Hypervirulence and cross-resistance to a clinical antifungal are induced by an environmental fungicide in Cryptococcus gattii.

The increasing human population requires ongoing efforts in food production. This is frequently associated with an increased use of agrochemicals, leading to environmental contamination and altering microbial communities, including human fungal pathogens that reside in the environment. Cryptococcus gattii is an environmental yeast and is one of the etiological agents of cryptococcosis. Benomyl (BEN) is a broad-spectrum fungicide used on several crops. To study the effects of agrochemicals on fungal pathogens, we first evaluated the susceptibility of C. gattii to BEN and the interactions with clinical antifungals. Antagonistic interaction between BEN and fluconazole was seen and was strain- and concentration-dependent. We then induced BEN-resistance by culturing strains in increasing drug concentrations. One strain demonstrated to be more resistant and showed increased multidrug efflux pump gene (MDR1) expression and increased rhodamine 6G efflux, leading to cross-resistance between BEN and fluconazole. Morphologically, BEN-adapted cells had a reduced polysaccharide capsule; an increased surface/volume ratio; increased growth rate in vitro and inside macrophages and also higher ability in crossing an in vitro model of blood-brain-barrier. BEN-adapted strain demonstrated to be hypervirulent in mice, leading to severe symptoms of cryptococcosis, early mortality and higher fungal burden in the organs, particularly the brain. The parental strain was avirulent in murine model. In vivo cross-resistance between BEN and fluconazole was observed, with mice infected with the adapted strain unable to present any improvement in survival and behavior when treated with this antifungal. Furthermore, BEN-adapted cells cultured in drug-free media maintained the hypervirulent and cross-resistant phenotype, suggesting a persistent effect of BEN on C. gattii. In conclusion, exposure to BEN induces cross-resistance with fluconazole and increases the virulence of C. gattii. Altogether, our results indicate that agrochemicals may lead to unintended consequences on non-target species and this could result in severe healthy problems worldwide.

N-acetylcysteine reduces amphotericin B deoxycholate nephrotoxicity and improves the outcome of murine cryptococcosis.

Cryptococcosis is a life-threatening fungal infection, and its current treatment is toxic and subject to resistance. Drug repurposing represents an interesting approach to find drugs to reduce the toxicity of antifungals. In this study, we evaluated the combination of N-acetylcysteine (NAC) with amphotericin B (AMB) for the treatment of cryptococcosis. We examined the effects of NAC on fungal morphophysiology and on the macrophage fungicidal activity 3 and 24 hours post inoculation. The therapeutic effects of NAC combination with AMB were investigated in a murine model with daily treatments regimens. NAC alone reduced the oxidative burst generated by AMB in yeast cells, but did not inhibit fungal growth. The combination NAC + AMB decreased capsule size, zeta potential, superoxide dismutase activity and lipid peroxidation. In macrophage assays, NAC + AMB did not influence the phagocytosis, but induced fungal killing with different levels of oxidative bursts when compared to AMB alone: there was an increased reactive oxygen species (ROS) after 3 hours and reduced levels after 24 hours. By contrast, ROS remained elevated when AMB was tested alone, demonstrating that NAC reduced AMB oxidative effects without influencing its antifungal activity. Uninfected mice treated with NAC + AMB had lower concentrations of serum creatinine and glutamate-pyruvate transaminase in comparison to AMB. The combination of NAC + AMB was far better than AMB alone in increasing survival and reducing morbidity in murine-induced cryptococcosis, leading to reduced fungal burden in lungs and brain and also lower concentrations of pro-inflammatory cytokines in the lungs. In conclusion, NAC + AMB may represent an alternative adjuvant for the treatment of cryptococcosis.

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.

Environmental Triazole Induces Cross-Resistance to Clinical Drugs and Affects Morphophysiology and Virulence of Cryptococcus gattii and C. neoformans.

Cryptococcus gattii and Cryptococcus neoformans are environmental fungi that cause cryptococcosis, which is usually treated with amphotericin B and fluconazole. However, therapeutic failure is increasing because of the emergence of resistant strains. Because these species are constantly isolated from vegetal materials and the usage of agrochemicals is growing, we postulate that pesticides could be responsible for the altered susceptibility of these fungi to clinical drugs. Therefore, we evaluated the influence of the pesticide tebuconazole on the susceptibility to clinical drugs, morphophysiology, and virulence of C. gattii and C. neoformans strains. The results showed that tebuconazole exposure caused in vitro cross-resistance (CR) between the agrochemical and clinical azoles (fluconazole, itraconazole, and ravuconazole) but not with amphotericin B. In some strains, CR was observed even after the exposure ceased. Further, tebuconazole exposure changed the morphology, including formation of pseudohyphae in C. neoformans H99, and the surface charge of the cells. Although the virulence of both species previously exposed to tebuconazole was decreased in mice, the tebuconazole-exposed colonies recovered from the lungs were more resistant to azole drugs than the nonexposed cells. This in vivo CR was confirmed when fluconazole was not able to reduce the fungal burden in the lungs of mice. The tolerance to azoles could be due to increased expression of the ERG11 gene in both species and of efflux pump genes (AFR1 and MDR1) in C. neoformans Our study data support the idea that agrochemical usage can significantly affect human pathogens present in the environment by affecting their resistance to clinical drugs.

Investigating cross-contamination by yeast strains from dental solid waste to waste-handling workers by DNA sequencing.

Trying to widen the discussion on the risks associated with dental waste, this study proposed to investigate and genetically compare yeast isolates recovered from dental solid waste and waste workers. Three samples were collected from workers' hands, nasal mucosa, and professional clothing (days 0, 30, and 180), and two from dental waste (days 0 and 180). Slide culture, microscopy, antifungal drug susceptibility, intersimple sequence repeat analysis, and amplification and sequencing of internal transcribed spacer regions were performed. Yeast strains were recovered from all waste workers' sites, including professional clothes, and from waste. Antifungal susceptibility testing demonstrated that some yeast recovered from employees and waste exhibited nonsusceptible profiles. The dendrogram demonstrated the presence of three major clusters based on similarity matrix and UPGMA grouping method. Two branches displayed 100% similarity: three strains of Candida guilliermondii isolated from different employees, working in opposite work shifts, and from diverse sites grouped in one part of branch 1 and cluster 3 that included two samples of Candida albicans recovered from waste and the hand of one waste worker. The results suggested the possibility of cross-contamination from dental waste to waste workers and reinforce the need of training programs focused on better waste management routines.

Atorvastatin as a promising anticryptococcal agent.

Cryptococcosis caused by Cryptococcus gattii leads to pneumonia and meningoencephalitis, and has a high mortality rate worldwide due to the inadequacy of available therapy and increasing drug resistance. There is a need to develop effective treatments, and drug repositioning is an interesting alternative to achieve new strategies to treat cryptococcosis. Atorvastatin (ATO), a statin currently used to treat hypercholesterolaemia, was tested in this study as an adjuvant to control infections caused by C. gattii. Several aspects of the effect of ATO on the host and the yeast were evaluated, with particular focus on the association of ATO with fluconazole (FLC), which (i) reduced ergosterol content in the cell membrane and altered properties of the polysaccharide capsule of C. gattii; (ii) increased the production of reactive oxygen species by macrophages; and (iii) reduced yeast phagocytosis and the intracellular proliferation rate. In an animal model, infected mice treated with ATO + FLC showed increased survival, improved clinical condition, and reduced fungal burden in the lungs and brain. This study is the first to perform in vivo tests with ATO + FLC for the treatment of cryptococcosis. The results suggest that ATO may be an important adjuvant for the treatment of cryptococcosis.

Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire.

UNLABELLED: Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the ∼ 1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE: APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.