Enhanced Solubility and Bioavailability of Clotrimazole in Aqueous Solutions with Hydrophobized Hyperbranched Polyglycidol for Improved Antifungal Activity.

The poor solubility of clotrimazole in the aqueous medium and the uncontrolled removal of the drug-loaded suppository content limit its effectiveness in the treatment of vulvovaginal candidiasis. We present here the aqueous formulations of clotrimazole in the form of non-Newtonian structured fluids, i.e., Bingham plastic or pseudoplastic fluids constructed of hyperbranched polyglycidol, HbPGL, with a hydrophobized core with aryl groups such as phenyl or biphenyl. The amphiphilic constructs were obtained by the modification of linear units containing monohydroxyl groups with benzoyl chloride, phenyl isocyanate, and biphenyl isocyanate, while the terminal 1,2-diol groups in the shell were protected during the modification step, followed by their deprotection. The encapsulation of clotrimazole within internally hydrophobized HbPGLs using a solvent evaporation method followed by water addition resulted in structured fluids formation. Detailed Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) analyses performed for aryl-HbPGLs with clotrimazole revealed the difference in drug compatibility among polymers. Clotrimazole in biphenyl-enriched HbPGL, unlike phenyl derivatives, was molecularly distributed in both the dry and the hydrated states, resulting in transparent formulations. The shear-thinning properties of the obtained fluid formulations make them injectable and thus suitable for the intravaginal application. Permeability tests performed with the usage of the Franz diffusion cell showed a 5-fold increase in the permeability constant of clotrimazole compared to drugs loaded in a commercially available disposable tablet and a 50-fold increase of permeability in comparison to the aqueous suspension of clotrimazole. Furthermore, the biphenyl-modified HbPGL-based drug liquid showed enhanced antifungal activity against both Candida albicans and Candida glabrata that was retained for up to 7 days, in contrast to the phenyl-HbPGL derivatives and the tablet. With their simple formulation, convenient clotrimazole/biphenyl-HbPGL formulation strategy, rheological properties, and enhanced antifungal properties, these systems are potential antifungal therapeutics for gynecological applications. This study points in the synthetic direction of improving the solubility of poorly water-soluble aryl-enriched pharmaceuticals.

Evaluation of the antidermatophytic activity of potassium salts of N-acylhydrazinecarbodithioates and their aminotriazole-thione derivatives.

Nowadays, dermatophyte infections are relatively easy to cure, especially since the introduction of orally administered antifungals such as terbinafine and itraconazole. However, these drugs may cause side effects due to liver damage or their interactions with other therapeutics. Hence, the search for new effective chemotherapeutics showing antidermatophyte activity seems to be the urge of the moment. Potassium salts of N-acylhydrazinecarbodithioates are used commonly as precursors for the synthesis of biologically active compounds. Keeping that in mind, the activity of a series of five potassium N-acylhydrazinecarbodithioates (1a-e) and their aminotriazole-thione derivatives (2a-e) was evaluated against a set of pathogenic, keratinolytic fungi, such as Trichophyton ssp., Microsporum ssp. and Chrysosporium keratinophilum, but also against some Gram-positive and Gram-negative bacteria. All tested compounds were found non-toxic for L-929 and HeLa cells, with the IC30 and IC50 values assessed in the MTT assay above 128 mg/L. The compound 5-amino-3-(naphtalene-1-yl)-4,5-dihydro-1H-1,2,4-triazole-5-thione (2d) was found active against all fungal strains tested. Scanning Electron Microscopy (SEM) revealed inhibition of mycelium development of Trichophyton rubrum cultivated on nail fragments and treated with 2d 24 h after infection with fungal spores. Transmission Electron Microscopy (TEM) observation of mycelium treated with 2d showed ultrastructural changes in the morphology of germinated spores. Finally, the RNA-seq analysis indicated that a broad spectrum of genes responded to stress induced by the 2d compound. In conclusion, the results confirm the potential of N-acylhydrazinecarbodithioate derivatives for future use as promising leads for new antidermatophyte agents development.

Dispersal of Aphanoascus keratinophilus by the rook Corvus frugilegus during breeding in East Poland.

The process of dispersal of the potentially disease-causing, geophilic and keratinolytic fungal strain Aphanoascus keratinophilus (the perfect, sexual stage of Chrysosporium keratinophilum) by the rook Corvus frugilegus was studied. The source of A. keratinophilus strains was pellets of the rook, thus far not considered a carrier of this particular opportunistic pathogen. Pellets collected from breeding colonies of rooks were analysed in terms of the occurrence of keratinolytic fungi with the application of the native keratin bait method. Among the 83 rook pellets analysed, 24 (29%) were infected by keratinophilic fungi. Pure cultures of the fungi were identified to species based on traditional morphological features. Traditional mycological identification was verified by the PCR-RFLP molecular identification method as well as DNA sequencing. The obtained results showed the presence of 90 Aphanoascus keratinophilus strains, 6 Chrysosporium tropicum strains, and 3 Chrysosporium pannicola strains. The PCR melting profile (PCR-MP) method was used to identify intraspecies variations of the 90 analysed A. keratinophilus strains. The dispersal of genotypes and possible pathways of A. keratinophilus dispersal and infection via rook pellets were analysed.

Metabolomic analysis of Trichophyton rubrum and Microsporum canis during keratin degradation.

Keratin is important and needed for the growth of dermatophytes in the host tissue. In turn, the ability to invade keratinised tissues is defined as a pivotal virulence attribute of this group of medically important fungi. The host-dermatophyte interaction is accompanied by an adaptation of fungal metabolism that allows them to adhere to the host tissue as well as utilize the available nutrients necessary for their survival and growth. Dermatophyte infections pose a significant epidemiological and clinical problem. Trichophyton rubrum is the most common anthropophilic dermatophyte worldwide and its typical infection areas include skin of hands or feet and nail plate. In turn, Microsporum canis is a zoophilic pathogen, and mostly well known for ringworm in pets, it is also known to infect humans. The aim of the study was to compare the intracellular metabolite content in the T. rubrum and M. canis during keratin degradation using liquid chromatography system coupled with tandem mass spectrometer (LC-MS/MS). The metabolite "fingerprints" revealed compounds associated with amino acids metabolism, carbohydrate metabolism related to the glycolysis and the tricarboxylic acid cycle (TCA), as well as nucleotide and energy metabolism. The metabolites such as kynurenic acid, L-alanine and cysteine in case of T. rubrum as well as cysteine and riboflavin in case of M. canis were detected only during keratin degradation what may suggest that these compounds may play a key role in the interactions of T. rubrum and M. canis with the host tissue. The metabolomic results were completed by qPCR gene expression assay. Our findings suggest that metabolomic analysis of T. rubrum and M. canis growing in culture media that mimic the dermatophyte infection could allow the understanding of processes involved in the pathogenesis of dermatophytes.

Selection and validation of reference genes for qPCR in the human dermatophyte Trichophyton rubrum exposed to different carbon sources which promote adhesion-inducing conditions.

OBJECTIVE: The present study aimed to identify reference genes for qPCR analysis of T. rubrum growth in culture media which promote adhesion-inducing conditions to the host tissue. METHODS: We investigated the suitability of six candidate reference genes: ß-act, ß-tub, ef1-α, gapdh, sdha and rpl2 in reference strain of Trichophyton rubrum in response to different environmental stimuli. The stability of these genes was determined by NormFinder, geNorm and BestKeeper software. RESULTS: Our data obtained from the three algorithms revealed that mRNA expression levels of two candidate reference genes, ef1-α and ß-tub, remained the most stable in response to different carbon sources, while different sample sets had their own most stable reference genes, highlighting the importance of the choice of internal controls in qPCR experiments. We then checked the stability of ef1-α and ß-tub reference genes expression in different T. rubrum strains, suggesting that these two genes are reliable for normalisation of qPCR. Finally, we validated the suitability of selected reference genes as internal controls for target gene (SUB3) using the 2-ΔΔCt method. The best result indicating an increase of SUB3 transcript of T. rubrum was found when the two the most stable reference (ef1-α and ß-tub ) genes were used, as revealed by all three algorithms. CONCLUSIONS: We recommend the use of ef1-α and ß-tub as reference genes for qPCR analysis of target gene expression in T. rubrum exposed to different carbon sources which promote adhesion-inducing conditions.

A new molecular marker for species-specific identification of Microsporum canis.

Species identification of dermatophytes by conventional mycological methods based on macro- and microscopy analysis is time-consuming and has a lot of limitations such as slow fungal growth or low specificity. Thus, there is a need for the development of molecular methods that would provide reliable and prompt identification of this group of medically important fungi. The are many reports in the literature concerning PCR identification of dermatophyte species, but still, there are not many PCR assays for the separate detection of members of the genera Microsporum, especially Microsporum canis (zoophilic species) and Microsporum audouinii (anthropophilic species). The correct distinction of these species is important to determine the source of infection to implement the appropriate action to eliminate the path of infection transmission. In this paper, we present such a PCR-based method targeting velB gene that uses a set of two primers-Mc-VelB-F (5'-CTTCCCCACCCGCAACATC-3') and Mc-VelB-R (5'-TGTGGCTGCACCTGAGAGTGG-3'). The amplified fragment is specific due to the presence of (CAGCAC)8 microsatellite sequence only in the velB gene of M. canis. DNA from 153 fungal samples was used in PCR assay followed by electrophoretic analysis. The specificity of the designed set of primers was also confirmed using the online BLAST-Primer tool. The positive results were observed only in the case of M. canis isolates, and no positive results were obtained neither for other dermatophytes and non-dermatophyte fungi nor for other Eukaryotes, including the human genome sequence, as well as the representatives of bacterial and viral taxa. The developed PCR assay using the proposed Mc-VelB-F and Mc-velB-R primers can be included in the algorithm of M. canis detection in animals and humans.

Reference genes for accurate evaluation of expression levels in Trichophyton interdigitale grown under different carbon sources, pH levels and phosphate levels.

Tinea pedis is a type of dermatophytosis caused by anthropophilic keratinolytic fungi such as Trichophyton interdigitale. Quantitative reverse transcription PCR (RT-qPCR) is a reliable and reproducible technique for measuring changes in target gene expression across various biological conditions. A crucial aspect of accurate normalization is the choice of appropriate internal controls. To identify reference genes for accurate evaluation of expression levels in T. interdigitale, the transcription levels of eight candidate reference genes (adp-rf, ß-act, ef1-α, gapdh, psm1, sdha, rpl2 and ubc) and one target gene (Tri m4) were analysed by RT-qPCR after growing the dermatophyte under different environmental conditions. The results obtained from expression stability evaluations with NormFinder, geNorm, BestKeeper, and RefFinder software demonstrated that adp-rf and psm1 were the most stable internal control genes across all experimental conditions. The present study constitutes the first report of the identification and validation of reference genes for RT-qPCR normalization for T. interdigitale grown under different environmental conditions resembling the conditions encountered by fungi during invasion of skin.

Selection and validation of reference genes for qRT-PCR analysis of gene expression in Microsporum canis growing under different adhesion-inducing conditions.

Dermatophytes are the group of filamentous fungi infecting keratinized structures such as skin, hair, and nails. Knowledge about genes and molecular mechanisms responsible for pathogenicity, as well as other biological properties of Microsporum canis is still relatively poor. The qRT-PCR is a reliable technique for quantifying gene expression across various biological processes, and choosing a set of suitable reference genes to normalize the expression data is a crucial step of this technique. We investigated the suitability of nine candidate reference genes: ß-act, ß-tub, adp-rf, ef1-α, sdha, rpl2, mbp1, psm1, and rGTPa for gene expression analysis in the dermatophyte M. canis in response to different carbon sources, phosphate levels, and pH shifts - factors that are extremely important and necessary for growth of dermatophyte in the host tissue. The transcription stability of these genes was evaluated using NormFinder, geNorm, BestKeeper, and RefFinder software. Regarding expression stability, mbp1, ß-act, and sdha were the most stable housekeeping genes which we recommend for future qRT-PCR studies on M. canis strains. To the best of our knowledge this is the first study on selection and validation of reference genes for qRT-PCR data normalization in M. canis growth in culture media which promote adhesion-inducing conditions.

Bioinformatic survey of ABC transporters in dermatophytes.

ATP binding cassette (ABC) transporters constitute a very large and ubiquitous superfamily of membrane proteins. They are responsible for ATP hydrolysis driven translocation of countless substrates. Being a very old and diverse group of proteins present in all organisms they share a common feature, which is the presence of an evolutionary conservative nucleotide binding domain (NBD)--the engine that drives the transport. Another common domain is a transmembrane domain (TMD) which consists of several membrane-spanning helices. This part of protein is substrate-specific, thus it is much more variable. ABC transporters are known for driving drug efflux in many pathogens and cancer cells, therefore they are the subject of extensive studies. There are many examples of conferring a drug resistance phenotype in fungal pathogens by ABC transporters, however, little is known about these proteins in dermatophytes--a group of fungi causing superficial mycoses. So far only a single ABC transporter has been extensively studied in this group of pathogens. We analyzed available genomic sequences of seven dermatophyte species in order to provide an insight into dermatophyte ABC protein inventory. Phylogenetic studies of ABC transporter genes and their products were conducted and included ABC transporters of other fungi. Our results show that each dermatophyte genome studied possesses a great variety of ABC transporter genes. Detailed analysis of selected genes and their products indicates that relatively recent duplication of ABC transporter genes could lead to novel substrate specificity.

Application of microsatellite-primed PCR (MSP-PCR) and PCR melting profile (PCR-MP) method for intraspecies differentiation of dermatophytes.

In this study, two PCR-based methods (MSP-PCR and PCR-MP) were compared for their abilities to identify intraspecies variations of 23 isolates of Trichophyton rubrum, 78 isolates of Trichophyton interdigitale and 22 isolates of Microsporum canis, obtained mainly from patients in Lódz city. The results allowed to distinguish four types (containing two subtypes) characteristic for T. interdigitale and three types characteristic for T. rubrum using PCR-MP method. Analysis conducted using MSP-PCR with (GACA)4 primer revealed four types for T. rubrum and three types (containing one subtype) for T. interdigitale and with (GTG), primer showed two types (containing one subtype) for T. rubrum and six types (containing one subtype) for T. interdigitale. No differentiation was observed for the M. canis isolates with either method.

Microsatellite-primed PCR for intra-species genetic relatedness in Trichophyton ajelloi strains isolated in poland from various soil samples.

Trichophyton ajelloi is a geophilic dermatophyte that specializes in the decomposition of native keratin. It exists in soil with a permanent influx of keratin matter. In the present study, two PCR-based methods were used for the identification and intra-species differentiation of T. ajelloi strains isolated from 3 types of soils with different physicochemical properties. The first method, employed for molecular identification, was PCR amplification of the 5.8S rRNA gene and its flanking regions encoding internal transcribed spacers (ITSs), followed by restriction enzyme digestion using endonuclease HinfI. The second method, employed for molecular differentiation, was microsatellite-primed PCR (MSP-PCR) using the repetitive oligonucleotide (GACA)4. All the T. ajelloi strains were also identified using a traditional culture method. Our results showed that molecular identification using the PCR-restriction fragment length polymorphism (PCR-RFLP) method agreed with the identification made using the traditional approach. On the other hand, PCR-RFLP results showed no strain differentiation, while MSP-PCR using the (GACA)4 primer identified different varieties among the T. ajelloi strains. The reasons for the intra-species differentiation of T. ajelloi have been discussed.

Identification of dermatophyte species using genomic in situ hybridization (GISH).

A genomic in situ hybridization (GISH)-based method for dermatophyte identification has been developed. Using specific GISH probes, discrimination between Trichophyton interdigitale, Trichophyton rubrum and Microsporum canis has been conducted. Moreover, GISH has been found particularly helpful when proper dermatophyte identification was difficult due to ambiguous PCR-RFLP patterns.