Nitrogen concentration affects amphotericin B and fluconazole tolerance of pathogenic cryptococci.

Environmental stress often causes phenotypic changes among pathogenic cryptococci, such as altered antifungal susceptibility, changes in capsule and melanin formation, as well as altered levels of the membrane sterol and antifungal target, ergosterol. We therefore hypothesised that nitrogen limitation, a prevalent environmental stress in the natural habitat of these yeasts, might affect virulence and antifungal susceptibility. We tested the effect of different nitrogen concentrations on capsule, melanin and ergosterol biosynthesis, as well as amphotericin B (AmB) and fluconazole (FLU) susceptibility. This was achieved by culturing cryptococcal strains representing Cryptococcus neoformans and Cryptococcus gattii in media with high (0.53 g/l), control (0.42 g/l) and low (0.21 g/l) NH4Cl concentrations. India ink staining was used to determine capsule thickness microscopically, while melanin and ergosterol content were determined spectrophotometrically. We found that lower nitrogen concentrations enhanced both ergosterol and capsule biosynthesis, while a variable effect was observed on melanisation. Evaluation of drug tolerance using time-kill methodology, as well as tests for FLU heteroresistance, revealed that the low nitrogen cultures had the highest survival percentages in the presence of both AmB and FLU, and showed the highest frequency of FLU heteroresistance, suggesting that nitrogen concentration may indeed influence drug tolerance.

Tracing Genetic Exchange and Biogeography of Cryptococcus neoformans var. grubii at the Global Population Level.

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically human immunodeficiency virus/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterize the major subdivisions, their relative diversity, and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii (serotype AA) and of hybrids with C. neoformans var. neoformans (serotype AD) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil reveal that the previously "African" VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions; demonstrating the highly dispersive nature of this pathogen.

Soil properties that impact yeast and actinomycete numbers in sandy low nutrient soils.

To explore the beneficial qualities or detrimental consequences of cultureable soil yeasts, it is important to understand which physicochemical soil properties most impact populations of these unicellular fungi in their natural habitat. The goal of this study was to determine which soil properties dictate yeast numbers in pristine sandy, low nutrient soils within a semi-arid region. A correlation matrix of the data obtained for 19 different environmental variables indicated a negative correlation between soil pH and yeast numbers. Using general regression models, it was demonstrated that soil pH and copper concentration were the 2 variables that correlated best with soil yeast counts in these soils. However, soil moisture content was found to be the environmental factor with the most impact on cultureable actinomycetes and heterotrophic microbes. The study also demonstrated that divalent cation availability might impact the size of both yeast and prokaryote populations in these soils.