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Background: Opportunistic fungal infections are increasingly common, with Candida albicans being the most common etiological agent, however, in recent years, episodes of candidiasis caused by non-albicans Candida species have emerged. Plants belonging to the Lauraceae family have shown remarkable antifungal effects. This study assessed the anti-Candida activity of Ocotea glomerata extracts and fractions, time of death and the synergistic effects with conventional antifungals. The possible mechanism of action was also addressed. Methods: Minimal inhibitory concentrations (MIC) were determined by broth microdilution technique, and the mechanism of action was assessed by ergosterol, sorbitol, cell viability, reactive oxygen species (ROS) generation and phosphatidylserine externalization tests. Results: All the tested extracts evidenced antifungal activity, but the methanol extract was revealed to be the most effective (MIC = 3.12 &mu, g/mL) on C. krusei. The combination of methanol extract with ketoconazole and fluconazole revealed a synergistic effect for C. krusei and C. albicans, respectively. Fractions 1 and 5 obtained from the methanol extract had fungicidal activity, mainly against C. krusei. Methanol extract did not reveal effects by ergosterol and sorbitol assays, however, it led to an increase in intracellular ROS levels, decreased cell viability, and consequently, cell death. Conclusion: O. glomerata methanol extract may be viewed as a rich source of biomolecules with antifungal activity against Candida spp.

Reactive oxygen species (ROS) cause cell damage and death. To reverse these effects, cells produce substances such as reduced glutathione (GSH) that serve as substrates for antioxidant enzymes. One way to combat microbial resistance includes nullifying the effect of glutathione in microbial cells, causing them to die from oxidative stress. The compound 2-((5-nitrothiophen-2-yl)methylene)-N-(pyridin-3-yl) hydrazine carbothioamide (L10) is a new thiophene-thiosemicarbazone derivative with promising antifungal activity. The aim of this study was to evaluate its mechanism of action against Candida albicans using assays that evaluate its effects on redox balance. Treatment with L10 promoted significant changes in the minimum inhibitory concentration (MIC) values in ascorbic acid and GSH protection tests, the latter increasing up to 64-fold of the MIC. Using nuclear magnetic resonance, we demonstrated interaction of L10 and GSH. At concentrations of 4.0 and 8.0 μg/mL, significant changes were observed in ROS production and mitochondrial membrane potential. The cell death profile showed characteristics of initial apoptosis at inhibitory concentrations (4.0 μg/mL). Transmission electron microscopy data corroborated these results and indicated signs of apoptosis, damage to plasma and nuclear membranes, and to mitochondria. Taken together, these results suggest a possible mechanism of action for L10 antifungal activity, involving changes in cellular redox balance, ROS production, and apoptosis-compatible cellular changes.

The Candida parapsilosis complex has been associated with highly refractory infections mainly due to the presence of biofilms. High glucose levels enable the development of this virulence factor which can aggravate the clinical condition of patients with diabetes mellitus, those using parenteral nutrition, with invasive medical device, including others. Combined antifungal therapy, such as azole and cyclooxygenase inhibitors, may be an alternative in such infections since they modulate prostaglandin production favoring the adhesion and development of biofilms. Thus, the present study aimed to evaluate the influence of glucose supplementation in the formation and detection of Candida parapsilosis complex biofilms and to treat them using fluconazole and a cyclooxygenase inhibitor in combination. Protein spectra evaluation allowed the differentiation between species from the complex (score > 2) in our studies. All isolates were able to form active biofilms at different glucose concentrations. In addition, a significant reduction in biofilm formation was observed when fluconazole and acetylsalicylic acid were combined. The ultrastructural analysis presented typical biofilm characteristics by species from the complex. These data support new combined therapies for the treatment of fungal infections, especially with those which are resistant and therapeutic failure is associated with virulence factors.

Trends in epidemiology and antifungal susceptibility of Candida species in Brazil between 2019–2020 are reported. A total of 22 isolates diagnosed from candidemia episodes were analyzed. Candida species were identified by specie specific primer and/or sequencing of 28S rDNA. Antifungal susceptibility was determined by CLSI method. C. tropicalis accounted for 11 (50%) cases followed by C. albicans (n=5, 22.73%), C. parapsilosis (n=4, 18.19%), Issatchenkia orientalis (n= 1, 4.54%), and Saccharomyces cerevisiae (n= 1, 4.54%). All Candida isolates were susceptible to amphotericin B and micafungin, and one was dose-dependent to anidulafungin. Eight of 20 Candida isolates were resistant to fluconazole and four sensitive doses dependent. Sixteen of 20 Candida isolates were resistant to voriconazole, and one resistant and one sensitive dose-dependent on caspofungin. Besides, the respective MIC50 and MIC90 values ​​were calculated with fluconazole (MIC50 and MIC90 of 4 and 64µg/mL, respectively) and voriconazole (MIC50 and MIC90 of 16µg/mL) showing the lowest potencies. The spread of fluconazole-resistant in one of major concerns, especially to C. tropicalis. The high use of antifungal drug may be a possible cause related to this scenario. Thus, the susceptibility profile test may be used to know the best way to introduce appropriateness empirical antibiotics therapy. Changing trends in incidence and antifungal susceptibility patterns of six Candida species causing candidemia in Kuwait between 2006–2017 are reported. A total of 2075 isolates obtained from 1448 patients were analyzed. Identity of Candida species isolates was determined by phenotypic methods and confirmed by PCR amplification/PCR-sequencing of rDNA and/or MALDI-TOF MS. Antifungal susceptibility was determined by Etest. C. albicans accounted for 539 (37.22%) cases followed by C. parapsilosis (n = 502, 34.67%), C. tropicalis (n = 210, 14.5%), C. glabrata (n = 148, 10.22%), C. krusei (n = 27, 1.81%) and C. dubliniensis (n = 22, 1.5%). The comparative percent distribution of Candida species causing candidemia between 2006–2011 and 2012–2017 was as follows: C. albicans 41.8% and 33.1%, C. parapsilosis complex 32.01% and 37.04%, C. tropicalis 13.59% and 15.31%, and C. glabrata 8.77% and 11.51%, C. krusei 2.0% and 1.7%, and C. dubliniensis 1.75 and 1.3%, respectively. Three of 371 C. albicans isolates during 2006–2011 and five of 363 during 2012–2017 were resistant to fluconazole. Among C. parapsilosis isolates, one of 310 during 2006–2011 and 21 of 446 during 2012–2017 were resistant to this drug. Furthermore, at an epidemiologic cutoff value (ECV) of ≤0.5 μg/ml, 70.1% C. albicans isolates were wild-type for fluconazole during 2006–2011 as compared to 58.1% during 2012–2017. Likewise, at an ECV of ≤2 μg/ml, 98.0% of C. parapsilosis isolates were wild-type during 2006–2011 as compared to 93.4% during 2012–2017. Clonal spread of fluconazole-resistant C. parapsilosis in one major hospital was documented. An 8.8% shift in favor of non-albicans Candida species with concomitant increase in MICs between the two periods preludes emergence of fluconazole-resistant candidemia cases in Kuwait.Changing trends in incidence and antifungal susceptibility patterns of six Candida species causing candidemia in Kuwait between 2006–2017 are reported. A total of 2075 isolates obtained from 1448 patients were analyzed. Identity of Candida species isolates was determined by phenotypic methods and confirmed by PCR amplification/PCR-sequencing of rDNA and/or MALDI-TOF MS. Antifungal susceptibility was determined by Etest. C. albicans accounted for 539 (37.22%) cases followed by C. parapsilosis (n = 502, 34.67%), C. tropicalis (n = 210, 14.5%), C. glabrata (n = 148, 10.22%), C. krusei (n = 27, 1.81%) and C. dubliniensis (n = 22, 1.5%). The comparative percent distribution of Candida species causing candidemia between 2006–2011 and 2012–2017 was as follows: C. albicans 41.8% and 33.1%, C. parapsilosis complex 32.01% and 37.04%, C. tropicalis 13.59% and 15.31%, and C. glabrata 8.77% and 11.51%, C. krusei 2.0% and 1.7%, and C. dubliniensis 1.75 and 1.3%, respectively. Three of 371 C. albicans isolates during 2006–2011 and five of 363 during 2012–2017 were resistant to fluconazole. Among C. parapsilosis isolates, one of 310 during 2006–2011 and 21 of 446 during 2012–2017 were resistant to this drug. Furthermore, at an epidemiologic cutoff value (ECV) of ≤0.5 μg/ml, 70.1% C. albicans isolates were wild-type for fluconazole during 2006–2011 as compared to 58.1% during 2012–2017. Likewise, at an ECV of ≤2 μg/ml, 98.0% of C. parapsilosis isolates were wild-type during 2006–2011 as compared to 93.4% during 2012–2017. Clonal spread of fluconazole-resistant C. parapsilosis in one major hospital was documented. An 8.8% shift in favor of non-albicans Candida species with concomitant increase in MICs between the two periods preludes emergence of fluconazole-resistant candidemia cases in Kuwait. Se informan las tendencias epidemiológicas y de susceptibilidad antifúngica de especies de Candida en Brasil, entre 2019-2020. Se analizaron un total de 22 aislamientos diagnosticados de episodios de candidemia. Las especies de Candida se identificaron mediante cebador específico y/o secuenciación del dominio D1/D2, región 28S del rDNA. La susceptibilidad antifúngica se determinó mediante el método CLSI. Candida tropicalis fue responsable de 11 (50%) casos, seguida de C. albicans (n=5, 22,73%), C. parapsilosis (n=4, 18,19%), Issatchenkia orientalis (n=1, 4,54 %) y Saccharomyces cerevisiae (n=1, 4,54%). Todos los aislados de Candida fueron sensibles a la anfotericina B y la micafungina, y uno fue sensible dosis dependiente a la anidulafungina. Ocho de los 20 aislados de Candida fueron resistentes al fluconazol y cuatro sensibles dosis dependiente. Dieciséis de los 20 aislados de Candida fueron resistentes al voriconazol, uno resistente y uno sensible dosis dependiente a la caspofungina. Además, se calcularon los valores respectivos de MIC50 y MIC90 con fluconazol (MIC50 y MIC90 de 4 y 64 µg/mL, respectivamente) y voriconazol (MIC50 y MIC90 de 16 µg/mL) que presentaron las potencias más bajas. La propagación de especies resistentes al fluconazol es una preocupación importante, especialmente para C. tropicalis. El uso indiscriminado de antifúngicos puede ser una posible causa relacionada con este escenario. Por lo tanto, la prueba del perfil de susceptibilidad se puede utilizar para encontrar la mejor manera de introducir una terapia antibiótica empírica adecuada. Tendências epidemiológicas e de susceptibilidade antifúngica de espécies de Candida no Brasil, entre 2019-2020, são relatadas. Um total de 22 isolados diagnosticados a partir de episódios de candidemia foram analisados. As espécies de Candida foram identificadas por meio de primer específico e/ou sequenciamento do domínio D1/D2, região 28S do rDNA. A susceptibilidade antifúngica foi determinada pelo método do CLSI. Candida tropicalis foi responsável por 11 (50%) casos, seguido por C. albicans (n=5, 22,73%), C. parapsilosis (n=4, 18,19%), Issatchenkia orientalis (n=1, 4,54%) e Saccharomyces cerevisiae (n=1, 4,54%). Todos os isolados de Candida foram susceptíveis à anfotericina B e micafungina, e um foi dose-dependente à anidulafungina. Oito dos 20 isolados de Candida foram resistentes ao fluconazol e quatro susceptível dose dependente. Dezesseis dos 20 isolados de Candida foram resistentes ao voriconazol, um resistente e um susceptível dose-dependente a caspofungina. Além disso, os respectivos valores de MIC50 e MIC90 foram calculados com fluconazol (MIC50 e MIC90 de 4 e 64µg/mL, respectivamente) e voriconazol (MIC50 e MIC90 de 16µg/mL) que apresentaram as potências mais baixas. A disseminação de espécies resistentes ao fluconazol é uma das principais preocupações, especialmente para C. tropicalis. O uso indiscriminado do antifúngico pode ser uma possível causa relacionada a esse cenário. Assim, a testagem do perfil de susceptibilidade pode ser usado para saber a melhor maneira de introduzir a terapia antibiótica empírica adequada.

In the present work, twelve N-substituted 2-(5-nitro-thiophene)-thiosemicarbazones derivatives (L1-12) were synthesized, characterized and their in vitro cytotoxic and antifungal activities were evaluated against Candida sp. and Cryptococcus neoformans. The probable mechanisms of action have been investigated by sorbitol and ergosterol assays. Additionally, ultrastructural study by Scanning Electron Microscopy was performed with the L10 compound. All compounds were obtained in good yield and their chemical structures were characterized on basis of their physico-chemical and Nuclear Magnetic Resonance - NMR, Spectrophotometric Absorption in the Infrared - IR and High-resolution Mass Spectrometry - HRMS data. The results showed that all strains were more sensitive to the compound L10 except Candida tropicalis URM 6551. On the other hand, the cytotoxicity assay by incorporation of tritiated thymidine showed moderate cytotoxic activity on L8 of the 50 μg/mLat which had the best MIC-cytotoxicity relationship. Concerning the study of the possible mechanism of action, the compounds were not able to bind to ergosterol in the membrane, do not act by inhibiting the synthesis of fungal cell wall (sorbitol assay). However, the Scanning Electron Microscopy - SEM analysis shows significant morphological changes in shape, size, number of cells and hyphae, and cell wall indicating a possible mechanism of action by inhibition of enzymes related to the ergosterol biosynthesis pathway. Our results demonstrate that N-substituted 2-(5-nitro-thiophene)-thiosemicarbazones derivatives are potential antifungal agents with activity associated with inhibition of enzymes related to biosynthesis of ergosterol.