isolation-culturable-yeasts-molds-from-soils-to-investigate-fungal

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

McMaster University

Aspergillus fumigatus is a saprophytic fungus that can cause lethal invasive aspergillosis in immunocompromised patients. Recent studies have shown that Eurasian and North American populations of A. fumigatus often consist of genetically diverse strains. However, very little is known about African populations of A. fumigatus. Here, we characterise the genetic diversity and triazole susceptibility of A. fumigatus in Cameroon, West Africa. A total of 495 soil samples were obtained from nine collection sites in three Cameroonian regions. Nine microsatellite markers were used to genotype all 51 identified A. fumigatus isolates. In vitro susceptibility to itraconazole and voriconazole was tested using micro broth dilution. The 51 Cameroonian A. fumigatus isolates belonged to 45 genotypes. Consistent with recombination, 32 of 36 possible pairwise loci combinations are phylogenetically incompatible. Interestingly, evidence for geographic sub-structuring was found within Cameroon and the sub-population with the most evidence of recombination was also the least susceptible sub-population to the triazole antifungals tested. Furthermore, the Cameroonian sample was significantly differentiated from those in Eurasia and North America. Overall, our results indicate the genetic uniqueness of Cameroonian A. fumigatus populations and that additional novel genetic diversity likely exist in other parts of Africa.

Evidence of unique genetic diversity in Aspergillus fumigatus isolates from Cameroon.

Aspergillus fumigatus is a ubiquitous opportunistic fungal pathogen that can cause aspergillosis in humans. Over the last decade there have been increasing global reports of treatment failure due to triazole resistance. An emerging hypothesis states that agricultural triazole fungicide use causes clinical triazole resistance. Here we test this hypothesis in Hamilton, Ontario, Canada, by examining a total of 195 agricultural, urban, and clinical isolates using 9 highly polymorphic microsatellite markers. For each isolate, the in vitro susceptibilities to itraconazole and voriconazole, 2 triazole drugs commonly used in the management of patients, were also determined. Our analyses suggested frequent gene flow among the agricultural, urban environmental, and clinical populations of A. fumigatus and found evidence for widespread sexual recombination within and among the different populations. Interestingly, all 195 isolates analyzed in this study were susceptible to both triazoles tested. However, compared with the urban population, agricultural and clinical populations showed significantly reduced susceptibility to itraconazole and voriconazole, consistent with ecological niche-specific selective pressures on A. fumigatus populations in Hamilton. Frequent gene flow and genetic recombination among these populations suggest greater attention should be paid to monitor A. fumigatus populations in Hamilton and other similar jurisdictions.

Limited evidence of fungicide-driven triazole-resistant Aspergillus fumigatus in Hamilton, Canada.

Amphotericin B (AMB) is one of the major antifungal drugs used in the management of aspergillosis and is especially recommended for treating triazole-resistant strains of . However, relatively little is known about the AMB susceptibility patterns of  in many parts of the world. This study aims to describe the AMB susceptibility patterns in Hamilton, Ontario, Canada.

Widespread amphotericin B-resistant strains of  in Hamilton, Canada.

Halocins are antimicrobial peptides or proteins that are produced by halophilic archaea. Although their function in inhibiting the growth of closely related haloarchaeal strains is well known, other physiological functions of halocins have also been proposed in recent years. To unveil the possible function and mechanism of halocins in DNA uptake, the halocin H4 producing strain  DF50-ΔEPS (incapable of EPS production) was used in this study. We found that deletion of the  resulted in the strain DF50-ΔEPSΔ which exhibited loss of natural DNA uptake ability. Moreover, supernatants of the halocin producing strain were capable of inducing the ability to uptake DNA. Obviously, halocin is likely responsible for inducing DNA uptake. Cell surface ultrastructures of these strains are varied from strains DF50-ΔEPS to DF50-ΔEPSΔ. The cell surface of strain DF50-ΔEPS is rough due to numerous pinholes, while that of the strain DF50-ΔEPSΔ is smooth without visible pinholes. The morphology of the  complemented strain, DF50-ΔEPSΔ, shows an intermediate phenotype between strains DF50-ΔEPS and DF50-ΔEPSΔ. We speculate that halocin H4 may accelerate DNA uptake by perforating the cell surface ultrastructure. The halocin H4 may represent a novel inducer or activator of DNA uptake in .

A Halocin Promotes DNA Uptake in .

 is a ubiquitous fungus and the main agent of aspergillosis, a common fungal infection in the immunocompromised population. Triazoles such as itraconazole and voriconazole are the common first-line drugs for treating aspergillosis. However, triazole resistance in  has been reported in an increasing number of countries. While most studies of triazole resistance have focused on mutations in the triazole target gene , >70% of triazole-resistant strains in certain populations showed no mutations in . To identify potential non- mutations associated with triazole resistance in , we analyzed the whole genome sequences and triazole susceptibilities of 195 strains from 12 countries. These strains belonged to three distinct clades. Our genome-wide association study (GWAS) identified a total of six missense mutations significantly associated with itraconazole resistance and 18 missense mutations with voriconazole resistance. In addition, to investigate itraconazole and pan-azole resistance, Fisher's exact tests revealed 26 additional missense variants tightly linked to the top 20 SNPs obtained by GWAS, of which two were consistently associated with triazole resistance. The large number of novel mutations related to triazole resistance should help further investigations into their molecular mechanisms, their clinical importance, and the development of a comprehensive molecular diagnosis toolbox for triazole resistance in .

Genome-Wide Association Analysis for Triazole Resistance in .

 is a ubiquitous saprophytic mold that can cause a range of clinical syndromes, from allergic reactions to invasive infections. Amphotericin B (AMB) is a polyene antifungal drug that has been used to treat a broad range of systemic mycoses since 1958, including as a primary treatment option against invasive aspergillosis in regions with high rates (≥10%) of environmental triazole resistance. However, cases of AMB-resistant  strains have been increasingly documented over the years, and high resistance rates were recently reported in Brazil and Canada. The objective of this study is to identify candidate mutations associated with AMB susceptibility using a genome-wide association analysis of natural strains, and to further investigate a subset of the mutations in their putative associations with differences in AMB minimum inhibitory concentration (MIC) and in growths at different AMB concentrations through the analysis of progeny from a laboratory genetic cross. Together, our results identified a total of 34 candidate single-nucleotide polymorphisms (SNPs) associated with AMB MIC differences-comprising 18 intergenic variants, 14 missense variants, one synonymous variant, and one non-coding transcript variant. Importantly, progeny from the genetic cross allowed us to identify putative SNP-SNP interactions impacting progeny growth at different AMB concentrations.

Genetic Analyses of Amphotericin B Susceptibility in .

 is a saprophytic mold and an opportunistic pathogen with a broad geographic and ecological distribution.  is the most common etiological agent of aspergillosis, affecting over 8,000,000 individuals worldwide. Due to the rising number of infections and increasing reports of resistance to antifungal therapy, there is an urgent need to understand  populations from local to global levels. However, many geographic locations and ecological niches remain understudied, including soil environments from arctic regions. In this study, we isolated 32 and 52  strains from soils in Iceland and the Northwest Territories of Canada (NWT), respectively. These isolates were genotyped at nine microsatellite loci and the genotypes were compared with each other and with those in other parts of the world. Though significantly differentiated from each other, our analyses revealed that  populations from Iceland and NWT contained evidence for both clonal and sexual reproductions, and shared many alleles with each other and with those collected from across Europe, Asia, and the Americas. Interestingly, we found one triazole-resistant strain containing the TR /L98H mutation in the  gene from NWT. This strain is closely related to a triazole-resistant genotype broadly distributed in India. Together, our results suggest that the northern soil populations of  are significantly influenced by those from other geographic regions.

Gregory Korfanty

Department of Biology

McMaster University

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure