Dynamic and heterogeneous mutations to fluconazole resistance in Cryptococcus neoformans.

Infections with the human pathogenic basidiomycetous yeast Cryptococcus neoformans are often treated with fluconazole. Resistance to this antifungal agent has been reported. This study investigated the patterns of mutation to fluconazole resistance in C. neoformans in vitro. The MIC of fluconazole was measured for 21 strains of C. neoformans. The MICs for these 21 strains differed (0.25 to 4.0 microg/ml), but the strains were selected for this study because they exhibited no growth on plates of yeast morphology agar (YMA) containing 8 microg of fluconazole per ml. To determine their mutation rates, six independent cultures from a single original colony were established for each of the 21 strains. Each culture was then spread densely on a YMA plate with 8 microg of fluconazole per ml. A random set of putative mutants was subcultured, and the MIC of fluconazole was determined for each mutant. The 21 strains evinced significant heterogeneity in their mutation rates. The MICs of the putative mutants ranged widely, from their original MIC to 64 microg of fluconazole per ml. However, for this set of 21 strains, there was no significant correlation between the original MIC for a strain and the mutation rate of that strain; the MIC for the mutant could not be predicted from the original MIC. These results suggest that dynamic and heterogeneous mutational processes are involved in generating fluconazole resistance in C. neoformans.

Uniparental mitochondrial transmission in sexual crosses in Cryptococcus neoformans.

Restriction fragment length polymorphism (RFLP) in the large ribosomal RNA region of the mitochondrial DNA (mtDNA) was developed as a genetic marker for investigating mitochondrial transmission in sexual crosses of the human pathogenic basidiomycetous yeast Cryptococcus neoformans. Strain JEC20 of C. neoformans var. neoformans (mat a) was mated with six strains of C. neoformans var. grubii (mat alpha). Successful mating was indicated by the formation of hyphae and basidiospores. These basidiospores were examined for mtDNA RFLP genotypes. All 570 basidiospores examined from the six crosses showed the mtDNA genotype of strain JEC20. The failure to recover the C. neoformans var. grubii mtDNA in any cross indicates that the C. neoformans var. grubii mtDNA is either selectively eliminated in the newly formed dikaryon or selectively excluded in the immediate dikaryotic hyphae of the newly formed dikaryon.

Mushroom poisoning.

The majority of cases of mushroom poisoning occur in children and involve benign gastrointestinal irritants. Critical poisonings most frequently occur in adults who ingest Amanita phalloides or other mushrooms containing amanitin. Critical versus noncritical poisonings can be diagnosed with a high degree of confidence by the patient's history and initial symptoms. The most promising new medical treatment for Amanita mushroom poisoning is silibinin. In suspected cases of mushroom poisoning, it is important to obtain specimens of the ingested mushrooms, if possible, since treatment is specific to the species.

Extensive genetic divergence associated with speciation in filamentous fungi.

Little is known about genetic differentiation during speciation in fungi. The Collybia dryophila complex (Basidiomycetes: Tricholomataceae) contains several closely related groups of fungi at various levels of evolutionary divergence. Mating compatibility studies show there are several intersterile groups within the complex, three of which are distributed over two or more continents. Genetic relationships among five intersterility groups were compared by the method of DNA.DNA hybridization. Clustering techniques were used to reconstruct phylogenetic relationships of these fungi. Genic identity based on DNA.DNA hybridization corresponds well with mating compatibility. Reduced genic identity was observed between geographically isolated populations of a single mating group. This evidence suggests an allopatric mode of evolution for the C. dryophila group. These results indicate extensive genic divergence is associated with the appearance of intersterility barriers in basidiomycetous fungi. The levels of divergence observed in these and in other fungi are significantly higher than that observed in many other eukaryotes, and this suggests that the rate of molecular or morphological evolution in fungi may differ from that found in other organisms.