Phylogeny, Antifungal Susceptibility, and Point Mutations of SQLE Gene in Major Pathogenic Dermatophytes Isolated From Clinical Dermatophytosis.

Drug resistance is one of the major challenges to skin fungal infections, especially in tropical and subtropical infections caused by dermatophytes. This study aimed to determine the antifungal susceptibility of clinically dermatophytes and evaluate point mutations in terbinafine-resistant isolates. A total number of 123 clinical dermatophyte isolates in eight species were evaluated in terms of sensitivity to seven major antifungals. Furthermore, the point mutation in squalene epoxidase (SQLE) gene responsible for terbinafine resistance was studied. The dermatophytes species were identified by morphological characteristics and confirmed by the ITS sequencing. Also, the phylogenetic tree was drawn using the RAxML analyses for 123 dermatophytes isolates. A new XXIX genotype was also found in 4 Trichophyton mentagrophytes isolates. Based on the results obtained, terbinafine was the most effective antifungal drug followed by itraconazole and voriconazole. Trichophyton rubrum and Trichophyton tonsurans were the most susceptible species (MIC50 = 0.01, 0.09 µg/ml), and T. mentagrophytes was the most resistant species (MIC50 = 0.125 µg/ml) to terbinafine. Of the 123 dermatophytes isolates, six isolates showed reduced susceptibility to terbinafine, and only Trichophyton indotineae had a mutation in SQLE gene as a Phe397Leu substitution. Overall, the antifungal susceptibility test is necessary for managing dermatophytosis. These results help physicians to control the course of the disease and provide further insights to select effective drugs for patients with dermatophytosis, especially in tropical and subtropical regions of the world, where dermatophytosis is still a public health problem.

Identification of Intestinal Fungal Microflora and Bacterial Pathogens in the Collected Adult Ixodes ricinus from the Northern Provinces of Iran.

Background: Ticks are vectors of many pathogens that involve various important diseases in humans and animals, they have several diverse hosts consequently can retain a diverse group of indigenous microbes, from bacteria to fungi. Little is known about the prevalence and diversity of tick microflora colonizing the midgut and their effects on ticks and their interaction. This information is important for development of vector control strategies. Methods: This study was carried out in northern Iran during autumn 2019. Ticks, Ixodes ricinus caught alive on the bodies of domestic animals in the fall. The tick homogenate was prepared. The identification of fungal isolates was carried out according to a combination of macro and microscopic morphology and molecular sequencing. Pathogenic bacteria of the family Borreliaceae, Francisella tularensis, Borrelia burgdorferi and Coxiella burnetii were tested by real-time PCR. Results: A total of 133 mature I. ricinus ticks were collected from domestic animals, including 71.5% cattle and 28.5% sheep. The tick frequency rates were 87.21% for Mazandaran, 8.28% for Golestan and 4.51% for Gilan Provinces. Total prevalence of fungal tick contamination was 53.4% (75/133) of which Trichoderma harzianum (57%) was the most prevalent species followed by Aspergillus spp. (42%), Mortierella alpine (19%) and Penicillium polonicum (14%). All tick samples were negative for three pathogenic bacteria including Francisella tularensis, Coxiella burnetii, and Borrelia burgdorferi by real-time PCR analysis. Conclusion: These results show a first picture of the microbial diversity of ticks and highlight the importance of microbiota and their role in host-pathogen interaction.

Olorofim Effectively Eradicates Dermatophytes In Vitro and In Vivo.

Superficial fungal infections are prevalent worldwide, with dermatophytes as the most common cause. Various antifungal agents including azoles and allylamines are commonly used to treat dermatophytosis. However, their overuse has yielded drug-resistant strains, calling for the development of novel antimycotic compounds. Olorofim is a newly developed antifungal compound that targets pyrimidine biosynthesis in molds. The purpose of this study was to determine the in vitro and in vivo antifungal effects of olorofim against common dermatophytes. The in vitro activity of olorofim against dermatophytes was assessed by microtiter broth dilution method. Bioinformatic analysis of olorofim binding to dihydroorotate dehydrogenase (DHODH) of dermatophytes was also performed, using Aspergillus fumigatus DHODH as a template. The in vivo efficacy of the drug was investigated, using a guinea pig model, experimentally infected with Microsporum gypseum. Microtiter assays confirmed the high in vitro sensitivity of dermatophytes to olorofim (MIC = 0.015-0.06 mg/liter). Amino acid sequence analysis indicated that DHODH is highly conserved among dermatophytes. The critical residues, in dermatophytes, involved in olorofim binding were similar to their counterparts in A. fumigatus DHODH, which explains their susceptibility to olorofim. Typical skin lesions of dermatophyte infection were observed in the guinea pig model at 7 days postinoculation. Following 1 week of daily topical administration of olorofim, similar to the clotrimazole group, the skin lesions were resolved and normal hair growth patterns appeared. In light of the in vitro and in vivo activity of olorofim against dermatophytes, this novel agent may be considered as a treatment of choice against dermatophytosis.

Toxigenicity and phylogeny of Aspergillus section Flavi in poultry feed in Iran.

BACKGROUND AND PURPOSE: This study was conducted to evaluate the presence of aflatoxigenic strains and level of aflatoxin in poultry feed. Aflatoxigenic strains were investigated in corn and soybean meal as the ingredients of poultry feed, as well as in two types of commercial feed, namely pellet and mash. The gene sequencing was performed to identify the species of Aspergillus section Flavi. MATERIALS AND METHODS: All samples were randomly collected from feed storage silos located in Iran in 2018. The samples were cultured on specialized media for 2 weeks at 28ºC. Identification of Aspergillus section Flavi isolates was based on macro- and microscopic morphological criteria and molecular analysis. The thin-layer chromatography (TLC) was applied to confirm the aflatoxigenic isolates. In addition, the level of aflatoxin B1 (AFB1) produced by these isolates was determined by high-performance liquid chromatography. The strains were subjected to sequence analysis, and Bt2 PCR products were purified by the QIAquick PCR purification kit. At the final stage, the phylogenetic tree was built. RESULTS: Among 54 isolates identified as Aspergillus section Flavi, 20 (37%) isolates were found to produce aflatoxin at a range of 11.28±1.18 to 2239.92±92.26 µg/g fungal dry weight. The aflatoxigenic isolates had the frequencies of 45%, 40%, 10%, and 5% in the corn, pellet, soybean meal, and mash samples, respectively. Furthermore, the mean concentrations of AFB1 were significantly higher in the corn samples (707.04±39.05) than that of other poultry feed samples (P<0.05). A total of 34 (63%) isolates were detected as non-aflatoxigenic on the yeast extract-sucrose broth in TLC analysis. The toxigenic isolates produced the highest (2232.62±55.49) and lowest (11.28±1.18) levels of AFB1 in the corn samples, compared to other feedstuffs. Furthermore, the mean level of AFB1 in mash product was 554.09±10.36 µg/g, compared to a mean level of 229.22±11.09 µg/g in pellets. The isolates were randomly selected, sequenced, and then analyzed. Subsequently, the phylogenetic tree of Aspergillus section Flavi was plotted. CONCLUSION: The process of converting raw ingredients to compound poultry feed is more hazardous when there is not enough time and temperature provided to eliminate aflatoxigenic isolates. Therefore, Aspergillus section Flavi in poultry feed can pose a threat to the poultry industry and poultry products, thereby affecting the health status of humans. Unprocessed/processed materials, such as corns and pelleted feed, need further monitoring, especially when conditions are not optimal for destroying the fungus.