新生隐球菌的AFLP分析

目的评估AFLP-DNA指纹技术在新生隐球菌分类中应用情况。方法新生隐球菌基因组DNA用双酶酶切,双链接头连于其酶切末端,用与接头和酶切位点互补的引物扩增DNA片段,其产物在高分辨的变性聚丙酰胺凝胶上电泳分离,然后进行银染。结果分析来自5种血清型和临床分离株的18株新生隐球菌,可见有30多条大小在30～500bp的DNA-AFLP指纹,相同的血清型有不同的指纹图谱,来自同一患者不同病期的两株分离株和来自同一患者患者的不同部位的两株分离株都显示出相同的带型。结论显示了AFLP的高分辨率,是适用于新生隐球菌流行病学调查的有力工具。     

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.     

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.     

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.     

Mating types and serotypes of Cryptococcus neoformans isolated in Japan

Thirty-two isolates of Cryptococcus neoformans from patients and one from the wild obtained in Japan were characterized for their serotype, self-fertility, and mating behaviour by crossing them with two mating types of Filobasidiella neoformans var. neoformans and F. neoformans var. bacillispora. Of the 32 isolates from patients, 31 were of serotype A and the remaining one was of serotype D. Although these 32 isolates were all self-sterile, 23 serotype A and one serotype D isolates produced a complete sexual state when mixed with the alpha mating type of F. neoformans var. neoformans. The one natural isolate was of serotype A-D and self-fertile. The Japanese clinical isolates of C. neoformans appear to be predominantly serotype A and alpha mating type of F. neoformans var. neoformans as is the case in the U.S.A.     

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.     

Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var. grubii) or serotype D (C. neoformans var. neoformans). In addition, serotype AD strains, which are hybrids of these two varieties, are commonly isolated from clinical and environmental samples. While most isolates of serotype A and serotype D are haploid, AD strains are diploid or aneuploid, and contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type (Aalpha); however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele (Aa). We previously described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by scoring amplified fragment length polymorphisms, and discovered that AD hybrid strains possessing the rare serotype A MATa allele (genotype AaDalpha) cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele (AalphaDa and AalphaDalpha) cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrid strains are more resistant to UV irradiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in sub-Saharan Africa from a cross between strains of serotypes A and D; and (ii) this fusion produced hybrid strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to survive, emigrate, and propagate throughout the world.     

新生隐球菌的生态学、流行病学、分子生物学及临床研究

在我国,对新生隐球菌(Cryptococcus neoformans)已进行了较系统的研究。生态学方面,,由鸽粪分离的环境株具有表型的多态性,包括新生变种的A、D血清型及尿素酶阴性株,而这些多态性菌株均已在临床发现。分子生物学方面,G+Cmol％和核型已被进行分析。,由PFGE分析所得到的有意义的信息是两个变种和5种血清型新生隐球菌株具有明显不同的核型谱。临床方面,研制的一种新的可同时检测酚氧化酶和尿素酶的培养基可用于该菌的临床鉴定。使用一种高渗培养基诱导出于该菌的L-型,提示在感染期间L-型的形成可能与该病的慢性过程与复发有关。已证实氟康唑对治疗新生隐球菌病有良好的效果。进一步应重视联合治疗的可能性,例如使用氟康唑加二性霉素B脂质体。总之在我国对新生隐球菌的系统性研究已经初步开始进行,许多有意义的结果已经得到并且将进一步得到。     

Isolation of Cryptococcus neoformans var. grubii (serotype A) from pigeon droppings in Seoul, Korea

Seventy-two pigeon dropping samples were collected from 26 different localities in Seoul and investigated for the occurrence of Cryptococcus neoformans. Seventeen samples from 8 different localities were found to be positive for C. neoformans. All isolates were obtained from withered pigeon droppings. Identification and serotyping of the isolates were determined by means of serological testing and DNA fingerprinting. All isolates belonged to C. neoformans var. grubbi (serotype A).     

Species-specific banding patterns of restriction endonuclease-digested mitochondrial DNA from the genusPythium

Restriction endonuclease-digested mitochondrial DNA from 29Pythium spp. showed distinctly different species-specific electrophoretic banding patterns. Numerical comparisons among species were conducted by calculating the percentage of restriction fragments having the same apparent molecular size. The greatest interspecific similarity in banding patterns (67%) was observed betweenHindIII digests ofPythium heterothallicum andP. sylvaticum. However, comparisons among other species generally revealed similarities of less than 50%, and often less than 30%. The lack of similarity of restriction banding patterns was observed even with several species that share many common morphological features:P. arrhenomanes vsP. graminicola (20%),P. myriotylum vsP. aristosporum (28%), andP. torulosum vsP. vanterpoolii (32%). In contrast to the fragment size heterogeneity among different species, isolates of the same species have highly conserved restriction patterns. Ten isolates ofP. oligandrum, collected from the United States, South Africa, and Czechoslovakia, had a minimum of 86% similarity inHindIII banding patterns. Similar results were observed with eight isolates ofP. ultimum, five ofP. acanthicum, six ofP. spinosum, five ofP. sylvaticum, and eight ofP. irregulare. However, two isolates ofP. irregulare exhibited a higher degree of heterogeneity and shared only 64 to 76% comigrating bands with the eight other isolates of this species.     

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.     

Restriction pattern of the major outer-membrane protein gene provides evidence for a homogeneous invasive group among ruminant isolates of Chlamydia psittaci.

Thirty-six ruminant isolates of Chlamydia psittaci, previously classified as invasive or non-invasive in a mouse model of virulence, were compared by analysing AluI restriction patterns of the major outer-membrane protein (MOMP) gene after DNA amplification by the polymerase chain reaction. The 24 invasive isolates, although from various origins, all belonged to serotype 1 and represented a strictly homogeneous group sharing a specific MOMP-gene restriction pattern that was not observed in the non-invasive strains. On the other hand, the 12 non-invasive strains, although all belonging to serotype 2, constituted a heterogeneous group with eight distinct MOMP-gene restriction patterns. However, all eight patterns shared a 180 bp fragment or the corresponding restricted fragments of 110 and 70 bp. MOMP-gene restriction patterns also clearly distinguished the ruminant strains from an avian C. psittaci isolate, a C. pneumoniae isolate and two C. trachomatis isolates which were studied for comparison. The homogeneous character of the invasive C. psittaci strains argues strongly for their genetic relatedness. Our results illustrate the usefulness of the MOMP-gene restriction mapping in typing chlamydiae.     

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.     

Serotype and PCR-fingerprints of Clinical and Environmental isolates of Cryptococcus neoformans in Chiang Mai,Thailand

From May 1999 to April 2000, serotypes of clinical and environmental isolates of Cryptococcus neoformans were studied in Chiang Mai province, northern Thailand. Three hundred and eighty-five environmental samples, of which 100 were dove droppings, 55 pigeon droppings and 230 eucalyptus flower, were collected from 7 Amphoes in Chiang Mai. C. neoformans was isolated from 45 of 100 (45.0%) dove dropping samples, 9 of 55 (16.4%) pigeon dropping samples and 2 of 230 (0.9%) eucalyptus flower samples. Serotypes of 56 environmental isolates and 75 clinical isolates of C. neoformans,obtained during the same period, were determined by the slide agglutination test. Fifty-six environmental and 74 clinical isolates belonged to C. neoformans serotype A (C. neoformans var. grubii), and only one clinical isolate belonged to C. neoformans serotype AD. The isolation of C. neoformans var. grubii from eucalyptus flower samples suggests contamination of avian droppings. PCR-fingerprinting, using (GACA)4 as a primer, discriminated 131 clinical and environmental isolates into 2 groups (group I and II). Seventy-five clinical and 54 environmental isolates were of group I, which had two major specific bands of approximately 1,250 and 960 base pairs. Two environmental isolates, one from pigeon excreta and the other from a eucalyptus flower sample were of group II, which had two major specific bands of approximately 1,180 and 500 base pairs.     

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.     

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.     

Genetic analyses of a hybrid cross between serotypes A and D strains of the human pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans has two varieties, var. grubii and var. neoformans, that correspond to serotypes A and D, respectively. Molecular phylogenetic analyses suggest that these two varieties have diverged from each other for approximately 18 million years. The discovery of pathogenic serotype AD hybrid strains in nature indicates that intervariety mating in C. neoformans occurs in the natural environment. However, little is known about the genetic consequences of hybridization in C. neoformans. Here, we analyzed a hybrid population of 163 progeny from a cross between strains of serotypes A (CDC15) and D (JEC20), using 114 codominant nuclear PCR-RFLP markers and 1 direct PCR marker. These markers were distributed on all 14 chromosomes of the sequenced strain JEC21 that was isogenic to one of the parents (JEC20) in our cross. Our analyses identified that of the 163 progeny, 5 were heterozygous at all 115 loci, 1 was completely homozygous and identical to one of the parents (CDC15), and the remaining 157 each contained at least 1 heterozygous locus. Because all 163 progeny inherited mitochondria from the MATa parent JEC20, none of the progeny had a genotype identical to either of the two parents or to a composite of the two parents. All 115 nuclear loci showed three different genotypes in the progeny population, consistent with Mendelian segregation during meiosis. While the linkage analysis showed independent reassortment among loci on different linkage groups, there were significant differences in recombination frequencies among chromosomes and among regions within certain chromosomes. Overall, the linkage-map length from this hybrid cross was much shorter and the recombination frequency much lower than those constructed using serotype D strains, consistent with suppressed recombination in the intervariety cross between strains of serotypes A and D. We discuss the implications of our results in our understanding of the speciation and evolution of the C. neoformans species complex.     

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.