Experimental infection of almond trees seedlings (Terminalia catappa) with an environmental isolate of Cryptococcus neoformans var. gattii, serotype C

Recently, our laboratory reported the isolation of Cryptococcus neoformans var. gattii, serotype C for the first time from almond trees (Terminalia catappa) detritus. The aim of the present study was to establish the survival of C. neoformans in almond trees seedlings. Thirty seedlings were infected in the stems and samples were taken and processed at different times and by different techniques. No morphological alterations (macro or microscopic) were observed in the infected plants. However, C. neoformans was found to be viable for at least 100 days after infection. These data constitute our first approach towards the understanding of the yeast interactions with a host-plant.     

Diversity among strains of Cryptococcus neoformans var. gattii as revealed by a sequence analysis of multiple genes and a chemotype analysis of capsular polysaccharide.

We demonstrated the diversity of Cryptococcus neoformans var. gattii strains by a sequence analysis of multiple genes: (i) the intergenic spacer (IGS) 1 and 2 regions of the rRNA gene; (ii) the internal transcribed spacer (ITS) region, including 5.8S of the rRNA gene; (iii) TOP1 (topoisomerase); and (iv) CAP59. In these studies, we compared C. neoformans var. gattii with varieties grubii, and neoformans of C. neoformans. Phylogenetic analysis indicated that both C. neoformans var. grubii and C neoformans var. neoformans are monophyletic, but C. neoformans var. gattii showed polyphyletic. C. neoformans var. gattii can be divided into three phylogenetic groups, I, II, and III, with high bootstrap support. Phylogenetic group I contains serotype B and C strains, and groups II and III include serotype B strains. Because the serotype B strains of C. neoformans var. gattii exhibited more genetic divergence, the serological characteristics and chemotypes of their capsular polysaccharide were further investigated. No remarkable difference among the serotype B strains was found in the reactivities to factor serum 5, which is specific for serotype B. The NMR spectra of the capsular polysaccharide from serotype B strains could be divided into three characteristic patterns, but the chemical shifts were very similar. These results suggested that the serotype B strain of C. neoformans var. gattii has more genetic diversity than the serotype C strain of C. neoformans var. gattii or the varieties grubii and neoformans of C. neoformans, but there was no correlation between genotype and chemotype.     

Isolation and characterization of Cryptococcus neoformans var. gattii from samples of Eucalyptus camaldulensis in Mexico city.

The habitat of Cryptococcus neoformans var. gattii is not fully known in Mexico. We investigated the relationship of the yeast with Eucalyptus camaldulensis soil in three main Avenues of the city. A total of 135 trees of the species E. camaldulensis, were selected. Samples were taken in duplicate from the ground containing vegetable debris, tree cortex, leaves and flowers. Isolation of the yeast was made on Guizotia abyssinica media, using Staib technique. The identification was accomplished by biochemical and morphologic tests, and caraterization of the variety was made by bromotimol canavanine-glycine-blue (CGB) and D-proline tests. Isolation of 87 strains of Cryptococcus spp. was acomplished and eight of them were identified as C. neoformans var. gattii. These findings confirmed the close relationship of C. neoformans var. gattii and E. camaldulensis. To our knowledge this is the first report concerning the isolation of this variety from E. camaldulensis trees in Mexico.     

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiania, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.     

Phenotype characterization of environmental Cryptococcus neoformans isolates

Cryptococcosis is caused by the three varieties of C. neoformans with physiological and virulence differences, some of which have been studied to determine biological aspects of this microorganism. The phenotypical aspects of environmental isolates from varieties grubii and gattii were evaluated to establish differences associated with their life cycle and virulence. To this end, 28 and 31 strains of C. neoformans serotypes A (var. grubii) and C (var. gattii) were studied. The microscopic and macroscopic morphology on Sabouraud agar and soils, growth rate at 37 degrees C, production of 22 extracellular enzymes, haploid fructification, mating type, killer toxin sensitivity patterns and virulence in BALB/c mice were evaluated. No differences were observed between the two varieties regarding microscopic and macroscopic morphology or growth at 37 degrees C (p > 0.05). However, a decrease in the cellular and capsular sizes of yeast in soil, as compared to Sabouraud, was observed (p < 0.05). Additionally, higher enzimatic activity of proteases, phospholipases, phenoloxidase and beta-glucosidase was observed in var. grubii isolates as compared to var. gattii (p < 0.05). In both varieties, structures related with haploid fruitification were observed and all isolates were mating type alpha. Killer toxin sensitivity patterns of the isolates of var. grubii were I and II; in contrast, in var. gattii, seven different patterns were found: I, V, IX-XIII. In the animal model we found that 12 of 22 (54.5%) isolates of var. grubii caused the death of the mice during the observation period, while none of the 14 var. gattii isolates caused it. The decrease in capsular and cellular sizes of the yeast in soil and the frequency of mating type alpha with structures related to haploid fructification suggest an important mechanism of production of infectious particles in nature. Additionally, greater enzimatic activity of var. grubii can be associated with the virulence in the animal model.     

Diversity of laccase among Cryptococcus neoformans serotypes

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.     

Molecular sequence analyses of the intergenic spacer (IGS) associated with rDNA of the two varieties of the pathogenic yeast, Cryptococcus neoformans

The pathogen Crytococcus neoformans has been traditionally grouped in two varieties, C. neoforrmans var. neoformans (serotypes A, D and AD) and C. neoformans var. gattii (serotypes B and C). A recent taxonomic evaluation of C. neoformans var. neoformans described C. neoformans var. grubii as a new variety represented by serotype A isolates. Despite immunological, biochemical, ecological and molecular differences the three varieties are classified within one species. We examined the genetic variability of one hundred and five clinical and environmental isolates that included all varieties and serotypes. Sequence analysis of the intergenic spacer (IGS) associated with rDNA revealed significant differences in nucleotide composition between and within the varieties. Parsimony analysis showed five different genotypes representing distinct genetic lineages. Although there was a high degree of relatedness between serotype and genotype this relatedness was not exclusive as serotypes were not restricted to one particular genotypic group. Serotyping and sequence analyses indicate that C. neoformans var. grubii (serotype A) should not be recognized as a separate variety. Based on this study we propose to accept two separate species, C. neoformans (serotypes A, D and AD) and C. bacillisporus (serotypes B and C synonymous with C. neoformans var. gattii).     

Varietes and serotypes of Cryptococcus neoformans clinical isolates in Colombia

The aim of this study was to contribute to the knowledge of the geographic distribution of Cryptococcus neoformans varieties, in Colombian patients, and to determine the mating types of such varieties. A total of 370 clinical isolates were studied. C. neoformans var. neoformans was identified in 95.2% of them. C. neoformans var. gattii in 4.8%, 4.5% being serotype B and 0.3%, serotype C. Fifty-five of the 74 (74%) isolates studied were mating type "alpha", all of them C. neoformans var. neoformans. Serotype C had been previously reported only once in South American countries.     

Identification of Novel Hybrids Between Cryptococcus neoformans var. grubii VNI and Cryptococcus gattii VGII

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C. neoformans molecular type VNI and C. gattii molecular type VGII.     

Isolation and characterisation of the phospholipase B gene of Cryptococcus neoformans var. gattii

Cryptococcus neoformans var. gattii (serotypes B and C) is a human pathogen, ecologically, biochemically, clinically and genetically different from C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D). The phospholipase B (PLB1) gene from serotypes B and C was isolated and characterised. It resembled the serotype A and D genes, with an overall sequence homology of more than 85%. The respective open reading frames were 2236 bp (serotype B) and 2239 bp (serotype C) in length. Each contained six introns and encoded a 68-kDa protein destined for secretion. PLB1 was located on the second smallest chromosome in both serotypes. Gene expression, measured as mRNA, was not regulated by temperature, pH or exogenous nutrients.     

The internal transcribed spacers and 5.8S rRNA gene show extensive diversity among isolates of the Cryptococcus neoformans species complex

Sequences of the internal transcribed spacer (ITS) region including the 5.8S rRNA gene delineated seven genotypes within the three varieties of Cryptococcus neoformans via specific combinations of eight nucleotide differences located at positions 10, 11, 15, 19, 108 (ITS1), 221 (5.8S), 298 and 346 (ITS2). The ITS types correlated to polymerase chain reaction fingerprint/random amplification of polymorphic DNA (RAPD) molecular types: with ITS type 1 (ATACTAGC)=C. neoformans var. grubii, molecular types VNI+VNII and the serotype A allele of the AD hybrid, VNIIIA; ITS type 2 (ATATAGGC)=the serotype D allele of the AD hybrid, VNIIIB, and C. neoformans var. neoformans, VNIV; and ITS type 3 (GCGCTGGC) and ITS type 7 (ACGCTGGC)=VGI=RAPD type III, ITS type 4 (ACACTGAC)=VGII=RAPD type II, ITS type 5: (ACACTGGG)=VGIII=RAPD type I, ITS type 6 (ACACTGGC)=VGIV=RAPD type IV, all corresponding to C. neoformans var. gattii. Cloned sequences from serotype AD revealed that the hybrid serotype is diploid at the ITS1-5.8S-ITS2 locus carrying the ITS type 1 (ATACTAGC) and the ITS type 2 (ATATAGGC) alleles. ITS sequencing is a useful technique for genotyping the three C. neoformans varieties and for subtyping within C. neoformans var. gattii.     

Cryptococcus neoformans/Cryptococcus gattii Species Complex in Southern Italy: An Overview on the Environmental Diffusion of Serotypes, Genotypes and Mating-Types

Given the lack of comprehensive molecular epidemiology studies in Reggio Calabria and Messina, Italy, we decided to perform an extensive environmental sampling to describe the current molecular epidemiology of C. neoformans/C. gattii species complex in southern Italy. In this study, we report the occurrence of serotypes, genotypes and mating-types of isolates of the C. neoformans/C. gattii species complex recovered from environmental sources. In addition, a number of environmental C. neoformans var. grubii strains, isolated in 1997 by our laboratory, were also retrospectively examined in order to compare their genotypes with those recently found and to infer the possible epidemiological changes in our country. One hundred and twenty-two isolates were identified as being C. neoformans, whereas only one was found to belong to C. gattii serotype B, genotype VGI and mating-type alpha. Our data revealed that all environmental isolates of C. neoformans recovered here as well as those previously isolated in 1997 belong to serotype A and genotype VNI and posses a mating-type alpha allele.     

Characterization of Cryptococcus neoformans by random DNA amplification

Random amplified polymorphic DNA (RAPD) was optimized and used to distinguish between the varieties and serotypes of Cryptococcus neofirmans . The RAPD technique distinguished between serotypes A, D or AD within C. neoformans var. neoformans , and revealed further differentiation within each serotype. Four RAPD profiles were clearly recognizable within C. neofirmans var. gattii , although its two serotypes, B and C, were only differentiated with one primer combination out of seven.     

Chromosome length polymorphism in Cryptococcus neoformans clinical and environmental isolates

A protocol for intact DNA preparation from the basidiomycetous yeast Cryptococcus neoformans has been developed and applied to karyotyping C. neoformans isolates displaying different degrees of capsule formation. A total of 46 strains have been analyzed: 23 (50%) isolated from environmental samples (pigeon droppings), all of them belonging to C. neoformans var. neoformans; and 23 (50%) from clinical samples (human and veterinarian) including 10 isolates of C. neoformans var. neoformans and 13 isolates of C. neoformans var. gattii. Our results showed a global genome size ranging from 14.2 to 20.9 Mb for variety neoformans and from 7.9 to 16.8 Mb for variety gattii. The karyotype diversity was very high for variety neoformans (29 different patterns for the 33 analyzed strains) and lower for variety gattii (six different patterns for 13 strains). No grouping among variety neoformans strains from the same origin was found indicating very high genome diversity for this variety, irrespectively of the origin of the strains.     

In vitro antifungal susceptibility of clinical isolates of Cryptococcus neoformans var. neoformans and C. neoformans var. gattii

One of the differences observed between the two varieties of Cryptococcus neoformansis the greater difficulty to achieve an adequate therapeutical response in patients affected by C. neoformans var. gattii, an observation that has been validated in vitro only rarely. The aim of this work was to study the susceptibility patterns of 35 Colombian clinical isolates of C. neoformans, 20 of which belonged to the var. neoformans and 15 to the var. gattii. The minimal inhibitory concentration (MIC) was determined by broth microdilution, according to a modification of the methodology proposed by the National Committee for Clinical Laboratory Standards (NCCLS), using the breakpoints recently suggested by Nguyen et al. (Antimicrob Agents Chemother 1998; 42: 471-472). The antifungals tested were amphotericin B, fluconazole and itraconazole. Most of the isolates were susceptible to the three antimycotics tested regardless of the variety. Resistance to amphotericin B (MIC=2 microg/ml) was documented in two (10%) C. neoformans var. neoformans isolates; additionally, five (33%) C. neoformans var. gattii isolates felt in the category of fluconazole susceptible but dose dependent (MIC 16 microg/ml). In general, all C. neoformans var. gattii isolates proved susceptible only to the higher concentrations of the antifungals tested. For amphotericin B, seven (47%) isolates of this variety had MICs of 1 microg/ml, for fluconazole there were seven (47%) with MICs of 8 microg/ml and in the case of itraconazole, 10 isolates (66%) had MICs > 0.03 microg/ml. The data showed that although these isolates would be classified as susceptible, they actually require greater concentrations of the antifungals to be inhibited. This finding may well correlate both with the difficulty to attain therapeutic success in patients affected with C. neoformans var. gattii and with the need for more prolonged treatment courses in such cases.     

Cryptococcus neoformans mates on pigeon guano: implications for the realized ecological niche and globalization

The ecological niche that a species can occupy is determined by its resource requirements and the physical conditions necessary for survival. The niche to which an organism is most highly adapted is the realized niche, whereas the complete range of habitats that an organism can occupy represents the fundamental niche. The growth and development of Cryptococcus neoformans and Cryptococcus gattii on pigeon guano were examined to determine whether these two species occupy the same or different ecological niches. C. neoformans is a cosmopolitan pathogenic yeast that infects predominantly immunocompromised individuals, exists in two varieties (grubii [serotype A] and neoformans [serotype D]), and is commonly isolated from pigeon guano worldwide. By contrast, C. gattii often infects immunocompetent individuals and is associated with geographically restricted environments, most notably, eucalyptus trees. Pigeon guano supported the growth of both species, and a brown pigment related to melanin, a key virulence factor, was produced. C. neoformans exhibited prolific mating on pigeon guano, whereas C. gattii did not. The observations that C. neoformans completes the life cycle on pigeon guano but that C. gattii does not indicates that pigeon guano could represent the realized ecological niche for C. neoformans. Because C. gattii grows on pigeon guano but cannot sexually reproduce, pigeon guano represents a fundamental but not a realized niche for C. gattii. Based on these studies, we hypothesize that an ancestral Cryptococcus strain gained the ability to sexually reproduce in pigeon guano and then swept the globe.     

High frequency transformation of Cryptococcus neoformans and Cryptococcus gattii by Agrobacterium tumefaciens

Cryptococcus neoformans and Cryptococcus gattii are the caus-ative agents of cryptococcal meningoencephalitis and are amenable to genetic manipulations, making them important models of pathogenic fungi. To improve the efficiency of Agrobacterium tumefaciens mediated transformation (ATMT) in C. neoformans, we optimized various co-cultivation conditions including incubation time and temperature, and bacteria to yeast ratio. ATMT was also applied to both serotypes (B and C) of C. gattii. Transformation efficiency by ATMT in C. neoformans was comparable to either electroporation or biolistic transformation and gave superior efficiencies in serotypes B and C, but unlike Saccharomyces cerevisiae, adenine auxotrophy did not increase ATMT efficiency in C. neoformans or C. gattii. All transformants tested were stable, with a majority containing only a single T-DNA insertion; however, homologous recombination was not observed. Additionally, we isolated adenine auxotrophs containing a single T-DNA insertion in the ADE2 gene for representative serotype B and C strains.     

First environmental isolation of Cryptococcus neoformans var. neoformans and var. gatti from the Gharbia Governorate, Egypt

Flowers from two Eucalyptus camaldulensis trees in the Qutur area and one tree from the Tanta area yielded three isolates of Cryptococcus neoformans var. gattii. Pigeon and sparrow droppings were also investigated for the occurrence of C. neoformans within the study area. Ninety five isolates of the neoformans variety of C. neoformans were recovered from 550 samples of avian droppings.     

Electrophoretic karyotype of the pathogenic yeast Cryptococcus neoformans

The electrokaryotype of the pathogenic yeast Cryptococcus neoformans is described for the first time. Three different patterns were seen: (a) serotypes B and C (variety gattii) are similar and consist of nine chromosome mobility groups of greater than 580 kb; (b) serotype A (variety neoformans) revealed eight chromosome-like groups greater than 700 kb; (c) serotype D (the second serotype of variety neoformans) not only differs from those described above, but each D isolate tested showed a different distribution of bands. The discrepancy, and the importance of electrokaryotyping as a taxonomic tool, are discussed.