In vitro activity of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> antifungal protein (PAF) and its combination with fluconazole against different dermatophytes

Strains of five dermatophyte species (Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum and Trichophyton tonsurans) were selected for testing against Penicillium chrysogenum antifungal protein (PAF) and its combination with fluconazole (FCZ). Inhibition of microconidia germination and growth was detected with MICs of PAF ranging from 1.56 to 200 mug ml(-1) when it was used alone, or at constant concentration (100 mug ml(-1)) in combination with FCZ at from 0.25 to 32 mug ml(-1). The MICs for FCZ were found to be between 0.25 and 128 mug ml(-1). PAF caused a fungicidal effect at 200 mug ml(-1) and reduced growth at between 50 and 200 mug ml(-1). Total growth inhibition with fungistatic activity was detected at 64 mug ml(-1) of FCZ for M. gypseum, T. mentagrophytes, and T. tonsurans, and at 32 mug ml(-1) FCZ for M. canis and T. rubrum. PAF and FCZ acted synergistically and/or additively on all of the tested fungi except M. gypseum, where no interactions were detected.     

Insecticidal activity of inhibitors of polyamine biosynthesis on Spodoptera litura F. larvae.

The inhibitors of polyamine (PA) biosynthesis such as alpha-difluoromethylornithine (DFMO), methylglyoxal bis (guanylhydrazone) (MGBG) and bis (cyclohexylammonium) sulphate (BCHA) have been used to protect crop plants from pathogenic fungi. In this communication the insecticidal activity of these inhibitors on tobacco caterpillar, S. litura has been reported. All the inhibitors exhibited insecticidal activity; MGBG being more effective than others. The results suggest, for the first time, a possible avenue for the control of insect pests by specific inhibition of insect PA biosynthesis.     

Dermatophytes isolated from patients in University Hospital,Kuala Lumpur,Malaysia

A total of 576 dermatophytes were isolated from patients with a variety of skin infections from January 1993 to May 2000. Ten species of dermatophytes were identified: Epidermophyton floccosum (0.7%), Microsporum audouinii (1.1%), M. canis (3.1%), M. gypseum (0.3%), Trichophyton concentricum (3.5%), T. equinum (0.2%), T. mentagrophytes (36.1%), T. rubrum (53.8%), T. verrucosum (0.2) and T. violaceum (1.0%). The body sites most frequently affected by dermatophytes were the buttocks, nails and trunk. Anthropophilic dermatophytes made up 60.1% of the isolates; the most common species was T. rubrum, T. mentagrophytes and M. canis were the two main zoophilic dermatophytes. T. mentagrophytes was isolated from all body sites except the scalp. M. canis was found to be associated with domestic dogs and was not isolated from ethnic Malays. The only geophilic dermatophyte was M. gypseum, an uncommon dermatophyte associated with tinea pedis.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Antifungal activities of the essential oils in Syzygium aromaticum (L.) Merr. Et Perry and Leptospermum petersonii Bailey and their constituents against various dermatophytes

This study was carried out in order to investigate the potential of using plant oils derived from Leptospermum petersonii Bailey and Syzygium aromaticum L. Merr. Et Perry as natural antifungal agents. The antifungal effects of essential oils at concentrations of 0.05, 0.1, 0.15, and 0.2 mg/ml on the dermatophytes Microsporum canis (KCTC 6591), Trichophyton mentagrophytes (KCTC 6077), Trichophyton rubrum (KCCM 60443), Epidermophyton floccosum (KCCM 11667), and Microsporum gypseum were evaluated using the agar diffusion method. The major constituents of the active fraction against the dermatophytes were identified by gas chromatography-mass spectrometry and high-performance liquid chromatography analysis. The antifungal activities of S. aromaticum oil (clove oil) against the dermatophytes tested were highest at a concentration of 0.2 mg/ml, with an effectiveness of more than 60%. Hyphal growth was completely inhibited in T. mentagrophytes, T. rubrum, and M. gypseum by treatment with clove oil at a concentration of 0.2 mg/ml. Eugenol was the most effective antifungal constituent of clove oil against the dermatophytes T. mentagrophytes and M. canis. Morphological changes in the hyphae of T. mentagrophytes, such as damage to the cell wall and cell membrane and the expansion of the endoplasmic reticulum, after treatment with 0.11 mg/ml eugenol were observed by transmission electron microscopy (TEM). At a concentration of 0.2 mg/ml, L. petersonii oil (LPO) was more than 90% effective against all of the dermatophytes tested, with the exception of T. rubrum. Geranial was determined to be the most active antifungal constituent of L. petersonii oil. Taken together, the results of this study demonstrate that clove and tea tree oils exhibited significant antifungal activities against the dermatophytes tested in this study.     

Keratinolysis by poultry farm soil fungi

Two hundred and eleven dogs (including strictly house and stray dogs) and 170 cattle in and around the city of Madras, India were screened for the presence of dermatophytosis. 106 strains of dermatophytes (89 strains from dogs and 17 strains from bovines) were isolated. 57/106 strains were Trichophyton mentagrophytes var. mentagrophytes and 42/106 strains were of the Microsporum gypseum complex. 5 strains of T. rubrum and 2 strains of T. simii were also obtained in culture. A predominance of M. gypseum complex isolates was recorded in stray dogs and cattle and T. mentagrophytes var. mentagrophytes and T. rubrum in strictly house dogs. The family history of the owners of the most of the dogs had clear records of dermatophytosis. Further, the owners of the 11 dogs that yielded T. mentagrophytes var. mentagrophytes had either tinea corporis or tinea pedis. The etiological agent of all the 11 human cases was T. mentagrophytes var. interdigitale. Similarly the owners of 4 of the 5 dogs that yielded T. rubrum were known T. rubrum patients. All these patients responded to oral griseofulvin or ketaconozole, but the recurrence of lesions was noted with the cessation of treatment. None of the patients had onychomycosis and the family history of all the patients revealed no reports of T. rubrum infections. The pet dogs were presumed to be the source of re-infection. Reversed transmission of dermatophytes from humans to animals may be the reason for the selective predominance of these organisms in strictly house dogs. They also may act as sources of reinfection. Most of the animals had small, occult, scattered lesions. These lesions may either go unnoticed or are ignored by the owners of the animals. The taxonomic status of T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale was aligned to their teleomorph Arthroderma vanbreuseghemii. Our study suggests that the periodic screening and medication of all live-stock are essential for the prevention and management of the public health problem caused by dermatophytes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inhibition of polyamine biosynthesis and growth in plant pathogenic fungi in vitro

Polyamine (PA) biosynthesis inhibitors, difluoromethylornithine (DFMO), difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and bis-(cyclohexylammonium) sulphate (BCHA) have been tested for their effects on colony diameters at different intervals after inoculation of four plant pathogenic fungi (Helminthosporium oryzae, Curvularia lunata, Pythium aphanidermatum and Colletotrichum capsici). All these inhibitors, except DFMA had strongly retarded the growth of four fungi in a dose- and species-dependent fashion, and H. oryzae and C. lunata were found to be most sensitive to the effects of PA inhibitors. P. aphanidermatum and C. capsici were relatively insensitive and required rather high concentrations of inhibitors to get greater inhibition of mycelial growth, except DFMA which had stimulatory effect on the growth of these two fungi. However DFMA had greatly suppressed the growth of H. oryzae and C. lunata. The effect was generally more pronounced with MGBG than with DFMO and BCHA, and 1 mM Put completely prevented the inhibitory effects of 1 and 5 mM DFMO. Analysis of free and conjugated PAs in two sensitive fungi (H. oryzae and C. lunata) revealed that Put was present in highest concentrations followed by Spd and Spm and their levels were greatly reduced by DFMO application, and such inhibitions were totally reversed by exogenously supplied Put; in fact, PA titers were considerably increased by 1 mM Put alone and in combination with 1 mM DFMO. These results suggest that PA inhibitors, particularly DFMO and MGBG may be useful as target-specific fungicides in plants.     

Dermatophytes isolated from symptomatic dogs and cats in Tuscany,Italy during a 15-year-period

Between January, 1, 1986 and December, 31, 2000, dermatological specimens from 10.678 animals (7.650 cats and 3.028 dogs) were examined for dermatophytes. All the animals presented clinical signs of ringworm. Two thousand-four hundred fifty-six of the 10.678 (23%) examined animals scored positive for dermatophytes, 566 out of 3.028 canine (18.7%) and 1890 out of 7.650 feline specimens (24.7%). Microsporum canis constituted 83% and 97% of the isolated dermatophytes respectively in dogs and cats, M. gypseum represented 13% and 2.6% and T. mentagrophytes 5.5% and 0.2%. A sexual predisposition for mycotic infections was not observed. The animals with less than 1 year of age were more frequently infected. Canine toy breeds showed a significantly higher (P < 0.001) prevalence of infections by M. canis. Microsporum gypseum was mostly recorded from sporting (hunting) breeds [such as T. mentagrophytes (6.7%)]. Microsporum canis was isolated from long-haired cats with a ratio of 2:1 versus short-haired cats, while M. gypseum and T. mentagrophytes were never recovered from Persian cats. The annual distribution of the infections in dogs showed a significantly higher incidence for M. gypseum in summer versus winter and spring, while the recovery rate of M. canis from cats was very significantly higher in fall and winter than in summer and spring. Trichophyton mentagrophytes did not show a similar seasonal distribution.     

Antimicrobial activity of Hyptis ovalifolia towards dermatophytes

The essential oil and the aqueous, hexane and methanolic fractions from Hyptis ovalifolia leaves were evaluated for their antifungal activity in vitro against 60 strains of dermatophytes: 10 strains of Microsporum canis, 10 of M. gypseum, 20 of Trichophyton rubrum and 20 of T. mentagrophytes. The extracts inhibited growth of the dermatophytes tested at different concentrations. The most biologically active was the essential oil from the leaves which inhibited 57 isolates (95%) at a concentration of 500 g/ml.     

Inhibition of fungal growth and infection in maize grains by spice oils

Essential oils of 12 spices were tested for inhibitory activity against fungal infection and mycelial growth in post-harvest maize grains during storage. It was observed that the oils of cassia, clove (30 μl/g grain and above), star-anise (40 μl/g grain and above, geranium (30 μl/g grain and above) and basil (50 μl/g grain) inhibit the in viuo mycelial growth of established seed-borne infections of Aspergillus flavus, Curvularia pallescens and Chaetomium indicum as well as preventing infection following inoculation with A. flavus, A. glaucus, A. niger and A. sydowi . These oils also preserved the grain from natural A. flavus infection during the experimental period. Nutmeg, ginger and cumin oil (all at 50 μ1/g grain) could check mould growth and grain infection for only a brief period.     

Synthesis, characterization and antifungal activity of a series of manganese(II) and copper(II) complexes with ligands derived from reduced N,N'-O-phenylenebis(salicylideneimine)

A series of manganese(II) and copper(II) complexes with reduced Schiff bases derived from o-phenylenediamine has been prepared and characterized by elemental analysis, TG measurements, ESR, magnetic measurements, FTIR, UV-Visible spectra and conductivity. These complexes were found to be [MnL(H2O)n] and [CuL](H2O)n species with n=0-2. Their antifungal activity was evaluated on different human fungi including yeasts of the Candida genus (C. albicans, C. glabrata, C. tropicalis and C. parapsilopsis) some opportunistic moulds belonging to the Aspergillus (A. fumigatus, A. terreus and A. flavus), Scedosporium genus (S. apiospermum and S. prolificans) and some dermatophytes (M. gypseum, M. persicolor, T. mentagrophytes, M. canis and T. tonsurans). The manganese complexes showed a significant growth inhibition of the dermatophytes tested and fungi of the genus Scedosporium. This is very interesting as these fungi are usually poorly susceptible to current antifungal including Amphotericin B and Itraconazole chosen as reference in this study.     

Culture medium alkalinization by dermatophytes

95 dermatophyte strains (12 of Trichophyton mentagrophytes, 12 of T. tonsurans, 11 of T. rubrum, 12 of T. megninii, 12 of T. violaceum, 2 of T. schoenleinii, 1 of T. soudanense, 12 of M. canis, 8 of Microsporum gypseum, 1 of M. ferrugineum and 12 of Epidermphoyton floccosum). 1 of Aspergillus niger, 1 of A. ochraceus, 1 of Paecilomyces sp., 1 of Penicillium sp. and 1 of Candida albicans were grown in Sabouraud liquid medium for the study of pH variation over 6 weeks at room temperature and after 4 weeks at 37 degrees C. All the dermatophyte strains alkalinized the medium. The highest pH values after 2-3 weeks' development in room temperature were produced by T. mentagrophytes, M. gypseum and M. canis, and the lowest by T. rubrum and T. violaceum. After the 4th week the alkalinizing activity became more marked for T. mentagrophytes and remained stationary for T. violaceum, In the 5th week the values produced by T. tonsurans were higher than those for M. gypseum, and those for E. floccosum and T. megninii were higher than those of M. canis. A similar behaviour was observed for T. rubrum and T. megninii and for M. canis, M. gypseum and M. ferrugineum. There seems to be a relation between the alkalinizing capacity and the rapidity and amount of the growth. At 37 degrees C both alkalinization and range of variation of the pH values of the medium became more noticeable for the strains of each species.     

In vivo inhibition of polyamine biosynthesis and growth in tobacco ovary tissues

Post fertilization growth of tobacco ovary tissues treated with inhibitors of polyamine (PA) biosynthesis was examined in relation to endogenous PA titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17). DL-alpha-Difluoromethylornithine (DFMO) and DL-alpha-difluoromethylarginine (DFMA), specific, irreversible ("suicide") inhibitors of ODC and ADC in vitro, were used to modulate PA biosynthesis in excised flowers. ODC represented >99% of the total decarboxylase activity in tobacco ovaries. In vivo inhibition of ODC with DFMO resulted in a significant decrease in PA titers, ovary fresh weight and protein content. Simultaneous inhibition of both decarboxylases by DFMO and DFMA produced only a marginally greater depression in growth and PA titers, indicating that ODC activity is rate-limiting for PA biosynthesis in these tissues. Paradoxically, DFMA alone inhibited PA biosynthesis, not as a result of a specific inhibition of ADC, but primarily through the inactivation of ODC. In vivo inhibition of ODC by DFMA appears to result from arginase-mediated hydrolysis of this inhibitor to urea and DFMO, the suicide substrate for ODC. Putrescine conjugates in tobacco appear to function as a storage form of this amine which, upon hydrolysis, may contribute to Put homeostasis during growth.     

Basic carriers of Trichophyton mentagrophytes var. gypseum in the Sverdlovsk region

Tests for myococarriers were made on 4.297 wild small mammals. Clinical examinations only were carried out on 1,204 animals, both clinical and laboratory examinations on 3,093 animals. The suppurative form of trichophytosis was diagnosed in only one specimen of Microtus oeconomus out of 1,204 animals examined. T. ectothrix microides was found in the hair taken from the focus. T. mentagrophytes var. gypseum was obtained by culture on nutrient media. No clinical signs of mycosis were found in 3,093 animals, but seeding of the hair 175 animals (5.65%) on Sabouraud's agar with chlortetracycline and acti-dione produced cultures of T. mentagrophytes var. gypseum. The fungus was most frequently isolated in clinically healthy water rats, common field voles and field mice (Arvicola terrestris, Microtus arvalis, Apodemus agrarius). Mycocarriers were most frequently found among small mammals living in corn fields (5.9%), less frequently in water meadows (0.9) and sporadically in forests and bushes (0.1%). The possibility of bringing mycocarriers from the fields into inhabited areas during the transport of forage was reported.     

Inhibition of Aspergillus growth and aflatoxin release by derivatives of benzoic acid.

A study was conducted to determine the effects of o-nitrobenzoate, p-aminobenzoate, benzocaine (ethyl aminobenzoate), ethyl benzoate, methyl benzoate, salicylic acid (o-hydroxybenzoate), trans-cinnamic acid (beta-phenylacrylic acid), trans-cinnamaldehyde (3-phenylpropenal), ferulic acid (p-hydroxy-3-methoxycinnamic acid), aspirin (o-acetoxy benzoic acid), and anthranilic acid (o-aminobenzoic acid) upon growth and aflatoxin release in Aspergillus flavus NRRL 3145 and A. parasiticus NRRL 3240. A chemically defined medium was supplemented with various concentrations of these compounds and inoculated with spores, and the developing cultures were incubated for 4, 6, and 8 days at 27 degree C in a mechanical shaker. At the beginning of day 8 of incubation, aflatoxins were extracted from cell-free filtrates, separated by thin-layer chromatography, and quantitated by ultraviolet spectrophotometry. The structure of these aromatic compounds appeared to be critically related to their effects on mycelial growth and aflatoxin release. At concentrations of 2.5 and 5.0 mg per 25 ml of medium, methyl benzoate and ethyl benzoate were the most effective in reducing both mycelial growth and aflatoxin release by A. flavus and A. parasiticus. Inhibition of mycelial growth and aflatoxin release by various concentrations of the above-named aromatic compounds may indicate the possibility of their use as fungicides.     

Effect of dibutyryl cyclic AMP on lipid synthesis in Microsporum gypseum

The influence of intracellular levels of cAMP on the lipid synthesis of Microsporum gypseum has been examined by exogenous supplementation of dibutyryl cAMP and its activators/inhibitors. Incorporation of [14C]acetate into various lipid fractions of M. gypseum was markedly enhanced in the presence of dibutyryl cAMP and its modulators, probably as a consequence of increased intracellular cAMP levels, which, in turn, affected the lipid biosynthesis. Increased activities of phosphatidic acid phosphatase, glycerol kinase, ethanolamine kinase and choline kinase in the presence of these additives supports the enhanced synthesis of phospholipids and suggests that lipid biosynthesis is being controlled by cAMP in M. gypseum.     

In vitro survival of human pathogenic fungi in Hawaiian beach sand.

In vitro studies utilizing 4 pathogenic fungi, Trichosporon cutaneum, Candida albicans, Microsporum gypseum and Trichophyton mentagrophytes, all known from Hawaiian beaches, indicate that they survive in the fluctuating beach habitat where they can serve as potential sources of infection for significant periods of time. Survival was measured by testing the viability of propagules at intervals for 6 months. All species survived 6 months under 1 or more experimental conditions. Survival patterns showed both increases and decreases depending upon the given parameters designed to simulate various beach conditions. Propagules inoculated on hair and horn (keratinized inoculum) did not remain viable longer than propagules from pure culture suspensions (non-keratinized). Microbial antagonism was not a major factor in survival. All species survived at least 1 month in non-sterile sand inoculated with keratinized propagules. This condition approximated the natural sand habitat. Alternate wetting and drying of sand caused an overall decrease in survival time except for M. gypseum (non-keratinized inoculum) at 37 degrees C in sterile sand and T. mentagrophytes (keratinized inoculum) at 37 degrees C in non-sterile sand. Temperature was important: increasing temperature resulted in a general decrease in survival time; 45 degrees C was definitely inhibitory, with the exception of T. cutaneum which survived that level for 6 months (keratinized inoculum). Salinity did not influence survival.     

Dermatophytoses due to Microsporum gypseum: report of eight cases and literature review

Microsporum gypseum is a geophilic fungus infrequent agent of human dermatophytoses and world-wide in distribution. In Cadiz, Spain, between 1997 and 2003, a study of 133 positive cases showed that the fifth more isolated dermatophyte was M. gypseum (6.0%), followed by Trichophyton mentagrophytes (24,8%), Microsporun canis (24,6%), Trichophyton rubrum (21,8%) y Trichophyton violaceum (6,8%). During 2003 the infection due to this fungus has been repeatedly observed in our area (17.5%). We report herein eight new cases of infection by M. gypseum. Our epidemiological data were compared with those obtained by other authors in other regions of Spain and in those reported in other countries.     

Synthesis and in vitro microbiological evaluation of novel diethyl 6,6'-(1,4-phenylene)bis(4-aryl-2-oxo-cyclohex-3-enecarboxylates)

Novel bis cyclohexenone ester derivatives 14-19 were synthesized and characterized by their spectral data. In vitro microbiological evaluations were carried out for all the novel compounds 14-19 against clinically isolated bacterial and fungal strains. Compounds 15, 16, 18 against Staphylococcus aureus, 14, 15 against β-Haemolytic streptococcus, 15, 19 against Micrococcus luteus, 17, 18 against Salmonella typhii, 14, 17 against Shigella flexneri, 15 against Escherichia coli, 16 against Pseudomonas aeruginosa, 15, 18, 19 against Klebsiella pneumonia exhibited potent antibacterial activity at an minimum inhibitory concentration (MIC) value of 6.25 μg/ml, whereas compound 16 against Aspergillus flavus, 17 against A. niger, 16, 18 against Mucor indicus, 15, 17-19 against Microsporum gypseum revealed excellent antifungal activity at an MIC value of 6.25 μg/ml.     

Ecological determinants for germination and growth of some Aspergillus and Penicillium spp. from maize grain

This study compared the effect of temperature (5-45 degrees C), water availability (water activity, aw; 0.995-0.75) and their interactions on the temporal rates of germination and mycelial growth of three mycotoxigenic strains of Aspergillus ochraceus and one isolate each of A. flavus, A. niger, Penicillium aurantiogriseum and P. hordei in vitro on a maize extract medium. Germination was very rapid at > 0.90 aw with an almost linear increase with time for all species. However, at < 0.90 aw, the germination rates of A. flavus and P. hordei were slower. The aw minima for germination were usually lower than for growth and varied with temperature. The effect of aw x temperature interactions on the lag phases (h), prior to germination, and on the germination rates (h(-1)), were predicted for the first time for these fungi using the Gompertz model modified by Zwietering. This showed that A. flavus, A. niger and the two Penicillium spp. had very short lag times between 0.995-0.95 aw over a wide temperature range. At marginal temperatures, these were significantly higher, especially at < 10 degrees C for Aspergillus spp. and > 30 degrees C for Penicillium spp. There were also statistically significant differences between lag phases and germination rates for three different isolates of A. ochraceus. The Aspergillus spp. also germinated faster than the Penicillium spp. The temperature x aw profiles for mycelial growth varied considerably between species, both in terms of rates (mm d(-1)) and tolerances. Predictions of the effects of important environmental factors such as temperature, aw and their interactions on lag times to germination, germination rates and mycelial growth are important in the development of hurdle technology approaches to predicting fungal spoilage in agricultural and food products.