Gattii Species Complex Research Articles

Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism

ObjectiveCryptococcosis is a major opportunistic fungal infection caused by members of the genus Cryptococcus, mainly those belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. Here, we report a comprehensive molecular epidemiological study of the environmental distribution of Cryptococcus isolates in Shiraz, Iran with review of litreature. MethodA total of 406 samples were obtained from Eucalyptus trees and 139 samples from pigeon droppings. Cryptococcus species identification and genotyping were performed by amplified fragment length polymorphism (AFLP) fingerprinting sequencing and sequencing of the ITS rDNA region. ResultsMajority of the isolates belonged to the Naganishia taxon (n=69) including N. albida (formerly C. albidus, n=62), N. globosa (formerly C. saitoi, n=4), N. adeliensis (formerly C. adeliensis, n=2), N. diffluens (formerly C. diffluens, n=1), and the identified C. neoformans isolates (n=25) belonged to genotype AFLP1/VNI (n=22) and AFLP1B/VNII (n=3). ConclusionMore research efforts should be employed to isolate C. gattii species complex from environmental niches in Iran and provide additional evidence related to novel molecular types.

Banana blossom agar (BABA), a new medium to isolate members of the Cryptococcus neoformans/Cryptococcus gattii species complex useful for resource limited countries.

Isolation of representatives of the Cryptococcus neoformans/Cryptococcus gattii species complex can be made using dopamine containing media, such as Niger seed agar and l-DOPA agar. Here, we describe an alternative medium that uses banana flowers. Banana is a dopamine containing fruit and is widely available in tropical and subtropical countries that have high numbers of cryptococcosis patients. This banana blossom-based agar is useful for the enrichment of isolates of the C.neoformans/C.gattii species complex from environmental and clinical materials. The banana blossom agar (BABA) with and without creatinine can differentiate between the melanin forming isolates of the C.neoformans/C.gattii species complex from other yeasts that do not form melanin.

Butenafine and analogues: An expeditious synthesis and cytotoxicity and antifungal activities

The incidence of fungal infections is considered a serious public health problem worldwide. The limited number of antimycotic drugs available to treat human and animal mycosis, the undesirable side effects and toxicities of the currently available drugs, and the emergence of fungal resistance emphasizes the urgent need for more effective antimycotic medicines. In this paper, we describe a rapid, simple, and efficient synthetic route for preparation of the antifungal agent butenafine on a multigram scale. This novel synthetic route also facilitated the preparation of 17 butenafine analogues using Schiff bases as precursors in three steps or less. All the synthesized compounds were evaluated against the yeast, Cryptococcus neoformans/C. gattii species complexes and the filamentous fungi Trichophyton rubrum and Microsporum gypseum. Amine 4bd, a demethylated analogue of butenafine, and its corresponding hydrochloride salt showed low toxicity in vitro and in vivo while maintaining inhibitory activity against filamentous fungi.

Characterization by PCR-RFLP of the Cryptococcus neoformans and Cryptococcus gattii species complex in Venezuela

Abstract. Cryptococcosis is a sub-acute or chronic mycosis caused by opportunistic yeasts of the Cryptococcus genus, mainly by the C. neoformans and C.gattii species complex. This disease tends to position itself as one of the first opportunistic conditions associated with AIDS, with a high morbidity in these patients, who frequently develop meningoencephalitis. To date, there are techniques that allow the identification of the eight molecular types associated with these species complex, but there is little information about their circulation in Venezuela. The purpose of this work was to perform the molecular characterization of the C. neoformans and C.gattii species complex by PCR-RFLP. A total of 80 strains were used, 65 of the C. neoformans complex (CNC) and 15 of the C. gattii complex (CGC), following the protocol described by Escandon et al., carrying out the amplification of the URA5 gene and subsequent digestion with the Sau96I and HhaI restriction enzymes, that generated band patterns that allowed the identification of different molecular types. The most prevalent molecular type was VNI with 68.75% (n=55), similar to that reported worldwide;secondly, VGII molecular type with con 15% (n=12), which corresponds to that reported for Latin America, followed by VNII with 11.25% (n=9), VGIII with 2.5% (n=2), VNIII with 1.25% (n=1) and VGI with 1.25% (n=1)). Ninety-two point three percent of CCN (n=60) and 66.7% (n=10) of GCC isolates were from patients with HIV/AIDS. This study provided important epidemiological information on circulating molecular types and allowed to deepen the knowledge of this opportunistic mycosis in Venezuela.

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment.

Cryptococcus neoformans and Cryptococcus gattii species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, C. neoformans H99 and C. gattii R265. Deletion of AFR1 in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when AFR2 or MDR1 was deleted in both strains. However, when these genes were deleted in combination with AFR1, a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in afr1Δ mutants of both strains, but rhodamine 6G accumulation increased only in the mdr1Δ mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. AFR1 and AFR2 expression was upregulated in H99 and R265 when treated with fluconazole. However, MDR1 expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

Differential In Vitro Cytokine Induction by the Species of Cryptococcus gattii Complex.

Cryptococcal species vary in capsule and cell size, thermotolerance, geographic distribution, and affected populations. Cryptococcus gattii sensu stricto and C. deuterogattii affect mainly immunocompetent hosts; however, C. bacillisporus, C. decagattii, and C. tetragattii cause infections mainly in immunocompromised hosts. This study aimed to compare the capacities of different species of the C. gattii species complex to induce cytokines and antimicrobial molecules in human peripheral blood mononuclear cells (PBMCs). Cryptococcus bacillisporus and C. deuterogattii induced the lowest levels of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 among the five species of the C. gattii complex. Cryptococcus deuterogattii induced higher levels of IL-22 than those induced by C. tetragattii and the environmental species C. flavescens In addition, C. bacillisporus and C. gattii sensu stricto proliferated inside human monocyte-derived macrophages after 24 h of infection. All Cryptococcus species were able to generate reactive oxygen species (ROS) in human PBMCs, with C. bacillisporus and C. deuterogattii being more efficient than the other species. In conclusion, C. bacillisporus and C. deuterogattii induce lower levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 and higher ROS levels than those induced by the other species. Species of the Cryptococcus gattii complex have different abilities to induce cytokine and ROS production by human PBMCs.

A New screening medium for environmental recovering of Cryptococcus neoformans/C. gattii species complex

A New screening medium for environmental recovering of Cryptococcus neoformans/C. gattii species complex

Adaptive Immunity to Cryptococcus neoformans Infections

The Cryptococcus neoformans/Cryptococcus gattii species complex is a group of fungal pathogens with different phenotypic and genotypic diversity that cause disease in immunocompromised patients as well as in healthy individuals. The immune response resulting from the interaction between Cryptococcus and the host immune system is a key determinant of the disease outcome. The species C. neoformans causes the majority of human infections, and therefore almost all immunological studies focused on C. neoformans infections. Thus, this review presents current understanding on the role of adaptive immunity during C. neoformans infections both in humans and in animal models of disease.

Ecoepidemiology of Cryptococcus gattii in Developing Countries.

Cryptococcosis is a systemic infection caused by species of the encapsulated yeast Cryptococcus. The disease may occur in immunocompromised and immunocompetent hosts and is acquired by the inhalation of infectious propagules present in the environment. Cryptococcus is distributed in a plethora of ecological niches, such as soil, pigeon droppings, and tree hollows, and each year new reservoirs are discovered, which helps researchers to better understand the epidemiology of the disease. In this review, we describe the ecoepidemiology of the C. gattii species complex focusing on clinical cases and ecological reservoirs in developing countries from different continents. We also discuss some important aspects related to the antifungal susceptibility of different species within the C. gattii species complex and bring new insights on the revised Cryptococcus taxonomy.

Presence of pathogenic cryptococci on trees situated in two recreational areas in South Africa

Abstract Knowledge of the environmental prevalence of members of the Cryptococcus neoformans/Cryptococcus gattii species complex is important, since cryptococcal infection is acquired from the environment. We determined whether trees located in two South African recreational areas harboured pathogenic cryptococci and compared the isolates to clinical isolates obtained from Western Cape hospitals with molecular typing techniques. The majority of isolates originating from trees in a public park in Cape Town (PPCT) were C. gattii sensu stricto, followed by C. neoformans sensu stricto genotype AFLP1/VNI. The PPCT trees might be a source of infection, since all genotype AFLP1/VNI isolates from these trees and one clinical isolate belonged to the same sequence type (ST), i.e. ST23. Recombination and basidiospore production might be occurring in PPCT trees that contained C. gattii s.s. isolates belonging to both mating types. The presence of C. gattii s.s. in PPCT trees might therefore pose a risk to human health.

Heteroresistance to Fluconazol in Clinical and Environmental Brazilian Strains of Cryptococcus neoformans/C. gattii Species Complex

To review the literature on heteroresistance to fluconazole (FLC) and investigate the level of heteroresistance to FLC (LHF), we analyzed 100 clinical and environmental Brazilian Cryptococcus strains. Heteroresistance is a phenomenon described as the emergence of resistant subpopulation cells within a single susceptible strain that can tolerate higher concentrations of fluconazole above the minimal inhibitory concentration (MIC) level. We found lower FLC-MICs (0.12–64 mg/L) than LHF (8–128 mg/L). Highly heteroresistant adapted subpopulations (256 mg/L) was found in minority (9%) strains, but importantly, 33% showed low FLC-MIC (8 mg/L). We concluded for similar LHF in both species, but higher LHF in clinical strains in comparison to environmental ones. Our findings stressed that the LHF is not correlated to species and pretty is strain-dependent and alert about high heteroresistant subpopulations that hardly reverts to the original LHF even upon the removal of drug pressure.

Molecular characterization of Cryptococcus neoformans and Cryptococcus gattii from environmental sources and genetic comparison with clinical isolates in Apulia, Italy

The present study investigated the environmental distribution of Cryptococcus neoformans and C. gattii species complex molecular types, mating types and sequence types in Apulia, a region of Southern Italy. A total of 2078 specimens from arboreal and animal sources were analyzed. The percentage of positive samples was similar among both arboreal and animal specimens: 4.2% vs. 5.1% for C. neoformans species complex and 0.6% vs. 1.4% for C. gattii species complex. Molecular typing identified 78 isolates as VNI (76 αA and two aA), one as AD-hybrid αADa, and 16 as VGI aB. VNI isolates presented 10 different sequence types (STs) and VGI isolates two. The most frequent STs among C. neoformans and C. gattii species complex isolates were ST23 (51%) and ST156 (90%), respectively. Comparison with molecular types and STs results obtained from 21 clinical isolates collected in Apulia showed that one C. neoformans VNI clinical isolate shared an identical sequence type of one arboreal isolate (ST61) and that one C. gattii VGI clinical isolate matched with the main ST (ST156) present in the environment. In addition, molecular type VNIV was found only among clinical isolates and was absent in the investigated environmental area. In conclusion, the present study identified which C. neoformans and C. gattii species complex genotypes are circulating in Apulia, defined their ecological niches and revealed the relationship with clinical cases. It represents a basal study for addressing future investigations and public health interventions in the region.

Nutritional Requirements and Their Importance for Virulence of Pathogenic Cryptococcus Species

Cryptococcus sp. are basidiomycete yeasts which can be found widely, free-living in the environment. Interactions with natural predators, such as amoebae in the soil, are thought to have promoted the development of adaptations enabling the organism to survive inside human macrophages. Infection with Cryptococcus in humans occurs following inhalation of desiccated yeast cells or spore particles and may result in fatal meningoencephalitis. Human disease is caused almost exclusively by the Cryptococcus neoformans species complex, which predominantly infects immunocompromised patients, and the Cryptococcus gattii species complex, which is capable of infecting immunocompetent individuals. The nutritional requirements of Cryptococcus are critical for its virulence in animals. Cryptococcus has evolved a broad range of nutrient acquisition strategies, many if not most of which also appear to contribute to its virulence, enabling infection of animal hosts. In this review, we summarise the current understanding of nutritional requirements and acquisition in Cryptococcus and offer perspectives to its evolution as a significant pathogen of humans.

Molecular characterization of Cryptococcus neoformans isolates from HIV patients, Guayaquil, Ecuador

Neurocryptococcosis is an opportunistic fungal infection that represents a high cost in human lives and for the economy of countries. Its causative agent, the Cryptococcus neoformans/Cryptococcus gattii species complex, has a sexual and an asexual phase, four major serotypes and seven molecular varieties with phenotypic, clinical-epidemiological and antifungal susceptibility differences. To characterize by molecular methods clinical isolates of C. neoformans from Guayaquil, Ecuador. We determined mating types, serotypes and molecular varieties by PCR and RFLP in 27 yeast isolates previously identified as C. neoformans by conventional methods. The isolates were recovered from cerebrospinal fluid of HIV seropositive patients with neurological syndrome admitted at "Dr. José Daniel Rodríguez Maridueña" Hospital from December, 2013, to January, 2015. We established a wide prevalence of C. neoformans serotype A, MATα and genotype VNI among the studied isolates. These data are similar to those obtained in other countries and the first identified by molecular characterization in Guayaquil, Ecuador. Therefore, they constitute an important contribution to the knowledge on cryptococcosis in this country.

Importance of Resolving Fungal Nomenclature: the Case of Multiple Pathogenic Species in the Cryptococcus Genus.

Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C.neoformans were raised to species level, and the same was done for five genotypes within C.gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature "C. neoformans species complex" and "C. gattii species complex." Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

Heterocycle Thiazole Compounds Exhibit Antifungal Activity through Increase in the Production of Reactive Oxygen Species in the Cryptococcus neoformans-Cryptococcus gattii Species Complex.

Human cryptococcosis can occur as a primary or opportunistic infection and develops as an acute, subacute, or chronic systemic infection involving different organs of the host. Given the limited therapeutic options and the occasional resistance to fluconazole, there is a need to develop novel drugs for the treatment of cryptococcosis. In this report, we describe promising thiazole compounds 1, 2, 3, and 4 and explore their possible modes of action against Cryptococcus To this end, we show evidence of interference in the Cryptococcus antioxidant system. The tested compounds exhibited MICs ranging from 0.25 to 2 μg/ml against Cryptococcus neoformans strains H99 and KN99α. Interestingly, the knockout strains for Cu oxidase and sarcosine oxidase were resistant to thiazoles. MIC values of thiazole compounds 1, 2, and 4 against these mutants were higher than for the parental strain. After the treatment of C. neoformans ATCC 24067 (or C. deneoformans) and C. gattii strain L27/01 (or C. deuterogattii) with thiazoles, we verified an increase in intracellular reactive oxygen species (ROS). Also, we verified the synergistic interactions among thiazoles and menadione, which generates superoxides, with fractional inhibitory concentrations (FICs) equal to 0.1874, 0.3024, 0.25, and 0.25 for the thiazole compounds 1, 2, 3, and 4, respectively. In addition, thiazoles exhibited antagonistic interactions with parasulphonatephenyl porphyrinato ferrate III (FeTPPS). Thus, in this work, we showed that the action of these thiazoles is related to an interference with the antioxidant system. These findings suggest that oxidative stress may be primarily related to the accumulation of superoxide radicals.

Central nervous system infection due to Cryptococcus gattii sensu lato in India: Analysis of clinical features, molecular profile and antifungal susceptibility.

Cryptococcus gattii species complex has evolved as a pathogen in the last two decades causing infection among both immunocompetent and immunocompromised hosts. We aimed to analyse the clinical features of CNS infection caused by C.gattii sensu lato, molecular and antifungal susceptibility profile of this pathogen. Cases diagnosed to have CNS cryptococcosis were included in the study. Cryptococcus recovered from patient's specimen was identified by standard protocol. Species confirmation, mating type and molecular type determination were performed by PCR based methods. Antifungal susceptibility was tested in VITEK2C to amphotericin B, 5-flucytosine, fluconazole and voriconazole. Among 199 cases, 20 (10%) were due to C.gattii, comprising of 75% cryptococcal meningitis and 25% cryptococcoma cases. Young adult males were commonly affected. Headache and vomiting were prominent symptoms and 50% were immunocompromised. Among the isolates, 75%, 20% and 5% were C.tetragattii, C.gattii sensu stricto and C.bacillisporus respectively and all had mating type α. Four (20%) isolates of C.tetragattii and the only isolate of C.bacillisporus were resistant to fluconazole. The most common species isolated from south India is C.tetragattii. The study contributes to the epidemiology of C.gattii and reiterates the need for genotyping and antifungal susceptibility testing.

Differential Antifungal Activity of Human and Cryptococcal Melanins with Structural Discrepancies.

Melanin is a pigment found in all biological kingdoms, and plays a key role in protection against ultraviolet radiation, oxidizing agents, and ionizing radiation damage. Melanin exerts an antimicrobial activity against bacteria, fungi, and parasites. We demonstrated an antifungal activity of synthetic and human melanin against Candida sp. The members of the Cryptococcus neoformans and C. gattii species complexes are capsulated yeasts, which cause cryptococcosis. For both species melanin is an important virulence factor. To evaluate if cryptococcal and human melanins have antifungal activity against Cryptococcus species they both were assayed for their antifungal properties and physico-chemical characters. Melanin extracts from human hair and different strains of C. neoformans (n = 4) and C. gattii (n = 4) were investigated. The following minimum inhibitory concentrations were found for different melanins against C. neoformans and C. gattii were (average/range): 13.7/(7.8–15.6) and 19.5/(15.6–31.2) μg/mL, respectively, for human melanin; 273.4/(125–>500) and 367.2/(125.5–>500) μg/mL for C. neoformans melanin and 125/(62.5–250) and 156.2/(62–250) μg/mL for C. gattii melanin. Using Scanning Electron Microscopy we observed that human melanin showed a compact conformation and cryptococcal melanins exposed an amorphous conformation. Infrared spectroscopy (FTIR) showed some differences in the signals related to C-C bonds of the aromatic ring of the melanin monomers. High Performance Liquid Chromatography established differences in the chromatograms of fungal melanins extracts in comparison with human and synthetic melanin, particularly in the retention time of the main compound of fungal melanin extracts and also in the presence of minor unknown compounds. On the other hand, MALDI-TOF-MS analysis showed slight differences in the spectra, specifically the presence of a minor intensity ion in synthetic and human melanin, as well as in some fungal melanin extracts. We conclude that human melanin is more active than the two fungal melanins against Cryptococcus. Although some physico-chemical differences were found, they do not explain the differences in the antifungal activity against Cryptococcus of human and cryptococcal melanins. More detailed studies on the structure should be considered to associate structure and antifungal activity.

Continental Drift and Speciation of the Cryptococcus neoformans and Cryptococcus gattii Species Complexes.

ABSTRACTGenomic analysis has placed the origins of two human-pathogenic fungi, the Cryptococcus gattii species complex and the Cryptococcus neoformans species complex, in South America and Africa, respectively. Molecular clock calculations suggest that the two species separated ~80 to 100 million years ago. This time closely approximates the breakup of the supercontinent Pangea, which gave rise to South America and Africa. On the basis of the geographic distribution of these two species complexes and the coincidence of the evolutionary divergence and Pangea breakup times, we propose that a spatial separation caused by continental drift resulted in the emergence of the C. gattii and C. neoformans species complexes from a Pangean ancestor. We note that, despite the spatial and temporal separation that occurred approximately 100 million years ago, these two species complexes are morphologically similar, share virulence factors, and cause very similar diseases. Continuation of these phenotypic characteristics despite ancient separation suggests the maintenance of similar selection pressures throughout geologic ages.

The Case for Adopting the "Species Complex" Nomenclature for the Etiologic Agents of Cryptococcosis.

ABSTRACTCryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii. Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using “Cryptococcus neoformans species complex” and “C. gattii species complex” as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion.