An improved medium for the isolation of Cryptococcus neoformans from pigeon droppings

Isolation of Cryptococcus neoformans is enhanced when methyl violet at 2 mg l-1 is added to the usual Guizotia abyssinica medium. In preliminary tests the medium has also proved useful for isolating C. neoformans from other sources.     

Phenotypic switching and its implications for the pathogenesis of Cryptococcus neoformans

Phenotypic switching has been described in several strains of Cryptococcus neoformans. It occurs in vivo during chronic infection and is associated with differential gene expression and changes in virulence. The switch involves changes in the polysaccharide capsule and cell wall that affect the yeast's ability to resist phagocytosis. In addition, the phenotypic switch variants elicit qualitatively different inflammatory responses in the host. The host's immune response ultimately affects selection of the switch variants in animal models of chronic cryptococcosis. The biological relevance of phenotypic switching is demonstrated in several murine infection models and further underlines the importance of phenotypic switching in the setting of human disease. This includes the association of switching and poor outcome in chronic infection, the ability of the mucoid variant of strain RC-2 (RC-2 MC) but not the smooth variant (RC-2 SM) to promote increased intracranial pressure in a rat model, and lastly the observation that antifungal interventions can promote the selection of more virulent switch variants during chronic murine infection.     

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.     

Relation of the pigeon to cryptococcosis: Natural carrier state,heat resistance and survival of Cryptococcus neoformans

Summary The feral pigeon in New York City was found to serve as a mechanical carrier of pathogenic strains ofCryptococcus neoformans. Of 94 feral pigeons freshly trapped in the city, 7 were found to carryC. neoformans on their beaks and feet, while their rectal swabs were negative. Following crop instillation ofC. neoformans in 3 feral pigeons, the fungus survived passage through the gastrointestinal tract and appeared in the fresh feces within one hour after inoculation and was still present 24 hours later. The internal body (rectal) temperatures of 57 feral pigeons recorded soon after capture in two seasons of the year, ranged from 41.5° C to 43.3° C and averaged 42.5° ± 0.39° C. The birds were able to maintain their high temperatures in the face of sustained cold, indicating the presence of a strong thermoregulating mechanism. A study of the growth and survival of 60 human strains ofC. neoformans on Sabouraud dextrose yeast extract agar slants revealed that 100 % of the cultures were able to grow at 39° C, 92 % at 40° C, 30 % at 41° C, 17 % at 42° C and 8 % at 43° C. Despite exposure to these temperatures for 7 days, 100 % of the human strains survived at 40° C, 97 % at 41° C, 95 % at 42° C, 91 % at 43° C, 88 % at 44° C, 47 % at 45° C and none at 47° C. Seventy strains ofC. neoformans of pigeon excreta origin showed a similar pattern of heat resistance, except that 11 % of these strains survived 47° C. The inability ofC. neoformans to grow at 44° C, a property shared by all 130 strains, provides a new species characteristic.C. neoformans was found to multiply rapidly in moist pigeon excreta extract, reaching counts up to 60 million per ml within 3 weeks, and to be still viable in moist as well as dessicated pigeon excreta extract after more than two years of storage at room temperature.Presented in part at the 26th VA-Armed Forces Pulmonary Disease Research Conference, Cleveland, Ohio, Jan. 23–26, 1967, and the 67th Annual Meeting American Society for Microbiology, New York, April 30–May 4, 1967. Supported by Grants GB-4722 from the National Science Foundation and U-1367 from The Health Research Council of the City of New York.Acknowledgment is gratefully given toThomas J. Dalton, Division of Environmental Sanitation, Department of Health, New York City andMabel Halloran for their valuable assistance and to the ASPCA of Manhattan and Brooklyn for their cooperation in this study.     

The contest of microbial pigeon neighbors: Interspecies competition between Serratia marcescens and the human pathogen Cryptococcus neoformans

In nature, microorganisms often exhibit competitive behavior for nutrients and limited space, allowing them to alter the virulence determinants of pathogens. The human pathogenic yeast Cryptococcus neoformans can be found organized in biofilms, a complex community composed of an extracellular matrix which confers protection against predation. The aim of this study was to evaluate and characterize antagonistic interactions between two cohabiting microorganisms: C. neoformans and the bacteria Serratia marcescens. The interaction of S. marcescens with C. neoformans expressed a negative effect on biofilm formation, polysaccharide capsule, production of urease, and melanization of the yeast. These findings evidence that competition in mixed communities can result in dominance by one species, with direct impact on the physiological modulation of virulence determinants. Such an approach is key for understating the response of communities to the presence of competitors and, ultimately, rationally designing communities to prevent and treat certain diseases.     

Looking for sex in the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii

Why are we interested in understanding the mode of reproduction being used by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii? Empirical evidence has finally supported the long-held assumption that, by increasing the rate of adaptive evolution, sex increases the chances of long-term survival. Understanding the ability of pathogenic organisms to adapt to diagnostic and treatment regimes is also important in the fight against the diseases caused by these organisms. This review looks at the different approaches used to identify population structure in C. neoformans and C. gattii. These are sexual species; however, recombination in natural populations has only recently been found. We highlight the importance of population selection and the value of both indirect molecular analysis and direct biological evidence for sexual recombination, when looking for the mode of reproduction in these fungal pathogens.     

人类环境病原真菌——新生隐球菌的社会行为

人类病原真菌代表了病原微生物一个重要类群,主要感染免疫缺陷、受损和受抑制的患者,导致很高的死亡率。绝大多数的人类病原真菌属于环境病原微生物,它们与人类宿主并无长期共同进化史以及紧密的共生关系,这类真菌被称为环境病原真菌。新生隐球菌作为环境病原真菌的模式种,每年导致约60万人死亡。与其他环境病原真菌相似,该菌的致病能力并不由宿主因素驱动,而是衍生于其对于生境压力出色的适应性策略。近几年来的研究表明,群体或社会行为可能作为一个关键环境适应策略,广泛参与新生隐球菌的感染、耐药以及有性生殖过程。本文以新生隐球菌为例,针对生物被膜以及生物被膜样群落,总结其中的群体或社会调控方式,并对其研究前景和临床应用提出了一些思考。     

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.     

Towards a vaccine for Cryptococcus neoformans: principles and caveats

In the Damage-response framework of microbial pathogenesis, infectious diseases are one outcome of a host-microorganism interaction in a susceptible host. In cryptococcal disease, damage to the host is caused by Cryptococcus neoformans virulence determinants, the nature of the host response, or both. Further, the disease may be acute or reactivated from a latent state. Hence, a vaccine for C. neoformans would need to prevent disease resulting from either acute or reactivated infection. The evidence to support the development of a vaccine for C. neoformans that induces antibody-mediated immunity is discussed herein.     

Unique hybrids between the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii

Cryptococcus neoformans and Cryptococcus gattii are yeasts that cause meningoencephalitis, but that differ in host range and geographical distribution. Cryptococcus neoformans occurs world-wide and mostly infects immunocompromised patients, whereas C. gattii occurs mainly in (sub)tropical regions and infects healthy individuals. Anomalous C. neoformans strains were isolated from patients. These strains were found to be monokaryotic, and diploid or aneuploid. Amplified Fragment Length Polymorphism (AFLP) and sequence analyses indicated that AFLP genotypes 2 (C. neoformans) and 4 (C. gattii) were present. The strains were serologically BD. Mating- and serotype-specific PCR reactions showed that the strains were MATa-serotype D/MATalpha-serotype B. This study is the first to describe naturally occurring hybrids between C. neoformans and C. gattii.     

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.     

Cryptococcus neoformans: the yeast that likes it hot

The ability of fungi to grow well at mammalian body temperatures is a fundamental characteristic of invasive human fungal pathogens. Cryptococcus neoformans, with its genetics, molecular biology, robust animal models and clinical importance, has become a premier fungal model system for molecular fungal pathogenesis studies. There has been over a half century of study into how C. neoformans grows at high temperatures. However, recently the understanding of high-temperature growth at a molecular level has dramatically accelerated. Many strategies have been used to identify genes and over a dozen genes have already been identified to be necessary for high-temperature growth. It is likely that there are many more to discover. It is clear that, as further studies understand how this encapsulated yeast is able to withstand the stresses of high temperature at a genetic and molecular basis, we will also know more about how it and other fungal pathogens have evolved into well-established human pathogens.     

Essential Metals in Cryptococcus neoformans: Acquisition and Regulation

Transition metals such as iron, zinc, and copper play key roles in numerous biological processes, including innate immunity in vertebrates. In fact, control of the availability of these essential metals is an important component of the immune response to limit the growth of pathogens during infection. In turn, pathogens must overcome the extreme limitation of essential metals and potential metal toxicity to successfully cause disease. Recent work illuminates the mechanisms by which the pathogenic fungus Cryptococcus neoformans manages iron, copper, and zinc availability in support of proliferation in vertebrate hosts. In particular, the fungus makes use of high-affinity metal transport systems and complex regulatory networks that play critical roles in the virulence of the fungus.     

Nitrogen fixation by Vibrio parahaemolyticus and its implications for a new ecological niche

A Vibrio parahaemolyticus strain isolated from the rhizosphere of the ecosystem dominant estuarine grass, Spartina alterniflora, was characterized and shown to carry nifH, the gene encoding the nitrogenase iron protein, and to fix N(2). Nitrogen fixation may contribute substantially to the adaptability, niche breadth, and ecological significance of V. parahaemolyticus.     

新生隐球菌对角质形成细胞活力的作用研究

目的研究新生隐球菌体外对角质形成细胞活力的影响。方法将新生隐球菌父代标准株与子代荚膜缺陷株于体外分别与角质形成细胞分别共培养,同时设立热灭活的菌体、空白对照,再分别设立菌体与细胞直接接触与不接触共培养相互对照,分别作用0.5 h、1 h和2 h后,采用流式细胞仪检测隐球菌角质形成细胞的调亡率。结果随着时间延长,与空白对照组及热灭活组比较,实验组角质形成细胞的凋亡率逐渐增加。无荚膜株与父代有荚膜株比较,无荚膜株对细胞活力的影响在作用后1 h、2 h明显低于有荚膜株。2种菌株不直接接触培养使细胞的凋亡率明显下降;不直接接触的有荚膜株与热灭活的菌体之间比较差异不显著。结论虽然有荚膜株与无荚膜株隐球菌均可以使角质形成细胞活性明显降低,但荚膜可以显著增强菌体对细胞活力的影响;角质形成细胞活力的降低主要是通过与菌体接触培养后产生的,诱导细胞调亡需要菌体与细胞的直接接触。     

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.