Effects of hydroxypropyl-ß-cyclodextrin on the growth and morphology of Absidia coerulea.

Cyclodextrin has been found to be an attractive novel solubilizer due to its unique material properties. Absidia coerulea is widely used in steroid bioconversion. The effects of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on the growth, morphology, and steroid-converting activity of A. coerulea CICC 40302 were systematically studied. HP-ß-CD affected A. coerulea growth, resulting in changes in its spore morphology and mycelial morphology. It induced an increase in the spore germination rate and a decrease in cell biomass at the stationary phase. Optical microscopy revealed that HP-ß-CD altered the mycelial morphology and reduced the pellet compactness of A. coerulea. A convenient and feasible computing method was used to measure pellet compactness, and it demonstrated that the compactness degree of the pellet decreased as HP-ß-CD increased, which could be attributed to the modification of the physical properties of the fermentation medium. Moreover, the changing of mycelial morphology influenced steroid-converting activity. The results showed that HP-ß-CD had multiple concentration-dependent effects on A. coerulea cells. HP-ß-CD in the proper concentration range holds great potential as a biocompatible solubilizer.

Differential growth of the fungus Absidia cylindrospora on 13C/15N-labelled media.

Many studies utilise enrichment of stable isotopes as tracers to follow the interactions occurring within soil food webs and methods have been developed to enrich bacteria, soil fauna and plant litter, Here for the first time we attempt to enrich a soil fungus to 99 atom% with (13)C and (15)N stable isotopes. In this study our objectives were to (a) assess whether the saprotrophic zygomycete fungus Absidia cylindrospora could grow on a medium enriched to 99 atom% with (13)C-glucose and (15)N-ammonium chloride, (b) to determine the level of enrichment obtained, and (c) to examine the change in growth rate of this fungus while it was growing on the dually enriched medium. To achieve this, the fungus was grown on agar enriched with (13)C and (15)N to 99 atom% and its growth rate monitored. The results showed that A. cylindrospora would grow on the highly labelled growth medium, but that its rate of growth was affected compared with the rate on either natural abundance media or media highly enriched with a single isotope ((13)C or (15)N). The implications of these results is that although the fungus is able to utilise these heavier isotopes, the biochemical processes involved in growth are affected, and consideration should be given to these differences when using stable isotope tracers in, for example, soil food web studies.

Absidia parricida plays a dominant role in biotrophic fusion parasitism among mucoralean fungi (Zygomycetes): Lentamyces, a new genus for A. parricida and A. zychae.

Within the order Mucorales (Zygomycetes), the facultative parasites Parasitella parasitica, Chaetocladium brefeldii, Chaetocladium jonesii and Absidia parricida are known to initiate biotrophic fusion parasitic interactions on a wide variety of other mucoralean hosts. Their phylogenetic relationship within the Mucorales and their ability to form parasitic structures with several known host species was examined. Together with interspecific reactions between the mycoparasites, this study found: (i) no evident differences in the spectrum of non-parasitic hosts tested within the study; (ii) A. parricida parasitises all other fusion parasites; (iii) A. parricida is ancestral to all other parasites; (iv) A. parricida is reported for the first time as phylogenetically basal to all other mucoralean fungi except the Umbelopsidaceae and (v) based on phylogenetic analyses and physiological and morphological characteristics, the slow-growing species A. parricida and Absidia zychae are removed from the genus Absidia and reclassified in the newly described genus Lentamyces.

Interaction of amphotericin B lipid formulations and triazoles with human polymorphonuclear leucocytes for antifungal activity against Zygomycetes.

The frequency of zygomycosis has increased considerably over recent years mainly in immunocompromised and diabetic patients. Little is known about the effects of host innate immunity against different Zygomycetes especially under the influence of antifungal agents. The antifungal activity of human polymorphonuclear leucocytes (PMN) in combination with liposomal amphotericin B (LAMB), amphotericin B lipid complex (ABLC), voriconazole (VRC) and posaconazole (PSC) against Rhizopus oryzae and Rhizopus microsporus, frequently isolated Zygomycetes, were studied and compared with Absidia corymbifera, a less pathogenic Zygomycete. Antifungal activity was evaluated as per cent of hyphal damage using the XTT metabolic assay. While A. corymbifera was more susceptible to PMN than the other two Zygomycetes, R. microsporus appeared to be the most susceptible to combined effects of amphotericin B formulations and VRC with PMN. LAMB exhibited synergistic activity with PMN in inducing hyphal damage to R. microsporus but not to the other fungi. In contrast, ABLC exhibited synergistic or additive activity with PMN against all three fungi. Among triazoles, only VRC exhibited additive effect with PMN against R. microsporus. Lipid formulations of amphotericin B and particularly ABLC interact with PMN predominantly in inducing augmented hyphal damage to three different species of Zygomycetes.

Sexual reproduction in subcultures of Absidia blakesleeana after years of preservation under mineral oil.

Two subcultures of Absidia blakesleeana IOC No. 2425 (-) mating type strain preserved in 1959 and 1981 on a thick layer of potato dextrose agar (PDA) medium under sterile mineral oil and maintained at room temperature in the Fungal Cultural Collection of Institute Oswaldo Cruz--IOC--were paired with the defined Absidia blakesleeana URM-UFP No. 2076 (+) mating type strain. Portions of the line where mycelia of the two strains met were observed under light microscopy. Zygospores belonging to Subgenus Mycocladus were observed. The results demonstrated conservation and re-establishment of the physiological and genetic metabolic processes of two subcultures preserved under mineral oil.

[Lipid Composition in Cell Walls and in Mycelial and Spore Cells of Mycelial Fungi].

Qualitative and quantitative differences were found between the lipids of cell walls (CW) and of whole mycelial cells and dormant cells of mucoraceous and ascomycete fungi. Thus, whole mycelial cells (WC) contained more lipids than CW. Unlike sporangiospores and conidia (exogenous dormant spores), zygotes were found to have the highest content of triacylglycerol lipids (70%). Cell walls of mucoraceous fungi contained more triacylglycerols (TAG) and less polar lipids than ascomycete lipids. While all CW and WC studied were similar in fatty acid (FA) composition, their ratio was specific for each structure: linoleic acid predominated in mycelial CW and WC, while oleic acid was predominant in the spores; this difference was especially pronounced in conidial WC. Unlike WC, in CW massive lipids may be represented not by phosphatidylethanolamine (PEA) and phosphatidylcholine (PC), but by free fatty acids (FFA), free (FSt) and etherified sterols (ESt), phosphatidic acid (PA), fatty acid methyl esters (FAME), and glycolipids (GL), which is an indication of a special functional role of CW.

Microbiological evolution of hay and relapse in patients with farmer's lung.

BACKGROUND: Recent studies in France have shown that Absidia corymbifera and, to a lesser degree Eurotium amstelodami and Wallemia sebi, play a role in farmer's lung disease (FLD), but that Saccharopolyspora rectivirgula, classically incriminated, does not. Little is known about farmers' reactions to these fungi or the circumstances which lead to exposure. AIMS: To investigate the conditions which favour the development of these microorganisms in hay and to analyse the relation between their concentration and the risk of occurrence of FLD. METHODS: Sequential microbiological analyses of each batch of hay stored in 10 farms at risk for FLD and a serological survey of 10 farmers (five with a past history of FLD). RESULTS: Exposure to microorganisms varied widely according to farms and periods. These microorganisms usually reached a peak in January and proliferated when harvesting conditions favoured excessive humidity in hay (rain during harvest, soil in the hay). Three of the five FLD patients presented with FLD respiratory recurrence and positive serology for A corymbifera during the winter (2000-01), after exposure to a significantly higher amount of A corymbifera than other farmers. Similar, but less significant, results were found for E amstelodami exposure, but not with W sebi. CONCLUSIONS: Results contribute to confirming A corymbifera as a major aetiological agent of FLD in Doubs, and encourage further studies with a view to implementing preventive measures.

Lethal invasive mucormycosis: case report and recommendations for treatment.

A case of lethal invasive mucormycosis (IM), a rare fungal infection which predominantly affects immunocompromised patients, is reported in a 73-year-old female patient who presented with a cervical abscess. The patient had asthma treated with steroids and had previously undiagnosed diabetes mellitus. Despite surgical treatment and parenteral antibiotic therapy, there was fatal progression of the condition. The pathogenesis, histological appearances and treatment of mucormycosis are discussed, particularly the importance of urgent histological examination of debrided tissue to distinguish this condition from necrotizing fasciitis (NF) earlier than microbiological culture alone would allow, thus permitting the early introduction of appropriate antifungal therapy.

Influence of fowl uropygial gland and its secretory lipid components on the growth of skin surface fungi of fowl.

Fungal species, which were shown to colonize consistently on the skin surface of the breast region of adult (1 year old) white leghorn fowl, were identified as Aspergillus sydowii, A. tamarii, A. rugulosus and Absidia corymbifera. Of these, A. sydowii and A. tamarii were the dominant forms. Two species of fungi, namely, Aspergillus niger and Scopulariopsis brevicaulis were shown to be present in the cultures of the scrubbings from breast skin surface after 60 days of captivity of the fowls. Extirpation of the uropygial gland resulted in encouragement of the in vitro population growth of all species of fungi except that of A. rugulosus. The effect was found to be very conspicuous for A. sydowii and A. tamarii, particularly after 60 days of gland removal. Addition of total lipids and the wax diester component of free-flowing uropygial secretion as 0.2% suspension in Sabouraud's agar medium of individual fungal isolates caused marked suppression of the population growth of A. sydowii, A. tamarii, Absidia corymbifera and to some extent of S. brevicaulis. Other components of secretory lipids, such as wax alcohols (2,3-alkane-diols), wax acids, triglycerides and hydrocarbons (including squalene) when supplemented separately to culture medium of individual fungi at identical concentration, were also shown to cause inhibition of the growth of most of fungal species at different degrees.

Lactones 35 [1]. Metabolism of iodolactones with cyclohexane ring in Absidia cylindrospora culture.

The metabolism of δ-iodo-γ-lactones containing cyclohexane ring with an increasing number of methyl substituents in Absidia cylindrospora was studied and seven metabolites were isolated as the products of biotransformations of these substrates. They were formed as the result of various dehalogenation pathways and four of them (hydroxylactones and epoxylactone) turned out to be new compounds. The conversion of substrates ranged from 60% to 90% and the highest conversion was observed for the iodolactone with an unsubstituted cyclohexane ring. The products were fully characterized by the spectroscopic methods and for the hydroxylactone with gem-dimethyl group at C-5 and hydroxylactone with trimethylcyclohexane system the crystal structures were obtained. The main products formed in the process of hydrolytic dehalogenation were δ-hydroxy-γ-lactones with the hydroxy group located cis in relation to lactone moiety. In case of lactone with 4,4,6-trimethylcyclohexane system the dehydrohalogenation followed by the epoxidation of double bond was also observed. One of the metabolites 4,5-epoxy-2,2,6-trimethyl-9-oxabicyclo[4.3.0]nonan-8-one was formed in the sequence of three reactions: hydroxylation at C-5, translactonization and intramolecular nucleophilic substitution of the iodine by the hydroxy group. Some of the isolated products of transformation of the iodolactone with trimethylcyclohexane system were obtained as the single enantiomers. The application of fungi studied to the dehalogenation of iodolactones could be a useful method in the production of new metabolites with oxygen-containing functional groups with antifeedant activity.

Mycophagous growth of Collimonas bacteria in natural soils, impact on fungal biomass turnover and interactions with mycophagous Trichoderma fungi.

Bacteria of the genus Collimonas are widely distributed in soils, although at low densities. In the laboratory, they were shown to be mycophagous, that is, they are able to grow at the expense of living hyphae. However, so far the importance of mycophagy for growth and survival of collimonads in natural soil habitats is unknown. Using a Collimonas-specific real-time PCR assay, we show here that the invasion of field soils by fungal hyphae (Absidia sp.) resulted in a short-term, significant increase (average fourfold) of indigenous collimonads. No such responses were observed for other soil bacteria studied (Pseudomonas, Burkholderia, PCR-denaturing gradient gel electrophoresis patterns of total bacteria and Burkholderia). Hence, it appears that the stimulation of growth of Collimonas bacteria by fungal hyphae is not common among other soil bacteria. In the same field soils, Trichoderma, a fungal genus known for mycophagous (mycoparasitic) growth, increased upon introduction of Absidia hyphae. Hence, mycophagous growth by Collimonas and Trichoderma can occur in the same soils. However, in controlled experiments (sand microcosms), collimonads appeared to have a negative effect on mycophagous growth of a Trichoderma strain. The effect of mycophagous growth of collimonads on fungal biomass dynamics was studied in sand microcosms using the same Absidia sp. as a test fungus. The growth of collimonads did not cause a significant reduction in the Absidia biomass. Overall, the study indicates that mycophagous nutrition may be important for collimonads in natural soils, but the impact on fungal biomass turnover is likely to be minor.

Revision of the genus Absidia (Mucorales, Zygomycetes) based on physiological, phylogenetic, and morphological characters; thermotolerant Absidia spp. form a coherent group, Mycocladiaceae fam. nov.

The genus Absidia comprises ubiquitously distributed soil fungi inhabiting different growth temperature optima ranging from 20-42 degrees C. Some of the mesophilic species are important biotechnologically in the biotransformation of steroids or as producers of rennin-like components, whereas species with higher growth temperature optima are of clinical relevance as opportunistic human pathogens. The aim of this study was to investigate the phylogenetic relationships between these species and to establish a revision of their systematics. For this purpose single and combined genealogies based on distance, MP, ML, and Bayesian analyses of aligned nucleotide sequences of the nuclear-encoded genes for actin (act) and for the 5.8S ribosomal RNA flanked by the ITS regions 1 and 2 (comprising 807 and 828 characters, respectively) of 16 Absidia species were reconstructed. The phylogenetic reconstructions suggest a trichotomy of the Absidia genus consisting of a mesophilic, a fast-growing thermotolerant, and a slowly-growing mycoparasitic Absidia group. The trichotomous phylogenetic grouping is concordant with the morphology of the zygospores, which are zygotes resulting from sexual conjugation between two compatible mating partners. Whereas the mesophilic group comprises the majority of absidiaceaeous species forming sterile hair-like, mycelial appendages on the suspensors of their zygospores, the thermotolerant group is characterised by the formation of smooth-walled zygospores, and the mycoparasitic group, namely Absidia parricida and A. zychae, by Mucor-like rough-walled zygospores. Based on the phylogenetic coherence of mesophilic and thermotolerant Absidia species, we propose that the two groups are separated into two distinct genera, Absidia for the mesophilic Absidia species resembling the Absidiaceae and Mycocladus for the thermotolerant species A. corymbifera, A. blakesleeana and A. hyalospora. Because Mycocladus is physiologically, phylogenetically, and morphologically distinct from the Absidiaceae s. str. we suggest that they are classified as a separate family, Mycocladiaceae fam. nov., which comprises the three species M. corymbifer, M. blakesleeanus and M. hyalospora.

Posaconazole prophylaxis in experimental systemic zygomycosis.

Three isolates of zygomycetes belonging to two different genera (Rhizopus oryzae and Absidia corymbifera) were used to produce a systemic infection in neutropenic mice. On days -2 and -1 and at 2 h prior to infection, the mice received either posaconazole (POS) at doses ranging from 20 to 80 mg/kg of body weight/day or amphotericin B (AMB) at 1 mg/kg/day. Antifungal drug efficacy was assessed by determination of the prolongation of survival, determination of the percentage of infected organs (brain, lung, spleen, and kidney), and histological examination for the number of infection foci and their sizes in brain and kidney tissues. AMB significantly prolonged the survival of mice infected with all isolates. POS significantly prolonged the survival of mice infected with zygomycetes. Cultured organs from mice infected with R. oryzae were all positive, while treated mice challenged with A. corymbifera generally showed lower percentages of infected organs compared with the percentages for the controls. Zygomycete isolates established an active infection (the presence of hyphae) in the brains and the kidneys of all controls. In mice challenged with R. oryzae, both antifungal drugs were effective at reducing the number and the size of infection foci in the kidneys. Only AMB reduced the numbers, but not the sizes, of infection foci in the brain. Finally, both drugs significantly reduced the numbers and the sizes of infection foci in both tissues of mice infected with A. corymbifera. Our data suggest that prophylaxis with POS has some potential to prevent zygomycosis.

Effect of media composition and in vitro activity of posaconazole, caspofungin and voriconazole against zygomycetes.

OBJECTIVES: The effect of different media and composition on the in vitro activity of posaconazole, caspofungin and voriconazole against 59 zygomycetes species was determined. METHODS: The media tested were RPMI 1640 medium with and without 2% glucose, antibiotic medium 3 (AM3) with and without 2% glucose, and high resolution (HR) medium. RESULTS: Posaconazole was significantly more active than caspofungin and voriconazole, both in RPMI 1640 medium with 2% glucose and in HR medium. Adding glucose improved the determination of end points, but had only minor influence on the MICs. MICs evaluated in AM3 were lower than in RPMI 1640 medium or HR medium. CONCLUSIONS: The in vivo effect of posaconazole in zygomycosis needs further evaluation.