Bacillus methylotrophicus DCS1: Production of Different Lipopeptide Families, In Vitro Antifungal Activity and Suppression of Fusarium Wilt in Tomato Plants.

The present work aims to quantitatively and qualitatively monitor the production of lipopeptide mixtures by Bacillus methylotrophicus DCS1 strain in Landy medium and to investigate the antifungal activities of DCS1 strain and its produced lipopeptides. The in vitro activities were tested by the direct confrontation and agar well diffusion methods, while the in vivo study was carried out in order to test the efficiency of DCS1 bacterial suspension in the control of Fusarium wilt in tomato plants. Identification of lipopeptides by mass spectrometry (LC/MSD-TOF) showed that lipopeptide isoforms produced during the first 24 h and 48 h of fermentation are identical, belonging to bacillomycin D and fengycins A and B homologues with a difference in the yield of production. After 72 h of fermentation corresponding to the end of incubation period, B. methylotrophicus DCS1 is able to produce a mixture of surfactin, pumilacidin, iturin A/mycosubtilin, iturin C1, bacillomycin D and fengycins A and B isoforms. The results of in vitro antifungal experiments suggest that B. methylotrophicus DCS1 has a significant potential as a biocontrol agent, owing to lipopeptides produced, endowed with antifungal activity against several phytopathogenic fungi. The curative treatment of tomato plants with DCS1 bacterial suspension was more effective in the protection against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) than the preventive treatment by comparing the average number of leaves remaining healthy after 30 days of each treatment and the appearance of tomato plants roots. The results indicate that B. methylotrophicus DCS1 exhibit a significant suppression of Fusarium wilt symptoms in tomato plants comparable to that of commercial fungicides and could be an alternative to chemically synthesized pesticides.

The inhibitory effects of carvacrol, nystatin, and their combination on oral candidiasis isolates.

BACKGROUND: Candida, a common oral microbiota, can cause opportunistic fungal infections. With rising Candida infections and limited effective antifungals, new treatments are needed. This study investigates carvacrol essential oil's effect on oral candidiasis, alone and with nystatin, compared to nystatin alone. MATERIALS AND METHODS: In this study, oral samples were collected from dental clinic patients, especially denture users. The presence of Candida was confirmed and cultured from these samples. Candidiasis was detected by observing Candida colonies. Drug sensitivity was tested on 100 positive samples. The minimum concentration of inhibition and lethality of each isolate was evaluated using nystatin and carvacrol. The results were compared using two-way analysis of variance. Finally, the minimum inhibitory concentration (MIC) of nystatin and carvacrol was calculated individually and in combination. RESULTS: The present study found that Candida albicans and non-albicans species were equally prevalent. Carvacrol showed significant biological activity against all Candida species, with an average MTT of 50.01%. The average MIC value of carvacrol was 24.96 µg/ml, indicating its potential to inhibit Candida growth. The mean Minimum Fungicidal Concentration (MFC) value of carvacrol was 23.48 µg/ml, suggesting its effectiveness in killing the fungi. CONCLUSION: The study's findings reveal that the MIC of carvacrol was significantly lower than that of nystatin and the combination of nystatin and carvacrol. This suggests that carvacrol holds potential as an effective herbal remedy for candidiasis.

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.

Synergistic action of synthetic peptides and amphotericin B causes disruption of the plasma membrane and cell wall in Candida albicans.

The objective of this work was to evaluate the combination of synthetic peptides based on the γ-core motif of defensin PvD1 with amphotericin B (AmB) at different concentrations against Candida albicans. We applied the checkerboard assay using different concentrations of the commercial drug AmB and the synthetic peptides γ31-45PvD1++ and γ33-41PvD1++ against C. albicans, aiming to find combinations with synergistic interactions. Between these two interactions involving γ31-45PvD1++ and AmB, an additive effect was observed. One such interaction occurred at concentrations of 0.009 µM of peptide γ31-45PvD1++ and 13.23 µM of AmB and another condition of 0.019 µM of peptide γ31-45PvD1++ and 6.61 µM of AmB. The other two concentrations of the interaction showed a synergistic effect in the combination of synthetic peptide γ31-45PvD1++ and AmB, where the concentrations were 1.40 µM peptide γ31-45PvD1++ and 0.004 µM AmB and 0.70 µM γ31-45PvD1++ peptide and 0.002 µM AmB. We proceeded with analysis of the mechanism of action involving synergistic effects. This examination unveiled a range of impactful outcomes, including the impairment of mitochondrial functionality, compromise of cell wall integrity, DNA degradation, and a consequential decline in cell viability. We also observed that both synergistic combinations were capable of causing damage to the plasma membrane and cell wall, causing leakage of intracellular components. This discovery demonstrates for the first time that the synergistic combinations found between the synthetic peptide γ31-45PvD1++ and AmB have an antifungal effect against C. albicans, acting on the integrity of the plasma membrane and cell wall.

Discovery of Antifungal Norsesquiterpenoids from a Soil-Derived Streptomyces microflavus: Targeting Biofilm Formation and Synergistic Combination with Amphotericin B against Yeast-like Fungi.

Thirty-five norsesquiterpenoids were isolated from the fermentation broth of Streptomyces microflavus from the forest soil of Ailaoshan in China. The structures of new compounds (1-5, 10-26) were elucidated by comprehensive spectroscopic analysis including data from experimental and calculated ECD spectra, as well as Mosher's reagent derivatives method. Norsesquiterpenoids showed different levels of antifungal activity with MIC80 values ranging from 25 to 200 µg/mL against Candida albicans, Candida parapsilosis, and Cryptococcus neoformans. The combining isolated norsesquiterpenoids with amphotericin B resulted in a synergistic interaction against test yeast-like fungi with a fractional inhibitory concentration index < 0.5. Compound 33 significantly inhibited biofilm formation and destroyed the preformed biofilm of fungi. Moreover, 33 downregulated the expression of adhesion-related genes HWP1, ALS1, ALS3, ECE1, EAP1, and BCR1 to inhibit the adhesion of C. albicans. Findings from the current study highlight the potential usage of norsesquiterpenoids from soil-derived Streptomyces for antifungal leads discovery.

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.

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.

Assessment of phenolic and flavonoid contents, antioxidant and antimicrobial activities of Moroccan propolis.

Background: Despite the extensive literature focused on propolis extract, few data exists on the bioactive compounds and biological activities in the Moroccan propolis and its economic value is low. Objective: In this research, the aim was to evaluate the total content of phenols and flavonoids as well as the antioxidant, antibacterial and antifungal activities of Moroccan propolis. Material and Methods: The polyphenol and flavonoid content of the Moroccan propolis from three geographic regions, was quantified in the ethanolic extract by colorimetric methods using folin-ciocalteu and aluminum chloride. The antioxidant activity was evaluated by the DPPH test and expressed as IC50. Disk diffusion and broth microdilution methods were used to examine in vitro antimicrobial activity against known human microorganism pathogens. Results: The obtained data revealed that Moroccan propolis samples presented significant variations in total polyphenols and flavonoids. All samples showed significant antioxidant activity with IC50 values ranging from 4.23±0.5 to 154±0.21 µg/ mL. A strong correlation between total phenolic activity, flavonoids and antioxidant activity was found. The in vitro study of antibacterial activity showed that the propolis samples exhibited a range of growth inhibitory actions against all bacterial strains tested with the highest activity against gram-positive bacteria. Only propolis from the Sidi Bennour region demonstrated an antifungal activity. Conclusion: The study data show that Moroccan propolis extracts have a promising content of antioxidant and antimicrobial compounds that could be exploited to prevent certain diseases linked to oxidative stress and pathogenic infections.

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.

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.

Comprehensive analysis of different solvent extracts of Ferula communis L. fruit reveals phenolic compounds and their biological properties via in vitro and in silico assays.

Although giant fennel is recognized as a "superfood" rich in phytochemicals with antioxidant activity, research into the antibacterial properties of its fruits has been relatively limited, compared to studies involving the root and aerial parts of the plant. In this study, seven solvents-acetone, methanol, ethanol, ethyl acetate, chloroform, water, and hexane-were used to extract the chemical constituents of the fruit of giant fennel (Ferula communis), a species of flowering plant in the carrot family Apiaceae. Specific attributes of these extracts were investigated using in silico simulations and in vitro bioassays. High-performance liquid chromatography equipped with a diode-array detector (HPLC-DAD) identified 15 compounds in giant fennel extract, with p-coumaric acid, 3-hydroxybenzoic acid, sinapic acid, and syringic acid being dominant. Among the solvents tested, ethanol demonstrated superior antioxidant activity and phenolic and flavonoid contents. F. communis extracts showed advanced inhibition of gram-negative pathogens (Escherichia coli and Proteus mirabilis) and variable antifungal activity against tested strains. Molecular docking simulations assessed the antioxidative, antibacterial, and antifungal properties of F. communis, facilitating innovative therapeutic development through predicted compound-protein interactions. In conclusion, the results validate the ethnomedicinal use and potential of F. communis. This highlights its significance in natural product research and ethnopharmacology.

Synergistic antifungal mechanism of cinnamaldehyde and nonanal against Aspergillus flavus and its application in food preservation.

Aspergillus flavus colonization on agricultural products during preharvest and postharvest results in tremendous economic losses. Inspired by the synergistic antifungal effects of essential oils, the aims of this study were to explore the mechanism of combined cinnamaldehyde and nonanal (SCAN) against A. flavus and to evaluate the antifungal activity of SCAN loading into diatomite (DM). Shriveled mycelia were observed by scanning electron microscopy, especially in the SCAN treatment group. Calcofluor white staining, transmission electron microscopy, dichloro-dihydro-fluorescein diacetate staining and the inhibition of key enzymes in tricarboxylic acid cycle indicated that the antifungal mechanism of SCAN against A. flavus was related to the cell wall damage, reactive oxygen species accumulation and energy metabolism interruption. RNA sequencing revealed that some genes involved in antioxidation were upregulated, whereas genes responsible for cell wall biosynthesis, oxidative stress, cell cycle and spore development were significantly downregulated, supporting the occurrence of cellular apoptosis. In addition, compared with the control group, conidia production in 1.5 mg/mL DM/cinnamaldehyde, DM/nonanal and DM/SCAN groups were decreased by 27.16%, 48.22% and 76.66%, respectively, and the aflatoxin B1 (AFB1) contents decreased by 2.00%, 73.02% and 84.15%, respectively. These finding suggest that DM/SCAN complex has potential uses in food preservation.

Genome-wide analysis of in vivo-evolved Candida auris reveals multidrug-resistance mechanisms.

Candida auris, an emerging and multidrug-resistant fungal pathogen, has led to numerous outbreaks in China. While the resistance mechanisms against azole and amphotericin B have been studied, the development of drug resistance in this pathogen remains poorly understood, particularly in in vivo-generated drug-resistant strains. This study employed pathogen whole-genome sequencing to investigate the epidemiology and drug-resistance mutations of C. auris using 16 strains isolated from two patients. Identification was conducted through Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and antimicrobial susceptibilities were assessed using broth microdilution and Sensititre YeastOne YO10. Whole-genome sequencing revealed that all isolates belonged to the South Asian lineage, displaying genetic heterogeneity. Despite low genetic variability among patient isolates, notable mutations were identified, including Y132F in ERG11 and A585S in TAC1b, likely linked to increased fluconazole resistance. Strains from patient B also carried F214L in TAC1b, resulting in a consistent voriconazole minimum inhibitory concentration of 4 µg/mL across all isolates. Furthermore, a novel frameshift mutation in the SNG1 gene was observed in amphotericin B-resistant isolates compared to susceptible ones. Our findings suggest the potential transmission of C. auris and emphasize the need to explore variations related to antifungal resistance. This involves analyzing genomic mutations and karyotypes, especially in vivo, to compare sensitive and resistant strains. Further monitoring and validation efforts are crucial for a comprehensive understanding of the mechanisms of drug resistance in C. auris.

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.

Pan-azole- and multi-fungicide-resistant Aspergillus fumigatus is widespread in the United States.

Aspergillus fumigatus is an important global fungal pathogen of humans. Azole drugs are among the most effective treatments for A. fumigatus infection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistant A. fumigatus has been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance in A. fumigatus are tandem repeats (TR) in the cyp51A promoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistant A. fumigatus has been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistant A. fumigatus in the U.S., we collected isolates from agricultural sites in eight states and tested 202 isolates for sensitivity to azoles. We found azole-resistant A. fumigatus in agricultural environments in seven states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades, and TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow indicating recombination between clades highlighting the potential for azole-resistance to continue spreading in the U.S.IMPORTANCEAspergillus fumigatus is a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris, and compost. Azoles are the first line of defense antifungal drugs against A. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistant A. fumigatus has been a problem in Europe and Asia for 20 years and has recently been reported in patients in the United States (U.S.). Until this study, we did not know much about azole-resistant A. fumigatus in agricultural settings in the U.S. In this study, we isolated azole-resistant A. fumigatus from multiple states and compared it to isolates from around the world. We show that A. fumigatus which is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.

Exploring the Therapeutic Potential of Scorpion-Derived Css54 Peptide Against Candida albicans.

Candida albicans (C. albicans) is one of the most common opportunistic fungi worldwide, which is associated with a high mortality rate. Despite treatment, C. albicans remains the leading cause of life-threatening invasive infections. Consequently, antimicrobial peptides (AMPs) are potential alternatives as antifungal agents with excellent antifungal activity. We previously reported that Css54, found in the venom of Centrurodies suffusus suffusus (C. s. suffusus) showed antibacterial activity against zoonotic bacteria. However, the antifungal activity of Css54 has not yet been elucidated. The objective of this study was to identify the antifungal activity of Css54 against C. albicans and analyze its mechanism. Css54 showed high antifungal activity against C. albicans. Css54 also inhibited biofilm formation in fluconazole-resistant fungi. The antifungal mechanism of action of Css54 was investigated using membrane-related assays, including the membrane depolarization assay and analysis of the membrane integrity of C. albicans after treatment with Css54. Css54 induced reactive oxygen species (ROS) production in C. albicans, which affected its antifungal activity. Our results indicate that Css54 causes membrane damage in C. albicans, highlighting its value as a potential therapeutic agent against C. albicans infection.

Modes of action and potential as a peptide-based biofungicide of a plant defensin MtDef4.

Due to rapidly emerging resistance to single-site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation-tolerant MtDef4 variant was generated that bound to ß-glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation-tolerant variant as a peptide-based fungicide.

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.