Evaluation of the Experimental Inoculation of <Emphasis Type="Italic">Cryptococcus albidus</Emphasis> and <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> in Normal Mice: Virulence Factors and Molecular Profile Before and After Animal Passage

The genus Cryptococcus includes free-developing species, a few of which are of medical importance. Some, such as C. neoformans and C. gattii, cause infections in man frequently and C. albidus and C. laurentii cause less so. The aims of this study were to evaluate organ colonization after inoculation of C. albidus and C. laurentii isolates in normal BALB/c mice, the virulence factors (growth at 37°C, capsule, melanin, proteinase, and phospholipase production) and the molecular profile (PCR-fingerprinting) of the yeasts before and after infection. The importance of different profiles (virulence and molecular) was considered in relation to the distribution in different organs and to the time intervals of isolation from organs. C. albidus was isolated from animal organs 2 to 10 days after inoculation and C. laurentii from 2 to 120 days. Most isolates of the two species kept the virulence factors showed before inoculation. The high homogeneity of the molecular profile of C. albidus and the high heterogeneity of C. laurentii were kept through the passages in animals. It is concluded that most isolates of both species were recovered from the animal organs after 5 or more days, and phenotypes were not altered by inoculation. No molecular alteration was detected and the virulence factors were not related to the time intervals before isolation from organs.     

Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species

The Heteroduplex Mobility Assay (HMA) method using the PCR amplified D1/D2 region of the 26S rDNA was tested for the differentiation of clinically relevant Candida species. Strains belonging to the same species are not expected to form heteroduplexes in this assay when their PCR products are mixed. D1/D2 HMA experiments between all Candida type strains tested showed heteroduplex formation, including Candida albicans and Candida dubliniensis. There was no heteroduplex formation when most clinical and non-type strains were tested against the type strain of their presumptive species, except when C. albicans WVE and C.␣dubliniensis TAI were analysed. Additional HMA experiments, phenotypic characterisation, and D1/D2 sequencing identified these isolates as Candida tropicalis and Candida parapsilosis, respectively. HMA provides a rapid and relatively simple molecular tool for the differentiation of potentially pathogenic Candida species.     

Isolation of Cryptococcus laurentii from Canada Goose guano in rural upstate New York

Cryptococcus neoformans and Cryptococcus gattii are etiologic agents of cryptococcal pneumonia and meningitis, potentially lethal syndromes associated with AIDS. A related species, Cryptococcus laurentii, has recently been implicated in several cases of human disease. Guano from Canada Goose (Branta canadensis), an organism that lives closely beside man and inhabits recreational space in rural and suburban areas, might be a significant environmental reservoir of Cryptococcus organisms in non-urban areas. Cryptococcal organisms were isolated from Canada Goose guano from a site in rural northern New York, with identification based upon colony and microscopic morphology, ability to metabolize l-Dopa to melanin, and positive reaction with a commercial anti-cryptococcal capsular polysaccharide latex bead agglutination test. DNA sequences from five positive isolates were identical to each other, and identical to the ITS1-5.8S-ITS2 sequences of C. laurentii strain CBS7140 (Accession AY315665) across a 511 bp sequence. All five isolates of C. laurentii possess three of the known virulence factors common to cryptococcal organisms that cause human disease: capsule, ability to grow at 37 °C, and laccase activity.     

Occurrence of Yeasts in Cloacae of Migratory Birds

Several species of yeast have been reported as pathogens in humans based on increases in immunodeficiency syndromes and as a result of immunosuppressant chemotherapy in cancer treatment. Domestic and wild birds are known to act as carriers of human pathogenic fungi. To gain additional information on the yeasts present in the cloacae of some species of migratory birds, 421 wild birds (24.39% out of 1726 birds caught in Romania, Hungary and Bulgaria) were sampled with the permission of the local judicial authority. The state of conservation of the birds (i.e. post-mortem alterations, colour of the mucosae etc.), along with their age and sex were determined. Samples were collected directly from the cloacae and cultured, and colonies were identified in each positive sample. Yeasts were isolated from 15.7% of the animals sampled, with the highest percentage found in coots (Fulica atra −58.8%) and the lowest in quails (Coturnix coturnix −1.7%). A total of 131 isolates belonging to 15 species of yeast were identified. Rhodotorula rubra was the yeast with the highest number of isolates (28.2%), followed by Cryptococcus albidus (18.4%), Candida albicans (9.2%), Trichosporon cutaneum (8.4%), Candida guilliermondii (6.1%), Candida tropicalis (6.1%) and other species. The present study represents the first survey on the occurrence of yeasts in the cloacae of migratory birds. The prevalence and species of yeasts isolated is discussed on the basis of the ecology, diet, and habitat of the birds.     

Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in Brazil

In this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and β-d-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) β-d-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant β-d-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular β-d-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) β-d-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and/or its hydrolysis products (xylo-oligosaccharides and xylose). Xylanolytic yeasts are able to secrete xylanolytic enzymes mainly when induced by xylan and present different strategies (intra- and/or extracellular hydrolysis) for the metabolism of xylo-oligosaccharides. Some of the unique xylanolytic traits identified here should be further explored for their applicability in specific biotechnological processes.     

Molecular Identification of <Emphasis Type="Italic">Candida orthopsilosis</Emphasis> Isolated from Blood Culture

The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.     

Yeast diversity in a mesotrophic lake on the karstic plateau of Lagoa Santa,MG-Brazil

The yeast diversity in a paleo-karstic lake of the Lagoa Santa plateau was studied during March 1986–March 1987. Water samples were collected monthly at five stations and the yeasts were isolated at 25 °C on Sabourad dextrose agar supplemented with 0.5% of yeast extract and 10 mg% of chloramphenicol. August and February showed the highest numbers of isolates, coinciding, respectively with the beginning and the ending of stratification. Of 56 isolated species, Aureobasidium pullulans, Candida famata, Cryptococcus albidus, Cryptococcus laurentii, Rhodotorula glutinis, Rhodotorula rubra and Trichosporon cutaneum occurred at the highest frequencies. A. pullulans showed greatest prevalence during the dry period (winter), whereas C. famata, Cr. albidus, Cr. laurentii and Rh. rubra were prevalent during the rainy season (summer). Tr. cutaneum was distributed between July and November. Most isolates yeasts are associated with plants and soils, and are important in decomposition of aquatic macrophytes. Furthermore, a possible role of these species as indicators of pollution is discussed.     

Repeated isolation of Cryptococcus laurentii from the oropharynx of an immunocompromized patient

Cryptococcus laurentii is one of the non-neoformans cryptococci that have rarely been isolated from humans. We report a case of repeated colonization of the oropharynx by Cr. laurentii in a patient with erythroleukaemia. The isolate was identified by phenotypic and genotypic tests and showed resistance to fluconazole. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

Biological Control of Postharvest Diseases of Apple and Pear under Semi-commercial and Commercial Conditions Using Three Saprophytic Yeasts

The yeastsCryptococcus laurentii(strain HRA5),Cryptococcus infirmominiatus(strain YY6), andRhodotorula glutinis(strain HRB6) were tested as biocontrol agents of postharvest diseases of apple and pear in semi-commercial and commercial trials. The yeasts effectively controlled decay when applied in a drench or line spray. The yeasts were not adversely affected when treated fruits were stored in a controlled atmosphere consisting of 1% oxygen and 99% nitrogen. In a commercial trial, the most effective treatments for control of blue mold of pear were a combination ofC. laurentiiandC. infirmo-miniatus(91% control) and the commercially recommended high rate (528 μg/ml) of thiabendazole (88% control). In the commercial apple trial, the most effective treatments for blue mold wereC. infirmo-miniatuscombined with 264 μg/ml thiabendazole (91% control),C. infirmo-miniatuscombined withC. laurentii(84% control), and thiabendazole alone at 528 μg/ml (79% control). The combination ofC. laurentiiwith 264 μg/ml of thiabendazole was significantly more effective for control of blue mold on pear than thiabendazole at 528 μg/ml whenever any thiabendazole-resistant spores were present in the inoculum.     

Isolation and identification ofMicrococcus roseus andPlanococcus sp. from schirmacher oasis,antarctica

Five cultures isolated from soil samples collected in Schirmacher oasis, Antarctica, have been identified as members of the familyMicrococcaceae, with 3 belonging to the genusMicrococcus and two toPlanococcus. The 3Micrococcus isolates (37R, 45R and 49R) were red-pigmented and h a d ∼ 75 mol% G + C in their DNA; they were identified asMicrococcus roseus. The twoPlanococcus isolates (30Y and Lz3OR) were yellow and orange in colour, and had 43.5 and 40.9 mol % G + C in their DNA respectively; they were identified asPlanococcus sp.     

Extracellular enzymatic activity in 11 Cryptococcus species

The extracellular enzymatic activity of 36 strains of yeast belonging to 11 species of the genus Cryptococcus, has been investigated, using the API-ZYM (BioMérieux, France) commercial system, with the objective of determining the differences in the enzymatic profiles of the various species. The strains studied were : 9 of C. neoformans, 7 of C. albidus, 6 of C. laurentii, 5 of C. uniguttulatus, 3 of C. humicolus, and 1 each of C. ater, C. curvatus, C. dimennae, C. hungaricus, C. infirmo-miniatus and C. magnus. All the strains showed enzymatic activity with positivity to Phosphatase alkaline, Esterase lipase C8, Leucine arylamidase, Phosphatase acid and Naphthol-AS-BI-phosphohydrolase, and negativity to Lipase C14, Trypsin, Chemotrypsin, β-galactosidase, β-glucuronidase and α-manosidase. Variable enzymatic activity was shown to Esterase C4, Valine arylamidase, Cystine arylamidase, α-galactosidase,α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and α-fucosidase. This allowed 11 separate enzymatic patterns to be established. The species C. neoformans and C. laurentii each presented two distinct patterns; C. uniguttulatus, C. hungaricus andC. magnus shared the same pattern; C. albidus, C. ater, C. curvatus,C. dimennae, C. humicolus and C. infirmo-miniatus presented an individual enzymatic pattern. The results obtained suggest that the API-ZYM system could be useful for the identification of species of the genus Cryptococcus and for the differentiation of the enzymotypes for epidemiological purposes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Prevalence of yeast-like fungi and evaluation of several virulence factors from feral pigeons in Seoul, Korea

Aims: Studies of pigeon‐borne yeasts have tended to focus on species, such as Cryptococcus neoformans and Candida albicans, with scant attention to feral pigeons in Korea. We studied the prevalence of yeasts from faecal samples of feral pigeons obtained in various public places in Seoul, Korea, and assessed their potential capacity as human pathogens. Methods and Results: Three hundred and six pigeon faeces samples were collected at city squares and parks in 21 localities in Seoul and Seoul Grand Park and analysed for yeast with conventional methods. Of the 306 samples, 126 (41·2%) were positive for yeast. Seventeen species of yeast were identified. The most frequent species were Candida glabrata (34·1%), Candida famata (12·7%), Cryptococcus albidus (14·3%) and Cryptococcus laurentii (7·9%). The yeast isolates were tested for virulence. Of the 116 isolates (ten isolates missing), 70·7% (n = 82) grew at 37°C. All the Cryptococcus spp. isolates possessed a capsule, 16·4% (n = 19) produced melanin, and 33·6% (n = 39) produced proteinase. Two Ca. glabrata, a Ca. famata and Ca. albicans as well as three C. neoformans, a C. laurentii and Ca. albicans isolates had three virulence factors. Accordingly, 29·3% (n = 34) isolates possessed more than two virulence factors except capsule formation. Conclusions: These results of this study indicate that feral pigeons harbour a variety of yeasts and are a reservoir of human pathogenic fungi. Significance and Impact of the Study: This study is the first time about the microflora (fungi) presents in faecal samples collected from a variety of public areas throughout Seoul, Korea.     

Biocontrol of Botrytis cinerea in apple fruit by Cryptococcus laurentii and indole-3-acetic acid

This study evaluated the effect of a yeast antagonist Cryptococcus laurentii and a plant regulator indole-3-acetic acid (IAA) on inhibition of Botrytis cinerea infection in harvested apple fruit. The results showed that the combined treatment with C. laurentii and IAA at 20 μg/ml was a more effective approach to reduce the gray mold rot in apple wounds than the C. laurentii alone. After 4 days of incubation, gray mold incidence in the combined treatment with C. laurentii and IAA was about 18%, which was a 50% reduction in incidence compared to the treatment with C. laurentii alone. Although IAA had no direct antifungal activity against B. cinerea infection when the time interval between IAA treatment and pathogen inoculation was within 2 h, application of IAA strongly reduced gray mold infection when IAA was applied 24 h prior to inoculation with B. cinerea in apple fruit wounds. Moreover, combination of IAA and C. laurentii stimulated the activities of superoxide dismutase, catalase and peroxidase with above 1.5-fold higher than that treatment with C. laurentii alone at 48 h. Therefore, combination of C. laurentii with IAA, which integrated the dual biological activity from the antagonistic yeast and plant regulator, might be developed to be a useful approach to control gray mold in harvested apple fruit.     

Oxidation of amines by yeasts grown on 1-aminoalkanes or putrescine as the sole source of carbon,nitrogen and energy

The maximum growth rate of Trichosporon cutaneum CBS 8111 in chemostat cultures was 0.185 h^-1 on ethylamine and 0.21 h^-1 on butylamine, that of Candida famata CBS 8109 was 0.32 h^-1 on putrescine.The amine oxidation pattern of the ascomycetous strains studied, viz. Candida famata CBS 8109, Stephanoascus ciferrii CBS 4856 and Trichosporon adeninovorans CBS 8244 was independent of the amine that had been used as the growth substrate. It resembled that of benzylamine/putrescine oxidase found in other ascomycetous yeasts. However, differences in pH optimum and substrate specificity were observed between the amine-oxidizing systems of these three species.The amine oxidation pattern of cell-free extracts of Trichosporon cutaneum CBS 8111 varied with the amine that was used as growth substrate. The enzyme system produced by Cryptococcus laurentii CBS 7140 failed to oxidize isobutylamine and benzylamine, and showed a high pH optimum.The synthesis of amine oxidase in the four yeast strains studied was not repressed by ammonium chloride and was weakly repressed by glucose but was strongly repressed if both compounds were present in the growth medium.     

Use of CHROMagar in the Differentiation of Common Species of <Emphasis Type="Italic">Candida</Emphasis>

CHROMagar has been reported to be useful for the rapid and accurate identification of Candida species. We tested 135 isolates of Candida species isolated from oropharyngeal candidiasis in HIV patients and found that it was useful in the presumptive identification of Candida albicans and Candida krusei. Occasional strains of C. tropicalis produced colonies with a greenish tinge making it difficult to differentiate from C. albicans.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

The first clinical isolates of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in Slovakia

Candida dubliniensis, yeast closely related to Candida albicans, is a new pathogen associated mainly with infections of immunocompromised hosts. In this study, we report the first isolation of three isolates of C. dubliniensis in Slovakia. The first selection of both C. albicans and C. dubliniensis from the other Candida species was done on the basis of specific green color of primoculture grown on CHROMagar Candida. The presumptive identification was completed by supplemental tests: germ-tube formation, production of chlamydospores, ability or inability to grow at 42 and 45,°C and by commercial set API 20C AUX. Parallely, the discrimination between both species was performed by PCR assay using primers specific for Candida dubliniensis     

<Emphasis Type="Italic">Candida patagonica</Emphasis> sp. nov., a new species of yeast from cellar surfaces

A novel anamorphic yeast species belonging to the genus Candida has been isolated from cellar surfaces in North Patagonia. Morphological and physiological observation and phylogenetic analysis were performed. Pseudomycelium was plentifully produced. No sexual reproduction was observed. From sequence analysis of the 26S rDNA D1/D2 region, Candida bituminiphila and Zygoascus hellenicus were the closest species with 40 and 79 bp substitutions, respectively. C. bituminiphila differed physiologically from the novel species in its ability to assimilate sucrose and erythritol, in not fermenting any sugars, in growing without some vitamin compounds, and in growing at 40°C. All these data support the hypothesis that the new yeast, named Candida patagonica, is a novel species related to C. bituminiphila. The type strain is UNCOMA 159.5 (= CECT 12029 = CBS 10443).