Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains (original) (raw)
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Infection and Immunity
Four strains of Cryptococcus neoformans var. gattii originating from Eucalyptus camaldulensis, three from Australia and one from San Francisco, were tested for their serotype, virulence for mice, and a number of genetic and molecular characteristics. All were found to be serotype B and showed significantly higher virulence for mice than did the type strains of C. neoformans var. gattii and Filobasidiella neoformans var. bacilispora, which were obtained from human cryptococcosis cases. Electrophoretic karyotypes of the strains from Australia were identical, although they were collected from sites at least 15 to 500 km apart. The electrophoretic karyotype of the strain from San Francisco was the same as that of the Australian isolates except for the mobility of one chromosome. On the contrary, no two isolates of serotype B (of a total of 11) from clinical sources were the same, regardless of their geographic origin. Furthermore, none of the clinical isolates showed a chromosomal banding pattern identical to that of Eucalyptus-originated strains. The Eucalyptusoriginated strains failed to form dikaryons when crossed with the tester strains of the two varieties of F. neoformans. Hybridization analysis with a nucleic acid probe (AccuProbe C. neoformans Culture Confirmation Test; Gen-Probe Inc., San Diego, Calif.), however, showed signals of equal intensity for clinical strains and the Eucalyptus-originated strains. Various fungi phylogenetically related to C. neoformans, including a phenol oxidase-positive strain of Cryptococcus laurentii obtained from E. camaldulensis, were negative in the nucleic acid hybridization test. These observations confirm that, in spite of karyotypic differences and the lack of dikaryon formation with the tester strains ofF. neoformans, Eucalyptus-originated C. neoformans var. gattii is the same organism as those isolated from cases of human infection. Furthermore, the C. neoformans culture confirmation test using a commercial nucleic acid probe is specific for C. neoformans.
Infection and immunity, 1998
Cryptococcus neoformans is a major fungal pathogen for patients with debilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence for C. neoformans laboratory strains. A serendipitous observation in our laboratory that one isolate of C. neoformans ATCC 24067 (strain 52D) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor chromosome siz...
Molecular Identification of Cryptococcus neoformans Serotypes
Journal of Clinical Microbiology, 2007
Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections primarily in immunocompromised hosts. Based on the genetic characteristics and serologic properties of capsular polysaccharides, three varieties and five serotypes have been defined: C. neoformans var. neoformans (serotype D), C. neoformans var. grubii (serotype A), hybrid serotype AD, and C. neoformans var. gattii (serotypes B and C). Epidemiologic features, such as geographic distribution and ecologic niche, and clinical characteristics have been shown to be associated with serotypes. At the present time, serotyping is based on agglutination tests with either commercial or "homemade" antisera or on immunofluorescence assays using a monoclonal antibody directed against the capsule polysaccharide. In this paper, we describe two molecular methods (PCR-restriction enzyme analysis and length polymorphism analysis) for C. neoformans serotype identification. Both are based on the sequence characteristics of a fragment of the CAP59 gene required for capsule biosynthesis. Testing of 72 C. neoformans strains including representatives of the five serotypes demonstrated the reliability of these methods.
Comparative Analysis of Environmental and Clinical Populations of Cryptococcus neoformans
Journal of Clinical Microbiology, 2005
Cryptococcus neoformans is a major, global cause of meningoencephalitis in immunocompromised patients. Despite advances in the molecular epidemiology of C. neoformans, its population structure and mode of reproduction are not well understood. In the environment, it is associated with avian guano or vegetation. We collected nearly 800 environmental isolates from three locations in the United States (viz., North Carolina, California, and Texas) and compared them with one another and with clinical isolates from North Carolina. As expected, they consisted of the most prevalent serotypes, serotypes A and D, as well as serotype AD hybrids. The majority of environmental isolates were obtained from pigeon excreta. All environmental and clinical isolates of serotype A or D had the MAT␣ mating-type allele. However, the AD hybrids included MATa alleles typical of serotypes A and D. Using an amplified fragment length polymorphism fingerprinting technique with two primer pairs, we identified 12 genotypes among the isolates of serotype A. Six of these genotypes were present in both the clinical and the environmental populations. However, one of the most prevalent environmental genotypes was absent from the clinical samples, and two other genotypes were isolated only from patients. The combined molecular data suggest that this environmental population of C. neoformans is predominantly clonal, although there was evidence for recent or past recombination. Cryptococcus neoformans is an exogenous, opportunistic pathogen capable of causing life-threatening infections, especially in persons with cellular immunodeficiencies, such as patients with AIDS, transplants, or hematologic malignancies (9). This yeast is ubiquitous in the environment, where it is usually associated with avian guano or vegetative debris (9, 11, 29, 40). C. neoformans is a haploid basidiomycetous species with a bipolar mating system and two alternative mating alleles, MATa and MAT␣. Strains of the opposite mating type are capable of sexual reproduction in the laboratory. However, most clinical populations are dominated by isolates of only one mating type, MAT␣, and it is unknown whether this fungus undergoes sexual recombination in nature (20, 26, 41, 42). On the basis of differences in ecology, physiology, capsular polysaccharide, and clinical manifestations, three varieties and five serotypes of C. neoformans have been recognized. The most common variety, C. neoformans var. grubii, represents isolates of serotype A, primarily infects immunocompromised individuals, and causes more than 90% of all cryptococcal infections and more than 99% of the cases of cryptococcosis in patients with AIDS (9, 24). Strains of serotype D, C. neoformans var. neoformans, also infect immunocompromised patients; however, they cause fewer cases of disease and are considered less pathogenic (9, 24). Hybrid serotype AD strains have been isolated from the environment and patients in North America and Europe (6, 18, 43, 47). Isolates of the other two serotypes, serotypes B and C, are represented by C. neoformans var. gattii, and they tend to infect immunocompetent individuals. This variety is usually con
Mycoses, 1998
Schlusselworter. Cyptococcus neofomans, DNA-Extraktion Summary. The mucopolysaccharide capsule of Cgpococcus neOfOnnans and other pathogenic yeasts prevent the extraction of DNA from these important zoonotic agents. We report that the use of a lysis buffer containing a high concentration of urea is an easy, efficient and time-saving technique to obtain high yields of good-quality DNA for molecular diagnosis. The use of urea also prevents the degradation of DNA during storage of samples at room temperature for up to 6 months. Zusarnmenfassung. Die Mukopolysaccharid-Kapsel von Cgpococcus negormans und anderen pathogenen Hefepilzen verhindern die DNA-Extraktion dieser wichtigen Erreger. Wir belegen, da8 der Einsatz eines lytischen Puffers mit hoher Harnstoff-Konzentration eine einfache, effiziente und zeitsparende Technik darstellt, um eine hohe DNA-Ausbeute guter Qualitat fur die molekulare Diagnose zu erhalten. Der Einsatz von Harnstoff verhindert zudem die Degradierung der DNA wahrend der Proben-Lagerung bei Zimmertemperatur bis zu sechs Monaten.
Molecular Taxonomy of Cryptococcus Neoformans Varieties Displaying Phenotypic Similarities
Biotechnology & Biotechnological Equipment, 2006
Cryptococcus neoformans is a pathogenic fungus and can cause life-threatening infections in humans, especially in immunocompromised patients. According to the current classification, the species consist of three varieties. The taxonomic position of these varieties is debated. We applied fluorescent Amplified Fragment Length Polymorphism (FAFLP) genotyping to analyze clinical isolates, serotypes A and D of C. neoformans. The FAFLP genotyping suggested a considerable genetic divergence between the varieties of serotypes A, D and the clinical isolates. Our FAFLP typing strategy confirms the divergence between the varieties. The FAFLP analysis might prove to be a reliable method for taxonomy, identification and typing of Cryptococcus neoformans varieties on the basis of their specific FAFLP pattern.
Molecular methods for the diagnosis and characterization of Cryptococcus : a review
Canadian Journal of Microbiology, 2010
Cryptococcosis is a fungal infection caused by yeasts of the genus Cryptococcus, with Cryptococcus neoformans and Cryptococcus gattii as the primary pathogenic species. This disease is a threat to immunocompromised patients, especially those who have AIDS. However, the disease has also been described in healthy individuals. The tests used to identify these microorganisms have limitations that make final diagnosis difficult. However, currently there are specific gene sequences that can be used to detect C. neoformans and C. gattii from clinical specimens and cultures. These sequences can be used for identification, typing, and the study of population genetics. Among the main identification techniques are hybridization, which was the pioneer in molecular identification and development of specific probes for pathogen detection; PCR and other PCR-based methods, particularly nested PCR and multiplex PCR; and sequencing of specific genomic regions that are amplified through PCR, which is especially useful for diagnosis of cryptococcosis caused by unconventional Cryptococcus sp. Concerning microorganism typing, the following techniques have shown the best ability to differentiate between fungal serotypes and molecular types: PCR fingerprinting, PCR-RFLP, AFLP, and MLST. Thus, the accumulation of data generated by molecular methods can have a positive impact on monitoring resistant strains and treating diseases.
Mycoses, 1998
Analysis of ribosomal DNA (rDNA) restriction fragment-length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) was used to investigate the genetic variability and biogeographic distribution of clinical and environmental strains of Cryptococcus neoformans isolated from a limited area of southern Italy, where the selection of a predominant cryptococcal genotype could be expected. All isolates belonged to the species Cr. neoformans variety neoformans serotype A. RFLP analysis of a specific rDNA fragment allowed the distinction of strains of Cr. neoformans from closely related fungal reference species, but neither intraspecies nor intravarieties polymorphism was detected. On the contrary, RAPD fingerprints produced by priming with four different primers [(GTG)5, (GACA)4, M13 core sequence and the 8-mer oligonucleotide (GCGGACGG)] were able to characterize the isolates up to the individual level, indicating the presence of marked heterogeneity among Cr. neoformans serotype A strains in southern Italy.