Cathelicidin boosts the antifungal activity of neutrophils and improves prognosis during Aspergillus fumigatus keratitis.

Aspergillus fumigatus (A. fumigatus) is one of the common pathogens of fungal keratitis. Fungal growth and invasion cause excessive inflammation and corneal damage, leading to severe vision loss. Neutrophils are the primary infiltrating cells critical for fungal clearance. Cathelicidin [LL-37 in humans and cathelicidin-related antimicrobial peptide (CRAMP) in mice], a natural antimicrobial peptide, can directly inhibit the growth of many pathogens and regulate immune responses. However, the role of cathelicidin and its effect on neutrophils in A. fumigatus keratitis remain unclear. By establishing A. fumigatus keratitis mouse models, we found that cathelicidin was increased in A. fumigatus keratitis. It could reduce fungal loads, lower clinical scores, and improve corneal transparency. Restriction of CRAMP on fungal proliferation was largely counteracted in CD18-/- mice, in which neutrophils cannot migrate into infected sites. When WT neutrophils were transferred into CD18-/- mice, corneal fungal loads were distinctly reduced, indicating that neutrophils are vital for CRAMP-mediated resistance. Furthermore, cathelicidin promoted neutrophils to phagocytose and degrade conidia both in vitro and in vivo. CXC chemokine receptor 2 (CXCR2) was reported to be a functional receptor of LL-37 on neutrophils. CXCR2 antagonist SB225002 or phospholipase C (PLC) inhibitor U73122 weakened LL-37-induced phagocytosis. Meanwhile, LL-37 induced PLC γ phosphorylation, which was attenuated by SB225002. SB225002 or the autophagy inhibitors Bafilomycin-A1 and 3-Methyladenine weakened LL-37-induced degradation of conidia. Transmission electron microscopy (TEM) observed that LL-37 increased autophagosomes in Aspergillus-infected neutrophils. Consistently, LL-37 elevated autophagy-associated protein expressions (Beclin-1 and LC3-II), but this effect was weakened by SB225002. Collectively, cathelicidin reduces fungal loads and improves the prognosis of A. fumigatus keratitis. Both in vitro and in vivo, cathelicidin promotes neutrophils to phagocytose and degrade conidia. LL-37/CXCR2 activates PLC γ to amplify neutrophils' phagocytosis and induces autophagy to eliminate intracellular conidia.

N-3-Methylbutyl-benzisoselenazol-3(2H)-one Exerts Antifungal Activity In Vitro and in a Mouse Model of Vulvovaginal Candidiasis.

In the present work, we evaluated the antifungal activities of two novel ebselen analogs, N-allyl-benzisoselenazol-3(2H)-one (N-allyl-bs) and N-3-methylbutylbenzisoselenazol-3(2H)-one (N-3mb-bs). Colorimetric and turbidity assays were performed to determine the minimum inhibitory concentration (MIC) of these compounds in S1 (fluconazole-sensitive) and S2 (fluconazole-resistant) strains of C. albicans. N-3mb-bs was more active than the N-allyl-bs compound. It is noteworthy that the concentration of N-3mb-bs observed to inhibit fungal growth by 50% (18.2 µM) was similar to the concentration observed to inhibit the activity of the yeast plasma membrane H+-ATPase (Pma1p) by 50% (19.6 µM). We next implemented a mouse model of vulvovaginal candidiasis (VVC) using the S1 strain and examined the mouse and yeast proteins present in the vaginal lavage fluid using proteomics. The yeast proteins detected were predominately glycolytic enzymes or virulence factors associated with C. albicans while the mouse proteins present in the lavage fluid included eosinophil peroxidase, desmocollin-1, and gasdermin-A. We then utilized the N-3mb-bs compound (12.5 mg/kg) in the mouse VVC model and observed that it significantly reduced the vaginal fungal burden, histopathological changes in vagina tissue, and expression of myeloperoxidase (MPO). All in all, the present work has identified a potentially promising drug candidate for VVC treatment.

Application and antagonistic mechanisms of atoxigenic Aspergillus strains for the management of fungal plant diseases.

This review covers, for the first time, all methods based on the use of Aspergillus strains as biocontrol agents for the management of plant diseases caused by fungi and oomycetes. Atoxigenic Aspergillus strains have been screened in a variety of hosts, such as peanuts, maize kernels, and legumes, during the preharvest and postharvest stages. These strains have been screened against a wide range of pathogens, such as Fusarium, Phytophthora, and Pythium species, suggesting a broad applicability spectrum. The highest efficacies were generally observed when using non-toxigenic Aspergillus strains for the management of mycotoxin-producing Aspergillus strains. The modes of action included the synthesis of antifungal metabolites, such as kojic acid and volatile organic compounds (VOCs), secretion of hydrolytic enzymes, competition for space and nutrients, and induction of disease resistance. Aspergillus strains degraded Sclerotinia sclerotiorum sclerotia, showing high control efficacy against this pathogen. Collectively, although two Aspergillus strains have been commercialized for aflatoxin degradation, a new application of Aspergillus strains is emerging and needs to be optimized.

Evaluation of 1021Bp, a close relative of Pseudomonas eucalypticola, for potential of plant growth promotion, fungal pathogen suppression and boxwood blight control.

BACKGROUND: Pseudomonas eucalypticola, a new species of the P. fluorescens group that generates most Pseudomonas-based biocontrol agents, has not been found in any plants other than Eucalyptus dunnii leaves. Except for antagonism to the growth of a few fungi, its features in plant growth promotion and disease control have not been evaluated. Here, we identified a similar species of P. eucalypticola, 1021Bp, from endophyte cultures of healthy leaves of English boxwood (Buxus sempervirens 'Suffruticosa') and investigated its antifungal activity, plant growth promotion traits, and potential for boxwood blight control. RESULTS: Colorimetric or plate assays showed the properties of 1021Bp in nitrogen fixation, phosphate solubilization, and production of indole-3-acetic acid (IAA) and siderophores, as well as the growth suppression of all five plant fungal pathogens, including causal agents of widespread plant diseases, gray mold, and anthracnose. Boxwood plant leaves received 87.4% and 65.8% protection from infection when sprayed with cell-free cultural supernatant (CFS) but not the resuspended bacterial cells at 108-9/mL of 1021Bp at one and seven days before inoculation (dbi) with boxwood blight pathogen, Calonectria pseudonaviculata, at 5 × 104 spores/mL. They also received similarly high protection with the 1021Bp cell culture without separation of cells and CFS at 14 dbi (67.5%), suggesting a key role of 1021Bp metabolites in disease control. CONCLUSIONS: Given the features of plant growth and health and its similarity to P. eucalypticola with the P. fluorescens lineage, 1021Bp has great potential to be developed as a safe and environmentally friendly biofungicide and biofertilizer. However, its metabolites are the major contributors to 1021Bp activity for plant growth and health. Application with the bacterial cells alone, especially with nonionic surfactants, may result in poor performance unless survival conditions are present.

A Novel Secreted Protein of Fusarium oxysporum Promotes Infection by Inhibiting PR-5 Protein in Plant.

Fusarium oxysporum, an important soilborne fungal pathogen that causes serious Fusarium wilt disease, secretes diverse effectors during the infection. In this study, we identified a novel secreted cysteine-rich protein, FolSCP1, which contains unknown protein functional domain. Here, we characterized FolSCP1 as a secreted virulence factor that promotes the pathogen infection of host plants by inhibiting diverse plant defence responses. FolSCP1 interacted with the pathogenesis-related 5 (PR-5) protein SlPR5, a positive regulator of tomato plant immunity against multiple tomato pathogens, and effectively attenuated the antifungal activity of the tomato PR-5 protein. FoSCP1, a homologue of FolSCP1, was secreted by a F. oxysporum isolate from infected tobacco and targeted the tobacco PR-5 protein NtPR5 to suppress plant defence for further infection. In summary, our study revealed a fungal virulence strategy in which F. oxysporum secrete effectors that interfere with plant immunity by binding to the PR-5 protein of the host plant and inhibiting its biological activity, thereby promoting fungal infection.

Antifungal activity of Serratia plymuthica against the phytopathogenic fungus Alternariatenuissima.

The antifungal activity of Serratia plymuthica CCGG2742, a bacterial strain isolated from grapes berries skin, against a phytopathogenic fungus isolated from blueberries was evaluated in vitro and in vivo. In order to characterize the wild fungal isolate, phylogenetic analysis using concatenated DNA sequences from the RPB2 and TEF1 genes and of the ITS region was performed, allowing the identification of the fungal isolate that was called Alternaria tenuissima CC17. Hyphae morphology, mycelium ultrastructure, conidia and reproductive structures were in agreement with the phylogenetic analysis. The antifungal activity of the S. plymuthica strain was dependent on the composition of the culture medium. The greatest inhibition of mycelial growth of A. tenuissima CC17 by S. plymuthica CCGG2742 was observed on YTS medium, which lacks of an easily assimilable carbon source. Fungal growth medium supplemented with 50 % of bacterial supernatant decreased the conidia germination of A. tenuissima CC17 up to 32 %. Preventive applications of S. plymuthica CCGG2742 to blueberries and tomato leaves at conidia:bacteria ratio of 1:100, protected in 77.8 ± 4.6 % and 98.2 ± 0.6 % to blueberries and tomato leaves from infection caused by A. tenuissima CC17, respectively. To the best of our knowledge, this is the first report on the antifungal activity of S. plymuthica against A. tenuissima, which could be used as a biological control agent of plant diseases caused by this fungal species. In addition, the results of this work could be a starting point to attribute the real importance of A. tenuissima as a pathogen of blueberries in Chile, which until now had been considered almost exclusively to A. alternata. Likewise, this research could be relevant to start developing highly effective strategies based on S. plymuthica CCGG2742 for the control of this important phytopathogenic fungus.

GC-MS analysis and antifungal potential of flower extract of Acacia nilotica subsp. Indica against Macrophomina phaseolina.

Macrophomina phaseolina is a wide host ranged soil-borne fungal plant pathogen. It infects more than 500 host plant species belonging to 100 families. Many important oil-seed and leguminous crops are known to be attacked by this devastating plant pathogen. In the present study, antifungal potential of flowers of a leguminous tree Acacia nilotica subsp. indica, was assessed against this pathogen through bioassays guided fractionation. Initially, methanolic extracts of 1 %-5 % of leaf, flower, root-bark and stem-bark of the plant species under consideration were evaluated for their antifungal potential against the target pathogen. Among these, the best antifungal activity was shown by flower extract. The reduction in growth of the test fungal strain was 27-49 %, 4-40 % and 2-27 % due to flower, root-bark and leaf extracts, respectivey, over control. Flower extract was partitioned using n-hexane, chloroform, ethyl acetate and n-butanol as the solvents. Bioassays guided study of these fractions of methanolic extract of flower revealed that high antifungal potential was shown by n-hexane and chloroform fractions against M. phaseolina causing 26-53 % and 28-50 % decline in fungal biomass, respectively, as compared to that of control. GC-MS analysis of chloroform fraction revealed the presence of 27 compounds in this fraction. Among these cyclopentanol,-1-methyl (10.93 %) was the predominant compound followed by methyl, 4,4-dimethyl butanoate (7.04 %), 1-pentanol (6.80 %), 2-propanol, 1-cyclopropyl (6.11 %), 1H,imidazole-4-5-dihydro-2-methyl (5.93 %), trichloroethane (5.91 %), carbonic acid-ethyl hexyl ester (4.59 %), 1,4-butandiol,2,3-bis(methylene)- (4.54 %) and [S]-3,4-dimethyl pentanol (4.48 %).

Thamnostylum piriforme, a novel etiological agent of superficial mycosis.

Fungi are opportunistic eukaryotic entities often taking advantage of susceptibilities offered by a host due to its immunocompromised status, changed microbiome, or ruptured physical barriers and eventually cause infections. They either invade the skin superficially or are deep-seated. Superficial mycosis affects the skin, hair, and nails inhabiting the outermost layer, stratum corneum. In the present study, we report a case of superficial mycosis (onychomycosis in particular) in a 45-year-old immunocompetent man who was an ex-defense personnel and presently serving as a security guard at the University of Jammu, District Jammu, Jammu and Kashmir, India. The infection evolved 17 years ago and negatively affected the quality of life of the patient. For the identification of the causal agent, direct microscopy, cultural, micro-morphological, molecular characterization (ITS sequencing), and phylogenetic analysis were taken into account. A mucoralean fungal species, Thamnostylum piriforme, was isolated from the fingernails (left hand) of the investigated patient, which represents a new global report as the causal agent of superficial mycosis. In vitro antifungal susceptibility testing showed T. piriforme sensitivity to itraconazole, amphotericin B and ketoconazole while resistance to fluconazole. Careful selection of optimal therapy for fungal infection based primarily on correct identification and antifungal susceptibility testing could provide effective results during treatment against these opportunistic human fungal pathogens.

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.

Imidazolium-based ionic liquids disrupt saccharomyces cerevisiae cell membrane integrity.

Ionic liquids (ILs) are interesting chemical compounds that have a wide range of industrial and scientific applications. They have extraordinary properties, such as the tunability of many of their physical properties and, accordingly, their activities; and the ease of synthesis methods. Hence, they became important building blocks in catalysis, extraction, electrochemistry, analytics, biotechnology, etc. This study determined antifungal activities of various imidazolium-based ionic liquids against yeast Saccharomyces cerevisiae via minimum inhibitory concentration (MIC) estimation method. Increasing the length of the alkyl group attached to the imidazolium cation, enhanced the antifungal activity of the ILs, as well as their ability of the disruption of the cell membrane integrity. FTIR studies performed on the S. cerevisiae cells treated with the ILs revealed alterations in the biochemical composition of these cells. Interestingly, the alterations in fatty acid content occurred in parallel with the increase in the activity of the molecules upon the increase in the length of the attached alkyl group. This trend was confirmed by statistical analysis and machine learning methodology. The classification of antifungal activities based on FTIR spectra of S. cerevisiae cells yielded a prediction accuracy of 83%, indicating the pharmacy and medicine industries could benefit from machine learning methodology. Furthermore, synthesized ionic compounds exhibit significant potential for pharmaceutical and medical applications.

Ethanolic extract from fruiting bodies of Cordyceps militaris HL8 exhibits cytotoxic activities against cancer cells, skin pathogenic yeasts, and postharvest pathogen Penicillium digitatum.

Cordyceps militaris is a well-known medicinal mushroom in Asian countries. This edible fungus has been widely exploited for traditional medicine and functional food production. C. militaris is a heterothallic fungus that requires both the mating-type loci, MAT1-1 and MAT1-2, for fruiting body formation. However, recent studies also indicated two groups of C. militaris, including monokaryotic strains carrying only MAT1-1 in their genomes and heterokaryotic strains harboring both MAT1-1 and MAT1-2. These strain groups are able to produce fruiting bodies under suitable cultivating conditions. In previous work, we showed that monokaryotic strains are more stable than heterokaryotic strains in fruiting body formation through successive culturing generations. In this study, we report a high cordycepin-producing monokaryotic C. militaris strain (HL8) collected in Vietnam. This strain could form normal fruiting bodies with high biological efficiency and contain a cordycepin content of 14.43 mg/g lyophilized fruiting body biomass. The ethanol extraction of the HL8 fruiting bodies resulted in a crude extract with a cordycepin content of 69.15 mg/g. Assays of cytotoxic activity on six human cancer cell lines showed that the extract inhibited the growth of all these cell lines with the IC50 values of 6.41-11.51 µg/mL. Notably, the extract significantly reduced cell proliferation and promoted apoptosis of breast cancer cells. Furthermore, the extract also exhibited strong antifungal activity against Malassezia skin yeasts and the citrus postharvest pathogen Penicillium digitatum. Our work provides a promising monokaryotic C. militaris strain as a bioresource for medicine, cosmetics, and fruit preservation.

Synthesis and toxicological study of chitosan-pirul (Schinus molle L.) essential oil nanoparticles on Aspergillus flavus.

In recent years, the study of essential oils as antifungal alternatives and their encapsulation to increase their properties for greater effects has been tested. In this work, nanoparticles of chitosan-Schinus molle L. essential oil (CS-PEO-Np) with a size of 260 ± 31.1 nm were obtained by ionic gelation and evaluated in some growth phases of Aspergillus flavus, a toxigenic fungus. At a concentration of 250 µg/mL of CS-PEO-Np, the A. flavus mycelial growth was inhibited at 97.1% with respect to control, at 96 h of incubation; the germination and viability of spores were inhibited at 74.8 and 40%, respectively, after exposure to 500 µg/mL of these nanomaterials, at 12 h of incubation. The fluorescence images of stained spores with DAPI showed the affectations caused by nanoparticles in the cell membrane, vacuoles and vacuolar content, cell wall, and nucleic acids. For both nanoparticles, CS-Np and CS-PEO-Np, no mutagenic effect was observed in Salmonella Typhimurium; also, the phytotoxic assay showed low-to-moderate toxicity toward seeds, which was dependent on the nanoparticle's concentration. The acute toxicity of CS-PEO-Np to A. salina nauplii was considered low in comparison to CS-Np (control), which indicates that the incorporation of Schinus molle essential oil into nanoparticles of chitosan is a strategy to reduce the toxicity commonly associated with nanostructured materials. The nanoparticulated systems of CS-PEO-Np represent an effective and non-toxic alternative for the control of toxigenic fungi such as A. flavus by delaying the initial growth stage.

Identification of chitinase from Bacillus velezensis strain S161 and its antifungal activity against Penicillium digitatum.

Previous studies have demonstrated the presence of chitinase in Bacillus velezensis through extensive genomic sequencing and experimental analyses. However, the detailed structure, functional roles, and antifungal activity of these chitinases remain poorly characterized. In this study, genomic screening identified three genes-chiA, chiB, and lpmo10-associated with chitinase degradation in B. velezensis S161. These genes encode chitinases ChiA and ChiB, and lytic polysaccharide monooxygenase LPMO10. Both ChiA and ChiB contain two CBM50 binding domains and one catalytic domain, whereas LPMO10 includes a signal peptide and a single catalytic domain. The chitinases ChiA, its truncated variant ChiA2, and ChiB were heterologously expressed in Escherichia coli. The purified enzymes efficiently degraded colloidal chitin and inhibited the spore germination of Penicillium digitatum. Notably, even after losing one CBM50 domain, the resultant enzyme, consisting of the remaining CBM50 domain and the catalytic domain, maintained its colloidal chitin hydrolysis and antifungal activity, indicating commendable stability. These results underscore the role of B. velezensis chitinases in suppressing plant pathogenic fungi and provide a solid foundation for developing and applying chitinase-based biocontrol strategies.

Biochemical characterization and antifungal activity of a recombinant ß-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659.

ß-1,3-glucanases can degrade ß-1,3-glucoside bonds in ß-glucan which is the main cell-wall component of most of fungi, and have the crucial application potential in plant protection and food processing. Herein, a ß-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 composed of 333 amino acids with a predicted molecular mass of 36.6 kDa was expressed in Escherichia coli BL21, purified and characterized. The deduced amino acid sequence of FlGluA showed the high identity with the ß-1,3-glucanase belonging to glycoside hydrolase (GH) family 16. Enzymological characterization indicated FlGluA had the highest activity on zymosan A, with a specific activity of 3.87 U/mg, followed by curdlan (1.16 U/mg) and pachymaran (0.88 U/mg). It exhibited optimal catalytic activity at the pH 5.0 and 40 °C, and was stable when placed at 4 °C for 12 h in the range of pH 3.0-8.0 or at a temperature below 50 °C for 3 h. Its catalytic activity was enhanced by approximately 36 % in the presence of 1 mM Cr3+. The detection of thin-layer chromatography and mass spectrometry showed FlGluA hydrolyzed zymosan A mainly to glucose and disaccharide, and trace amounts of tetrasaccharide and pentasaccharide, however, it had no action on laminaribiose, indicating its endo-ß-1,3-glucanase activity. The mycelium growth of F. oxysporum treated by FlGluA was inhibited, with approximately 37 % of inhibition rate, revealing the potential antifungal activity of the enzyme. These results revealed the hydrolytic properties and biocontrol activity of FlGluA, laying a crucial foundation for its potential application in agriculture and industry.

Antifungal properties of abnormal cannabinoid derivatives: Disruption of biofilm formation and gene expression in Candida species.

Abnormal cannabinoids (including comp 3) are a class of synthetic lipid compounds with non-psychoactive properties and regioisomer configurations, but distinct from traditional cannabinoids since they do not interact with the established CB1 and CB2 receptors. Previous research showed the cardioprotective and anti-inflammatory potentials of comp 3 and more recently its antimicrobial effect on methicillin-resistant Staphylococcus aureus (MRSA). Given the escalating challenges posed by Candida infections and the rise of antifungal drug resistance, the exploration of novel therapeutic avenues is crucial. This study aimed to assess the anti-Candida properties of newly synthesized AbnCBD derivatives. AbnCBD derivatives were synthesized by acid catalysis-induced coupling and further derivatized. We evaluated the potential of the AbnCBD derivatives to inhibit the growth stages of various Candida species. By in vitro colorimetric assays and in vivo mice experiments, we have shown that AbnCBD derivatives induce differential inhibition of Candida growth. The AbnCBD derivatives, especially comp 3, comp 10, and comp 9 significantly reduced the growth of C. albicans, including FLC-resistant strains, and of C. tropicalis and C. parapsilosis but not of C auris compared to their controls (FLC and 0.5 % DMSO). Comp 3 also disrupted C. albicans biofilm formation and eradicated mature biofilms. Notably, other derivatives of AbnCBD disrupted the biofilm formation and maturation of C. albicans but did not affect yeast growth. In a murine model of VVC, comp 3 demonstrated significant fungal clearance and reduced C. albicans burden compared to vehicle and FLC controls. These findings highlight the potential of AbnCBDs as promising antifungal agents against Candida infections.

Amycolatopsis mongoliensis sp. nov., a novel actinobacterium with antifungal activity isolated from a coal mining site in Mongolia.

A novel filamentous actinobacterium designated strain 4-36T showing broad-spectrum antifungal activity was isolated from a coal mining site in Mongolia, and its taxonomic position was determined using polyphasic approach. Optimum growth occurred at 30 °C, pH 7.5 and in the absence of NaCl. Aerial and substrate mycelia were abundantly formed on agar media. The colour of aerial mycelium was white and diffusible pigments were not formed. Phylogenetic analyses based on 16S rRNA gene sequence showed that strain 4-36T formed a distinct clade within the genus Amycolatopsis. The 16S rRNA gene sequence similarity showed that the strain was mostly related to Amycolatopsis lexingtonensis DSM 44544T and Amycolatopsis rifamycinica DSM 46095T with 99.3 % sequence similarity. However, the highest digital DNA-DNA hybridization value to closest species was 44.1 %, and the highest average nucleotide identity value was 90.2 %, both of which were well below the species delineation thresholds. Chemotaxonomic properties were typical of the genus Amycolatopsis, as the major fatty acids were C15 : 0, iso-C16 : 0 and C16 : 0, the cell-wall diamino acid was meso-diaminopimelic acid, the quinone was MK-9(H4), and the main polar lipids were diphosphatidylglycerol, phosphatidylmethanolamine and phosphatidylethanolamine. The in silico prediction of chemotaxonomic markers was also carried out by phylogenetic analysis. The genome mining for biosynthetic gene clusters of secondary metabolites in strain 4-36T revealed the presence of 34 gene clusters involved in the production of polyketide synthase, nonribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptide, lanthipeptide, terpenes, siderophore and many other unknown clusters. Strain 4-36T showed broad antifungal activity against several filamentous fungi. The phenotypic, biochemical and chemotaxonomic properties indicated that the strain could be clearly distinguished from other species of Amycolatopsis, and thus the name Amycolatopsis mongoliensis sp. nov. is proposed accordingly (type strain, 4-36T=KCTC 39526T=JCM 30565T).

Synergistic activity between conventional antifungals and chalcone-derived compound against dermatophyte fungi and Candida spp.

The scarce antifungal arsenal, changes in the susceptibility profile of fungal agents, and lack of adherence to treatment have contributed to the increase of cases of dermatomycoses. In this context, new antimicrobial substances have gained importance. Chalcones are precursors of the flavonoid family that have multiple biological activities, have high tolerability by humans, and easy synthesis. In this study, we evaluated the in vitro antifungal activity, alone and in combination with conventional antifungal drugs, of the VS02-4'ethyl chalcone-derived compound against dermatophytes and Candida spp. Susceptibility testing was carried out by broth microdilution. Experiments for determination of the target of the compound on the fungal cell, time-kill kinetics, and toxicity tests in Galleria mellonella model were also performed. Combinatory effects were evaluated by the checkerboard method. Results showed high activity of the compound VS02-4'ethyl against dermatophytes (MIC of 7.81-31.25 µg/ml). The compound targeted the cell membrane, and the time-kill test showed the compound continues to exert gradual activity after 5 days on dermatophytes, but no significant activity on Candida. Low toxicity was observed at 250 mg/kg. Excellent results were observed in the combinatory test, where VS02-4'ethyl showed synergistic interactions with itraconazole, fluconazole, terbinafine, and griseofulvin, against all isolates tested. Although further investigation is needed, these results revealed the great potential of chalcone-derived compounds against fungal infections for which treatments are long and laborious.

In vitro antifungal activity analysis of Streptomyces sp. strain 196 against Candida albicans and Aspergillus flavus.

Numerous bioactive compounds have been reported to be produced by the members of the genus Streptomyces. During our previous studies, Streptomyces sp. strain 196 was tested for its antimicrobial activity, and bioactive compounds produced by this strain were characterized LC-MS and 1H NMR. To examine the antifungal potential of strain 196 is the goal of the current investigation. Present investigation is focused on exploring antifungal activity of extract of strain 196 (196EA) on membrane disruption potential against two fungi Candida albicans ATCC 90028 and Aspergillus flavus ITCC 5599. Results revealed that the MIC value is higher for A. flavus than for C. albicans which is 450 µg/mL and 250 µg/mL, respectively. Disc diffusion and spot assay also correspond to the values of the MIC for their respective pathogen. In growth curve analysis, lag and log phase are significantly affected by the extract of strain 196. The effects of extract from strain 196 on plasma membrane disruption of Candida albicans and Aspergillus flavus were analyzed in terms of ergosterol quantification assay, cellular leakage, proton efflux measurement (PM-ATPase), plasma membrane integrity assay (PI), and DNA damage assay (DAPI). Results shown that the extract of strain 196 has the potential to inhibit the cell membrane of the both pathogenic fungi which was further confirmed with the help of scanning electron microscopic (SEM) studies.

Investigating the antifungal potential of genetically modified hybrid chitinase enzymes derived from Bacillus subtilis and Serratia marcescens.

Chitinases are glycosyl hydrolase enzymes that break down chitin, an integral component of fungal cell walls. Bacteria such as Bacillus subtilis and Serratia marcescens produce chitinases with antifungal properties. In this study, we aimed to generate hybrid chitinase enzymes with enhanced antifungal activity by combining functional domains from native chitinases produced by B. subtilis and S. marcescens. Chitinase genes were cloned from both bacteria and fused together using overlap extension PCR. The hybrid constructs were expressed in E. coli and the recombinant enzymes purified. Gel electrophoresis and computational analysis confirmed the molecular weights and isoelectric points of the hybrid chitinases were intermediate between the parental enzymes. Antifungal assays demonstrated that the hybrid chitinases inhibited growth of the fungus Fusarium oxysporum significantly more than the native enzymes and also showed fungicidal activity against Candida albicans, Alternaria solani, and Rhizoctonia solani. The results indicate that hybrid bacterial chitinases are a promising approach to engineer novel antifungal proteins. This study provides insight into structure-function relationships of chitinases and strategies for generating biotherapeutics with enhanced bioactive properties. These hybrid chitinases result in a more potent and versatile antifungal agent.

Synergistic potential of essential oil combinations against Microsporum, Trichophyton, and Epidermophyton.

Dermatophyte infections globally account for 20 to 25% of fungal infections. Dermatophytes have begun exhibiting antifungal drug resistance, making it challenging to treat this particular infection. Essential oils could be used as alternative solutions as they have been used for a long period to treat different infections. The research has demonstrated the antifungal efficacy of cinnamon, clove, lemongrass, tea tree, thyme, and garlic essential oils, and the impact of their combinations was assayed against Microsporum canis, Trichophyton tonsurans, T. violaceum, T. verrucosum, and Epidermophyton floccosum. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) was used to identify the most prevalent M. canis. The accession number of M. canis was obtained as ON007275. All tested essential oils exhibited antidermatophytic action except garlic. A synergistic effect was attained by cinnamon + clove, cinnamon + lemongrass, clove + lemongrass, clove + tea tree, and thyme + tea tree combinations. Concerning antifungal activity, M. canis was the most susceptible dermatophytic species, except in the case of thyme T. violaceum, which was the most susceptible dermatophytic species. The maximum inhibition was recorded in the cases of cinnamon and cinnamon + lemongrass combination against M. canis. The least minimum inhibitory concentrations were attained by cinnamon and clove against M. canis, cinnamon + clove against M. canis and T. violaceum, and cinnamon + lemongrass against M. canis, T. violaceum, T. verrucosum, and E. floccosum. The least minimum fungicidal concentration showed by cinnamon against M. canis, cinnamon + clove against M. canis and T. violaceum, cinnamon + lemongrass against M. canis, T. violaceum, T. verrucosum, and E. floccosum, and clove + lemongrass against M. canis.