grown on standard concentrations of Multipeptone. Antisera to antigenic preparations from 2-week-old mycelia exhibited better and sometimes more lines than those of antigens prepared from 1- or 3-week-old mycelia, regardless of the nitrogen concentration in the medium. Dialysis and storage of the antigen produced no change in the quality of the precipitin lines, even though both processes involved considerable loss of Lowry protein. Immunofluorescence studies showed that young mycelia were more antigenic than the old mycelia, since a substantial degree of cell wall fluorescence was exhibited by the young mycelia, especially at the hyphal tips. Older mycelia lacked this fluorescence. An extracellular antigen was also found to be associated with the young mycelia, but cytoplasmic fluorescence was not observed.     

Antimycotic Activity Potentiation of Allium sativum Extract and Silver Nanoparticles against Trichophyton rubrum

A natural and biocompatible extract of garlic as a support, decorated with silver nanoparticles, is a proposal to generate an effective antifungal agent against dermatophytes at low concentrations. Silver nanoparticles (AgNPs) with a diameter of 26±7 nm were synthesized and their antimycotic activity was examined against Trichophyton rubrum (T. rubrum), inhibiting 94 % of growth at a concentration of 0.08 mg ml^?1. Allium sativum (garlic) extract was also obtained (AsExt), and its MIC was 0.04 mg ml^?1. To increase the antifungal capacity of those systems, AsExt was decorated with AgNPs, obtaining AsExt‐AgNPs. Using an AsExt concentration of 0.04 mg ml^?1 in independent experiments with concentrations from 0.01 to 0.08 mg ml^?1 of AgNPs, it was possible to inhibit T. rubrum at all AgNPs concentrations; it proves a synergistic effect between AgNPs and AsExt. Even if 1 % of the minimum inhibitory concentration of AsExt (0.0004 mg ml^?1) is used, it was possible to inhibit T. rubrum at all concentrations of AgNPs, demonstrating the successful antimycotic activity potentiation when combining AsExt and AgNPs.     

三种抗菌织物对红色毛癣菌和须癣毛癣菌生长抑制效应的实验研究

目的通过分析汉麻织物、含纳米银锦纶纤维织物和无机银抗菌整理后棉布对红色毛癣菌、须癣毛癣菌生长的影响,探讨抗菌织物对皮肤癣菌的抑制作用。方法制备红色毛癣菌、须癣毛癣菌的菌悬液,分别接种于上述抗菌织物,并紧贴在PDA培养基表面培养,每12 h观察菌落形态及大小,根据菌落平均直径绘制生长曲线。结果三种抗菌织物上24 h内均未见菌落生长。①汉麻布样上,红色毛癣菌和须癣毛癣菌菌落直径分别在36-84 h、36-72 h时间区间内小于棉布组,差异有显著性（P〈0.05）,之后与棉布组渐趋一致。②含纳米银锦纶织物上,在36-108 h内红色毛癣菌菌落直径小于棉布组,差异有显著性（P〈0.05）,120 h时已无显著差异（P〉0.05）,须癣毛癣菌在48 h后才开始生长,在60-108 h区间内菌落直径小于棉布对照（P〈0.05）。③无机银抗菌整理后棉布样上,红色毛癣菌和须癣毛癣菌分别在48 h和60 h后才开始生长,在120 h以内两种皮肤癣菌菌落直径均小于棉布组,差异有显著性（P〈0.05）。结论三种抗菌织物与棉布相比较,在一定时间区间内,均可显著抑制红色毛癣菌和须癣毛癣菌的生长,尤其是经无机银抗菌整理后棉布的抑菌更有效和持久。     

植物精油抑菌活性研究进展

本文综述了国内外植物精油在农用抑菌活性及抑菌活性成分研究方面所取得的成果。阐述了具有农用抑菌活性的31种植物精油及其抑菌效果,并列举了植物精油中13种具有开发潜力的抑菌活性成分,简要分析了植物精油及其主要活性成分的作用机理。     

The effect of microbial mycolytic agents on Trichophyton rubrum

Chitinolytic microorganisms isolated from forest soil and from healthy gypsy moth larvae (Porthetria dispar (L.) were screened for their ability to lyse Trichophyton rubrum mycelia. A few of these isolates were mycolytic on both autoclaved and on actively growing, intact, T. rubrum mycelia. Supernatants from these isolates, utilizing live T. rubrum as the sole carbon source, showed the same mycolytic ability. Assays of the supernatants for enzymatic activity revealed exocellular, stable enzymes that releases reducing substances including N-acetylglucosamine from the mycelia.     

Further environmental factors affecting the antigenicity of Trichophyton rubrum

Biochemical determinations performed on ammonium sulfate soluble and insoluble fractions of crude mycelial extracts ofTrichophyton rubrum indicated that these antigens were either carbohydrates or carbohydrates with peptide side chains. The antigens contained considerable amounts of galactose and mannose.Gel filtration techniques proved to be effective in separating these antigens. One antigen had a molecular weight greater than or equal to 2.0 × 10⁶. A smaller, more reactive antigen was also found; however, the elution time of this antigen varied with the concentration of dextrose in the medium.Quantitative precipitation tests used to differentiate the serological reactivity of crude antigens, recovered from mycelia grown on media containing variable concentrations of dextrose, indicated that the serological reactivity of the crude antigen was inversely proportional to the concentration of the carbohydrate source, with an optimum reactivity occurring with antigens prepared from mycelia grown in low dextrose concentrations.Nitrogen and carbohydrate concentrations performed on whole mycelia and cell free extracts demonstrated that the total nitrogen, soluble nitrogen, total carbohydrate and soluble carbohydrate concentrations were influenced by the concentration of the carbohydrate source. The optimum carbohydrate concentration necessary for the maximum ratio of protein and carbohydrates per gram of mycelium was 15.0 g/l. This is less than the amount used in most Sabouraud's dextrose media formulations. The effect of these environmental factors on the serological reactivity was discussed.Supported in part by NIH Environmental Health Tranining Grant ES00081-02. The help of Mrs. Gary Swecker is gratefully acknowledged for preparing the graphs.     

Antifungal activity of essential oils and their synergy with fluconazole against drug-resistant strains of Aspergillus fumigatus and Trichophyton rubrum

The aim of this study was to screen certain plant essential oils and active compounds for antifungal activity and their in vitro interaction with fluconazole against drug-resistant pathogenic fungi. The methods employed in this work included disc diffusion, broth macrodilution, time kill methods and checkerboard microtiter tests. Oil compositions were evaluated by gas chromatography-mass spectrometry (GC-MS) analysis. Transmission electron microscopy was used to assess the effect of essential oils on cellular structures of test fungi. Test fungal strains exhibited resistance to at least two drugs (fluconazole and itraconazole). Among the 21 essential oils or active compounds tested, ten showed promising antifungal activity. GC-MS analysis revealed the presence of major active compounds in the essential oils used. Cinnamaldehyde showed the most promising antifungal activity and killing potency against Aspergillus fumigatus MTCC2550 and Trichophyton rubrum IOA-9. Cinnamaldehyde showed strongest synergy with fluconazole against A. fumigatus and T. rubrum by reducing the minimum inhibitory concentration of fluconazole up to 8-fold. Zones of lysis of the cell wall and cell membrane appeared to be where cinnamaldehyde acted on fungi. This study highlights the broad spectrum antifungal activity of essential oils and active compounds and their synergy with fluconazole against drug-resistant fungi.     

Antifungal and Insect-Repellent Activity of Essential Oil of Zanthoxylum alatum

The essential oil of fruits of Zanthoxylum alatum (Rutaceae)proved repellent to the insect Allacophora foveicollis and fungistaticto 24 fungi, including aflatoxin-produting strains of Aspergillusflavus and A. parasiticus at a minimum dose of 2.0 x 10³ µll^–1.The fungistatic property of the oil was not affected by hightemperature, prolonged storage and increased inoculum. Essential oil, Zanthoxylum, antifungal, insect repellent     

A Study on Stability of Phenotype and Genotype of Trichophyton rubrum

Objective: To explore the stability of phenotype and genotype in Trichophyton rubrum. Methods: All the strains were cultured on Sabouraud’s dextrose agar slopes, and identified to species level. Strains isolated recently were subcultured on Sabouraud agar slopes four times at an interval of 4 weeks. DNA was extracted with CTAB method. A probe consisting of 3′ end of 18S rDNA, adjacent ITS1, 5.8S rDNA and ITS2 regions was amplified from template DNA of the T. rubrum standard strain using fungal universal primers NS5 and ITS4, labelled by P³² and hybridized with EcoR I-digested T. rubrum genomic DNA. Results: (1) Four phenotypes were isolated from 207 T. rubrum strains, with downy type (45.4%) in the first place, and granular type not found. After 1 year of conservation, 54 strains showed morphological variations with the total variation rate of 26.1%. (2) Eleven strains showed variations in colony morphology or pigment upon subculture. (3) AP-PCR analysis of 10 T. rubrum isolates and one T. rubrum standard strain showed similar DNA patterns with main bands at 2.2, 1.7, 1.3, 0.9 and 0.7 kb. No changes in DNA pattern were found upon subculture. (4) Hybridization analysis revealed that all the 11 T. rubrum strains presented three bands and were identified into two types (2.4, 3.9, 5.9 kb and 2.4, 4.4, 6.5 kb). No changes in band pattern were found upon subculture. Conclusions: Phenotype of T. rubrum was instable and the colonial morphology and pigment easily changed during conservation or subculture, while its genotype was relatively stable.     

Antifungal Activity of the Essential Oil of Hyssop (Hyssopus officinalis)

The antifungal and fungicidal effects of hyssop ( Hyssopus officinalis ) oil and its individual components were studied in a series of in vitro and in vivo experiments. Mycelial growth of the plant pathogenic fungi Pyrenophora avenae and Pyricularia oryzae was completely inhibited by 0.4% hyssop oil. Volatile components diffusing from agar medium containing 0.4% hyssop oil also completely inhibited the growth of these two fungi. Various components of hyssop oil ( L -bornyl acetate, isopinocampheol and pinocamphone), used individually, reduced growth of P. avenae and, where combinations of individual components were used, any mixture containing isopinocampheol completely inhibited fungal growth. Growth of P. oryzae was less affected by individual components of the oil. Hyssop oil reduced germination of Botrytis fabae conidia and uredospores of Uromyces viciae-fabae , but in contrast to the data from in vitro experiments, its effects on pathogen infection were less clear cut. Thus, although 0.05% hyssop oil reduced rust infection of broad bean when applied 1, 2 or 3 days before, or 1 or 2 days after inoculation, its effects against barley powdery mildew and apple powdery mildew were variable. It is suggested that this variability might be the result of the volatile components of the oil diffusing away from leaf surfaces, thus reducing the concentration of active components on the leaf surface.     

Antifungal Activity of the Essential Oil of Basil (Ocimum basilicum)

The antifungal and fungicidal effects of two chemotypes of basil (Ocimum basilicum) oil and its major individual components were studied in a series of in vitro and in vivo experiments. Mycelial growth of the plant pathogenic fungus Botrytis fabae was reduced significantly by both the methyl chavicol chemotype oil and the linalol chemotype oil, and the major individual components of the oils all reduced fungal growth, with methyl chavicol, linalol, eugenol and eucalyptol reducing growth significantly. Combining the pure oil components in the same proportions as found in the whole oil led to very similar reductions in fungal growth, suggesting that the antifungal effects of the whole oils were due primarily to the major components. When the fungus was exposed to the oils in liquid culture, growth was reduced by concentrations considerably smaller than those used in the Petri dish studies. Botrytis fabae and the rust fungus Uromyces fabae were also controlled in vivo, with the whole oils of both chemotypes, as well as pure methyl chavicol and linalol, reducing infection of broad bean leaves significantly. Most effective control of fungal infection was achieved if the treatments were applied 3 h postinoculation.     

Enzymic activities of Trichophyton rubrum and the chemotaxis of polymorphonuclear leucocytes

The enzymic activity of Trichophyton rubrum has been investigated in relation to the plasma-dependent chemotaxis of polymorphonuclear leucocytes (PMNs). In Boyden-type experiments use of a cytoplasmic extract of T. rubrum (CETr) produces neutrophil chemotactic factor (NCF) from plasma. CETr was shown to have activity for eight enzymes: heat treatment of CETr led to a partial loss of activity for seven enzymes and a significant reduction in the number of PMNs migrating. Addition of CETr to plasma and incubation for 18 h at 37 degrees C before use led to complete loss of chemotactic activity. The similar incubation of plasma with trypsin led to a complete loss of chemotactic activity. CETr has greater activity than trypsin in the production of NCF from plasma. The results are discussed in relation to reports on the importance of serine esterases in PMN chemotaxis. The failure of PMNs to migrate into keratinized tissue infected with T. rubrum is noted and it is suggested that the high enzymic activities necessary for the colonization of keratinized tissue effect a breakdown of NCF.