Molecular identification, antifungal resistance and virulence of Cryptococcus neoformans and Cryptococcus deneoformans isolated in Seville, Spain

Abstract

Cryptococcal meningitis is one of the leading causes of death in HIV/AIDS patients. Our aim was to in order to characterise the epidemiology, antifungal susceptibility pattern and virulence of 28 Cyptococcus sp. strains recovered from 12 AIDS patients during two years in a Spanish single institution. Antifungal susceptibility testing was performed according to the CLSI protocols. Clinical strains were molecularly characterised by serotyping, mating type, PCR fingerprinting (M13 and GACA microsatellites) and analysis of two rDNA regions (IGS1 and ITS). Sequencing of the ERG11 gene was used to explore mechanisms of fluconazole resistance. Differences in virulence between species were studied in a Galleria mellonella infection model. Cryptococcus deneoformans and C. deneoformans x Cryptococcus neoformans hybrids were the most frequent variety (65%) followed by C. neoformans (35%). Strains were categorised according to 13 microsatellite genotypes and mixed infections could be detected in three patients. Twenty-nine per cent of the strains were fluconazole resistant. In one of the patients, the fluconazole resistance phenotype was associated with a point mutation in the ERG11 gene responsible for the amino acid substitution G470R. C. neoformans strains were able to kill G. mellonella larvae more efficiently than C. deneoformans and hybrids between both species. Precisely molecular characterisation of C. neoformans species is important for an accurate patient's management.

Potential microbial drivers of biodegradation of polycyclic aromatic hydrocarbons in crude oil sludge using a composting technique

“…Qubit 2.0 Fluorometer (Invitrogen, Life Technologies, Randburg, South Africa) was used to quantify filtered DNA extracts and extracts were stored at −20 °C for further use . The universal fungal primers *ITS1 (5′‐TCC GTA GGT GAA CCT GCG G‐3′) and ITS4 (5′‐ TCC TCC GCT TAT TGA TAT GC‐3′ were used to amplify the ITS region of the fungal DNA using PCR . A total volume of 20 μL amplification reaction mix was prepared which comprised 1 μL (10 μmol L –1 ) of each primer, 10 μL 2× Thermo Scientific Phusion Flash High‐Fidelity PCR Master mix, 2 μL DNA templates and 6 μL of sterile distilled water.…”

Protection of Public Health from Microbial and Chemical Hazards in Swimming Pool Environments

“…In addition, there are concerns about the effects of climate change on pathogenic fungi: rising temperatures may increase the growth of waterborne pathogens, including fungi (de Roda Husman & Schets, 2010;van der Wielen et al, 2014). Other concerns are about the emergence of resistance to antifungal agents in pathogenic fungi over the past decades (Alcazar-Fuoli & Mellado, 2014;Gago et al, 2016).…”

A Novel Restraint Device for Injection of Galleria mellonella Larvae that Minimizes the Risk of Accidental Operator Needle Stick Injury

“…In recent years, they have been used for studying virulence mechanisms, investigating differences between clinical isolates as well as for preliminary investigation of the efficacy of antimicrobial compounds, for a wide range of both Gram-positive and Gram-negative bacteria (Joyce and Gahan, 2010; McLaughlin et al, 2012; Ramarao et al, 2012; Loh et al, 2013; Thomas et al, 2013; Williamson et al, 2014; Adamson et al, 2015; Champion et al, 2016; Johnston et al, 2016; Moreira et al, 2016; Nale et al, 2016; Yang et al, 2016), fungi (Alcazar-Fuoli et al, 2015; Forastiero et al, 2015; Borman et al, 2016; de Lacorte Singulani et al, 2016; Frenkel et al, 2016; Gago et al, 2016; Santos et al, 2016), and viruses (Garzon et al, 1978; Buyukguzel et al, 2007; Özkan and Coutts, 2015). The use of waxworms as a model host has many advantages.…”