Effect of Topical Antifungal Luliconazole on Hyphal Morphology of Trichophyton mentagrophytes Grown on in vitro Onychomycosis Model.

Luliconazole, recently launched in Japan, is a novel topical imidazole antifungal agent for the treatment of onychomycosis. Using in vitro onychomycosis model, the effect of luliconazole on the morphology of the growing hyphae of Trichophyton mentagrophytes was investigated by scanning electron microscopy (SEM). The model was produced by placing human nail pieces on an agar medium seeded with conidia of T. mentagrophytes. After incubating the agar medium for 3 days, luliconazole was applied to the surface of the nail in which hyphal growth was recognized, then cultured for up to 24 h. The initial change after treatment with the drug was the formation of fine wrinkles on the surface of the hyphae, eventually, the hyphae were flattened, and after that, no hyphal growth was observed. On the other hand, when the nails were pretreated with luliconazole for 1 h, no hyphal growth was observed even after culturing for 24 h. This study suggests that luliconazole has a strong antifungal activity by inhibiting the ability of fungi to grow and the drug has both excellent nail permeation and retention properties.

Novel Pathogenic Mucorales Identified Using the Silkworm Infection Model.

Mucormycosis, a rare but highly fatal infection, is caused by fungi of the order Mucorales. Due to their ubiquitous nature, reduced susceptibility to antifungals, acid tolerance, and ability to infect immunocompromised patients through rapid dissemination, these fungi have been frequently reported to infect the COVID-19 patients. In order to develop strategies to overcome mucormycosis, it is essential to understand and identify novel Mucorales present in the environment. In this study, we report the identification of four novel pathogenic Mucorales using the silkworm (Bombyx mori) model. The strains' phylogeny was analyzed using the genome sequence of the large subunit ribosomal ribonucleic acid (LSU rRNA) and the internal transcribed spacer (ITS) region, where strains 1-3, 5-3, and S286-1101 claded with Mucor orantomantidis, and strain 827-14 claded with Backusella lamprospora. All the strains had a cold-sensitive phenotype with their inability to grow prominently at 4 °C. Mucor sp. 1-3 and 5-3 were characterized by their filamentous and yeast-like growth under aerobic and anaerobic conditions, respectively. The yeast colonies of Mucor sp. 5-3 had multipolar budding cells often observed with cleaved cell surfaces under a scanning electron microscope. We further found that these strains were able to kill immunocompromised mice suggesting their pathogenicity to mammals. Our study established an invertebrate model-based screening system to identify novel pathogenic Mucorales from the natural environment and provided a clue towards the rapid increase in COVID-19 related mucormycosis.

Seven years of progress in determining fungal diversity and characterization of fungi isolated from the Japanese Experiment Module KIBO, International Space Station.

The International Space Station (ISS) is a closed facility that orbits the earth carrying not only its crew but also microorganisms. We have participated in microbiota analysis projects for the Japanese Experiment Module KIBO (ISS; operations nomenclature: Microbe-I, II, III, and IV) and were in charge of fungal screening. The interior of KIBO was sampled using swabs and microbe detection sheets (MDSs) for fungal detection. The dominant genera obtained by culture were Aspergillus and Penicillium. DNA analyses of the fungal biota using a clone library showed that KIBO was dominated by Malassezia, a fungal inhabitant of human skin. Three fungal species, Aspergillus sydowii, Penicillium palitans, and Rhodotorula mucilaginosa, which grew under microgravity in KIBO were observed under a field emission-scanning electron microscope on the ground. No novel phenotypic characteristics were noted. The results of antifungal susceptibility testing of all isolates did not differ significantly from previous reports of corresponding fungi. In Microbe-I (August 2009), MDSs were culture negative, while in the next stages the CFU of MDSs were 10 for Microbe-II (February 2011), 24 for Microbe-III (October 2012), and 151 for Microbe-IV (February 2015). These results indicated that fungi inside KIBO are increasing and expanding over time, and therefore continuous surveillance is crucial.

Antifungal activity and mechanism of action of Ou-gon (Scutellaria root extract) components against pathogenic fungi.

Ou-gon, an extract from Scutellaria baicalensis Georgi root, has been shown to exhibit pronounced antifungal activity. The present study aimed to identify antifungal components of Ou-gon and to determine their mechanism of action against pathogenic fungi. Antifungal activity was assessed by the microbroth dilution method using four common human pathogenic fungi, Trichophyton rubrum, Trichophyton mentagrophytes, Aspergillus fumigatus, and Candida albicans. Components of crude Ou-gon extract were separated by reversed-phase high-performance liquid chromatography. Active antifungal components were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Terminal deoxynucleotidyl transferase dUTP nick end-labelling assay, SYTOX® green uptake assay, determination of intracellular reactive oxygen species and mitochondrial membrane potential as well as microscopy (confocal laser microscopy, scanning and transmission electron microscopy) were used to probe the mode of action. Two components with potent antifungal activity, baicalein and wogonin, were identified in Ou-gon. Baicalein showed potent antifungal activity against the four fungi tested. Wogonin displayed antifungal activity against all four fungi except C. albicans. The components are considered to induce apoptosis-like programmed cell death via hyperproduction of reactive oxygen species. This study enhances our understanding of the antifungal activity of Kampo medicine, and may contribute to the development of new and safe antifungal therapeutics.

Lactobacillus paraplantarum 11-1 Isolated from Rice Bran Pickles Activated Innate Immunity and Improved Survival in a Silkworm Bacterial Infection Model.

Lactic acid bacteria (LAB) have high immune system-stimulating activity and are considered beneficial for human health as probiotics in the gut. The innate immune system is highly conserved between mammals and insects. Microbe-associated molecular patterns (e.g., peptidoglycan and ß-glucan) induce cytokine maturation, which, in silkworm larvae, leads to muscle contraction. The purpose of this study is to find a novel probiotic by using silkworm muscle contraction assay. In the present study, we isolated LAB derived from rice bran pickles. We selected highly active LAB to activate the innate immune system of the silkworm, which was assayed based on silkworm muscle contraction. Of various LAB, L. paraplantarum 11-1 strongly stimulated innate immunity in the silkworm, leading to stronger silkworm contraction than a dairy-based LAB. Silkworms fed a diet containing L. paraplantarum 11-1 exhibited tolerance against the pathogenicity of Pseudomonas aeruginosa. These findings suggest that L. paraplantarum 11-1 could be a useful probiotic for activating innate immunity.

Lactic acid bacteria of the Leuconostoc genus with high innate immunity-stimulating activity.

We screened lactic acid bacteria that exhibited high innate immunity-stimulating activity by monitoring muscle contraction activity in silkworms. Heat-treated fractions of lactic acid bacteria, Leuconostoc carnosum #7-2, Leuconostoc gelidum #4-2, and Leuconostoc mesenteroides 8/11-3, had high (250-460 units/mg) innate immunity-stimulating activity. These lactic acid bacteria proliferated in milk to concentrations of 1 × 106 colony forming unit/mL. The present findings suggest that the silkworm muscle contraction assay is useful for screening lactic acid bacteria with high innate immunity-stimulating activity, and that the assay can be used for the production of fermented foods made from milk.

Morphological Effect of the New Antifungal Agent ME1111 on Hyphal Growth of Trichophyton mentagrophytes, Determined by Scanning and Transmission Electron Microscopy.

The effects of ME1111, a novel antifungal agent, on the hyphal morphology and ultrastructure of Trichophyton mentagrophytes were investigated by using scanning and transmission electron microscopy. Structural changes, such as pit formation and/or depression of the cell surface, and degeneration of intracellular organelles and plasmolysis were observed after treatment with ME1111. Our results suggest that the inhibition of energy production by ME1111 affects the integrity and function of cellular membranes, leading to fungal cell death.

Genetic and Phenotypic analyses of Calcineurin A subunit in Arthroderma vanbreuseghemii.

Calcineurin is a serine/threonine protein phosphatase that consists of catalytic (calcineurin A) and regulatory (calcineurin B) subunits. The conserved protein plays important roles in various biological processes. Drug combination of fluconazole and the calcineurin inhibitor (FK506) showed synergistic effects against dermatophytes. In the current study, we identified the calcineurin A homologous gene (TmcanA) in the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes). Knockdown mutants were produced from A. vanbreuseghemii, resulting in a defection in growth properties in accordance with dose of the suppressing reagent. The TmcanA gene restored the ability of calcineurin A-deficient Cryptococcus neoformans strain to grow at elevated temperatures. Repression of TmcanA at 37°C resulted in severely stunted growth, suggesting that this protein plays a role in tolerance to elevated temperatures. In addition, TMCANA showed an interaction with high osmolarity glycerol (HOG) signalling pathway by governing the secretion of a secondary metabolite. Moreover, expression of the hydrophobin A gene (TmHF) decreased significantly under the TmcanA-repressive condition, suggesting that TMCANA is involved in its regulation. In conclusion, calcineurin A is a multifunctional gene that is involved in the regulation of several biological processes and therefore is worth being considered as a drug target for treatment of dermatophytoses.

Mechanism of action of efinaconazole, a novel triazole antifungal agent.

The mechanism of action of efinaconazole, a new triazole antifungal, was investigated with Trichophyton mentagrophytes and Candida albicans. Efinaconazole dose-dependently decreased ergosterol production and accumulated 4,4-dimethylsterols and 4α-methylsterols at concentrations below its MICs. Efinaconazole induced morphological and ultrastructural changes in T. mentagrophytes hyphae that became more prominent with increasing drug concentrations. In conclusion, the primary mechanism of action of efinaconazole is blockage of ergosterol biosynthesis, presumably through sterol 14α-demethylase inhibition, leading to secondary degenerative changes.

The effect of cinnamaldehyde on the growth and the morphology of Candida albicans.

We examined the viability and morphology of Candida albicans under experimental conditions after treatment with varying concentrations of cinnamaldehyde, the major component of cassia (Cinnamomum cassia), using XTT assay, fluorescent microscopy, scanning electron microscopy, and thin-section electron microscopy. At 10 µg/ml level, cinnamaldehyde inhibited mycelial growth, but did not affect the growth of yeast cells, metabolic activity, cell shape, or the ultrastructure of the cells. At 40 µg/ml level, cinnamaldehyde showed fungicidal activity accompanied by alteration of the membrane and interior of Candida cells. These findings indicate that cinnamaldehyde has both fungistatic and fungicidal activities against C. albicans and affects the structure of the cells.

Effect of cinnamaldehyde on hyphal growth of C. albicans under various treatment conditions.

This study investigated the effects of cinnamaldehyde in combatting the hyphal growth of Candida albicans under varying concentrations, treatment times, and temperatures to determine the potential benefits of applying this substance to anti-Candida foods or gargles. From the results of pretreatment with cinnamaldehyde against Candida hyphae, we found that its inhibitory activity seemed to be strengthened in parallel with prolonged pretreatment time and a rise in temperature, and that pretreatment of 2,000 µg/ml for only 1 minute significantly inhibited the hyphal growth of C. albicans. We also demonstrated by XTT assay that pretreatment with cinnamaldehyde affected the metabolic activity of Candida hyphal cells. These findings suggest that a warm drink or mouthwash containing cinnamaldehyde might be a candidate as a prophylactic or therapeutic tool against oral Candida infection.

Development of a tightly regulatable copper-mediated gene switch system in dermatophytes.

Targeted gene deletion is now available for molecular genetic research of dermatophytes, and the physiological roles of several genes have been elucidated. However, this method cannot be applied to essential genes, which can be potential drug targets. To overcome this limitation, we have developed a conditional gene knockdown system using a copper-responsive promoter. The promoter sequence of the copper transporter gene CTR4 (P(CTR4)) and that of the copper efflux pump gene CRP1 (P(CRP1)) derived from Trichophyton rubrum were examined for their response to copper in Arthroderma vanbreuseghemii. P(CTR4) was demonstrated to repress expression of a reporter gene in the presence of copper, while the activity of P(CRP1) was induced by addition of copper. Importantly, P(CTR4) regulated the gene expression more tightly. Furthermore, when P(CTR4) was applied to regulate the expression of the endogenous genes ERG1 and TRP5, their conditional mutants exhibited decreased growth activity under the repressive conditions. These results suggest that the P(CTR4)-based gene regulation system represents a powerful tool for identification and characterization of a broad range of genes, including essential genes, in dermatophytes.

Phenotypic characterization of Aspergillus niger and Candida albicans grown under simulated microgravity using a three-dimensional clinostat.

The living and working environments of spacecraft become progressively contaminated by a number of microorganisms. A large number of microorganisms, including pathogenic microorganisms, some of which are fungi, have been found in the cabins of space stations. However, it is not known how the characteristics of microorganisms change in the space environment. To predict how a microgravity environment might affect fungi, and thus how their characteristics could change on board spacecraft, strains of the pathogenic fungi Aspergillus niger and Candida albicans were subjected to on-ground tests in a simulated microgravity environment produced by a three-dimensional (3D) clinostat. These fungi were incubated and cultured in a 3D clinostat in a simulated microgravity environment. No positive or negative differences in morphology, asexual reproductive capability, or susceptibility to antifungal agents were observed in cultures grown under simulated microgravity compared to those grown in normal earth gravity (1 G). These results strongly suggest that a microgravity environment, such as that on board spacecraft, allows growth of potentially pathogenic fungi that can contaminate the living environment for astronauts in spacecraft in the same way as they contaminate residential areas on earth. They also suggest that these organisms pose a similar risk of opportunistic infections or allergies in astronauts as they do in people with compromised immunity on the ground and that treatment of fungal infections in space could be the same as on earth.

Microbe-I: fungal biota analyses of the Japanese experimental module KIBO of the International Space Station before launch and after being in orbit for about 460 days.

In addition to the crew, microbes also find their way aboard the International Space Station (ISS). Therefore, microbial monitoring is necessary for the health and safety of the crew and for general maintenance of the facilities of this station. Samples were collected from three sites in the Japanese experimental module KIBO on the ISS (air diffuser, handrail, and surfaces) for analysis of fungal biota approximately 1 year after this module had docked with the ISS. Samples taken from KIBO before launch and from our laboratory were used as controls. In the case of KIBO, both microbe detection sheet (MDS) and swab culture tests of orbital samples were negative. The MDS were also examined by field emission-scanning electron microscopy; no microbial structures were detected. However, fungal DNAs were detected by real-time PCR and analyzed by the clone library method; Alternaria sp. and Malassezia spp. were the dominant species before launch and in space, respectively. The dominant species found in specimens from the air conditioner diffuser, lab bench, door push panel, and facility surfaces on our laboratory (ground controls) were Inonotus sp., Cladosporium sp., Malassezia spp., and Pezicula sp., respectively. The fungi in the KIBO were probably derived from contamination due to humans, while those in our laboratory came from the environment (e.g., the soil). In conclusion, the cleanliness in KIBO was equivalent to that in a clean room environment on the ground.