Reactive oxygen and nitrogen species are crucial for the antifungal activity of amorolfine and ciclopirox olamine against the dermatophyte Trichophyton interdigitale.

Onychomycosis is a nail infection caused by Trichophyton interdigitale and other fungi, which can be treated with topical amorolfine (AMR) and ciclopirox olamine (CPX). Although these drugs are widely used, little is known about the role of reactive oxygen (ROS) and nitrogen (RNS) in their mechanism of action. To better understand the effects of AMR and CPX in dermatophytes, we evaluated whether they act through the production of ROS and peroxynitrite (PRN). We tested a set of strains, all susceptible to AMR and CPX, and these antifungals significantly reduced T. interdigitale viability within 24 h. This effect occurred concomitantly with reduced ergosterol, increased production of ROS and PRN, and consequently increased lipid peroxidation. Together, these mechanisms lead to cell damage and fungal death. These fungicidal effects were abolished when PRN and superoxide scavengers were used in the assays, demonstrating the role of these species in the mechanism of action. We also studied the antioxidant system when T. interdigitale was exposed to AMR and CPX. Interestingly, superoxide dismutase and catalase inhibition lead to altered ROS and PRN production, lipid peroxidation, and ergosterol levels. In fact, the combination of AMR or CPX with a superoxide dismutase inhibitor was antagonistic. Together, these data demonstrate the importance of ROS and PRN in the antifungal action of AMR and CPX against the evaluated T. interdigitale strains. LAY SUMMARY: Onychomycosis is a nail infection, which can be treated with amorolfine and ciclopirox olamine. Here we demonstrate that these drugs exhibit antifungal activity also through the production of oxidative and nitrosative radicals.

Acanthamoeba castellanii as an alternative interaction model for the dermatophyte Trichophyton rubrum.

BACKGROUND: Trichophyton rubrum (Tr) is the main aetiological agent of human dermatophytosis, being isolated from the environment and keratinised tissues. In the environment, Tr can interact with other organisms, such as free-living amoebas (FLA), which can act as an alternative host system to study the interaction between microbes and phagocytic cells. OBJECTIVES: To characterise the Acanthamoeba castellanii (ALX)-Tr interaction. METHODS: Interaction was characterised in three conditions: trophozoites (PYG), late (PYG/NES) and early (NES) encystation stimulus, evaluating encystation kinetics, phagocytosis, exocytosis and fungicidal activity dynamics. RESULTS: Tr was able to induce ALX encystation and be internalised by ALX. The number of internalised conidia was high at 1 hour, and ALX presented fungicidal activity with increased intracellular ROS production and exocytosis. In PYG/NES, phagocytosis and ROS production were reduced, with decreased ALX's fungicidal activity. However, in NES there was an increased fungal engulfment, and a reduced ROS production and higher fungal burden. Furthermore, exogenous mannose decreased phagocytosis of Tr conidia, and divalent cations induced ROS production and increased ALX's fungicidal activity. Interestingly, phagocytosis was reduced in the presence of cytoskeleton inhibitor, but exocytosis was increased, suggesting that Tr conidia may have alternative pathways to escape ALX's cells. CONCLUSION: A castellanii is a proper model for studying Tr-FLA interaction, since ALX can engulf, produce ROS and kill Tr, and all these parameters are influenced by an encystation stimulus and divalent cations. Moreover, this interaction is likely to occur in the environment implicating in the adaptation to environmental stressful conditions in both organisms.

17-ß-Estradiol increases macrophage activity through activation of the G-protein-coupled estrogen receptor and improves the response of female mice to Cryptococcus gattii.

Cryptococcus gattii (Cg) is one of the agents of cryptococcosis, a severe systemic mycosis with a higher prevalence in men than women, but the influence of the female sex hormone, 17-ß-estradiol (E2), on cryptococcosis remains unclear. Our study shows that female mice presented delayed mortality, increased neutrophil recruitment in bronchoalveolar lavage fluid, and reduced fungal load after 24 hr of infection compared to male and ovariectomised female mice (OVX). E2 replacement restored OVX female survival. Female macrophages have more efficient fungicidal activity, which was increased by E2 and reversed by the antagonist of G-protein-coupled oestrogen receptor (GPER), which negatively modulates PI3K activation. Furthermore, E2 induces a reduction in Cg cell diameter, cell charge, and antioxidant peroxidase activity. In conclusion, female mice present improved control of Cg infection, and GPER is important for E2 modulation of the female response.

A Study of the Disruptive Effect of the Acetate Fraction of Punica granatum Extract on Cryptococcus Biofilms.

Cryptococcosis, caused by yeasts of the genus Cryptococcus, is an infectious disease with a worldwide distribution. Cryptococcus neoformans and Cryptococcus gattii are the species that commonly cause this disease in humans; however, infections caused by Cryptococcus laurentii, especially in immunocompromised patients, are increasingly being reported. Owing to the increase in the resistance of fungi to antifungals, and a lack of treatment options, it is important to seek new therapeutic alternatives such as natural products. Among these are plant species such as Punica granatum, which is used in folk medicine to treat various diseases. This study aimed to evaluate the activity of the acetate fraction of P. granatum leaf extract against environmental and clinical isolates of Cryptococcus. Three environmental isolates of C. laurentii, PMN, PMA, and PJL II, isolated from soils of different municipalities in the state of Maranhão, a clinical isolate, C. gattii, from a patient with neurocryptococcosis, and a standard strain of C. gattii (ATCC 32068) were used. The minimum and fractional inhibitory concentrations (MIC and FIC, respectively) and time-kill curve of the extract and fluconazole were determined to assess the susceptibility profile of the fungal isolates. Larvae of Tenebrio molitor were infected with Cryptococcus strains, and the effects of acetate fraction of P. granatum extract and fluconazole on the survival and fungal burden were determined. The extract activity was tested against pre-formed biofilms. The acetate fraction of P. granatum extract showed promising antifungal activity against all the species of Cryptococcus evaluated in this study, with an MIC value lower than that of fluconazole. The indices obtained in the FIC test indicated that the antimicrobial effect of the combination of the extract and antifungal was indifferent for 80% of the isolates. The P. granatum acetate fraction reduced the pre-formed biofilm of some isolates, showing better activity than fluconazole, which is consistent with results from fluorescence microscopy. This is the first study on the use of P. granatum and its ability to inhibit Cryptococcus biofilms; therefore, further studies and tests are needed to investigate the components and mechanism of action of P. granatum against cryptococcosis agents.

Phylogenetic and Molecular Profile of Staphylococcus aureus Isolated from Bloodstream Infections in Northeast Brazil.

Staphylococcus aureus is a notorious human pathogen associated with serious nosocomial and community-acquired infections, such as pneumonia, meningitis, endocarditis, toxic shock syndrome, and sepsis, among others. The objective of this study was to investigate the molecular profile, antimicrobial resistance, and clonal diversity of S. aureus isolated from the bloodstream. The determination of the minimum inhibitory concentration (MIC) of the antimicrobial was performed by an automated method. The presence of several virulence and resistance genes was evaluated by PCR. In addition, multilocus sequence typing (MLST) was used to analyze the clonal diversity of S. aureus. A high resistance to oxacillin (78%), clindamycin (78%), erythromycin (70%), ciprofloxacin (61%), and gentamicin (52%) was observed among the isolates. In most of them, the following virulence genes were detected: hlb (83%), ebpS (61%), icaA (57%), fnbpA (17%), and clfA (13%). Only one isolate carried the pvl gene. MLST analysis identified five new sequence types (STs): 5429, 5430, 5431, 5432, and 5433, as well as another seven-ST5, ST97, ST398, ST101, ST30, ST461, and ST2779-among the remaining strains. These seven STs and the four new STs are clustered in four clonal complexes: CC1, CC2, CC7, and CC17. Phylogenetic analysis showed the genetic relationship of the five new ST strains with another 18 strains. Altogether, these analyses indicate the horizontal transfer acquisition of virulence factor genes and multidrug resistance.

Evaluation of in vitro Antifungal Activity of Xylosma prockia (Turcz.) Turcz. (Salicaceae) Leaves Against Cryptococcus spp.

Cryptococcus species are responsible for important systemic mycosis and are estimated to cause millions of new cases annually. The available therapy is limited due to the high toxicity and the increasing rates of yeast resistance to antifungal drugs. Popularly known as "sucará," Xylosma prockia (Turcz.) Turcz. (Salicaceae) is a native plant from Brazil with little information on its pharmacological potential. In this work, we evaluated in vitro anticryptococcal effects of the leaf ethanolic extract of X. prockia and its fractions against Cryptococcus gattii and Cryptococcus neoformans. We also evaluated phenotypic alterations caused by ethyl acetate fraction (EAF) (chosen according to its biological results). The liquid chromatography-mass spectrometry (LC-MS) analysis of EAF demonstrated the presence of phenolic metabolites that belong to three structurally related groups as majority compounds: caffeoylquinic acid, coumaroyl-glucoside, and caffeoyl-glucoside/deoxyhexosyl-caffeoyl glucoside derivatives. The minimum inhibitory concentration (MIC) values against C. gattii and C. neoformans ranged from 8 to 64 mg/L and from 0.5 to 8 mg/L, for ethanolic extract and EAF, respectively. The EAF triggered an oxidative burst and promoted lipid peroxidation. EAF also induced a reduction of ergosterol content in the pathogen cell membrane. These effects were not associated with alterations in the cell surface charge or in the thermodynamic fingerprint of the molecular interaction between EAF and the yeasts evaluated. Cytotoxic experiments with peripheral blood mononuclear cells (PBMCs) demonstrated that EAF was more selective for yeasts than was PBMCs. The results may provide evidence that X. prockia leaf extract might indeed be a potential source of antifungal agents.

Eugenol Induces Phenotypic Alterations and Increases the Oxidative Burst in Cryptococcus.

Eugenol is a phenolic compound and the main constituent of the essential oil of clove India. Although there are reports of some pharmacological effects of eugenol, this study is the first that proposes to evaluate the antifungal effects of this phenol against both Cryptococcus gattii and C. neoformans cells. The effect of eugenol against yeast cells was analyzed for drug susceptibility, alterations in cell diameter, capsule properties, amounts of ergosterol, oxidative burst, and thermodynamics data. Data demonstrated that there is no interaction between eugenol and fluconazole and amphotericin B. Eugenol reduced the cell diameter and the capsule size, increased cell surface/volume, changed positively the cell surface charge of cryptococcal cells. We also verified increased levels of reactive oxygen species without activation of antioxidant enzymes, leading to increased lipid peroxidation, mitochondrial membrane depolarization and reduction of lysosomal integrity in cryptococcal cells. Additionally, the results showed that there is no significant molecular interaction between eugenol and C. neoformans. Morphological alterations, changes of cellular superficial charges and oxidative stress play an important role in antifungal activity of eugenol against C. gattii and C. neoformans that could be used as an auxiliary treatment to cutaneous cryptococcosis.

Influenza A Virus as a Predisposing Factor for Cryptococcosis.

Influenza A virus (IAV) infects millions of people annually and predisposes to secondary bacterial infections. Inhalation of fungi within the Cryptococcus complex causes pulmonary disease with secondary meningo-encephalitis. Underlying pulmonary disease is a strong risk factor for development of C. gattii cryptococcosis though the effect of concurrent infection with IAV has not been studied. We developed an in vivo model of Influenza A H1N1 and C. gattii co-infection. Co-infection resulted in a major increase in morbidity and mortality, with severe lung damage and a high brain fungal burden when mice were infected in the acute phase of influenza multiplication. Furthermore, IAV alters the host response to C. gattii, leading to recruitment of significantly more neutrophils and macrophages into the lungs. Moreover, IAV induced the production of type 1 interferons (IFN-α4/ß) and the levels of IFN-γ were significantly reduced, which can be associated with impairment of the immune response to Cryptococcus during co-infection. Phagocytosis, killing of cryptococci and production of reactive oxygen species (ROS) by IAV-infected macrophages were reduced, independent of previous IFN-γ stimulation, leading to increased proliferation of the fungus within macrophages. In conclusion, IAV infection is a predisposing factor for severe disease and adverse outcomes in mice co-infected with C. gattii.

Heteroresistance and fungi.

The concept of heteroresistance refers to the heterogeneous susceptibility to an antimicrobial drug in a microorganism population, meaning that some clones may be resistant and others are susceptible. This phenomenon has been widely studied in bacteria, but little attention has been given to its expression in fungi. We review the available literature on heteroresistance in fungi and invite the reader to recognise this phenomenon as a fungal mechanism to adapt to environmental stress, which may interfere both in resistance and virulence. Finally, heteroresistance may explain the treatment failures to eradicate mycosis in some patients treated with a seemingly appropriate antifungal.

Annona glabra Flavonoids Act As Antimicrobials by Binding to Pseudomonas aeruginosa Cell Walls.

Pseudomonas aeruginosa is an important pathogen in opportunistic infections in humans. The increased incidence of antimicrobial-resistant P. aeruginosa isolates has highlighted the need for novel and more potent therapies against this microorganism. Annona glabra is known for presenting different compounds with diverse biological activities, such as anti-tumor and immunomodulatory activities. Although other species of the family display antimicrobial actions, this has not yet been reported for A. glabra. Here, we investigated the antimicrobial activity of the ethyl acetate fraction (EAF) obtained from the leaf hydroalcoholic extract of A. glabra. EAF was bactericidal against different strains of P. aeruginosa. EAF also presented with a time- and concentration-dependent effect on P. aeruginosa viability. Testing of different EAF sub-fractions showed that the sub-fraction 32-33 (SF32-33) was the most effective against P. aeruginosa. Analysis of the chemical constituents of SF32-33 demonstrated a high content of flavonoids. Incubation of this active sub-fraction with P. aeruginosa ATCC 27983 triggered an endothermic reaction, which was accompanied by an increased electric charge, suggesting a high binding of SF32-33 compounds to bacterial cell walls. Collectively, our results suggest that A. glabra-derived compounds, especially flavonoids, may be useful for treating infections caused by P. aeruginosa.