Urease testing and yeast taxonomy

When urease production was assayed by the hydrolysis of [14C]urea, all basidiomycetous yeasts tested, including the Cryptococcus vishniacii complex (previously reported urease negative), produced significant amounts of 14CO2. The Schizosaccharomycetaceae were the only urease-positive ascomycetous yeasts tested. Yarrowia lipolytica was urease negative. The stoichiometry of [14C]urea hydrolysis paralleled by Roberts' rapid urea hydrolysis (RUH) test indicated that causes of anomalous results in conventional urease testing include acidification and alkalinization of the test medium by products of endogenous metabolism and autolysis rather than urease activity. Anomalous results also occurred when cells were grown on media containing the chelating agent ethylenediaminetetraacetic acid (EDTA) prior to RUH. The addition of EDTA to a complex natural medium inhibited urease production in all yeasts reportedly growing at 35 degrees C (and all other yeasts tested), except Filobasidiella (Cr.) neoformans var. neoformans (NIH 12). The RUH test could differentiate at the varietal level: Fil. (Cr.) neoformans var. neoformans was about 10 times more resistant to EDTA in media used for the growth of cells prior to RUH testing than was Fil. neoformans var. bacillispora (Cr. neoformans var. gattii) (NIH 191). Urease production by Fil. neoformans var. bacillispora was specifically restored to half maximal activity by the addition of 22 microM Ni+2 (as NiCl2) to a growth medium containing 0.100 mM EDTA.     

Identification and Molecular Analysis of Pathogenic Yeasts in Droppings of Domestic Pigeons in Beijing, China

Feral pigeons are known as reservoirs of pathogenic yeasts that cause opportunistic infections in human. In the outskirts of Beijing, China, pigeons are more frequently raised at homes than are encountered in public areas. Many studies have focused on the presence of pathogenic yeasts in the excreta (fresh or withered) of a variety kinds of birds, pigeon crop and cloacae. One hundred and forty-three samples of fresh droppings were collected from three suburban pigeon-raising homes in an area of northern Beijing, China. The internal transcribed sequences (ITS) of all strains (except for 8 strains of Rhodotorula sp. ) were sequenced and compared with those of the databases of the National Center for Biotechnology Information website ( http://www.ncbi.nlm.nih.gov ) using the Basic Local Alignment Search Tool (BLAST). Yeasts representing 8 genera, Cryptococcus, Filobasidium, Rhodotorula, Candida, Debaryomyces, Saccaromyces, Trichosporon and Sporidiobolus, were identified from 120 isolates. Cryptococcus was the most prolific genera represented by eight species. The populations of yeast species isolated from fresh pigeon droppings were different among homes. Although it is well established that Cryptococcus neoformans exists mainly in old pigeon guano, several C. neoformans strains were still isolated from fresh pigeon excreta, providing a clue that live cryptococcal cells could move through the gastrointestinal tract of the pigeons. Eight genera identified from fresh droppings of domestic pigeons further confirm that pigeons serve as reservoirs, carriers and even spreaders of Cryptococcus species and other medically significant yeasts. The proportion of pathogenic yeasts in all isolates is more than 90 %.     

新生隐球菌的酚氧化酶及用于菌种鉴定的研究

采用4％玉米浸汁咖啡酸琼脂(CACA)培养基。观察了具不同生物学特性的新生隐球菌的酚氧化酶活性,并对临床常见的多种酵母和酵母样真菌作了该酶的检测。结果,受试的3个变种、5种血清型和尿素酶阴性新生隐球菌均呈明确的阳性反应;150株常见酵母和酵母样真菌中43株新生隐球菌全部呈酚氧化酶阳性。107株其它酵母和酵母样真菌全部阴性。具各种不同生物学特性的新生隐球菌均特异性地产生酚氧化酶,用检测该酶的方法作该菌鉴定的特异性和敏感性均为100％,且可于72小时内得到结果。此外,结合尿素酶试验可以准确的鉴定出尿素酶阴性的新生隐球菌。     

The PRP8 inteins in Cryptococcus are a source of phylogenetic and epidemiological information

Only two nuclear encoded inteins have been described. The first, SceVMA, was found in a vacuolar ATPase gene of Saccharomyces cerevisiae and related yeasts. The second, CnePRP8, was found in the PRP8 gene of Cryptococcus neoformans. CnePRP8 contains protein sequences associated with intein splicing but no endonuclease domain. We compared allelic mini-inteins in both varieties of C. neoformans (var. neoformans and var. grubii) and in the related primary pathogen C. gattii to study the evolution of both the mini-intein and the host. We also describe a full-length, endonuclease-containing intein in Cryptococcus laurentii, a moderately distant relation of C. neoformans. We did not detect an intein in the PRP8 gene of other species of Cryptococcus including species closely related to the C. neoformans/C. gattii group. It is therefore probable that the C. neoformans/C. gattii mini-intein was derived from horizontal transfer in which C. laurentii or another intein-containing species was the source.     

Cryptococcus neoformans varieties from material under the canopies of eucalyptus trees and pigeon dropping samples from four major cities in Jordan

To our best knowledge, any study related to the ecological distribution of Cryptococcus neoformans in Jordan does not exist in the medical literature. In order to determine the environmental occurrence of both varieties of Cryptococcus neoformans in Jordan, pigeon droppings and material under the canopies of eucalyptus trees were collected from four major cities of this country. For the isolation of Cryptococcus neoformans variety gattii from environmental sources, 500 samples of the mixed soil debris, including tree materials, under the eucalyptus trees from cities of Amman, Irbid, Jerash, and Ajlun were collected. Also, 509 samples of pigeon droppings were collected from the same cities for the isolation of Cryptococcus neoformans variety neoformans. After inoculating the samples onto modified Staib agar medium in Petri dishes, a total of 336 melanoid yeast colonies were picked up during screening process. At the end of serial mycological studies, none of these isolates was identified as Cryptococcus neoformans, but all were Cryptococcus species other than C. neoformans. For determining the exact status, more extensive environmental studies need to be done in the future.     

Laccase activity in Cryptococcus gattii strains isolated from goats

Cryptococcosis is a life-threatening infection in humans and animals caused by encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformans and Cryptococcus gattii are the main agents of this mycosis. Until 2002 C. gattii was classified as a variety of C. neoformans but now is accepted as an independent species. The laccase (phenoloxydase) enzyme produced by these yeasts is considered one of the main pathogenic factors for its ability to induce melanin from dihydroxyphenolic compounds. The vast majority of the studies in laccase and melanin synthesis have been developed using isolates of C. neoformans. The main objective of this study was to evaluate laccase activity in strains of C. gattii, serotype B isolated from immunocompetent goats that died of lung and disseminated cryptococcosis, in several outbreaks occurring in Spain. The laccase activities of these isolates were compared with those of other strains of C. gattii and C. neoformans. After fungal cell rupture, the supernatant of each isolate was analyzed for its laccase activity using as substrate an L-dopa 20 mM solution. The degree of enzymatic activity was assessed according to its absorbance at 450 nm and scored using Enzymatic Units (EU). The maximum values were observed in three strains of C. gattii from goats (EU > 12). The smallest values were observed in one environmental isolate of C. gattii serotype C (EU = 0.7). The highest recorded value for C. neoformans was 6.3 EU in a serotype A isolate from one human case of meningitis. C. gattii serotype B obtained from goats showed different degrees of laccase activity, being the highest in those isolated from severe outbreaks of cryptococcosis. This enzyme appears to represent a major, though nonexclusive, pathogenic factor for Cryptococcus gattii.     

Molecular characterization and evaluation of virulence factors of Cryptococcus laurentii and Cryptococcus neoformans strains isolated from external hospital areas

Cryptococcosis is a common opportunistic fungal infection that is mainly caused by the species Cryptococcus neoformans and Cryptococcus gattii, but there have recently been several reports of infection by non-neoformans Cryptococcus species. The aims of this study were to genetically characterize Cryptococcus spp. isolated from external hospital areas in Minas Gerais State, Brazil, and to evaluate their pathogenic potential, analyzing their phospholipase and melanin production and the capacity for capsule enlargement. Seventy-three different samples were collected: 62 from bird droppings and 11 from tree detritus. C.?neoformans alone was isolated from 43.8% of the samples, Cryptococcus laurentii alone from 23.3% and both fungi were found together in 10.9%. C. laurentii was exclusively isolated from 45% (5/11) of the tree samples (Anacardium occidentale, Guazuma ulmifolia, Mangifera indica and Ficus benjamina). Among the 51 C. neoformans isolates, 47 were classified as type VNI and four as type VNII. All of the C. neoformans isolates were of MATα type. Among the 21 isolates of C. laurentii genotyped using the URA5-RFLP technique, 16 amplified a 1.6kb amplicon which produced a specific restriction profile in 15 isolates. In C.?neoformans, 76.4% of the isolates were capable of capsule enlargement in the induction medium and 92.1% were phospholipase producers. In C. laurentii, 7.4% of the isolates were capable of capsule enlargement and 85.1% were phospholipase producers. Characterization of the genotypes and the pathogenic potential of the Cryptococcus spp. isolates studied may contribute towards better understanding of the epidemiology of cryptococcosis and the ecology of agents causing this disease in our region.     

Emendation of Dioszegia with redescription of Dioszegia hungarica and two new combinations,Dioszegia aurantiaca and Dioszegia crocea

During phylogenetic analyses of hymenomycetous yeasts based on 18S rDNA sequences, we found that Bullera armeniaca showed an extremely close phylogenetic relationship to Cryptococcus hungaricus. The analyses of internal transcribed spacer (ITS) regions of the two yeasts and the phylogenetically related species, Bullera aurantiaca and Bullera crocea, showed that B. armeniaca and C. hungaricus had identical sequences, indicating that these were conspecific. B. aurantiaca and B. crocea also showed high sequence similarity, 97.1% for ITS1, 100% for ITS2, and 98.7% for overall ITS regions. A DNA-DNA reassociation experiment revealed that B. armeniaca and C. hungaricus were conspecific and B. aurantiaca and B. crocea were two distinct species. These species occurred at a phylogenetically different lineage from that of Bulleromyces albus (anamorph: Bullera alba, type species of Bullera) and Filobasidiella neoformans (anamorph: Cryptococcus neoformans, neotype species of Cryptococcus). Based on these results, we emend the genus Dioszegia to include both ballistoconidium-forming and non-ballistoconidium-forming yeasts and redescribe the species Dioszegia hungarica. B. aurantiaca and B. crocea are also transferred to Dioszegia as Dioszegia aurantiaca comb. nov. and Dioszegia crocea comb. nov.     

Isolation of Cryptococcus neoformans from pigeon manure on two media inducing pigment formation

A newly described medium with esculin for identification of Cryptococcus neoformans was compared with Staib's Guizotia abyssinica extract-creatinine medium (GAEC) with and without diphenyl (DF). Twenty-seven samples of pigeon manure were examined. Cr. neoformans was found in 6 samples (22%) on GAEC plates (-DF); ESC medium (-DF) and malt extract agar allowed isolation from 2 and 3 samples respectively. Cr. neoformans was found in 0 to 2 samples when DF was added. Colonies of Cr. neoformans found on ESC plates had no distinctive pigmentation although inocula of pure cultures produced brown colonies. On GAEC plates some colonies of Cr. neoformans turned brown not until after 2 weeks of incubation. At 1 month the presence of pigmented colonies on GAEC plates (-DF) allowed the identification of 5 of the 6 samples from which Cr. neoformans was isolated. Other yeasts were grown from 26 samples (96%) and Torulopsis candida was found to be more frequent than Cr. neoformans.     

Comparison of Media for the Isolation of Cryptococcus neoformans

Four media, Staib's Guizotia abyssinica, trypan blue, and Staib's with 2 and 10 mg of methyl violet per liter, were compared for the selective and differential isolation of Cryptococcus neoformans from environmental samples. Trypan blue medium allowed for the differentiation of C. neoformans colonies from Candida albicans colonies several days earlier than did Staib's medium. The addition of methyl violet to Staib's medium was found to be inhibitory to some strains of all species tested. Diphenyl in Staib's medium inhibited the growth of 30 strains of C. neoformans and C. albicans.     

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.     

Alkalitolerant yeasts from natural biotopes

Using a solid nutrient medium containing alkaline buffer (pH 10) and an antibiotic, alkalitolerant yeasts were isolated from samples of soda-rich saline soils (solonchaks) of Armenia (Arazdayan) and the Transbaikal Region (the Kungur Steppe). The species diversity of the yeast populations of the tested soda-rich soils was relatively insignificant. They only contained alkalitolerant representatives of asporogenic capsulated yeasts belonging to the species Cryptococcus laurentii, C. albidus, Rhodotorula glutinis, R. mucilaginosa, and Sporobolomyces roseus. C. laurentii representatives clearly dominated the isolates obtained, their number exceeding that of the other species by 2-3 orders of magnitude. All of the isolates grew on acidic wort agar, suggesting that they did not include obligate alkaliphiles.     

<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Isolated from Passerine and Psittacine Bird Excreta in the State of Paraná, Brazil

Cryptococcus neoformans is an opportunistic basidiomycete yeast that causes life-threatening infections as meningoencephalitis primarily in immunocompromised hosts, generally associated with AIDS. The source of this organism is mainly pigeon excreta; however, other avian species' excreta are implicated as a source of this yeast. The occurrence of C. neoformans and Cryptococcus gattii in bird excreta in the state of Paraná in Brazil was determined in this study. A total of 141 samples of Passerine and Psittacine excreta from captive birds were collected. Additionally, 25 clinical samples from Hospital de Clínicas, in the state of Paraná were also analyzed. The determination of molecular and mating type of the isolates was performed by PCR fingerprinting, multiplex PCR, and mating type PCR. Cryptococcus neoformans var. grubii (VNI) was isolated from 36 (25.53%) of Passerine and Psittacine excreta samples. Almost all clinical samples, except one (C. gattii VGI), were classified as C. neoformans var. grubii (VNI). All environmental and clinical isolates were mating type alpha. These findings reinforce that, besides pigeon excreta, the excreta of these birds can also be a reservoir of C. neoformans in domestic and public environments and is of zoonotic importance to immunocompromised patients.     

Distribution of aquatic yeasts — Effect of incubation temperature and chloramphenicol concentration on isolation

Fresh (river), estuarine, and marine waters in and along the coastline of Connecticut were cultured by the membrane filter technique at 20 and 37°C on a complex medium containing 0–1000 mg/L of chloramphenicol. Using counts on medium with 500 mg/L antibiotic as a base, ratios of total and pink yeast counts were recorded for other chloramphenicol concentrations at both temperatures for the waters sampled. Variable results were obtained; in general, both total and pink yeast counts decreased with increasing antibiotic levels, being most apparent at > 400 mg/L chloramphenicol. Medium without antibiotic and with 100 mg/L always produced baterial overgrowth. A total of 209 white yeasts were isolated from all platings; the genera Torulopsis, particularly T. Candida, and Candida were dominant with lesser numbers of Cryptococcus, Trichosporon, sporogenous genera, and Kloeckera. Most species isolated were found on media at all chloramphenicol levels. Comparisons were made of yeast distributions in these temperate waters with reports from other areas.Contribution No. 101 from the University of Connecticut Marine Research Laboratory.     

Alkalitolerant Yeasts from Natural Biotopes

Using a solid nutrient medium containing alkaline buffer (pH 10) and an antibiotic, alkalitolerant yeasts were isolated from samples of soda-rich saline soils (solonchaks) of Armenia (Arazdayan) and the Transbaikal region (the Kungur Steppe). The species diversity of the yeast populations of the tested soda-rich soils was relatively insignificant. They only contained alkalitolerant representatives of asporogenic capsulated yeasts belonging to the species Cryptococcus laurentii, C. albidus, Rhodotorula glutinis, R. mucilaginosa,and Sporobolomyces roseus. C. laurentii representatives clearly dominated the isolates obtained, their number exceeding that of the other species by two to three orders of magnitude. All of the isolates grew on acidic wort agar, suggesting that they did not include obligate alkaliphiles.     

Cryptococcus spp. DNA extraction from environmental samples

The genus Cryptococcus encompasses 38 species, but only 3 are associated with disease in humans and animals, Cryptococcus laurentii, Cryptococcus albidus and Cryptococcus neoformans. The last one is the most frequently reported. The disease is acquired by the inhalation of infectious propagules present in the environment. The habitat has been established using extraction techniques with buffer supplemented with antibiotics and plating in selective media. The aim of this work was to evaluate several DNA extraction techniques for Cryptocococus spp. from environmental samples. The control isolates were C. neoformans, C. albidus, C. laurentii and Paracoccidiodes brasiliensis. We also used vermiculita and soil samples contaminated with different yeast concentrations (10 to 10(6) cells/g) and samples naturally contaminated with C. neoformans. DNA was extracted with physical and chemical methods and with a commercial kit, and the DNA was purified with agarose blocks and silica columns. For the PCR amplification we used the CN4-CN5 primers, which are specific for C. neoformans. Only the commercial kit allowed DNA extraction and amplification from contaminated soil samples up to a concentration of 10 cells/g and from one sample naturally colonized. With this work we extracted and amplified DNA from Cryptococcus spp. from environmental samples with appropriate PCR specificity, it will be a tool to establish the ecological areas of C. neoformans in our country.     

Modified Littman Oxgall Agar to Isolate Cryptococcus neoformans

Littman Oxgall Agar was modified by adding an extract of Guizotia abyssinica seeds with a water diluent for rehydration. In this medium, colonies of Cryptococcus neoformans became brown, but the color failed to develop in eight other yeasts and yeast phases of diphasic organisms. The depression of saprophytic fungi noted on Littman Oxgall Agar was not lost in the modification.     

Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.     

Fluorescent-Antibody Reagent for the Identification of Cryptococcus neoformans

A sensitive and diagnostically applicable conjugate for the rapid and accurate detection and identification of Cryptococcus neoformans has been developed. C. neoformans rabbit antisera were produced by 14 daily intravenous injections of 36 million cells for a total dosage of approximately 500 million cells. Cross-staining reactions with species of Cryptococcus other than C. neoformans, as well as with Candida species, were eliminated by adsorption of the C. neoformans conjugate with cells of C. diffluens and C. krusei.     

The isolation and characterization of virulence factors of Cryptococcus spp. from saprophytic sources in the city of Ribeirão Preto, São Paulo, Brazil

Yeasts of the Cryptococcus genus are distributed in nature associated to animal and vegetal organic residues. Occasionally, species other than C. neoformans may be responsible for infectious diseases in human and animals. This study aims to determine the occurrence of Cryptococcus species in the atmosphere and bird droppings in the city of Ribeir?o Preto, S?o Paulo, Brazil, and to evaluate three virulence factors: capsule formation, growth at 37 degrees C and melanin production. We analyzed 86 environmental samples (54 droppings and 32 air). Of the 41 strains isolated, 15 were C. neoformans var. neoformans (12 droppings and 3 air), 15 C. albidus (12 droppings and 3 air), 9 C. laurentii (7 droppings and 2 air) and 2 C. uniguttulatus (from droppings). Capsules were produced by 93.3% of C. neoformans var. neoformans, 66.7% of C. albidus, 88.9% of C. laurentii and 50% (1/2) of C. uniguttulatus. All strains of C. neoformans, 20% of C. albidus and 44.4% of C. laurentii were able to grow at 37 degrees C. The melanin production on DOPA agar was verified in C. neoformans (93.3%), C. albidus (26.7%) and C. laurentii (66.7%). We concluded that different Cryptococcus species coexist in the same ecological niche and they are able to produce virulence factors.