Pigment Production on L-Tryptophan Medium by Cryptococcus gattii and Cryptococcus neoformans

In recent years strains previously grouped within Cryptococcus neoformans have been divided into two species C. neoformans and C. gattii, with Cryptococcus neoformans comprising serotypes A, D, and AD and C. gattii comprising serotypes B and C. Cryptococcus neoformans have also been subdivided into two varieties C. neoformans var. grubii, serotype A, and C. neoformans var. neoformans, serotype D. We analyzed the growth and pigment production characteristics of 139 strains of Cryptococcus spp. in L-tryptophan containing media. Nearly all strains of Cryptococcus, including each variety and serotype tested produced a pink water-soluble pigment (molecular weight of 535.2 Da) from L-tryptophan. Consequently, the partial separation of the species was based on whether the pink pigment was secreted into the medium (extracellular) or retained as an intracellular pigment. On L-tryptophan medium C. neoformans var. grubii and serotype AD produced a pink extracellular pigment. In contrast, for C. gattii, the pink pigment was localized intracellularly and masked by heavy production of brown pigments. Pigment production by C. neoformans var. neoformans was variable with some strains producing the pink extracellular pigment and others retained the pink pigment intracellularly. The pink intracellular pigment produced by strains of C. neoformans var. neoformans was masked by production of brown pigments. Cryptococcus laccase mutants failed to produce pigments from L-tryptophan. This is the first report that the enzyme laccase is involved in tryptophan metabolism. Prior to this report Cryptococcus laccase produced melanin or melanin like-pigments from heterocyclic compounds that contained ortho or para diphenols, diaminobenzenes and aminophenol compounds. The pigments produced from L-tryptophan were not melanin.     

Presence of <Emphasis Type="Italic">C. albidus</Emphasis>, <Emphasis Type="Italic">C. laurentii</Emphasis> and <Emphasis Type="Italic">C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type="Italic">Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.     

Fungal diseases of the respiratory tract

The proportion ofCandida and non-Candida species in the clinical material from patients. with respiratory-tract diseases was determined.C. albicans was isolated in 102 cases. An additional 89 strains of yeasts, isolated in association with respiratory diseases, belonged to 10 non-albicans Candida spp. andCryptococcus spp. The prevailing species, which occurred in 47 cases, wasC. parapsilosis. C. tropicalis, C. glabrata, andC. guilliermondii were isolated in 12, 10, and 9 cases, respectively. Four strains ofC. krusei and three strains ofC. lusitaniae and one strain each ofC. freyschussii, C. robusta, C. zeylanoides, andCryptococcus neoformans were also isolated.     

Evaluation of SOC for the presumptive identification of Candida albicans and Cryptococcus neoformans

SOC, a fungal growth medium composed of Solryth^R, oxgall, and caffeic acid, was evaluated as a medium to provide rapid, differential identification of Candida albicans and Cryptococcus neoformans. Using a variety of common isolation media to produce the yeast inocula, the germ tube methods tested ranked in the following order of decreasing sensitivity: SOC (97%±1), serum (92%±5), rabbit coagulase plasma with EDTA in combination with tryptic soy broth (89%±5), TOC (89%±6), and rabbit coagulase plasma with EDTA (83%±4). In chlamydospore production, SOC also proved to be the most sensitive after 24 h incubation: SOC (96%±2), TOC (80%±2), and cornmeal-Tween 80 agar (14%±3). Other medically important yeasts showed normal patterns of growth within 24 h on SOC, thus assisting in their identification.Eighty strains of Cryptococcus neoformans showed characteristic brown pigmentation on SOC and TOC within 18 h, while all other species of the genus Cryptococcus and 229 Candida isolates did not show a change in pigmentation.     

Phylogenetic relationships ofCryptococcus neoformans and some related basidiomycetous yeasts determined from partial large subunit rRNA sequences

The genusCryptococcus was found to be heterogeneous on the basis of partial rRNA sequences. The human-pathogenic speciesC. neoformans, comprising 4 serotypes and havingFilobasidiella neoformans andF. bacillispora as teleomorphs, was found at a relatively large distance fromFilobasidium. Serotypes B and C had identical sequences, while in A and D they were different, with D closer to B and C than to A.Filobasidiella depauperata, which lacks a yeast-like anamorph, clustered withF. neoformans.The genusFilobasidium was clearly separated fromFilobasidiella and clustered withC. albidus, C. kuetzingii, C. gastricus, C. lupi, C. vishniaciae, C. bhutanensis, C. aerius, C. terreus andC. ater. The latter may represent the anamorph ofFilobasidium elegans. The organe to red species ofCryptococcus, as well asC. aquaticus andC. yarrowii, were found completely unrelated with these taxa,C. macerans being affiliated toCystofilobasidium capitatum.The genusTrichosporon was found relatively homogeneous; it includesC. humicola, C. curvatus and the filamentous speciesHyalodendron lignicola. Cryptococcus flavus andC. dimennae probably belong to the Tremellales, though distances between these species are large. The positions ofC. laurentii andC. luteolus remains to be determined.     

A DL-DOPA drop test for the identification of Cryptococcus neoformans

A simple melanin assay using DL.DOPA as the substrate was developed to aid in the identification of Cryptococcus neoformans. The DL-DOPA drop test assay was simple and efficient. The best results (100% of the C. neoformans isolates were positive) occurred when C. neoformans was grown for two days at room temperature on Sabouraud agar modified. One to three loopfuls of yeast cells were then transferred to a starvation medium for 18–24 hours. Two to three drops of 0.3% DL-DOPA solution was applied to the transferred yeast cells. Only C. neoformans produced a brown or blackgrey pigment within 24 hrs, with 85% of the isolates becoming brown or black-grey within thirty minutes.     

Candidal urinary tract infections caused by non-<Emphasis Type="Italic">albicans Candida</Emphasis> species

The incidence of non-albicans Candida and non-Candida species isolated from the urine of patients admitted to various departments of theFaculty Hospital of the Medical Faculty of Šafárik University in Košice was examined. From a total of 94 samples of analyzed urine 58 strains ofC. albicans and 36 strains of yeasts belonging to 6 species of non-albicans Candida and non-Candida spp. were detected:C. parapsilosis (n=23), C. tropicalis (6), C. krusei (3), C. robusta (2), C. catenulata (1) andCryptococcus neoformans (1). In relation to the diagnosis, the yeasts were isolated from patients suffering from a kidneys disease, epididymitis, diabetes, neoplastic diseases, urogenital anomalies, obstructive uropathy, cystitis, prostatitis, hemolytic-uremic syndrome, and others.     

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.     

Enhanced activity of antifungal drugs using natural phenolics against yeast strains of Candida and Cryptococcus

Aims: Determine whether certain, natural phenolic compounds enhance activity of commercial antifungal drugs against yeast strains of Candida and Cryptococcus neoformans. Methods and Results: Twelve natural phenolics were examined for fungicidal activity against nine reference strains of Candida and one of C. neoformans. Six compounds were selected for synergistic enhancement of antifungal drugs, amphotericin B (AMB), fluconazole (FLU) and itraconazole (ITR). Matrix assays of phenolic and drug combinations conducted against one reference strain, each, of Candida albicans and C. neoformans showed cinnamic and benzoic acids, thymol, and 2,3‐ and 2,5‐dihydroxybenzaldehydes (‐DBA) had synergistic interactions depending upon drug and yeast strain. 2,5‐DBA was synergistic with almost all drug and strain combinations. Thymol was synergistic with all drugs against Ca. albicans and with AMB in C. neoformans. Combinations of benzoic acid or thymol with ITR showed highest synergistic activity. Of 36 combinations of natural product and drug tested, none were antagonistic. Conclusions: Relatively nontoxic natural products can synergistically enhance antifungal drug activity, in vitro. Significance and Impact of the Study: This is a proof‐of‐concept, having clinical implications. Natural chemosensitizing agents could lower dosages needed for effective chemotherapy of invasive mycoses. Further studies against clinical yeast strains and use of animal models are warranted.     

Epidemiology of Cryptococcus neoformans

The concept of the epidemiology of Cryptococcus neoformans as the causative agent of cryptococcosis and as a basidiomycetous yeast is based on the fact that bird manure has been until now its only known habitat but not plant material which likewise harbours various non-pathogenic Cryptococcus species.It could be shown that the possible influence of nutritional factors on the morphology and morphogenesis earns attention not only in view of the epidemiology of C. neoformans but of its perfect states, too.     

Identification of Cryptococcus neoformans in a routine clinical laboratory

Summary Eight taxonomic tests were compared for their ability to distinguishCryptococcus neoformans from the non-pathogenic species ofCryptococcus. Eight isolates ofCryptococcus were obtained from the American Type Culture Collection and 43 isolates were obtained directly from human and natural sources. The tests which appeared to be most valuable to the routine diagnostic laboratory were growth at 37° C, characteristic growth on Guizotia seed agar and virulence for mice.     

The mycobiome of Australian tree hollows in relation to the Cryptococcus gattii and C. neoformans species complexes

Cryptococcosis is a fungal infection caused by members of the Cryptococcus gattii and C. neoformans species complexes. The C. gattii species complex has a strong environmental association with eucalypt hollows (particularly Eucalyptus camaldulensis), which may present a source of infection. It remains unclear whether a specific mycobiome is required to support its environmental survival and growth. Conventional detection of environmental Cryptococcus spp. involves culture on differential media, such as Guizotia abyssinica seed agar. Next‐generation sequencing (NGS)‐based culture‐independent identification aids in contextualising these species in the environmental mycobiome. Samples from 23 Australian tree hollows were subjected to both culture‐ and amplicon‐based metagenomic analysis to characterize the mycobiome and assess relationships between Cryptococcus spp. and other fungal taxa. The most abundant genera detected were Coniochaeta, Aspergillus, and Penicillium, all being commonly isolated from decaying wood. There was no correlation between the presence of Cryptococcus spp. in a tree hollow and the presence of any other fungal genus. Some differences in the abundance of numerous taxa were noted in a differential heat tree comparing samples with or without Cryptococcus‐NGS reads. The study expanded the known environmental niche of the C. gattii and C. neoformans species complexes in Australia with detections from a further five tree species. Discrepancies between the detection of Cryptococcus spp. using culture or NGS suggest that neither is superior per se and that, rather, these methodologies are complementary. The inherent biases of amplicon‐based metagenomics require cautious interpretation of data through consideration of its biological relevance.     

Induction by Klebsiella aerogenes of a Melanin-Like Pigment in Cryptococcus neoformans

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37°C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.     

Native trees of the Northeast Argentine: Natural hosts of the Cryptococcus neoformans–Cryptococcus gattii species complex

BackgroundIn Argentina, information about epidemiology and environmental distribution of Cryptococcus is scarce. The city of Resistencia borders with Brazil and Paraguay where this fungus is endemic. All these supported the need to investigate the ecology of the genus and the epidemiology of cryptococcosis in this area.AimsThe aim was to investigate the presence of species of Cryptococcus neoformans–Cryptococcus gattii complex and their genotypes in trees of the city of Resistencia.MethodsOne hundred and five trees were sampled by swabbing technique. The isolates were identified using conventional and commercial methods and genotyped by PCR-RFLP (Restriction Fragment Length Polymorphism).ResultsCryptococcus was found in 7 out of the total trees. 6 out of 7 Cryptococcus isolates were identified as C. neoformans and one as C. gattii. C. gattii was isolated from Grevillea robusta. C. neoformans strains were isolated from Tabebuia avellanedae and Peltophorum dubium. Genotyping showed that all C. neoformans belonged to the VNI type and C. gattii belonged to the VGI type.ConclusionsThis represents the first study on the ecology of Cryptococcus spp. associated to trees from northeastern Argentina, and the first report describing Grevillea robusta as a host of members of this fungal genus. Another finding is the isolation of C. neoformans from Tabebuia avellanedae and Peltophorum dubium, both tree species native to northeastern Argentina.     

Perilla frutescens seed agar, a new medium for identification of the Cryptococcus species complex: evaluation for all major molecular types

Here we present a new and simple medium made by Perilla frutescens seed as a tool for identification of the Cryptococcus species complex, namely Cryptococcus neoformans and Cryptococcus gattii. Its usefulness was evaluated for all major molecular types within the Cryptococcus species complex. As a result, this medium is better for identification of the species complex compared with Guizotia abyssinica or Helianthus annuus seed medium.     

<Emphasis Type="Italic">Cryptococcus gattii</Emphasis>: Clinical Importance and Emergence in North America

Cryptococcus gattii is an emerging fungal pathogen in the Pacific Northwest of North America, where it has caused more than 50 human infections since its emergence in 2004. Among residents of British Columbia, where the disease emerged in 1999 on Vancouver Island, many infections have occurred in immunocompetent persons. The cause for the emergence is currently unknown. The pathogenic profile of Cryptococcus gattii in North American patients appears to be different from that seen previously for C. gattii and from the profile of infection among patients with Cryptococcus neoformans. Treatment duration and the need for patient follow-up may be different between patients infected with C. gattii and C. neoformans. For this reason, physicians treating atypical patients with Cryptococcal spp infection, particularly HIV-uninfected patients, should obtain a travel history and obtain a species identity for Cryptococcus isolates.     

Anticandidial and anticryptococcal activity of <Emphasis Type="Italic">Euphorbia fusiformis</Emphasis>, a rare medicinal plant

The present work demonstrates the screening of extracts of the rare medicinal herb Euphorbia fusiformis for antifungal activity. The main aim was to investigate its antifungal properties against Candida albicans and Cryptococcus neoformans, the causative agents of human candidiasis and cryptococcosis, respectively. Aqueous and organic solvent extracts from the leaves and rootstock of the plant were tested against the fungi by the well-in-agar method. Almost all the organic solvent extracts exhibited an inhibitory effect against C. albicans and to some extent on C. neoformans, except for the aqueous extracts, which had no effect. The combined formulations of the extracts also had better activity against C. albicans than C. neoformans. This study thus concludes by demonstrating the antifungal properties of E. fusiformis and also the potential research in identifying the active principles, which may have future therapeutic value.     

Tongue lesion due to Cryptococcus neoformans as the first finding in an HIV-positive patient

BackgroundCryptococcosis is a severe universally distributed mycosis which mainly affects immunocompromised hosts. This mycosis is caused by yeasts of two species complex of the genus Cryptococcus: Cryptococcus neoformans and Cryptococcus gattii. Meningeal cryptococcosis is the most frequent clinical presentation of this disseminated mycosis. The oral mucosa involvement is extremely unusual.Case reportWe present a case of cryptococcosis with an unusual clinical form. The patient was assisted because she had an ulcerated lesion on the lingual mucosa. Encapsulated yeasts compatible with Cryptococcus were found in microscopic exams of wet preparations from lingual ulcer clinical samples obtained for cytodiagnosis and mycological studies. Cryptococcus neoformans (C. neoformans var. grubii VNI) was isolated in culture. This patient did not know her condition of HIV seropositive before the appearance of the tongue lesion.ConclusionsThe involvement of the oral mucosa is uncommon in this fungal infection, but is important to include it in the differential diagnosis in HIV positive patients.     

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall

Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment “ghosts” and applied 2D ¹³C-¹³C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics.