Are γ-terpinene, 1,8-cineole, p-coumaric acid, and quercetin active against wild-type and non-wild-type Sporothrix brasiliensis to itraconazole?

The emergence of itraconazole (ITZ)-resistant Sporothrix brasiliensis in feline and canine cases in southern Brazil has hampered the clinical cure of animal sporotrichosis, encouraging the search for therapeutic alternatives. The promising use of plants extracts from Lamiaceae family is known; however, there are no studies with its major compounds, as γ-terpinene (γTER), 1,8-cineole (1,8CIN), p-coumaric acid (pCOU), and quercetin (QUER). For the first time, we evaluated the antifungal, synergistic, cytotoxic activities and action mechanism of these compounds against S. brasiliensis. For this, 28 S. brasiliensis from cats (n = 24) and dogs (n = 4) and standard strains of S. brasiliensis and S. schenckii (n = 4) were tested by M38-A2 (CLSI), revealing non-wild-type (WT) isolates to ITZ on 54.2% (13/24) and 75% (03/04) of feline and canine isolates, respectively. Of the compounds, γTER stood out against all isolates (MIC/MFC 0.75 to > 3 mg/ml; MIC50 3 mg/ml). However, 1,8CIN, pCOU, and QUER showed little or no activity (MIC50 > 3 mg/ml). Thus, γTER was selected for checkerboard assay, whose combination with ITZ showed synergistic (WT isolates) and indifferent (non-WT isolates) interaction. For action mechanism (sorbitol protection and ergosterol effect), γTER acted in membrane by complexing with fungal ergosterol and at the cell wall level, showing two possible pathways as antifungal target. Finally, cytotoxicity (MTT assay) showed that γTER was the safest compound on MDBK cells, even at a concentration of 3 mg/ml (90.16%). Our findings support that γTER is a potent antifungal candidate for the control of sporotrichosis, including against non-WT S. brasiliensis.

In vivo protection of the marjoram (Origanum majorana Linn.) essential oil in the cutaneous sporotrichosis by Sporothrix brasiliensis.

Thirty Wistar rats subcutaneously infected by an itraconazole-resistant Sporothrix brasiliensis received the oral daily treatment (n = 10, each) of control (CTL, saline solution), itraconazole (ITZ, 10 mg/kg) and marjoram essential oil (MRJ, 80 mg/kg) for 30 days. Weekly, the clinical evaluation and euthanasia for histopathology and fungal burden were performed. Only animals from MRJ evolved to the remission of the cutaneous lesion with a mild to absent presence of yeasts in footpad, besides decreased the fungal burden in the systemic organs compared to CTL and ITZ (p < 0.05), preventing the fungal spread, mainly in the liver and spleen. The antifungal activity may have been attributed to the majority composition of terpinen-4-ol (34.09%), γ-terpinene (14.28%) and α-terpinene (9.6%), which the mode of action was at the level of ergosterol complexation. These findings highlighted the antifungal and the systemic protective effects of MRJ, supporting the promising use in the treatment of cutaneous sporotrichosis.

In vitro pharmacokinetics/pharmacodynamics modeling and efficacy against systemic candidiasis in Drosophila melanogaster of a bisaryloxypropanamine derivative.

The number of deaths due to systemic fungal infections is increasing alarmingly, which is aggravated by the limitations of traditional treatments and multidrug resistance. Therefore, the research and development of new therapeutic options against pathogenic fungi is an urgent need. To evaluate the fungicidal activity of a synthetic compound, 1,3-bis-(3,4-dichlorophenoxy)propan-2-aminium chloride (2j), through time-kill studies and pharmacokinetics/pharmacodynamics (PK/PD) modeling. The protective effect of the compound was also evaluated using the Drosophila melanogaster minihost model of candidiasis. Mathematical modeling of time-kill data of compound 2j was performed to obtain PD characteristics. Additionally, Toll-deficient D. melanogaster flies were infected with a Candida albicans strain and treated with 2j. We observed that compound 2j demonstrated a time- and dose-dependent fungicidal effect against Candida spp. and dermatophytes, even at low concentrations, and rapidly achieved kill rates reaching the maximum effect in less than one hour. The efficacy of the compound against systemic candidiasis in D. melanogaster flies was comparable to that achieved by fluconazole. These results support the potential of compound 2j as a systemic antifungal agent candidate and serve as a starting point for further studies involving mammalian animal models.

Antifungal resistance on Sporothrix species: an overview.

INTRODUCTION: The treatment of human and animal sporotrichosis is often performed with antifungal agents; however, the emergence of antifungal-resistant strains of Sporothrix species has been reported. We aimed to discuss the ability of Sporothrix species in developing resistance to the conventional antifungals and mechanisms for this. METHODOLOGY: Published data on databases (PubMed, Science Direct, Google Scholar) were investigated using a combination of keywords from 2008 to 2019 by the StArt tool. RESULTS: The minimal inhibitory concentrations values based on the Clinical and Laboratory Standards Institute (CLSI) from eight references were classified according to the epidemiological cutoff values in wild-type or non-wild-type strains. In this way, non-wild-type S. schenckii and, mainly, S. brasiliensis isolates were recognized on itraconazole, amphotericin B, terbinafine, and voriconazole, which are strains that deserve more attention toward antifungal control, with a probable risk of mutation to antifungal resistance. Among the few reviewed studied on antifungal resistance, the melanin production capacity (DHN-melanin, L-DOPA melanin, and pyomelanin), the low genetic diversity due to the abnormal number of chromosomes, and the mutation in cytochrome P450 are some of the factors for developing resistance mechanism. CONCLUSIONS: The emergence of Sporothrix species with in vitro antifungal resistance was evidenced and the possible mechanisms for resistance development may be due to the melanin production capacity, genetic diversity and mutations in cytochrome P450. Further studies should be carried out targeting gene expression for the development of antifungal resistance on Sporothrix species in order to prospect new therapeutic targets for human and veterinary use.

Fumonisin B1 induces toxicity in human leukocytes at low concentrations: Are computational studies effective to determine biosafety?

Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides and Fusarium proliferatum found in various crops, particularly maize. Besides carcinogenicity, other manifestations have been registered in different animals and in humans. In the case of humans, epidemiological studies have reported high prevalence of esophageal cancer in populations exposed to fumonisins. This study aimed to evaluate the minimum concentration of FB1 capable of inducing cytotoxicity (cell viability test), genotoxicity (comet assay) and mutagenicity (micronucleus) in cultured human leukocytes and to evaluate the effectiveness of in silico tests to predict FB1 toxicity. All concentrations analyzed (200; 100; 50; 5; 0.5; 0.05; 0.005 µg/mL and 300; 30; 3; 1; 0.1; 0.01 fg/mL) except the lowest demonstrated dose-dependent toxicity in all parameters analyzed (p < 0.05 to p < 0.0001). As for predictions, only the Lazar software showed carcinogenicity of FB1 for rats. Thus, it is evident that FB1 is able to induce dose-dependent damage at low concentrations, and that computational tests, although desirable for prediction, are not effective as biological tests to determine toxicity, at least of FB 1 and within the experimental conditions tested.

The Role of Molecular Tests in the Diagnosis of Disseminated Histoplasmosis.

Histoplasmosis is an emerging fungal disease, with global distribution. The disseminated form of the disease is a more severe infection, generally associated with AIDS. Classic diagnostic methods for histoplasmosis consist of microscopy, culture, and histopathology. More recently, the importance of Histoplasma antigen detection has dominated the literature on histoplasmosis diagnosis, but the relevance of molecular assays has not been as much studied. Here we describe the results of a systematic literature review focusing on studies that mainly compared immunological techniques (Histoplasma urine antigen detection) with molecular tests for the diagnosis of histoplasmosis. In addition to the review of comparative studies using such diagnostic techniques, the literature on polymerase chain reaction (PCR) tests in patients with disseminated histoplasmosis is also summarized. Two studies reported the comparison between immunological and molecular methods applied simultaneously for the diagnosis of disseminated histoplasmosis. PCR demonstrates a satisfactory performance assisting in the detection of Histoplasma spp. DNA in clinical samples.

Design, synthesis, and evaluation of novel 2-substituted 1,4-benzenediol library as antimicrobial agents against clinically relevant pathogens.

Development of new antimicrobial agents, capable of combating resistant and multidrug-resistant fungal and bacterial clinical strains, is necessary. This study presents the synthesis and antimicrobial screening of 42 2-substituted-1,4-benzenediols, being 10 novel compounds. In total, 23 compounds showed activity against fungi and/or bacteria. Benzenediol compounds 2, 5, 6, 8, 11, and 12 demonstrated broad spectrum antimicrobial actions, including resistant and multidrug-resistant species of dermatophytes (Trichophyton mentagrophytes), Candida spp. and the ESKAPE panel of bacteria. Minimum inhibitory concentrations of these compounds for fungi and bacterial strains ranged from 25 to 50 µg/ml and 8-128 µg/ml, respectively. The antifungal mechanism of action is related to the fungal cell wall of dermatophytes and membrane disruption to dermatophytes and yeasts, in the presence of compound 8. Specific structural changes, such as widespread thinning along the hyphae and yeast lysis, were observed by scanning electron microscopy. The effects of compound 8 on cell viability are dose-dependent; however they did not cause genotoxicity and mutagenicity in human leukocyte cells nor haemolysis. Moreover, the compounds were identified as nonirritant by the ex-vivo Hen's egg test-chorioallantoic membrane (HET-CAM). The furan-1,4-benzenediol compound 5 showed in vivo efficacy to combat S. aureus infection using embryonated chicken eggs. Therefore, the compounds 8, and 5 are promising as hits for the development of new antimicrobial drugs with reduced toxicity.

Reversal of fluconazole resistance induced by a synergistic effect with Acca sellowiana in Candida glabrata strains.

CONTEXT: The increased incidence of non-albicans Candida (NAC) resistant to fluconazole (FLZ) makes it necessary to use new therapeutic alternatives. Acca sellowiana (O.berg) Burret (Myrtaceae) is a guava with several proven biological activities. The interaction with fluconazole can be a feasible alternative to overcome this resistance. OBJECTIVE: This study evaluates the in vitro antifungal activity of fractions obtained from the lyophilized aqueous extract of the leaves of A. sellowiana against resistant strains of NAC. MATERIALS AND METHODS: The antifungal activity of the fractions was evaluated at 500 µg/mL by microdilution method. Checkerboard assay was performed to determine the effect of the combination of the F2 fraction and antifungal at concentrations: MIC/4, MIC/2, MIC, MIC × 2 and MIC × 4. RESULTS: Candida glabrata showed the lowest MIC values (500-3.90 µg/mL) and the F2 active fraction was the most effective. The association of F2 with FLZ showed a strong synergistic effect (FICI ≤ 0.5) against 100% of C. glabrata resistant isolates. Moreover, the F2 active fraction has demonstrated that probably acts in the cell wall of these yeasts. There was no observed acute dermal toxicity of lyophilized aqueous extract of leaves of A. sellowiana on pig ear skin cells. DISCUSSION AND CONCLUSION: The interaction between substances present in the F2 active fraction is possibly responsible for the antifungal activity presented by this fraction. This study is unprecedented and suggests that the combination of F2 active fraction and FLZ might be used as an alternative treatment for mucocutaneus infections caused by C. glabrata resistant.