Gene Expression of Diverse Cryptococcus Isolates during Infection of the Human Central Nervous System.

Cryptococcus neoformans is a major human central nervous system (CNS) fungal pathogen causing considerable morbidity and mortality. In this study, we provide the widest view to date of the yeast transcriptome directly from the human subarachnoid space and within cerebrospinal fluid (CSF). We captured yeast transcriptomes from C. neoformans of various genotypes in 31 patients with cryptococcal meningoencephalitis as well as several Cryptococcus gattii infections. Using transcriptome sequencing (RNA-seq) analyses, we compared the in vivo yeast transcriptomes to those from other environmental conditions, including in vitro growth on nutritious media or artificial CSF as well as samples collected from rabbit CSF at two time points. We ranked gene expressions and identified genetic patterns and networks across these diverse isolates that reveal an emphasis on carbon metabolism, fatty acid synthesis, transport, cell wall structure, and stress-related gene functions during growth in CSF. The most highly expressed yeast genes in human CSF included those known to be associated with survival or virulence and highlighted several genes encoding hypothetical proteins. From that group, a gene encoding the CMP1 putative glycoprotein (CNAG_06000) was selected for functional studies. This gene was found to impact the virulence of Cryptococcus in both mice and the CNS rabbit model, in agreement with a recent study also showing a role in virulence. This transcriptional analysis strategy provides a view of regulated yeast genes across genetic backgrounds important for human CNS infection and a relevant resource for the study of cryptococcal genes, pathways, and networks linked to human disease. IMPORTANCE Cryptococcus is the most common fungus causing high-morbidity and -mortality human meningitis. This encapsulated yeast has a unique propensity to travel to the central nervous system to produce disease. In this study, we captured transcriptomes of yeasts directly out of the human cerebrospinal fluid, the most concerning site of infection. By comparing the RNA transcript levels with other conditions, we gained insights into how the basic machinery involved in metabolism and environmental responses enable this fungus to cause disease at this body site. This approach was applied to clinical isolates with diverse genotypes to begin to establish a genotype-agnostic understanding of how the yeast responds to stress. Based on these results, future studies can focus on how these genes and their pathways and networks can be targeted with new therapeutics and possibly classify yeasts with bad infection outcomes.

Adaptation to Fluconazole via Aneuploidy Enables Cross-Adaptation to Amphotericin B and Flucytosine in Cryptococcus neoformans.

The high morbidity and mortality of cryptococcal meningitis is due to the limited range of therapeutic options: only three classes of antifungal drugs are available (polyenes [amphotericin B], azoles [fluconazole], and pyrimidine analogues [flucytosine]). Fluconazole is the most widely used antifungal drug in sub-Saharan Africa, where cryptococcal meningitis is a major cause of death in patients infected with HIV. In this study, we found that exposure to fluconazole, even for short times (48 h) at subinhibitory concentrations, drove rapid adaptation of Cryptococcus neoformans serotype A strain H99 via the acquisition of different aneuploid chromosomes. These aneuploidies conferred heteroresistance to fluconazole. Importantly, most of the adaptors were cross-tolerant to flucytosine. Some of the aneuploid adaptors were not heteroresistant to fluconazole but were tolerant to amphotericin B. Thus, exposure to one antifungal drug class can promote adaptation to two antifungal drug classes, highlighting the plasticity of the C. neoformans genome and raising concerns about the rapid reduction in the range of treatment options for cryptococcal infections. IMPORTANCE Cryptococcosis is a globally distributed invasive fungal infection caused by infections with Cryptococcus neoformans or Cryptococcus gattii. Only three classes of therapeutic drugs are clinically available for treating cryptococcosis: polyenes (amphotericin B), azoles (fluconazole), and pyrimidine analogues (flucytosine). Fluconazole is the primary drug available in resource-limited countries. Aneuploidy is a genomic state due to the gain or loss of chromosomes. We found that C. neoformans rapidly adapted to fluconazole by acquiring diverse aneuploidies and that specific aneuploidies enabled improved growth of isolates susceptible (tolerance) to amphotericin B and/or cross-tolerance to both fluconazole and flucytosine. Therefore, aneuploidy is an underlying mechanism of drug tolerance that not only arises rapidly during growth in fluconazole but can also confer tolerance to other antifungal drugs without prior exposure to those drugs. Resistant isolates have high MICs, and all cells grow similarly in medium with the drug, while tolerant isolates test as susceptible and grow slowly at drug concentrations above the MIC.

Cryptococcus depauperatus, a close relative of the human-pathogen C. neoformans, associated with coffee leaf rust (Hemileia vastatrix) in Cameroon.

The genus Cryptococcus is well known for its two species -Cryptococcus neoformans and C. gatii- that are etiological agents of cryptococcosis, an important fungal disease of mammals, including humans, and which is particularly common in immunocompromised patients. Nevertheless, Cryptococcus is a large and widely distributed genus of basidiomycetes occupying a broad range of niches, including mycoparasitism. One such mycoparasitic species is Cryptococcus depauperatus, which was firstly mistakenly described as a pathogen of scale insects under the name Aspergillus depauperatus. The "Aspergillus" conidiophores were later shown to be basidia of a Cryptococcus and the new combination C. depauperatus was proposed. Additionally, instead of an entomopathogen, the fungus was found to be a mycoparasite growing on the entomopathogen Akanthomyces (Lecanicillium) lecanii. Recently, during surveys for mycoparasites of coffee leaf rust (Hemileia vastatrix) in the context of a biocontrol project, white colonies covering rust pustules were observed in Cameroon. Upon close examination, instead of a member of the "white colony forming complex" of Ascomycetes, commonly collected growing on H. vastatrix, such colonies were found to represent a basidiomycete fungus with basidia-bearing chains of basidiospores, typical of the genus Cryptococcus. Morphological and molecular evidence was generated supporting the identification of the fungus on rust pustules as C. depauperatus. This is the first record of C. depauperatus from Africa and of its association with coffee leaf rust.

Genotypic diversity of Iranian Cryptococcus neoformans using multilocus sequence typing (MLST) and susceptibility to antifungals.

Cryptococcus species is an opportunistic yeast pathogen and classified into different molecular types according to typing techniques including multilocus sequence typing (MLST). The study aimed to investigate the genotypes of environmental Cryptococcus isolates using MLST and the relationship between the in vitro antifungal susceptibility and sequence types of isolates. Genotyping Cryptococcus isolates was performed by the MLST method at seven nuclear loci. Antifungal susceptibility was determined by using CLSI broth micro-dilution method for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine, and luliconazole. Seven sequence types (ST) were detected using MLST analysis, with the most frequent (50%) ST77, followed by ST4 (16.7%) among 30 C. neoformans isolates. All antifungals demonstrated excellent activity against isolates, except for itraconazole and amphotericin B that were non-wild type against 53.3% and 10% of isolates, respectively. Although seven sequence types belonging to C. neoformans isolates were detected, ST77 was the main sequence type in Ahvaz. Also, non-wild type isolates were only found against itraconazole and amphotericin B.

Amoeba Predation of Cryptococcus neoformans Results in Pleiotropic Changes to Traits Associated with Virulence.

Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as Cryptococcus neoformans; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged times and then analyzed surviving colonies phenotypically and genetically. Surviving colonies comprised cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of traits associated with virulence, such as capsule size, urease production, and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. Isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene encoding Pkr1 (AMP-dependent protein kinase regulator) but manifested reduced virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes. Given the myriad potential predators in soils, the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation.IMPORTANCECryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle. Previous studies showed that C. neoformans increases its pathogenicity after interacting with its environmental predator amoebae. Amoebae, like macrophages, are phagocytic cells that are considered an environmental training ground for pathogens to resist macrophages, but the mechanism by which C. neoformans changes its virulence through interactions with protozoa is unknown. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival, with this diversity suggesting a bet-hedging strategy to ensure that some forms survive.

Combining natural language processing and metabarcoding to reveal pathogen-environment associations.

Cryptococcus neoformans is responsible for life-threatening infections that primarily affect immunocompromised individuals and has an estimated worldwide burden of 220,000 new cases each year-with 180,000 resulting deaths-mostly in sub-Saharan Africa. Surprisingly, little is known about the ecological niches occupied by C. neoformans in nature. To expand our understanding of the distribution and ecological associations of this pathogen we implement a Natural Language Processing approach to better describe the niche of C. neoformans. We use a Latent Dirichlet Allocation model to de novo topic model sets of metagenetic research articles written about varied subjects which either explicitly mention, inadvertently find, or fail to find C. neoformans. These articles are all linked to NCBI Sequence Read Archive datasets of 18S ribosomal RNA and/or Internal Transcribed Spacer gene-regions. The number of topics was determined based on the model coherence score, and articles were assigned to the created topics via a Machine Learning approach with a Random Forest algorithm. Our analysis provides support for a previously suggested linkage between C. neoformans and soils associated with decomposing wood. Our approach, using a search of single-locus metagenetic data, gathering papers connected to the datasets, de novo determination of topics, the number of topics, and assignment of articles to the topics, illustrates how such an analysis pipeline can harness large-scale datasets that are published/available but not necessarily fully analyzed, or whose metadata is not harmonized with other studies. Our approach can be applied to a variety of systems to assert potential evidence of environmental associations.

An analysis of the population of Cryptococcus neoformans strains isolated from animals in Poland, in the years 2015-2019.

Fungi belonging to the Cryptococcus neoformans/C. gattii species complex (CNGSC) are pathogens causing severe infections in humans and animals, that for humans may result in a mortality rate ranging up to 70%. The CNGSC is divided into eight major molecular types, that may differ in their virulence and susceptibility. In order to fully understand the epidemiology of cryptococcosis, it is important to study the world distribution and population structure of these pathogens. The present study is the first presenting a population of strains isolated in Poland and one of the few using a multi-species animal group as a source of the specimen. The pathogen was present in 2.375% of the tested animals. The URA5-RFLP and MALDI-TOF MS analyses have revealed that the population consisted exclusively of C. neoformans strains, with a predominance of major molecular type VNIV (C. neoformans var. neoformans). The MALDI-TOF MS was used to perform the CNGSC strains identification on both the species and sub-species level. Despite the fact that the animals providing the specimens were not treated with 5-fluorocytosine, around 10% of the tested population presented MIC values exceeding 64 mg/L, indicating the existence of the 5-fluorocytosine-resistant strains in the environment.

Genomic epidemiology of a Cryptococcus neoformans case cluster in Glasgow, Scotland, 2018.

In 2018, a cluster of two cases of cryptococcosis occurred at the Queen Elizabeth University Hospital (QEUH) in Glasgow, Scotland (UK). It was postulated that these cases may have been linked to pigeon droppings found on the hospital site, given there have been previous reports of Cryptococcus neoformans associated with pigeon guano. Although some samples of pigeon guano taken from the site yielded culturable yeast from genera related to Cryptococcus, they have since been classified as Naganishia or Papiliotrema spp., and no isolates of C. neoformans were recovered from either the guano or subsequent widespread air sampling. In an attempt to further elucidate any possible shared source of the clinical isolates, we used whole-genome sequencing and phylogenetic analysis to examine the relationship of the two Cryptococcus isolates from the QEUH cases, along with two isolates from sporadic cases treated at a different Glasgow hospital earlier in 2018. Our work demonstrated that these four clinical isolates were not clonally related; while all isolates were from the VNI global lineage and of the same mating type (MATα), the genotypes of the two QEUH isolates were separated by 1885 base changes and belonged to different sub-lineages, recently described as the intercontinental sub-clades VNIa-93 and VNIa-5. In contrast, one of the two sporadic 2018 clinical isolates was determined to belong to the VNIb sub-lineage and the other classified as a VNIV/VNI hybrid. Our work demonstrated that the two 2018 QEUH isolates and the two prior C. neoformans clinical isolates were all genetically distinct. It was not possible to determine whether the QEUH genotypes stemmed from independent sources or from the same source, i.e. pigeons carrying different genotypes, but it should be noted that whilst members of allied genera within the Tremellomycetes were isolated from the hospital environment, there were no environmental isolations of C. neoformans.

Cryptococcal meningitis in non-HIV patients in the State of Amazonas, Northern Brazil.

Cryptococcosis is a life-threatening fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii species complex. Most cases are recorded in patients suffering from HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome). However, this infection also occurs in non-HIV patients with a proportion of 10-30% of all cases. The study aimed at the clinical and molecular characterization of non-HIV patients diagnosed with cryptococcosis at the Tropical Medicine Foundation (FMT-HVD) from July 2016 to June 2019. Medical records of respective patients were analyzed to describe the course of cryptococcosis in non-HIV patients. In addition, multi-locus sequence typing (MLST) was applied to identify the sequence types of the isolated Cryptococcus strains, to perform phylogenetic analysis, and to evaluate the isolates' genetic relationship to global reference strains. Antifungal susceptibility profiles to amphotericin B, fluconazole, and itraconazole were assessed by broth microdilution. From a total of 7 patients, 4 were female, the age range varied between 10 and 53 years (median of 36.3 years). Cryptococcal meningitis was the common clinical manifestation (100%). The period between onset of symptoms and confirmed diagnosis ranged from 15 to 730 days (mean value of 172.9 days), and the observed mortality was 57.1%. Of note, comorbidities of the assessed cryptococcosis patients comprised hypertension, diabetes mellitus, and intestinal tuberculosis. Genotyping applying PCR-RFLP of the URA5 gene identified all clinical isolates as C. gattii genotype VGII. Using MLST, it was possible to discriminate the sequence types ST20 (n = 4), ST5 (n = 3), and the newly identified sequence type ST560 (n = 1). The antifungals amphotericin B, fluconazole, and itraconazole showed satisfactory inhibitory activity (microdilution test) against all C. gattii VGII strains.

Genotypic diversity and antifungal susceptibility of environmental isolates of Cryptococcus neoformans from the Yangtze River Delta region of East China.

Although cryptococcosis is widely recognized as infection by Cryptococcus neoformans sensu lato from environmental sources, information concerning the characteristics of environmental isolates of C. neoformans s. l. and how they are related to clinical isolates is very limited, especially in East China. In this study, 61 environmental isolates of C. neoformans were recovered from pigeon (Columba livia) droppings from the Yangtze River Delta region of East China. These isolates were genotyped using the ISHAM-MLST consensus scheme and their antifungal drug susceptibilities were determined following the CLSI M27-A3 guidelines. The 61 isolates were found belonging to 13 sequence types (STs), including several novel STs such as ST254 and ST194. The dominant ST in this environmental sample was ST31, different from that of clinical strains (ST5) in this region. Azole-resistance, such as fluconazole (FLU)-resistance, was observed among our environmental C. neoformans isolates. The findings of this study expand our understanding of ecological niches, population genetic diversity, and azole-resistance characteristics of the yeast in East China. Our research lays the foundation for further comparative analysis the potential mechanisms for the observed differences between environmental and clinical populations of C. neoformans in China. LAY SUMMARY: Cryptococcosis is widely recognized as infection by Cryptococcus neoformans sensu lato from environmental sources. However, there is currently limited information about the genetic diversity and antifungal susceptibility of environmental C. neoformans s. l. isolates, including how they may differ from clinical samples. In this study, we collected 61 environmental C. neoformans isolates from domestic pigeon droppings from the Yangtze River Delta region of East China. These isolates were genotyped using multi-locus sequencing. We found a high genotypic diversity in this population of C. neoformans, with several novel genotypes and a distribution of genotypes different from that of clinical strains in this region. Azole-resistance, such as fluconazole (FLU)-resistance, was observed among our environmental C. neoformans isolates. The findings of this study expand our understanding of ecological niches, genetic diversity, and azole-resistance characteristics of the yeast in East China. Our research lays the foundation for phylogenomic analysis investigating why and how disparate population structures of C. neoformans isolates formed between environmental and clinical sources in the region.

Microbe Profile: Cryptococcus neoformans species complex.

Cryptococcus neoformans is a lethal fungus disguised in a polysaccharide coat. It can remain dormant in the host for decades prior to reactivation, causing systemic cryptococcosis in humans and other mammals. Cryptococcus deploys a multitude of traits to adapt to and survive within the host, including immunosuppression, an ability to replicate intra- and extra-cellularly in phagocytes, changes in morphology and ploidy, a predilection to infect the CNS, and the capacity to utilize neurotransmitters and unique carbon sources available in the brain. These pathogenic strategies displayed by this fungus might have evolved through its interactions with microbial predators in the environment.

New multilocus sequence typing primers to enable genotyping of AD hybrids within the Cryptococcus neoformans species complex.

Although AD hybrids within the Cryptococcus neoformans species complex represent about 20% of the isolates identified in Europe, phylogenetic and population genetic studies are lacking due to the inability to use the standardized typing method. The aim of the present study was to design new molecular type specific primers in order to apply the standard ISHAM consensus multilocus sequence typing (MLST) scheme to AD hybrids. The new primers are able to specifically amplify VNI and VNIV alleles of the seven MLST loci in both haploid and diploid or aneuploid hybrid strains. This study forms the basis for future molecular epidemiology studies of AD hybrids. LAY ABSTRACT: We designed and tested new specific primers to amplify the two alleles of each of the seven MLST loci in C. neoformans species complex hybrids. The sequences obtained from hybrids can be compared with those present in the Cryptococcus global MLST database for future molecular epidemiology studies.

New ST623 of Cryptococcus neoformans isolated from a patient with non-Hodgkin's lymphoma in the Brazilian Amazon.

BACKGROUND: Cryptococcosis is a disease of wide geographic distribution. It is most critical when it affects immunocompromised patients, with AIDS, tuberculosis or other diseases that require prolonged hospitalization. METHODS: This study described a case report, molecular epidemiology, the phylogenetic relationship, along with antifungal susceptibility test of a new ST 623 of C. neoformans isolated in a patient with non-Hodgkin's Lymphoma, from Manaus, Brazil. RESULTS: The new C. neoformans was susceptible to all antifungal drugs tested. Our results showed that ST623 new clone has no evident evolutionary proximity to any other ST of the VNI subtype group identified in Brazil. CONCLUSIONS: In the context of phylogenetic analysis, this new genotype belongs to VNI subtype, and subsequencing complete genome studies are necessary to better understand the phylogenetic relationships amongst STs in this group.

Multi-locus Sequence Typing and Whole Genome Sequence Analysis of Cryptococcus neoformans Isolated from Clinical Specimens in Vajira Hospital, Bangkok, Thailand.

The basidiomycete yeast Cryptococcus neoformans causes disease in immunocompromized patients. Whole genome sequencing (WGS) technology provides insights into the molecular epidemiology of C. neoformans. However, the number of such studies is limited. Here we used WGS and multilocus sequence typing (MLST) to determine the genetic diversity of C. neoformans isolates and genetic structures of their populations among patients admitted to a single hospital in Bangkok, Thailand. Seven isolates from six patients collected during 1 year were identified as C. neoformans sensu stricto according to colony morphology, microscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nucleotide sequence analysis of internal transcribed sequences. These isolates were sensitive to the antifungal drugs amphotericin B, fluconazole, 5-flucytosine, voriconazole, itraconazole and posaconazole and were mating type α and molecular type VNI. MLST analysis identified ST4, ST5 and ST6. We further employed WGS to determine the genetic diversity and relationships of C. neoformans isolated here combined with C. neoformans sequences data acquired from a public database (n = 42). We used the data to construct a phylogenetic tree. WGS provided additional genomics data and achieved high discriminatory power for identifying C. neoformans isolates isolated in Thailand. This report further demonstrates the applicability of WGS analysis for conducting molecular epidemiology and provides insight into the genetic diversity of C. neoformans isolates from one hospital in Thailand.

Clinical insights and epidemiology of central nervous system infection due to Cryptococcus neoformans/gattii species complexes: A prospective study from South India.

In the last two decades, central nervous system (CNS) cryptococcosis (CNSc) has emerged as a major opportunistic infection in the immunocompromised population of India. We have analyzed the clinical features of CNSc and epidemiology of Cryptococcus neoformans and Cryptococcus gattii. A total of 160 clinical isolates of C. neoformans/gattii recovered from CNSc patients were analyzed. The origin, clinical parameters, and imaging features of the patients were recorded, and clinical parameters were analyzed based on their human immunodeficiency virus (HIV) status and infecting species, namely, C. neoformans or C. gattii. Serotypes and mating types of the isolates were determined. Molecular typing was performed by polymerase chain reaction (PCR) fingerprinting using M13 microsatellite primer (GTG)5, and multilocus sequence typing (MLST). Majority of the patients were from Bangalore Urban, Karnataka. Among 160 cases 128 (80%) were HIV seropositive, and 32 (20%) were HIV negative. Middle-aged males (36-55 years) were highly affected. There were statistically significant differences in the clinical manifestations, imaging and CSF parameters of HIV coinfected and noninfected cases, whereas limited differences were observed in these parameters in the cases infected with C. neoformans and C. gattii. We identified 80% C. neoformans VNI, 8.75% VNII and 22.5% C. gattii (VGI), 8.75% C. tetragattii (VGIV) among clinical strains. This comprehensive study will contribute toward a better prognosis of CNS cryptococcosis patients during the hospital stay, treatment strategies for HIV coinfected and noninfected cases and will provide the molecular epidemiology of these two pathogenic fungal species in south India, which was unclear in this part of the country.

Genotypic diversity in clinical and environmental isolates of Cryptococcus neoformans from India using multilocus microsatellite and multilocus sequence typing.

BACKGROUND: Cryptococcus neoformans is the leading cause of cryptococcal meningitis in HIV/AIDS patients. As infections in humans are predominantly caused by the inhalation of basidiospores from environmental sources, therefore, analysing the population structure of both clinical and environmental populations of C neoformans can increase our understanding of the molecular epidemiology of cryptococcosis. OBJECTIVE: To investigate the genotypic diversity and antifungal susceptibility profile of a large collection of C neoformans isolates (n = 523) from clinical and environmental sources in India between 2001 and 2014. MATERIALS AND METHODS: Cryptococcus neoformans isolates were genotyped by AFLP, microsatellite typing (MLMT) and MLST. In vitro antifungal susceptibility for standard antifungals was undertaken using CLSI M27-A3. RESULTS: All isolates were C neoformans, AFLP1/VNI and exhibited mating-type MATα. MLMT revealed that the majority of isolates belonged to microsatellite cluster (MC) MC3 (49%), followed by MC1 (35%), and the remaining isolates fell in 11 other MC types. Interestingly, two-thirds of clinical isolates were genotype MC3 and only 17% of them were MC1, whereas majority of environmental strains were MC1 (54%) followed by MC3 (16%). Overall, MLST assigned 5 sequence types (STs) among all isolates and ST93 was the most common (n = 76.7%), which was equally distributed in both HIV-positive and HIV-negative patients. Geometric mean MICs revealed that isolates in MC1 were significantly less (P < .05) susceptible to amphotericin B, 5-flucytosine, itraconazole, posaconazole and isavuconazole than isolates in MC3. CONCLUSIONS: The study shows a good correlation between MLMT and MLST genotyping methods. Further, environmental isolates were genetically more diverse than clinical isolates.

Patterns of allele distribution in a hybrid population of the Cryptococcus neoformans species complex.

BACKGROUND: The sister yeast species Cryptococcus neoformans (serotype A) and Cryptococcus deneoformans (serotype D) are causative agents of deadly cryptococcosis and fungal meningoencephalitis. These haploid yeasts can hybridise in nature, giving rise to AD hybrids that are predominantly diploid or aneuploid. Despite their increasing prevalence in clinical settings, much remains unknown about the allelic distribution patterns in AD hybrid strains. OBJECTIVES: This study aims to characterise allele distributions in AD hybrids derived from the same basidium as well as from multiple basidia in a laboratory-derived C neoformans × C deneoformans hybrid cross. METHODS: We dissected a total of 1625 basidiospores from 31 basidia. The 297 basidiospores that successfully germinated were genotyped by molecular characterisation of 33 markers using PCR-RFLP, with at least two markers on each of the 14 chromosomes in the genome. RESULTS: Of the 297 strains, 294 contained at least one heterozygous locus, with a mean heterozygosity of ~30% per strain. Most hybrid genomes and chromosomes displayed significantly distorted allele distributions, with offspring originating from the same basidium tended to have alleles at different loci from the same parent. More basidia were skewed in favour of C deneoformans alleles, the mitochondria-donor parent, than the C neoformans alleles. CONCLUSIONS: The divergence between C neoformans and C deneoformans genomes has likely created co-adapted allelic combinations, with their co-segregation in hybrid offspring imparting a significant fitness benefit. However, the diversity of genotypes recovered here in a single hybridisation event indicates the enormous capacity of AD hybrids for adaptation and diversification.

Molecular epidemiology and antifungal susceptibility profiles of clinical Cryptococcus neoformans/Cryptococcus gattii species complex.

Introduction. Limited data regarding the epidemiology and susceptibility profiles of cryptococcosis are available in the Middle East.Aim. Our study aimed to evaluate the molecular diversity, mating types and antifungal susceptibility pattern of Cryptococcus species (n=14) isolated from 320 suspected patients with cryptococcosis.Methodology. The URA5 gene was subjected to restriction fragment length polymorphism and sequence analysis. In addition, in vitro antifungal susceptibility testing was performed by Clinical and Laboratory Standards Institute (CLSI) M27-A4 and M59 guidelines.Results. Overall, 14 (4.4 %) patients were confirmed as cryptococcosis. Based on molecular type, 85.7 and 14.3 % of the isolates were C. neoformans VN I and VN II, respectively. Phylogenetic analysis of URA5 gene sequences revealed clustering of VN I and VN II isolates into two distinct clades with a substantial difference within each molecular type. Voriconazole and 5-fluorocytosine, respectively, had the lowest (0.031 µg ml-1) and highest (8 µg ml-1) MICs. The epidemiological cutoff values (ECVs) for amphotericin B, fluconazole, voriconazole and 5-fluorocytosine encompassed ≥97 % of all 14 C. neoformans VN I species. However, according to the CLSI document M59, ECVs for itraconazole (7; 50 % of the isolates) and for posaconazole (1; 7.1 % of the isolate), were one log2 dilution higher than the wild type range. Combinations of amphotericin B with 5-fluorocytosine, amphotericin B with fluconazole and fluconazole with 5-fluorocytosine exhibited synergistic effects against 37, 31 and 12.5 % of the isolates, respectively.Conclusion. Our findings may significantly contribute to the development of management strategies for patients at a higher risk of cryptococcosis, particularly HIV-positive individuals.

Cryptococcus neoformans Recovered From Olive Trees (Olea europaea) in Turkey Reveal Allopatry With African and South American Lineages.

Cryptococcus species are life-threatening human fungal pathogens that cause cryptococcal meningoencephalitis in both immunocompromised and healthy hosts. The natural environmental niches of Cryptococcus include pigeon (Columba livia) guano, soil, and a variety of tree species such as Eucalyptus camaldulensis, Ceratonia siliqua, Platanus orientalis, and Pinus spp. Genetic and genomic studies of extensive sample collections have provided insights into the population distribution and composition of different Cryptococcus species in geographic regions around the world. However, few such studies examined Cryptococcus in Turkey. We sampled 388 Olea europaea (olive) and 132 E. camaldulensis trees from seven locations in coastal and inland areas of the Aegean region of Anatolian Turkey in September 2016 to investigate the distribution and genetic diversity present in the natural Cryptococcus population. We isolated 84 Cryptococcus neoformans strains (83 MATα and 1 MATa) and 3 Cryptococcus deneoformans strains (all MATα) from 87 (22.4% of surveyed) O. europaea trees; a total of 32 C. neoformans strains were isolated from 32 (24.2%) of the E. camaldulensis trees, all of which were MATα. A statistically significant difference was observed in the frequency of C. neoformans isolation between coastal and inland areas (P < 0.05). Interestingly, the MATaC. neoformans isolate was fertile in laboratory crosses with VNI and VNB MATα tester strains and produced robust hyphae, basidia, and basidiospores, thus suggesting potential sexual reproduction in the natural population. Sequencing analyses of the URA5 gene identified at least five different genotypes among the isolates. Population genetics and genomic analyses revealed that most of the isolates in Turkey belong to the VNBII lineage of C. neoformans, which is predominantly found in southern Africa; these isolates are part of a distinct minor clade within VNBII that includes several isolates from Zambia and Brazil. Our study provides insights into the geographic distribution of different C. neoformans lineages in the Mediterranean region and highlights the need for wider geographic sampling to gain a better understanding of the natural habitats, migration, epidemiology, and evolution of this important human fungal pathogen.

Whole-Genome Sequencing of an Uncommon Cryptococcus neoformans MLST43 Genotype Isolated in Nigeria.

Cryptococcosis is a human infection caused mainly by two species of the Cryptococcus genus, Cryptococcus neoformans and Cryptococcus gattii, whose populations contain several phylogenetically related haploid (VN/VG-types) and hybrid genotypes. Here, we report the whole-genome sequencing of a Nigerian C. neoformans VNII, Mat-α, strain with a rare multilocus-sequence-type (MLST) genotype (ST43).