Antifungal chemosensitization through induction of oxidative stress: A model for control of candidiasis based on the Lippia origanoides essential oil.

In this work, evaluated the antifungal chemosensitizing effect of the Lippia origanoides essential oil (EO) through the induction of oxidative stress. The EO was obtained by hydrodistillation and analyzed by GC-MS. To evaluate the antifungal chemosensitizing effect through induction of oxidative stress, cultures of the model yeast Saccharomyces cerevisiae ∆ycf1 were exposed to sub-inhibitory concentrations of the EO, and the expression of genes known, due be overexpressed in response to oxidative and mutagenic stress was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) method. Carvacrol and thymol were identified as the main components. The EO was effective in preventing or reducing the growth of the microorganisms tested. The gene expression profiles showed that EO promoted changes in the patterns of expression of genes involved in oxidative and mutagenic stress resistance. The combined use of the L. origanoides EO with fluconazole has been tested on Candida yeasts and the strategy resulted in a synergistic enhancement of the antifungal action of the azolic chemical product. Indeed, in association with EO, the fluconazole MICs dropped. Thus, the combinatorial use of L. origanoides EO as a chemosensitizer agent should contribute to enhancing the efficiency of conventional antifungal drugs, reducing their negative side effects.

Novel diarylated tacrine derivatives: Synthesis, characterization, anticancer, antiepileptic, antibacterial, and antifungal activities.

In this study, our goal was to synthesize novel aryl tacrine derivatives and assess their potential as anticancer, antibacterial agents, and enzyme inhibitors. We adopted a two-step approach, initiating with the synthesis of dibromotacrine derivatives 3 and 4 through the Friedlander reaction. These intermediates underwent further transformation into diarylated tacrine derivatives 3a-e and 4a-e using a Suzuki-Miyaura cross-coupling reaction. Thorough characterization of these novel diarylated tacrines was achieved using various spectroscopic techniques. Our findings highlighted the potent anticancer effects of these innovative compounds across a range of cancer cell lines, including lung, gynecologic, bone, colon, and breast cancers, while demonstrating low cytotoxicity against normal cells. Notably, these compounds surpassed the control drug, 5-Fluorouracil, in terms of antiproliferative activity in numerous cancer cell lines. Moreover, our investigation included an analysis of the inhibitory properties of these novel compounds against various microorganisms and cytosolic carbonic anhydrase enzymes. The results suggest their potential for further exploration as cancer-specific, enzyme inhibitory, and antibacterial therapeutic agents. Notably, four compounds, namely, 5,7-bis(4-(methylthio)phenyl)tacrine (3d), 5,7-bis(4-(trifluoromethoxy)phenyl)tacrine (3e), 2,4-bis(4-(trifluoromethoxy)phenyl)-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-11-amine (4e), and 6,8-dibromotacrine (3), emerged as the most promising candidates for preclinical studies.

Low toxicity contributes to Sporothrix globosa invade the skin of patients in low-epidemic areas of China.

OBJECTIVE: This study aims to assess the clinical characteristics of sporotrichosis in low-endemic areas of China, including the prevalence geography, genotypic traits of patients, clinical manifestations, and strain virulence and drug sensitivities. The objective is to improve the currently used clinical management strategies for sporotrichosis. METHODS: Retrospective data were collected from patients diagnosed with sporotrichosis through fungal culture identification. The isolates from purified cultures underwent identification using CAL (Calmodulin) gene sequencing. Virulence of each strain was assessed using a Galleria mellonella (G. mellonella) larvae infection model. In vitro susceptibility testing against commonly used clinical antifungal agents for sporotrichosis was conducted following CLSI criteria. RESULTS: In our low-endemic region for sporotrichosis, the majority of cases (23) were observed in middle-aged and elderly women with a history of trauma, with a higher incidence during winter and spring. All clinical isolates were identified as Sporothrix globosa (S. globosa). The G. mellonella larvae infection model indicated independent and dose-dependent virulence among strains, with varying toxicity levels demonstrated by the degree of melanization of the G. mellonella. Surprisingly, lymphocutaneous types caused by S. globosa exhibited lower in vitro virulence but were more common in affected skin. In addition, all S.globosa strains displayed high resistances to fluconazole, while remaining highly susceptible to terbinafine, itraconazole and amphotericin B. CONCLUSION: Given the predominance of elderly women engaged in agricultural labour in our region, which is a low-epidemic areas, they should be considered as crucial targets for sporotrichosis monitoring. S. globosa appears to be the sole causative agent locally. However, varying degrees of melanization in larvae were observed among these isolates, indicating a divergence in their virulence. Itraconazole, terbinafine and amphotericin B remain viable first-line antifungal options for treating S.globosa infection.

Green synthesis of Superparamagnetic Iron Oxide and Silver Nanoparticles in Satureja hortensis Leave Extract: Evaluation of Antifungal Effects on Botryosphaeriaceae Species.

In recent years, green synthesis methods of metallic nanoparticles (MNPs) have been attractive because of the more facile, cheaper, and appropriate features associated with biomolecules in MNPs biosynthesis. This research represented an easy, fast, and environmentally friendly method to biosynthesis of superparamagnetic iron oxide nanoparticles (SPIONPs) and silver nanoparticles (AgNPs) by the Satureja hortensis leaf extract as stabilizer and reducer. The SPIONPs synthesized in co-precipitation method. The biosynthesized SPIONPs and AgNPs were studied their antifungal effects against three Botryosphaeriaceae plant pathogens, Botryosphaeria dothidea, Diplodia seriata, and Neofusicoccum parvum. UV-visible spectra (UV-Vis), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), field emission scanning electron microscopy (Fe-SEM), energy-dispersive X-ray spectroscopy (EDX), and vibrating-sample magnetometer (VSM) analyses were used to evaluate the physicochemical properties and verify the formation of green synthesized SPIONPs and AgNPs. UV-Vis spectra revealed absorption peaks at 243 and 448 nm for SPIONs and 436 nm for AgNPs, respectively. Microscopic and XRD analysis showed that SPIONPs and AgNPs was found spherical in shape with an average particle size of SPIONPs and AgNPs 10 and 12 nm, respectively. The antifungal test against Botryosphaeriaceae species showed that SPIONPs and AgNPs possess antifungal properties against B. dothidea, D. seriata, and N. parvum. However, AgNPs exhibits greater antifungal activity than SPIONPs. The results of the cytotoxicity tests of SPIONs and AgNPs on the MCF-7 cell line showed that AgNPs was significantly more cytotoxic towards the MCF-7 cell line, whereas no significant cytotoxic effect was recorded by SPIONs. Therefore, these biosynthesized MNPs could be substituted for toxic fungicides that are extensively applied in agriculture and contribute to environmental health and food safety.

Characterization of Bacillus amyloliquefaciens PM415 as a potential bio-preserving probiotic.

Animal feed is vulnerable to fungal infections, and the use of bio-preserving probiotics has received increasing attention. In contrast to Lactobacillus and Bifidobacteria spp., fewer Bacillus spp. have been recognized as antifungal probiotics. Therefore, our objective was to screen antifungal strains and provide more Bacillus candidates to bridge this gap. Here, we screened 56 bacterial strains for cyclic lipopeptide genes and conducted an antifungal assay with Aspergillus niger as a representative fungus. We found that a Bacillus strain Bacillus amyloliquefaciens PM415, isolated from pigeon manure, exhibited the highest fungal inhibition activity as demonstrated by the confrontation assay and morphological observation under scanning electron microscope (SEM). Preliminary safety assessment and probiotic characterization revealed its non-pathogenic feature and stress tolerance capability. Whole genome sequencing of Bacillus amyloliquefaciens PM415 revealed a genome size of 4.16 Mbp and 84 housekeeping genes thereof were used for phylogenetic analysis showing that it is most closely related to Bacillus amyloliquefaciens LFB112. The in silico analysis further supported its non-pathogenic feature at the genomic level and revealed potential biosynthetic gene clusters responsible for its antifungal property. RNA-seq analysis revealed genome-wide changes in transportation, amino acid metabolism, non-ribosomal peptides (NRPs) biosynthesis and glycan degradation during fungal antagonism. Our results suggest that Bacillus amyloliquefaciens PM415 is a safe and effective probiotic strain that can prevent fungal growth in animal feeds.

Effect of ozone gas on cultures of Candida albicans and Aspergillus fumigatus: evaluation of two ozonation devices.

INTRODUCTION: Candida albicans and Aspergillus fumigatus are two important agents of Healthcare-associated infections. This study aimed to evaluate the antifungal activity of ozone (O3) gas produced by two commercial devices against cultures of these two species. METHODOLOGY: Sterile plastic plates were inoculated with C. albicans and A. fumigatus and placed on a countertop at three distances (30 cm, 1 m, and 2 m) and three positions in relation to the wall (near, middle, and away), considering the source of O3. Plates were exposed to O3 for one hour and incubated. After incubation, the counting of colony-forming units was performed. As a control, an inoculated plate was incubated, without being exposed to O3. Tests were carried out with two different devices (namely, Mod.I and Mod.II), with the air conditioner on and off, in triplicate. RESULTS: Both devices showed antifungal activity. Mod. I presented better results, due to a higher flow rate. The best activity was on plates at 30 cm, middle position. Contrarily, on plates at 2m, near the wall, the inhibition activity was lower. The best results were obtained with the air conditioner off. Candida albicans was more sensitive to O3 than A. fumigatus. CONCLUSIONS: This method of decontamination by O3 gas shows potential due to its fast and easy execution. The establishment of new protocols for hygiene and hospital disinfection using this approach should be considered, which may reduce environmental contamination by fungi and, consequently, the burden of fungal infections.

In vitro antifungal susceptibility of the dermatophytes: a single center study from Eastern Black Sea Region of Turkey.

OBJECTIVE: A large number of patients applying to the dermatology clinics are affected by fungal diseases, and a significant portion of which are superficial fungal infections. Dermatophyte infections are a notable public health concern and frequently encountered in clinical practice. Dermatophytosis not only compromises the quality of life but also predisposes individuals to various comorbidities due to its role as a gateway for secondary bacterial agents. This study aims to determine the species distribution of dermatophytes prevalent and assess their susceptibility to antifungal drugs. PATIENTS AND METHODS: Skin, nail, and hair samples were obtained from patients with a clinical diagnosis of dermatophytosis. Samples were all cultured to isolate and identify the species. In vitro liquid microdilution tests were conducted to assess the susceptibility of the isolated strains against terbinafine, fluconazole, griseofulvin, and butenafine. RESULTS: A total of 353 samples were obtained from the hair, skin, and nail lesions of 326 patients. Dermatophyte was isolated in 71 of the samples (20.1%). The cultured dermatophyte subtypes included Trichophyton rubrum (13.8% in 49 samples), Microsporum audouini (5.7% in 20 samples), and Trichophyton mentagrophytes (0.6% in 2 samples). Antifungal susceptibility testing revealed that terbinafine was the most effective antifungal drug against all dermatophyte species, while fluconazole exhibited the highest resistance. CONCLUSIONS: The most common dermatophytosis agent in our region is T. rubrum. The least antifungal resistance was found against terbinafine. Conducting antifungal susceptibility tests is crucial for selecting effective treatment regimens and early detection of resistance development.

Analysis of candidemia cases in a city hospital during the COVID-19 pandemic.

OBJECTIVE: The frequency and mortality of candidemia remain important. Non-albicans Candida species such as C. auris are increasing. PATIENTS AND METHODS: A retrospective review of adult patients diagnosed with bloodstream infection due to Candida species in the 17 months between July 1, 2020, and December 1, 2021, was performed. Yeast colonies grown in culture were identified by matrix-assisted laser desorption/ionization time-of-flight. Antifungal susceptibility tests of Candida strains were performed with Sensititre YeastOne (TREK Diagnostic Systems Inc., Westlake, Ohio) kits, and minimum inhibitory concentration values were evaluated according to the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints. RESULTS: In total, 217 patients (mean age 64.9±15.7 years) were included. C. albicans was the most common fungus (detected in 82 patients; 37.8%), followed by C. parapsilosis (17.1%), C. glabrata (15.2%), C. tropicalis (15.2%), and C. auris (9%). Candidemia developed in 175 (81.4%) of the cases during their intensive care unit stay. Fluconazole (41.0%) and caspofungin (36.4%) were the two most frequently used antifungal agents in antifungal therapy. There were 114 (52.3%) deaths in the study group. Mortality rates were found to be lower in patients infected with C. parapsilosis or C. auris. Age and previous COVID-19 infection were other important risk factors. When the 217 Candida spp. were examined, resistance and intermediate susceptibility results were higher when EUCAST criteria were used. While the two methods were found to be fully compatible only for fluconazole, a partial agreement was also observed for voriconazole. CONCLUSIONS: As our study observed, the COVID-19 pandemic brought increasing numbers of immunosuppressed patients, widespread use of antibacterials, and central venous catheters, increasing the frequency and mortality of candidemia cases. All health institutions should be prepared for the diagnosis and treatment of candidemia. In addition, C. auris, the frequency of which has increased in recent years, is a new factor that should be considered in candidemia cases.

Hospital distribution, seasonality, time trends and antifungal susceptibility profiles of all Aspergillus species isolated from clinical samples from 2015 to 2022 in a tertiary care hospital.

BACKGROUND: Aspergillus species cause a variety of serious clinical conditions with increasing trend in antifungal resistance. The present study aimed at evaluating hospital epidemiology and antifungal susceptibility of all isolates recorded in our clinical database since its implementation. METHODS: Data on date of isolation, biological samples, patients' age and sex, clinical settings, and antifungal susceptibility tests for all Aspergillus spp. isolated from 2015 to 2022 were extracted from the clinical database. Score test for trend of odds, non-parametric Mann Kendall trend test and logistic regression analysis were used to analyze prevalence, incidence, and seasonality of Aspergillus spp. isolates. RESULTS: A total of 1126 Aspergillus spp. isolates were evaluated. A. fumigatus was the most prevalent (44.1%) followed by A. niger (22.3%), A. flavus (17.7%) and A. terreus (10.6%). A. niger prevalence increased over time in intensive care units (p-trend = 0.0051). Overall, 16 (1.5%) were not susceptible to one azole compound, and 108 (10.9%) to amphotericin B, with A. niger showing the highest percentage (21.9%). The risk of detecting A. fumigatus was higher in June, (OR = 2.14, 95% CI [1.16; 3.98] p = 0.016) and reduced during September (OR = 0.48, 95% CI [0.27; 0.87] p = 0.015) and October as compared to January (OR = 0.39, 95% CI [0.21; 0.70] p = 0.002. A. niger showed a reduced risk of isolation from all clinical samples in the month of June as compared to January (OR = 0.34, 95% CI [0.14; 0.79] p = 0.012). Seasonal trend for A. flavus showed a higher risk of detection in September (OR = 2.7, 95% CI [1.18; 6.18] p = 0.019), October (OR = 2.32, 95% CI [1.01; 5.35] p = 0.048) and November (OR = 2.42, 95% CI [1.01; 5.79] p = 0.047) as compared to January. CONCLUSIONS: This is the first study to analyze, at once, data regarding prevalence, time trends, seasonality, species distribution and antifungal susceptibility profiles of all Aspergillus spp. isolates over a 8-year period in a tertiary care center. Surprisingly no increase in azole resistance was observed over time.

Curcumin: An innovative approach for postharvest control of Alternaria alternata induced black rot in cherry tomatoes.

Curcumin, a natural bioactive compound derived from Curcuma longa, has been widely recognized for its antifungal properties. In this study, we investigated the effects of curcumin on the phytopathogenic fungus Alternaria alternata and its pathogenicity in cherry tomato fruit. The results demonstrated that curcumin treatment significantly inhibited mycelial growth and spore germination of A. alternata in a dose-dependent manner. Scanning electron microscopy revealed alterations in the morphology of A. alternata mycelia treated with curcumin. Furthermore, curcumin treatment led to an increase in malondialdehyde and hydrogen peroxide contents, indicating cell membrane damage in A. alternata. Moreover, curcumin exhibited a remarkable inhibitory effect on the incidence and lesion diameters of black rot caused by A. alternata in cherry tomato fruit. Gene expression analysis revealed upregulation of defense-related genes (POD, SOD, and CAT) in tomato fruit treated with curcumin. Additionally, curcumin treatment resulted in decreased activity of exocellular pathogenic enzymes (polygalacturonase, pectin lyase, and endo-1,4-ß-d-glucanase) in A. alternata. Overall, our findings highlight the potential of curcumin as an effective antifungal agent against A. alternata, providing insights into its inhibitory mechanisms on mycelial growth, spore germination, and pathogenicity in cherry tomato fruit.

Total transcriptome response for tyrosol exposure in Aspergillus nidulans.

Although tyrosol is a quorum-sensing molecule of Candida species, it has antifungal activity at supraphysiological concentrations. Here, we studied the effect of tyrosol on the physiology and genome-wide transcription of Aspergillus nidulans to gain insight into the background of the antifungal activity of this compound. Tyrosol efficiently reduced germination of conidia and the growth on various carbon sources at a concentration of 35 mM. The growth inhibition was fungistatic rather than fungicide on glucose and was accompanied with downregulation of 2199 genes related to e.g. mitotic cell cycle, glycolysis, nitrate and sulphate assimilation, chitin biosynthesis, and upregulation of 2250 genes involved in e.g. lipid catabolism, amino acid degradation and lactose utilization. Tyrosol treatment also upregulated genes encoding glutathione-S-transferases (GSTs), increased specific GST activities and the glutathione (GSH) content of the cells, suggesting that A. nidulans can detoxify tyrosol in a GSH-dependent manner even though this process was weak. Tyrosol did not induce oxidative stress in this species, but upregulated "response to nutrient levels", "regulation of nitrogen utilization", "carbon catabolite activation of transcription" and "autophagy" genes. Tyrosol may have disturbed the regulation and orchestration of cellular metabolism, leading to impaired use of nutrients, which resulted in growth reduction.

Pathogenic Aspergillus Strains Identification and Antifungal Susceptibility Analysis of 452 Cases with Otomycosis in Jingzhou, China.

OBJECTIVE: To study the distribution of pathogenic Aspergillus strains of otomycosis in central China and the identification of their antifungal sensitivity. METHODS: We collected external ear canal secretions clinically diagnosed as otomycosis from April 2020 to January 2023 from the Department of Otolaryngology-Head and Neck Surgery in central China. The pathogenic Aspergillus strains were identified through morphological examination and sequencing. The antifungal sensitivity was performed using the broth microdilution method described in the Clinical Laboratory Standard Institute document M38-A3. RESULTS: In the 452 clinical strains isolated from the external ear canal, 284 were identified as Aspergillus terreus (62.83%), 92 as Aspergillus flavus (20.35%), 55 as Aspergillus niger (12.17%). In antifungal susceptibility tests the MIC of Aspergillus strains to bifonazole and clotrimazole was high,all the MIC90 is > 16 ug/mL. However, most Aspergillus isolates show moderate greatly against terbinafine, itraconazole and voriconazole. CONCLUSION: A. terreus is the most common pathogenic Aspergillus strain in otomycosis in central China. The selected topical antifungal drugs were bifonazole and clotrimazole; the drug resistance rate was approximately 30%. If the infection is persistent and requires systemic treatment, terbinafine and itraconazole can be used. The resistance of Aspergillus in otomycosis to voriconazole should be screened to avoid the systemic spread of infection in immunocompromised people and poor compliance with treatment. However, the pan-azole-resistant strain of Aspergillus should be monitored, particularly in high-risk patients with otomycosis.

Lethal metabolism of Candida albicans respiratory mutants.

The destructive impact of fungi in agriculture and animal and human health, coincident with increases in antifungal resistance, underscores the need for new and alternative drug targets to counteract these trends. Cellular metabolism relies on many intermediates with intrinsic toxicity and promiscuous enzymatic activity generates others. Fuller knowledge of these toxic entities and their generation may offer opportunities of antifungal development. From this perspective our observation of media-conditional lethal metabolism in respiratory mutants of the opportunistic fungal pathogen Candida albicans was of interest. C. albicans mutants defective in NADH:ubiquinone oxidoreductase (Complex I of the electron transport chain) exhibit normal growth in synthetic complete medium. In YPD medium, however, the mutants grow normally until early stationary phase whereupon a dramatic loss of viability occurs. Upwards of 90% of cells die over the subsequent four to six hours with a loss of membrane integrity. The extent of cell death was proportional to the amount of BactoPeptone, and to a lesser extent, the amount of yeast extract. YPD medium conditioned by growth of the mutant was toxic to wild-type cells indicating mutant metabolism established a toxic milieu in the media. Conditioned media contained a volatile component that contributed to toxicity, but only in the presence of a component of BactoPeptone. Fractionation experiments revealed purine nucleosides or bases as the synergistic component. GC-mass spectrometry analysis revealed acetal (1,1-diethoxyethane) as the active volatile. This previously unreported and lethal synergistic interaction of acetal and purines suggests a hitherto unrecognized toxic metabolism potentially exploitable in the search for antifungal targets.

Antifungal plant flavonoids identified in silico with potential to control rice blast disease caused by Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, poses a severe threat to rice production, particularly in Asia where rice is a staple food. Concerns over fungicide resistance and environmental impact have sparked interest in exploring natural fungicides as potential alternatives. This study aimed to identify highly potent natural fungicides against M. oryzae to combat rice blast disease, using advanced molecular dynamics techniques. Four key proteins (CATALASE PEROXIDASES 2, HYBRID PKS-NRPS SYNTHETASE TAS1, MANGANESE LIPOXYGENASE, and PRE-MRNA-SPLICING FACTOR CEF1) involved in M. oryzae's infection process were identified. A list of 30 plant metabolites with documented antifungal properties was compiled for evaluation as potential fungicides. Molecular docking studies revealed that 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin exhibited superior binding affinities compared to reference fungicides (Azoxystrobin and Tricyclazole). High throughput molecular dynamics simulations were performed, analyzing parameters like RMSD, RMSF, Rg, SASA, hydrogen bonds, contact analysis, Gibbs free energy, and cluster analysis. The results revealed stable interactions between the selected metabolites and the target proteins, involving important hydrogen bonds and contacts. The SwissADME server analysis indicated that the metabolites possess fungicide properties, making them effective and safe fungicides with low toxicity to the environment and living beings. Additionally, bioactivity assays confirmed their biological activity as nuclear receptor ligands and enzyme inhibitors. Overall, this study offers valuable insights into potential natural fungicides for combating rice blast disease, with 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin standing out as promising and environmentally friendly alternatives to conventional fungicides. These findings have significant implications for developing crop protection strategies and enhancing global food security, particularly in rice-dependent regions.

Deciphering antifungal and antibiofilm mechanisms of isobavachalcone against Cryptococcus neoformans through RNA-seq and functional analyses.

Cryptococcus neoformans has been designated as critical fungal pathogens by the World Health Organization, mainly due to limited treatment options and the prevalence of antifungal resistance. Consequently, the utilization of novel antifungal agents is crucial for the effective treatment of C. neoformans infections. This study exposed that the minimum inhibitory concentration (MIC) of isobavachalcone (IBC) against C. neoformans H99 was 8 µg/mL, and IBC dispersed 48-h mature biofilms by affecting cell viability at 16 µg/mL. The antifungal efficacy of IBC was further validated through microscopic observations using specific dyes and in vitro assays, which confirmed the disruption of cell wall/membrane integrity. RNA-Seq analysis was employed to decipher the effect of IBC on the C. neoformans H99 transcriptomic profiles. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis was performed to validate the transcriptomic data and identify the differentially expressed genes. The results showed that IBC exhibited various mechanisms to impede the growth, biofilm formation, and virulence of C. neoformans H99 by modulating multiple dysregulated pathways related to cell wall/membrane, drug resistance, apoptosis, and mitochondrial homeostasis. The transcriptomic findings were corroborated by the antioxidant analyses, antifungal drug sensitivity, molecular docking, capsule, and melanin assays. In vivo antifungal activity analysis demonstrated that IBC extended the lifespan of C. neoformans-infected Caenorhabditis elegans. Overall, the current study unveiled that IBC targeted multiple pathways simultaneously to inhibit growth significantly, biofilm formation, and virulence, as well as to disperse mature biofilms of C. neoformans H99 and induce cell death.

Inclusion Complexes of Litsea cubeba (Lour.) Pers Essential Oil into ß-Cyclodextrin: Preparation, Physicochemical Characterization, Cytotoxicity and Antifungal Activity.

The uses of natural compounds, such as essential oils (EOs), are limited due to their instability to light, oxygen and temperature, factors that affect their application. Therefore, improving stability becomes necessary. The objective of this study was to prepare inclusion complexes of Litsea cubeba essential oil (LCEO) with ß-cyclodextrin (ß-CD) using physical mixing (PM), kneading (KN) and co-precipitation (CP) methods and to evaluate the efficiency of the complexes and their physicochemical properties using ATR-FTIR, FT-Raman, DSC and TG. The study also assessed cytotoxicity against human colorectal and cervical cancer cells and antifungal activity against Aspergillus flavus and Fusarium verticillioides. The complexation efficiency results presented significant evidence of LCEO:ß-CD inclusion complex formation, with KN (83%) and CP (73%) being the best methods used in this study. All tested LCEO:ß-CD inclusion complexes exhibited toxicity to HT-29 cells. Although the cytotoxic effect was less pronounced in HeLa tumor cells, LCEO-KN was more active against Hela than non-tumor cells. LCEO-KN and LCEO-CP inclusion complexes were efficient against both toxigenic fungi, A. flavus and F. verticillioides. Therefore, the molecular inclusion of LCEO into ß-CD was successful, as well as the preliminary biological results, evidencing that the ß-CD inclusion process may be a viable alternative to facilitate and increase future applications of this EO as therapeutic medication, food additive and natural antifungal agent.

Design of Protease-Responsive Antifungal Liposomal Formulation Decorated with a Lipid-Modified Chitin-Binding Domain.

Candida albicans is a prevalent fungal pathogen that displays antibiotic resistance. The polyene antifungal amphotericin B (AmB) has been the gold standard because of its broad antifungal spectra, and its liposomal formulation, AmBisome, has been used widely and clinically in treating fungal infections. Herein, we explored enhancing the antifungal activity of AmBisome by integrating a small chitin-binding domain (LysM) of chitinase A derived from Pteris ryukyuensis. LysM conjugated with a lipid (LysM-lipid) was initially prepared through microbial transglutaminase (MTG)-mediated peptide tag-specific conjugation of LysM with a lipid-peptide substrate. The AmBisome formulation modified with LysM-lipid conjugates had a size distribution that was comparable to the native liposomes but an increased zeta potential, indicating that LysM-lipid conjugates were anchored to AmBisome. LysM-lipid-modified AmBisome exhibited long-term stability at 4 °C while retaining the capacity to bind chitin. Nevertheless, the antifungal efficacy of LysM-lipid-modified AmBisome against C. albicans was modest. We then redesigned a new LysM-lipid conjugate by introducing a peptide linker containing a thrombin digestion (TD) site at the C-terminus of LysM (LysM-TD linker-lipid), thereby facilitating the liberation of the LysM domain from AmBisome upon the addition of thrombin. This new AmBisome formulation anchored with LysM-TD linker-lipid exhibited superior performance in suppressing C. albicans growth in the presence of thrombin compared with the LysM-lipid formulation. These results provide a platform to design stimuli-responsive AmBisome formulations that respond to external environments and thus advance the treatment of pathogenic fungi infections.

Synthesis of Novel Triazine-Based Chalcones and 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines as Potential Leads in the Search of Anticancer, Antibacterial and Antifungal Agents.

This study presents the synthesis of four series of novel hybrid chalcones (20,21)a-g and (23,24)a-g and six series of 1,3,5-triazine-based pyrimido[4,5-b][1,4]diazepines (28-33)a-g and the evaluation of their anticancer, antibacterial, antifungal, and cytotoxic properties. Chalcones 20b,d, 21a,b,d, 23a,d-g, 24a-g and the pyrimido[4,5-b][1,4]diazepines 29e,g, 30g, 31a,b,e-g, 33a,b,e-g exhibited outstanding anticancer activity against a panel of 60 cancer cell lines with GI50 values between 0.01 and 100 µM and LC50 values in the range of 4.09 µM to >100 µM, several of such derivatives showing higher activity than the standard drug 5-fluorouracil (5-FU). On the other hand, among the synthesized compounds, the best antibacterial properties against N. gonorrhoeae, S. aureus (ATCC 43300), and M. tuberculosis were exhibited by the pyrimido[4,5-b][1,4]diazepines (MICs: 0.25-62.5 µg/mL). The antifungal activity studies showed that triazinylamino-chalcone 29e and triazinyloxy-chalcone 31g were the most active compounds against T. rubrum and T. mentagrophytes and A. fumigatus, respectively (MICs = 62.5 µg/mL). Hemolytic activity studies and in silico toxicity analysis demonstrated that most of the compounds are safe.

Comprehensive antifungal investigation of natural plant extracts against Neosartorya spp. (Aspergillus spp.) of agriculturally significant microbiological contaminants and shaping their metabolic profile.

Fungi belonging to the genus Neosartorya (teleomorph of Aspergillus spp.) are of great concern in the production and storage of berries and fruit-based products, mainly due to the production of thermoresistant ascospores that cause food spoilage and possible secretion of mycotoxins. We initially tested the antifungal effect of six natural extracts against 20 isolates of Neosartorya spp. using a traditional inhibition test on Petri dishes. Tested isolates did not respond uniformly, creating 5 groups of descending sensitivity. Ten isolates best representing of the established sensitivity clusters were chosen for further investigation using a Biolog™ MT2 microplate assay with the same 6 natural extracts. Additionally, to test for metabolic profile changes, we used a Biolog™ FF microplate assay after pre-incubation with marigold extract. All natural extracts had an inhibitory effect on Neosartorya spp. growth and impacted its metabolism. Lavender and tea tree oil extracts at a concentration of 1000 µg mL-1 presented the strongest antifungal effect during the inhibition test, however all extracts exhibited inhibitory properties at even the lowest dose (5 µg mL-1). The fungal stress response in the presence of marigold extract was characterized by a decrease of amino acids and carbohydrates consumption and an uptake of carboxylic acids on the FF microplates, where the 10 studied isolates also presented differences in their innate resilience, creating 3 distinctive sensitivity groups of high, average and low sensitivity. The results confirm that natural plant extracts and essential oils inhibit and alter the growth and metabolism of Neosartorya spp. suggesting a possible future use in sustainable agriculture as an alternative to chemical fungicides used in traditional crop protection.

Pore-forming peptide C14R exhibits potent antifungal activity against clinical isolates of Candida albicans and Candida auris.

Introduction: Invasive candidiasis is a global public health problem as it poses a significant threat in hospital-settings. The aim of this study was to evaluate C14R, an analog derived from peptide BP100, as a potential antimicrobial peptide against the prevalent opportunistic yeast Candida albicans and the emergent multidrug-resistant yeast Candida auris. Methods: Antifungal susceptibility testing of C14R against 99 C. albicans and 105 C. auris clinical isolates from Colombia, was determined by broth microdilution. Fluconazole was used as a control antifungal. The synergy between C14R and fluconazole was assessed in resistant isolates. Assays against fungal biofilm and growth curves were also carried out. Morphological alterations of yeast cell surface were evaluated by scanning electron microscopy. A permeability assay verified the pore-forming ability of C14R. Results: C. albicans and C. auris isolates had a geometric mean MIC against C14R of 4.42 µg/ml and 5.34 µg/ml, respectively. Notably, none of the isolates of any species exhibited growth at the highest evaluated peptide concentration (200 µg/ml). Synergistic effects were observed when combining the peptide and fluconazole. C14R affects biofilm and growth of C. albicans and C. auris. Cell membrane disruptions were observed in both species after treatment with the peptide. It was confirmed that C14R form pores in C. albicans' membrane. Discussion: C14R has a potent antifungal activity against a large set of clinical isolates of both C. albicans and C. auris, showing its capacity to disrupt Candida membranes. This antifungal activity remains consistent across isolates regardless of their clinical source. Furthermore, the absence of correlation between MICs to C14R and resistance to fluconazole indicates the peptide's potential effectiveness against fluconazole-resistant strains. Our results suggest the potential of C14R, a pore-forming peptide, as a treatment option for fungal infections, such as invasive candidiasis, including fluconazole and amphotericin B -resistant strains.