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Terbinafine resistance has become epidemic as an emerging problem in treatment of dermatohpytosis. This could be attributed in part to a point mutation in the squalene epoxidase (SQLE) gene. In this study, point mutations in the SQLE gene were studied in T. rubrum and T. mentagrophytes/T. interdigitale species complex as two main causative agents of dermatophytosis. Antifungal susceptibility of clinical isolates of T. rubrum (n = 27) and T. mentagrophytes/T. interdigitale (n = 56) was assessed using the M38-3rd edition CLSI method. The SQLE gene and ITS region were sequenced for all the fungal strains, and the mutation sites and genotypes of the terbinafine-resistant strains were characterized. The results demonstrated that, in T. rubrum, the minimum inhibitory concentration of terbinafine (MIC50 and MIC90) was 0.03 µg/ml, and the geometric mean (G mean) concentration was 0.02. For the T. mentagrophytes complex, the MIC50 and MIC90 were 0.03 and 1.0 µg/ml, respectively, and the G mean concentration was 0.04 µg/ml. Four out of the five resistant strains were T. indotineae harboring the F397L and Q408L mutations, while the last one was T. mentagrophytes genotype VII, which harbors the F397L mutation. T. indotineae was the prominent causative agent of terbinafine resistance, with 80 % of the isolates, and T. mentagrophytes genotype VII was introduced as a new genotype in the terbinafine-resistant T. mentagrophytes complex. Our findings further substantiate the importance of antifungal susceptibility testing in selecting the choice of drug for effective treatment of dermatophytosis and highlight the importance of screening dermatophyte species for point mutations responsible for newly developed resistant strains to improve the current knowledge of overcoming infections caused by resistant species.
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Dermatophytes show a wide geographic distribution and are the main causative agents of skin fungal infections in many regions of the world. Recently, their resistance to antifungal drugs has led to an obstacle to effective treatment. To address the lack of dermatophytosis data in Iraq, this study was designed to investigate the distribution and prevalence of dermatophytes in the human population and single point mutations in squalene epoxidase gene (SQLE) of terbinafine resistant isolates. The identification of 102 dermatophytes isolated from clinical human dermatophytosis was performed through morphological and microscopic characteristics followed by molecular analysis based on ITS and TEF-1α sequencing. Phylogeny was achieved through RAxML analysis. CLSI M38-A2 protocol was used to assess antifungal susceptibility of the isolates to four major antifungal drugs. Additionally, the presence of point mutations in SQLE gene, which are responsible for terbinafine resistance was investigated. Tinea corporis was the most prevalent clinical manifestation accounting for 37.24% of examined cases of dermatophytosis. Based on ITS, T. indotineae (50.98%), T. mentagrophytes (19.61%), and M. canis (29.41%) was identified as an etiologic species. T. indotineae and T. mentagrophytes strains were identified as T. interdigitale based on TEF-1α. Terbinafine showed the highest efficacy among the tested antifungal drugs. T. indotineae and T. mentagrophytes showed the highest resistance to antifungal drugs with MICs of 2-4 and 4 µg/mL, while M. canis was the most susceptible species. Three of T. indotineae isolates showed mutations in SQLE gene Phe397Leu substitution. A non-previously described point mutation, Phe311Leu was identified in T. indotineae and mutations Lys276Asn, Phe397Leu and Leu419Phe were diagnosed in T. mentagrophytes XVII. The results of mutation analysis showed that Phe397Leu was a destabilizing mutation; protein stability has decreased with variations in pH, and point mutations affected the interatomic interaction, resulting in bond disruption. These results could help to control the progression of disease effectively and make decisions regarding the selection of appropriate drugs for dermatophyte infections.
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Antifúngicos , Arthrodermataceae , Farmacorresistência Fúngica , Testes de Sensibilidade Microbiana , Mutação Puntual , Esqualeno Mono-Oxigenase , Tinha , Humanos , Antifúngicos/farmacologia , Iraque/epidemiologia , Tinha/microbiologia , Tinha/epidemiologia , Tinha/tratamento farmacológico , Farmacorresistência Fúngica/genética , Masculino , Arthrodermataceae/genética , Arthrodermataceae/efeitos dos fármacos , Arthrodermataceae/patogenicidade , Arthrodermataceae/isolamento & purificação , Feminino , Esqualeno Mono-Oxigenase/genética , Adulto , Filogenia , Terbinafina/farmacologia , Terbinafina/uso terapêutico , Pessoa de Meia-Idade , Adolescente , Adulto Jovem , Criança , Proteínas Fúngicas/genética , IdosoRESUMO
Recurrent vulvovaginal candidiasis (RVVC) due to fluconazole resistance in Candida albicans isolates causes a wide range of complications. A number of 63 Candida albicans isolates obtained from vulvovaginal candidiasis (VVC) were identified by Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (ITS-RFLP). Antifungal susceptibility testing was performed by broth microdilution method according to the CLSI protocol. The role of CDR1 and MDR1 genes in progress of VVC to RVVC was examined and the activity of virulence-related enzymes was assessed. Candida albicans was diagnosed in 62.4 % cases, of which 22.2 % were confirmed as RVVC. Voriconazole was the most active drug among five tested antifungals. The mean expression level of CDR1 and MDR1 was higher in RVVC isolates compared to multidrug azole-resistant VVC isolates. Our results demonstrated that the expression of CDR1 and MDR1 and the level of phospholipase and proteinase activities could be quite important to induce fluconazole resistance in C. albicans and to progress of VVC to become RVVC in involved patients.
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Candidíase Vulvovaginal , Feminino , Humanos , Candidíase Vulvovaginal/tratamento farmacológico , Candida albicans , Fluconazol/farmacologia , Regulação para Cima , Farmacorresistência Fúngica/genética , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Testes de Sensibilidade MicrobianaRESUMO
Aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins for humans and livestock that mainly produced by members of the genus Aspergillus in a variety of food commodities. In this study, the effect of S. rosmarinus, T. fruticulosum, and T. caucasicum essential oils (EOs) was studied on fungal growth, AFB1 production and aflR gene expression in toxigenic A. flavus IPI 247. The AFB1 producer A. flavus strain was cultured in YES medium in presence of various two-fold concentrations of the plant EOs (62.5-500 µg/mL) for 4 days at 28 °C. EO composition of plants was analyzed by Gas Chromatography/Mass Spectrometry (GC/MS). The amount of fungal growth, ergosterol content of fungal mycelia and AFB1 content of EO-treated and non-treated controls were measured. The expression of aflR gene was evaluated using Real-time PCR in the fungus exposed to minimum inhibitory concentration (MIC50) of EOs. The main constituents of the oils analyzed by GC/MS analysis were elemicin (33.80 %) and 2,3-dihydro farnesol (33.19 %) in T. caucasicum, 1,8-cineole (17.87 %), trans-caryophyllene (11.14 %), α and áº-pinene (10.92 and 8.83 %) in S. rosmarinus, and camphor (17.65 %), bornyl acetate (15.08 %), borneol (12.48 %) and camphene (11.72 %) in T. fruticulosum. The results showed that plant EOs at the concentration of 500 µg/mL suppressed significantly the fungal growth by 35.24-71.70 %, while mycelial ergosterol content and AFB1 production were inhibited meaningfully by 36.20-65.51 % and 20.61-89.16 %. T. caucasicum was the most effective plant, while T. fruticulosum showed the lowest effectiveness on fungal growth and AFB1 production. The expression of aflR in T. caucasicum and S. rosmarinus -treated fungus was significantly down-regulated by 2.85 and 2.12 folds, respectively, while it did not change in T. fruticulosum-treated A. flavus compared to non-treated controls. Our findings on the inhibitory activity of T. caucasicum and S. rosmarinus EOs toward A. flavus growth and AFB1 production could promise these plants as good candidates to control fungal contamination of agricultural crops and food commodities and subsequent contamination by AFB1. Down-regulation of aflR as the key regulatory gene in AF biosynthesis pathway warrants the use of these plants in AF control programs. Further studies to evaluate the inhibitory activity of studied plants EOs in food model systems are recommended.
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Óleos Voláteis , Rosmarinus , Salvia , Tripleurospermum , Humanos , Aspergillus flavus/metabolismo , Aflatoxina B1 , Óleos Voláteis/farmacologia , Rosmarinus/química , Tripleurospermum/genética , Expressão Gênica , Ergosterol/metabolismo , Ergosterol/farmacologia , Antifúngicos/farmacologiaRESUMO
Background: Understanding the microbiota of disease vectors can help for developing new strategies to prevent the transmission of vector pathogens. Ixodes ricinus is one of the most notorious tick vectors with increasing importance in Iran and other parts of the world while there is limited data on its microbiota. This study aimed to use metagenomics for identifying the I. ricinus tick's microbiota of Iran. Methods: A total of 39 adult ticks were collected from Mazandaran (21 females), Gilan (17 females), and Golestan (1 male). Five tick pools prepared from 39 adults of I. ricinus were subjected to metagenomics analysis. The data were analyzed by targeting the V6 region of the 16S rRNA gene by Illumina 4000 Hiseq sequencing. Results: Among hundreds of intestinal microbiota identified by metagenomics, various pathogenic microorganisms distributed in 30 genera and species including those responsible for tick-borne diseases resided in the genera Coxiella, Rickettsia, and Burkholderia were found. Conclusion: Our results indicated that metagenomics identifies bacteria genera and species which cannot be easily recognized by routine methods. The presence of such pathogenic bacteria indicates the importance of possible zoonotic diseases in this region which could affect public health. These results further substantiate the importance of advanced metagenomics analyses to identify neglected tick-borne pathogens which enable researchers to provide efficient mapping roads for the management of emerging and re-emerging infectious diseases.
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Pulmonary aspergillosis is a life-threatening fungal infection with worldwide distribution. In the present study, clinical epidemiology of pulmonary aspergillosis and antifungal susceptibility of etiologic Aspergillus species were evaluated in one-hundred fifty patients with special focus on the frequency of voriconazole resistance. All the cases were confirmed by the clinical pictures, laboratory findings, and isolation of etiologic Aspergillus species which belonged to two major species, i.e., A. flavus and A. fumigatus. Seventeen isolates displayed voriconazole MIC greater than or equal to the epidemiological cutoff value. Expression of cyp51A, Cdr1B, and Yap1 genes was analyzed in voriconazole-intermediate/resistant isolates. In A. flavus, Cyp51A protein sequencing showed the substitutions T335A and D282E. In the Yap1 gene, A78C replacement led to Q26H amino acid substitution that was not reported previously in A. flavus resistant to voriconazole. No mutations associated with voriconazole resistance were found in the three genes of A. fumigatus. The expression of Yap1 was higher than that of two other genes in both A. flavus and A. fumigatus. Overall, voriconazole-resistant strains of both A. fumigatus and A. flavus demonstrated overexpression of Cdr1B, Cyp51A, and Yap1 genes compared to voriconazole-susceptible strains. Although there are still ambiguous points about the mechanisms of azole resistance, our results showed that mutations were not present in majority of resistant and intermediate isolates, while all of these isolates showed overexpression in all three genes studied. As a conclusion, it seems that the main reason of the emergence of mutation in voriconazole-resistant isolates of A. flavus and A. fumigatus is previous or prolonged exposure to azoles.
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Aspergillus , Aspergilose Pulmonar , Humanos , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Aspergillus/efeitos dos fármacos , Aspergillus/genética , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Azóis , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Testes de Sensibilidade Microbiana , Aspergilose Pulmonar/tratamento farmacológico , Aspergilose Pulmonar/epidemiologia , Aspergilose Pulmonar/microbiologia , Voriconazol/farmacologia , Voriconazol/uso terapêuticoRESUMO
Background and Purpose: The current study aimed to report a multiplex polymerase chain reaction (PCR) assay as a monitoring technique to differentiate aflatoxigenic from non-aflatoxigenic strains of Aspergillus flavus isolated from pistachio orchards soil. Materials and Methods: In total, 25 A. flavus strains were isolated from soil samples of pistachio orchards. To test the strains for Aflatoxin B1 (AFB1)-producing ability, thin-layer chromatography (TLC) was used and the amounts of AFB1 were measured by high-performance liquid chromatography (HPLC). Multiplex PCR was used as a genome-based method to detect genes responsible for AFB1 production by A. flavus and the results were analyzed in terms of speed and specificity of detection. A set of four primers was designed specifically for the omtA, omtB, ver-1, and aflR genes which are commonly present in aflatoxin biosynthetic pathways. Results: The AFB1 production by the A. flavus strains ranged from 0 to 321 ρg/µl. Four-band patterns of the primer sets were observed only in AFB1-producing A. flavus strains. Moreover, 18 out of the 25 strains showed all four bands belonging to omtA, omtB, ver-1, and aflR, whereas 7 strains did not display omtA, or aflR-related bands, in non-toxigenic and low toxin-producing A. flavus. Conclusion: The multiplex PCR is a supplementary strategy to current conventional mycotoxin analytical techniques, such as TLC and HPLC. It could be used as an efficient method to differentiate aflatoxigenic from non-aflatoxigenic strains of A. flavus. This achievement is crucial to minimize fungal contamination of food, feed, and agricultural commodities, thereby reducing the risk of subsequent aflatoxin consumption.
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Vulvovaginal candidiasis (VVC) is a prevalent infection of the genitourinary tract affecting millions of women worldwide. In the present study, the importance of virulence factors, ERG11 gene mutations, ERG11 gene expression, and plasma membrane ergosterol content for fluconazole resistance in Candida species was investigated in 200 women suspected of vulvovaginitis. Isolated Candida species were identified using the ITS-restriction fragment length polymorphism (ITS-RFLP) technique. Antifungal susceptibility testing was performed according to the CLSI document. ERG11 gene expression was analyzed using real-time PCR. ERG11 gene mutation analysis was performed using sequencing methods, and the ergosterol content of the cell membrane was determined in fluconazole-resistant isolates. Furthermore, the production of phospholipase and proteinase enzymes was evaluated in recurrent and non-recurrent infections. VVC was diagnosed in 101 (50.5%) of the 200 clinical cases, of which 21 (20.8%) were confirmed as RVVC. Candida albicans was the most prevalent species, followed by C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, and C. guilliermondii. Ketoconazole and fluconazole were the most effective drugs against C. albicans among five tested antifungals with MIC ranges between 0.06 and 16 µg/mL and 0.25-64 µg/mL. Substitutions of A114S, Y257H, T123I and A114V were detected in fluconazole-resistant C. albicans. The ergosterol content of the fungal cell membrane and the mean levels of ERG11 gene expression transcript were higher in fluconazole-resistant C. albicans isolates obtained from RVVC than in those obtained from VVC cases. Phospholipase and proteinase were produced in different amounts in all Candida species isolated from VVC and RVVC cases. In this review, our results demonstrated that several molecular mechanisms, including ERG11 gene expression, changes in the cell membrane ergosterol content, and mutations in ERG11 gene alone or simultaneously involved in fluconazole resistance of C. albicans species and the recurrence of VVC.
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Antifúngicos , Candidíase Vulvovaginal , Proteínas Fúngicas/metabolismo , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida , Candida albicans , Candida glabrata , Candida parapsilosis , Candida tropicalis , Candidíase Vulvovaginal/microbiologia , Farmacorresistência Fúngica/genética , Ergosterol/farmacologia , Feminino , Fluconazol/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Mutação , Peptídeo Hidrolases/genética , Fosfolipases/genéticaRESUMO
Onychomycosis, a nail fungal infection, is normally caused by dermatophytes. However, yeasts and non-dermatophyte moulds (NDM) are among pathogens that cause nail disease. Regarding, this study aimed to describe the molecular epidemiology of Fusarium onychomycosis in the North of Iran. Two hundred and fifty seven nail samples collected from the patients clinically suspected of onychomycosis were subjected to direct microscopy, calcofluor white staining and culture. Fusarium isolates were identified at a species level through determination of multi-locus sequences for internal transcribed spacer and translation elongation factor 1 alpha. Based on the findings, Fusarium species were isolated from onychomycosis patients (n = 27). According to a previous partial genes analysis, the species in the recent study belonged to the members of F. fujikuroi species complex (n = 14), Fusarium incarnatum-equiseti species complex (n = 1) and F. solani species complex (n = 12). In this study, F. proliferatum was the dominant Fusarium species collected from the samples. The correct identification of Fusarium species is essential regarding the increased prevalence of Fusarium onychomycosis and the inherent resistance of these agents to a wide spectrum of antifungals. The obtained results indicated variation in the epidemiology of Fusarium species isolated from onychomycosis. Moreover, the minimum inhibitory concentration (MIC) of luliconazole and lanoconazole was in the range of 0.001-1 µg/ml, with the geometric mean of MICs obtained at 0.0103 and 0.0343 µg/ml against Fusarium species, respectively. These findings can increase researchers' knowledge regarding diversity of species, distribution of onychomycosis and the choice of a proper treatment.
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Fusarium , Onicomicose , Antifúngicos/farmacologia , Variação Genética , Humanos , Irã (Geográfico)/epidemiologia , Onicomicose/epidemiologia , Onicomicose/microbiologia , Fator 1 de Elongação de Peptídeos/genética , PrevalênciaRESUMO
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.
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Arthrodermataceae , Tinha , Antifúngicos/farmacologia , Arthrodermataceae/genética , Farmacorresistência Fúngica/genética , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Mutação Puntual , Esqualeno Mono-Oxigenase/genéticaRESUMO
In the present study, metal and metal oxide nanoparticles were successfully synthesized using Aspergillus kambarensis. UV-Vis spectroscopy showed maximum absorbance of 417 nm for silver (AgNPs), 542 nm for gold (AuNPs), 582 nm for copper (CuNPs) and 367 nm for zinc oxide (ZnONPs) nanoparticles. Fourier transform infrared spectroscopy indicated the presence of various mycochemicals with diverse functional groups in the fungal cell-free filtrate. Transmission electron microscopy revealed mono and poly dispersed particles with an estimate size of 50 nm and different shapes for synthesized manufacture metallic nanoparticles (MNPs. Dynamic light scattering confirmed that MNPs were dispersed in the size range less than 50 nm. Zeta potential analysis showed values of -41.32 mV (AgNPs), -41.26 mV (AuNPs), -34.74 mV (CuNPs) and 33.72 mV (ZnONPs). X-ray diffraction analysis demonstrated crystalline nature for MNPs. All the synthesized MNPs except AuNPs showed strong antifungal and antibacterial activity in disc diffusion assay with growth inhibition zones of 13.1-44.2 mm as well as anticancer activity against HepG-2 cancer cell line with IC50 in the range of 62.01-77.03 µg/ml. Taken together, the results show that biologically active MNPs synthesized by A. kambarensis for the first time could be considered as promising antimicrobial and anticancer agents for biomedical applications.
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Nanopartículas Metálicas , Antibacterianos/farmacologia , Aspergillus , Ouro , Química Verde , Extratos Vegetais , Prata/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
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.
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Due to the increasing rate of invasive fungal infections and emerging antifungal resistance, development of novel antifungal drugs has been an urgent necessity. Antifungal peptides (AFPs) have recently attracted attention due to their unique ability to evade drug-resistant fungal pathogens. In this study, a novel AFP, Cc-AFP1, with a molecular weight of ~3.759 kDa, was isolated from Carum carvi L., purified by ammonium sulfate precipitation and reversed-phase HPLC and finally identified by sequence analysis using Edman degradation. Peptide sequence analysis revealed a fragment of 36 amino acid residues as RVCFRPVAPYLGVGVSGAVRDQIGVKLGSVYKGPRG for Cc-AFP1 with a net charge of +5 and a hydrophobicity ratio of 38%. The antifungal activity of Cc-AFP1 was confirmed against Aspergillus species with MIC values in the range of 8-16 µg/ml. Cc-AFP1 had less than 5% hemolytic activity at 8-16 µg/ml on human red blood cells with no obvious cytotoxicity against the HEK293 cell line. Stability analysis showed that the activity of Cc-AFP1 was maintained at different temperatures (20°C to 80°C) and pH (8 to 10). The results of a propidium iodide uptake and transmission electron microscopy showed that the antifungal activity of Cc-AFP1 could be attributed to alteration in the fungal cell membrane permeability. Taken together, these results indicate that Cc-AFP1 may be an attractive molecule to develop as a novel antifungal agent combating fungal infections cause by Aspergillus species.
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Antifúngicos , Carum , Antifúngicos/farmacologia , Aspergillus , Células HEK293 , Humanos , Testes de Sensibilidade Microbiana , Peptídeos/farmacologiaRESUMO
BACKGROUND AND PURPOSE: This study aimed to investigate the effects of Allium cepa ethanolic extract (EAC) on Cryptococcus neoformans biological activities and LAC1 gene expression. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) of EAC was determined based on the Clinical and Laboratory Standards Institute M27-A4 method at a concentration range of 125-4000 µg/ml. The EAC synergism activity was determined in combination with fluconazole (FCZ) as an antifungal azole. Laccase activity, melanin production, and cell membrane ergosterol content of C. neoformans were assessed at the 0.5× MIC concentration of EAC (1000 µg/ml) and FCZ (64 µg/ml) by approved methods. The expression of the LAC1 gene was studied in the fungus exposed to 0.5× MIC concentration of EAC and FCZ using the real-time polymerase chain reaction. RESULTS: Based on obtained results, MIC of EAC and FCZ were 2000 and 128 µg/ml, respectively. A combinatory effect was reported for FCZ and EAC by a fractional inhibitory concentration index of 0.25. The cell membrane ergosterol content was inhibited in EAC- and FCZ-treated C. neoformans by 58.25% and 49.85%, respectively. The laccase activity and melanin production were reduced in EAC-treated C. neoformans by 45.37% and 51.57%, and in FCZ-treated fungus by 54.64% and 53.68%, respectively. The expression of fungal LAC1 at messenger RNA (mRNA) level was measured 0.46 and 0.58 folds and significantly decreased in both EAC- and FCZ-treated C. neoformans at the 0.5×MIC concentration, respectively (P<0.05). CONCLUSION: The findings revealed that EAC contains inhibitory compounds which interact with biological activities in C. neoformans and thereby, it could be considered as a potential source for the development of novel antifungal drugs.
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BACKGROUND: Dermatophytes are a homogeneous group of species with low genetic diversity, and there are still many uncertainties about the boundaries among species. OBJECTIVES: Aiming at clarifying the relationships among species in the genus and introducing suitable genes for multilocus sequence typing (MLST), a new MLST scheme approach was developed to characterize the major pathogenic dermatophytes. METHODS: We performed maximum parsimony (MP), MrBayes, RAxML, and eBURST analyses, based on the MLST scheme to scrutinize the evolution within 95 clinical isolates and four reference strains belonging to the four major dermatophytes species. Then, the discriminatory power, pairwise genetic distances, ratio dN/dS, and sequence types (STs) of these isolates were determined. Also, to study taxonomy, sequences of the internal transcribed spacer (ITS), Beta-tubulin (BT2 ), and translation elongation factor 1-α (TEF-1α) genes of other dermatophytes species available in the GenBank were analyzed. RESULTS: Findings of the present study indicated that three genes: BT2, ITS, and TEF-1α, which showed the greatest diversity among dermatophyte species, were suitable for MLST. The most prevalent STs were seen among the species of Trichophyton interdigitale. Also, two new genotypes, i.e., XXVII and XXVIII, were introduced for T. interdigitale and Trichophyton mentagrophytes. The least informative sites were found in Epidermophyton floccosum, Trichophyton rubrum, and T. mentagrophytes, while the most informative sites were observed in T. interdigitale. Furthermore, the most informative locus was TEF-1α. The phylogenetic tree, constructed by the combination of the three genes, shows a new topological pattern that confirms the derivation of the anthropophilic and zoophilic genera from the geophilic genus. Also, the phylogenetic analyses and pairwise distances of the combination of the three loci showed that Trichophyton tonsurans and Trichophyton equinum were a species complex, where T. equinum is derived from T. tonsurans. CONCLUSIONS: Results of this study showed that MLST is very effective in determining the boundaries between species and taxonomy. Considering that there is no database for MLST dermatophytes, further studies are needed to determine the suitable genes for MLST. Also, the determination of STs in epidemiological studies and raising epidemiological information are helpful. This study was a new starting point to determine the ST and a foundation for a dermatophyte MLST database.
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Cryptococcosis is an opportunistic fungal infection caused mainly by Cryptococcus neoformans. The aim of the present study was to evaluate the inhibitory effect of aureobasidin A on C. neoformans with special focus on its mode of action. The effect of aureobasidin A on cell membrane ergosterol content, cell wall permeability, membrane pumps activities, the total oxidant status (TOS) and melanin production was evaluated. Cytotoxicity and cell hemolysis, and laccase (LacI) and ß1,2-xylosyltransferase (Cxt1p) gene expression were also evaluated. Aureobasidin A reduced melanin production and increased extracellular potassium leakage at 0.5 × MIC concentration. This peptide has no effect on fungal cell wall integrity. Cell membrane ergosterol content was decreased by 29.1% and 41.8% at 0.5 × MIC and 1 × MIC concentrations (2 and 4 µL/mL) in aureobasidin A treated samples, respectively. TOS level was significantly increased without activation of antioxidant enzymes. Lac1 gene was over-expressed (11.7-fold), while Cxt1p gene was down regulated (0.2-fold) following treatment with aureobasidin A. Overall, our results indicated that aureobasidin A inhibits C. neoformans growth by targeting different sites in fungal cells and it may be considered as a promising compound to use as an antifungal in treatment of clinical cryptococcosis.
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Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Depsipeptídeos/farmacologia , Antifúngicos/síntese química , Antifúngicos/química , Depsipeptídeos/síntese química , Depsipeptídeos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Background: Zoonotic diseases as health concerns worldwide account for more than half of the emerging infectious diseases. Arachnids are powerful vectors to transmit several diseases to humans. Additionally, these emerging zoonotic diseases have been a considerable health threat in the Eastern Mediterranean Region of the WHO (EMRO) due to the large population living close to farms and international trade with nearby countries. Methods: This review study is based on the reported three tick-borne diseases, Lyme disease, Tularemia, and Q fever, from Iran and other EMRO countries. To this end, we searched PubMed central, ISI web of Science, and Google with the related keywords in English at any time. The reported data are then sorted by countries for each disease. Results: According to the published data, 15 countries in the region have one/more emerging infectious diseases. Q fever has been the most frequent infection in EMRO countries, while Lyme was less recorded. Furthermore, Iran is among the countries with documented history of all three investigated diseases. Conclusion: Tick-borne disease is popular among EMRO countries, indicating that they have natural conditions for infections in animals and humans. It appears necessary to develop a disease management strategy and control programs against tick-borne diseases (TBDs). Moreover, the disease-resistant animal could be bred instead of susceptible livestock. Therefore, research studies to control TBDs should be regarded as a top priority plan.
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Background and Purpose: This study aimed to assess the effect of Allium cepa ethanolic extract (ACE) loaded polyacrylonitrile (PAN) and polyvinyl pyrrolidone (PVP) nanofibers on Candida albicans (C. albicans) CDR1 and CDR2 genes expression. Materials and Methods: The minimum inhibitory concentrations (MICs) of ACE against C. albicans ATCC 10231 and clinical fluconazole (FLC)-resistant C. albicans PFCC 93-902 were determined using the Clinical and Laboratory Standards Institute (CLSI) protocol (M27-Ed4) at a concentration range of 45.3-5800 µg/ml. The nanofibers containing ACE (60 wt%) were fabricated using the electrospinning technique. The expression of the CDR1 and CDR2 genes was studied in the fungus exposed to ACE-loaded nanofibers and 0.5×MIC concentration of FLC using the real-time polymerase chain reaction. Results: MIC50 and MIC90 of ACE against FLC-resistant C. albicans were 725 and 1450 µg/mL, respectively. The expression of CDR1 (4.5-fold) and CDR2 (6.3-fold) were down-regulated after the exposure of FLC-resistant C. albicans to ACE-loaded nanofibers (P<0.05). Furthermore, the expression of CDR1 (2.8-fold) and CDR2 (3.2-fold) were up-regulated in FLC-treated C. albicans (P<0.05). Conclusion: The results revealed that nanofibers containing ACE interact with drug-resistant genes expressed in C. albicans. Further studies are recommended to investigate the mode of action and other biological activities of ACE-loaded nanofibers against C. albicans and other pathogenic fungi.