Comparative analysis of enzymatic profiles and biofilm formation in clinical and environmental Candida kefyr isolates.

The global landscape of Candida infections has seen a significant shift. Previously, Candida albicans was the predominant species. However, there has been an emergence of non-albicans Candida species, which are often less susceptible to antifungal treatment. Candida kefyr, in particular, has been increasingly associated with infections. This study aimed to investigate the profiles of enzymatic activity and biofilm formation in both clinical and non-clinical isolates of C. kefyr. A total of 66 C. kefyr isolates were analysed. The activities of proteinase and phospholipase were assessed using bovine serum albumin and egg yolk agar, respectively. Haemolysin, caseinolytic and esterase activities were evaluated using specific methods. Biofilm formation was investigated using crystal violet staining. The findings indicated that biofilm and proteinase activity were detected in 81.8% and 93.9% of all the isolates, respectively. Haemolysin activity was observed with the highest occurrence (95.5%) among normal microbiota isolates. Esterase activity was predominantly identified in dairy samples and was absent in hospital samples. Caseinase production was found with the highest occurrence (18.2%) in normal microbiota and hospital samples. Phospholipase activity was limited, found in only 3% of all the isolates. These findings reveal variations in enzyme activity between clinical and non-clinical C. kefyr isolates. This sheds light on their pathogenic potential and has implications for therapeutic strategies.

Evaluation of building washing machines as an extreme environment for potentially pathogenic fungi.

Washing machines are commonly used in households and are considered indispensable appliances for maintaining cleanliness and hygiene. Environmental conditions within household washing machines are ideal for fungal colonization, which may pose risks to human health and contribute to sick building syndrome. This study aimed to investigate the fungal species contamination in the building washing machines. A total of 50 building washing machines were swab-sampled at three locations: the detergent drawer, the inner and outer parts of the rubber door seal. The housekeeping conditions of these appliances were assessed through a questionnaire. The isolated fungi were identified using standard mycological diagnostic procedures and molecular analysis based on the ITS1/ITS4 and ß-tubulin gene regions. The possibility of fungal agents transferring from contaminated washing machines to autoclaved clothes during laundry cycles was investigated. Fungi were detected in 82% of the sampled appliances, with the inner rubber door seal being the most frequently colonized area. Using conventional and molecular techniques, we identified 122 fungal isolates, encompassing 17 diverse genera of molds, yeast-like, and yeast fungi. The mold fungi included 14 genera of hyaline and black genus. Among these, the most frequently identified genera of hyaline and black fungi were Aspergillus (27.7%), and Cladosporium (10.7%), respectively. This study demonstrates that building washing machines may serve as suitable ecological niches for fungal growth and transmission. Therefore, regular cleaning and disinfection of these devices are necessary.

Species distribution and antifungal susceptibility profiles of yeasts isolated from onychomycosis: a cross-sectional study with insights into emerging species.

Candida onychomycosis is a common fungal infection affecting the nails, primarily caused by Candida (C.) species. Regarding the increasing trend of Candida onychomycosis and the antifungal resistant phenomenon in recent years, this study aims to evaluate the epidemiological characteristics of Candida onychomycosis, the distribution of emerging species, and the antifungal susceptibility profiles of isolates. Onychomycosis caused by yeast species was confirmed through direct examination and culture of nail scraping among all individuals suspected to have onychomycosis and referred to a medical mycology laboratory between June 2019 and March 2022. Species of yeast isolates were identified using the multiplex PCR and PCR-RFLP methods. The antifungal susceptibility of isolates to common antifungal agents and imidazole drugs was evaluated according to the M-27-A3 CLSI protocol. Among 101 yeast strains isolated from onychomycosis, Candida parapsilosis complex (50.49%) was the most common species, followed by C. albicans (20.79%) and C. tropicalis (10.89%). Rare species of yeasts such as C. guilliermondii and Saccharomyces cerevisiae were also identified by molecular methods. Results obtained from antifungal susceptibility testing showed significant differences in MIC values of isoconazole, fenticonazole, and sertaconazole among different species. Overall, a fluconazole-resistant rate of 3% was found among Candida species. Moreover, there was a statistically significant difference in MICs of fenticonazole and clotrimazole between the two most prevalent causative species, C. parapsilosis complex and C. albicans. Correct identification of the causative agents of onychomycosis and performing susceptibility testing could be helpful in choosing the most appropriate antifungal therapy.

Direct molecular analysis of Malassezia species from the clinical samples of patients with pityriasis versicolor.

Background and Purpose: Species identification of Malassezia using culture-dependent methods is time-consuming due to their fastidious growth requirements. This study aimed to evaluate a rapid and accurate molecular method in order to diagnose the pityriasis versicolor (PV) and identify Malassezia species from direct clinical samples. Materials and Methods: Skin scraping or tape samples from patients with PV and healthy volunteers as the control group were collected. Diagnosis of PV was confirmed by direct microscopic examination. The DNA extraction was performed according to the steel-bullet beating method. Polymerase chain reaction-restriction fragment length polymorphism assay using HhaI restriction enzyme was applied for the identification and differentiation of Malassezia species. Results: The PCR method was able to detect Malassezia in 92.1% of specimens which were also confirmed with microscopic examination. Statistically, a significant association was observed between the results of the two assays (P < 0.001). Moderate agreement was identified between the two methods to diagnose the PV in both populations (Kappa: 0.55). Considering microscopic examination as the gold standard method for confirmation of PV, the sensitivity, specificity, positive predictive value, and negative predictive value values of the PCR assay for recognition of PV were 85%, 75%, 92%, and 60%, respectively. M. globosa and M. restricta were the most prevalent species isolated from patients. Conclusion: In this study, the two-step molecular method based on the amplification of the D1/D2 domain and digestion of the PCR product by one restriction enzyme was able to diagnose and identify Malassezia directly from clinical samples. Consequently, it can be said that the molecular-based method provides more facilities to identify fastidious species, such as M. restricta.

Chemical Composition and Antifungal and Antibiofilm Effects of Vitex pseudo-negundo Essential Oil against Pathogenic Fungal Strains.

Background: Vitex pseudo-negundo is a plant of the Lamiaceae family that grows in different parts of the world and the vicinity of seasonal rivers in Iran. Methods: The chemical composition of the Vitex pseudo-negundo essential oils was distilled and evaluated using gas chromatography/mass spectrometry. The antifungal activity of the essential oils against the fungal strains was analyzed by broth microdilution methods as suggested by the Clinical and Laboratory Standards Institute. Furthermore, the antibiofilm activity of the Vitex pseudo-negundo essential oils was assessed using the XTT reduction assay. Results: Based on GC/MS analysis, the major components of the Vitex pseudo-negundo essential oils were α-pinene, α-terpinyl acetate, limonene, and (E)-caryophyllene. The growth of tested yeasts was inhibited at concentrations ranging from 2 to 64 µl/mL. Vitex pseudo-negundo fruit essential oil was the most effective in inhibiting yeast growth. Moreover, the essential oils exhibited antifungal activity against filamentous fungi strains. Additionally, the biofilm formation of Candida albicans was inhibited by the leaf, flower, and fruit of the essential oils. Conclusion: Considering the significant antifungal activities of these essential oils, they can be considered a potential source for formulating novel agents to control fungal infections.

Phenyl-quinoline derivatives as lead structure of cholinesterase inhibitors with potency to reduce the GSK-3ß level targeting Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to cognitive decline and memory loss. Unfortunately, there is no effective treatment for this condition, so there is a growing interest in developing new anti-AD agents. In this research project, a series of phenyl-quinoline derivatives were designed as potential anti-AD agents. These derivatives were substituted at two different positions on benzyl and phenyl rings. The structures of the derivatives were characterized using techniques such as IR spectroscopy, 1H NMR, 13C NMR, and elemental analysis. During the in vitro screening, the derivatives were tested against both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). It was observed that most of the derivatives showed higher selectivity against BChE compared to AChE. Among the derivatives, analog 7n (with a methoxy group at R1 and a 4-bromine substituent at R2 exhibited the highest potency, with a 75-fold improvement in the activity compared to the positive control. Importantly, this potent analog demonstrated no toxicity at the tested concentration on SH-SY5Y cells, indicating its potential as a safe anti-AD agent. The level of GSK-3ß was also reduced after treatments with 7n at 50 µM. Overall, this study highlights the design and evaluation of phenyl-quinoline derivatives as promising candidates for developing novel anti-AD agents.

Species identification, antifungal susceptibility patterns, and vitamin D3 level in women with vaginal candidiasis: a case-control study in Iran.

Vulvovaginal candidiasis (VVC) is a fungal infection that is a global issue of women's health due to its association with morbidity, infertility, and economic costs. This study aimed to compare the vitamin D3 levels between women with VVC to healthy controls and determine the species distribution and susceptibility pattern of isolates. Species identification was performed using sequencing of the ITS-rDNA regions and amplification of the HWP1 gene. Antifungal susceptibility testing was determined by the disk diffusion method. Moreover, serum vitamin D3 levels were measured using a commercial ELISA (enzyme-linked immunosorbent assay) kit. Our results indicated that vitamin D3 level in women with VVC was lower than those of healthy women (p-value < .001). Candida albicans complex (62.8 percent) was the most common species, and most species were susceptible to fluconazole, itraconazole, ketoconazole, and nystatin. In conclusion, our study revealed a potential link between vitamin D3 deficiency and VVC in women. Although our findings showed significantly lower vitamin D3 levels in women with VVC, further research is needed to establish a definitive causative relationship between vitamin D3 deficiency and VVC. Nonetheless, our study highlights the potential importance of maintaining adequate levels of vitamin D3 and the need for further exploration in this area.

Dendritic hybrid materials comprising polyhedral oligomeric silsesquioxane (POSS) and hyperbranched polyglycerol for effective antifungal drug delivery and therapy in systemic candidiasis.

Systemic Candida infections are routinely treated with amphotericin B (AMB), a highly effective antimycotic drug. However, due to severe toxicities linked to the parenteral administration of conventional micellar formulations (Fungizone®), its clinical utility is limited. Hyperbranched polyglycerols (HPGs) are multi-branched three-dimensional hydrophilic macromolecules that can be used to lessen the toxicity of AMB while also increasing its aqueous solubility. In the current research, to improve the safety and therapeutic efficacy of AMB, we developed new polyhedral oligomeric silsesquioxane - hyperbranched polyglycerol dendrimers with cholesterol termini (POSS-HPG@Chol) using azide-alkyne click reaction. Compared with Fungizone®, the as-synthesized POSS-HPG@Chol/AMB had lower minimum inhibitory and fungicidal concentrations against almost all studied Candida spp., as well as much less hemolysis and cytotoxicity. POSS-HPG@Chol/AMB revealed total protection of Balb/C mice from severe Candida infections in an experimental model of systemic candidiasis and can effectively reduce or eliminate AMB liver and kidney tissue injuries. Thanks to their safety, biocompatibility, and unique therapeutic properties, the developed POSS-polyglycerol dendrimers could be viable nanostructures for the delivery of poorly soluble drugs like AMB.

Molecular strategy for the direct detection and identification of the most common fungal community in cerumen specimens by multiplex PCR.

Introduction. Otomycosis is a superficial fungal infection that is responsible for approximately 9-27 % of otitis externa. However, fungal communities in otomycosis are varied, but Aspergillus spp. and Candida spp. are the most common causes of this infection.Hypothesis Statement. The multiplex PCR assay is postulated to be able to directly detect more than one fungal genus in cerumen specimens.Aim. This study aimed to develop and evaluate the role of the multiplex PCR assay in detecting the most common genus of fungi that cause otomycosis directly from the cerumen specimens.Methodology. To detect Candida and Aspergillus/Penicillium genera, three pairs of primers, including pan-fungal, pan-Candida, and pan-Aspergillus/Penicillium, were used in a multiplex PCR. In order to evaluate the performance and reproducibility of the multiplex PCR. the cerumen of 140 patients suspected of otomycosis were investigated.Results. Pan-Candida and pan-Aspergillus/Penicillium primers were designed to amplify the ITS1-5.8S-ITS2 region and the ß-tubulin gene, respectively. In the multiplex PCR assay, 64 (47.40 %) and 118 (87.40 %) specimens were positive with pan-Candida and pan-Aspergillus/Penicillium primers, respectively. Double amplicon bands of Candida and Aspergillus were obtained in 51 (37.77 %) specimens. In the culture method, yeast (n=18, 13.33 %) and mould (n=117, 86.66 %) were isolated from 135 cerumen specimens. The sensitivity, specificity, and positive and negative predictive values of the multiplex PCR assays using culture method results as the gold standard were determined to be 94, 33, 97, and 22 %, respectively.Conclusion. In our study, multiplex PCR assays enabled simultaneous detection of two common genera of the causative agent of otomycosis in a cerumen specimen. Regarding the high sensitivity of the first step of the multiplex PCR assay, this assay may be used for the direct detection of Candida and Aspergillus genera in other clinical specimens.

Chitosan-based emulgel and xerogel film containing Thymus pubescens essential oil as a potential wound dressing.

Controlling the wound exudates accompanied by microbial wound infections has still remained as one the most challenging clinical issues. Herein, a chitosan/gelatin/polyvinyl alcohol xerogel film containing Thymus pubescens essential oil is fabricated for antimicrobial wound dressing application. The chemical and physical characteristics of the devised formulation is characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, atomic force microscope, and tensile tests. Moreover, swelling capability, water vapour transmission rate, water contact angle, solubility, moisture content, and release properties are also studied. The antimicrobial and antibiofilm tests are performed using the broth microdilution and XTT assay, respectively. The produced formulation shows excellent antimicrobial efficacy against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida species. It is also demonstrated that the obtained film can reduce (∼80 %) Candida albicans biofilm formation, and its biocompatibility is confirmed with MTT (∼100 %) and hemolysis tests. The antimicrobial activity can be correlated to the microbial membrane attraction for Candida albicans cells, illustrated by flow cytometry. This proposed film with appropriate mechanical strength, high swelling capacity in different pH values (∼200-700 %), controlled release property, and antimicrobial and antioxidant activities as well as biocompatibility can be used as a promising candidate for antimicrobial wound dressing applications.

Evaluating the Typing Power of Six Isoenzymatic Systems for Differentiation of Clinical and Standard Isolates of Candida Species.

Background: Due to the increasing prevalence of candidiasis, early detection of the causative agents may pave the way for the management of this infection. The present study aimed to assess the discriminative power of the six isoenzymatic systems for differentiating the Candida species. Materials and Methods: Sixteen standard Candida albicans and Candida dubliniensis strains and 30 fluconazole-sensitive and fluconazole-resistant clinical strains of Candida albicans were analyzed using a Multilocus Enzyme Electrophoresis (MLEE) method, including six enzymatic systems consisting of malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucose-phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGD), and malic enzyme (ME). Results: Among the six enzymatic systems, ME showed no diagnostic activity, whereas MDH provided the best species-specific pattern for species discrimination. In addition, the MDH and G6PD systems provided a discriminatory pattern for differentiating C. dubliniensis from C. albicans isolates. The same isoenzymatic activity was detected in all 36 standard and clinical isolates. Moreover, the results showed no correlation between the isoenzymatic profiles and drug resistance. Conclusion: Among the investigated MLEE systems, MDH was able to differentiate between Candida albicans and Candida dubliniensis. Although no association was detected between isoenzyme patterns and fluconazole resistance in this investigation, isoenzyme patterns are likely correlated with virulence factors between species and even within species. To answer these questions, additional studies should be done on more strains.

The hospitalization rate and clinical characteristics of mucormycosis prior and during COVID-19 pandemic: A single-center study.

INTRODUCTION: There have been some reports of the association between SARS-CoV-2 infection and mucormycosis. This study aims to compare the hospitalization rates and clinical characteristics of mucormycosis before and during the COVID-19 pandemic. METHODOLOGY: In this retrospective study, we compared the hospitalization rate of mucormycosis patients in Namazi hospital in Southern Iran for two periods of 40 months. We defined July 1st, 2018 to February 17th, 2020, as the pre-COVID-19 period and February 18th, 2020, to September 30th, 2021, as the COVID-19 period. In addition, a quadrupled group of hospitalized patients with age and sex-matched SARS-COV-2 infection without any sign of mucormycosis was selected as the control group for COVID-associated mucormycosis. RESULT: In the total of 72 mucormycosis patients in the COVID period, 54 patients had a clinical history and a positive RT-PCR, which confirms the diagnosis of SARS-COV2 infection. The hospitalization rate of mucormycosis showed an increase of + 306% (95% CI: + 259%, + 353%) from a monthly average value of 0.26 (95% confidence interval (CI): 0.14, 0.38) in the pre-COVID period to 1.06 in the COVID period. The use of corticosteroids prior to the initiation of hospitalization (p ≤ 0.01), diabetes (DM) (p = 0.04), brain involvement (p = 0.03), orbit involvement (p = 0.04), and sphenoid sinus invasion (p ≤ 0.01) were more common in patients with mucormycosis during the COVID period. CONCLUSIONS: In high-risk patients, especially diabetics, special care to avoid the development of mucormycosis must be taken into account in patients with SARS-COV-2 infection considered for treatment with corticosteroids.

[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives as new therapeutic candidates against urease positive microorganisms: design, synthesis, pharmacological evaluations, and in silico studies.

Regarding the important role of the urease enzyme as a virulence factor in urease-positive microorganisms in this study, new series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were designed and synthesized. All compounds evaluated against urease enzyme exhibiting IC50 values of 0.87 ± 0.09 to 8.32 ± 1.21 µM as compared with thiourea as the positive control (IC50 = 22.54 ± 2.34 µM). The kinetic evaluations of 6a as the most potent derivative recorded a competitive type of inhibition. Molecular dynamic simulations of the 6a derivative were also conducted, showing that 6a occupied the active site with closed state. Antimicrobial activities of all derivatives were performed, and 6f (R = 3-Cl), 6g (R = 4-Cl), and 6h (R = 3,4-diCl) analogs demonstrated significant antifungal activities with MIC values of 1, 2, and 0.5 µg/mL compared with fluconazole with MIC = 2 µg/mL. Synthesized analogs also exhibited potent urease inhibitory activities against C. neoformans (IC50 = 83.7-118.7 µg/mL) and P. mirabilis (IC50 = 74.5-113.7 µg/mL), confirming their urease inhibitory potential. The results demonstrated that the designed scaffold could be considered a suitable pharmacophore to develop potent urease inhibitors.

Encapsulation of Zataria multiflora essential oil in polyvinyl alcohol/chitosan/gelatin thermo-responsive hydrogel: Synthesis, physico-chemical properties, and biological investigations.

Zataria multiflora essential oil is a natural volatile plant product whose therapeutic applications require a delivery platform. Biomaterial-based hydrogels have been extensively used in biomedical applications, and they are promising platforms to encapsulate essential oils. Among different hydrogels, intelligent hydrogels have recently attracted many interests because of their response to environmental stimuli such as temperature. Herein, Zataria multiflora essential oil is encapsulated in a polyvinyl alcohol/chitosan/gelatin hydrogel as a positive thermo-responsive and antifungal platform. According to the optical microscopic image, the encapsulated spherical essential oil droplets reveal a mean size of 1.10 ± 0.64 µm, which are in consistent with the SEM imaging results. Encapsulation efficacy and loading capacity are 98.66 % and 12.98 %, respectively. These results confirm the successful efficient encapsulation of the Zataria multiflora essential oil within the hydrogel. The chemical compositions of the Zataria multiflora essential oil and the fabricated hydrogel are analyzed by gas chromatography-mass spectroscopy (GC-MS) and Fourier transform infrared (FTIR) techniques. It is found that thymol (44.30 %) and γ-terpinene (22.62 %) are the main constituents of the Zataria multiflora essential oil. The produced hydrogel inhibits the metabolic activity of Candida albicans biofilms (∼60-80 %), which can be related to the antifungal activity of the essential oil constituents and chitosan. Based on the rheological results, the produced thermo-responsive hydrogel shows a gel-sol viscoelastic transition at a temperature of 24.5 °C. This transition leads to a facile release of the loaded essential oil. The release test depicts that about 30 % of Zataria multiflora essential oil is released during the first 16 min. In addition, 2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay demonstrates that the designed thermo-sensitive formulation is biocompatible with high cell viability (over 96 %). The fabricated hydrogel can be deemed as a potential intelligent drug delivery platform for controlling cutaneous candidiasis due to antifungal effectiveness and less toxicity, which can be a promising alternative to traditional drug delivery systems.

Phenotypes characterization and ABC genotypes distribution of clinical Candida albicans isolates.

BACKGROUND: Candidemia and vaginitis are the most common types of candidiasis mostly caused by Candida albicans species. C. albicans has several genotypes and the potential ability to form different phenotype colonies on specific media. This study aimed to evaluate the genotype distribution of blood and vaginal C. albicans isolates and phenotype characteristics on Spider and yeast peptone dextrose agar medium. METHODS: A total of 40 clinical Candida albicans isolates comprising vagina (20) and blood (20) were used. ABC typing using CA-INT-R and CA-INT-L primers was performed to span the transposable group I intron of the 25S rDNA gene. For colony phenotypic characteristics, the Spider and YPDA media were used. RESULTS: Among the blood and vaginal isolates, genotype A (12/60%) and genotype C (10/50%) were the most common types, respectively. The highest phenotype shape frequency of the colonies in blood and vaginal samples was the ring and the lowest was the hat/ring. The dominant color phenotype in blood and vaginal samples was gray. There was a significant relationship between genotype and phenotype forms in the blood sample on YPDA medium (p = 0.02). In the Spider medium, there were no significant differences between genotypes and phenotypes. CONCLUSION: In this study, genotype A and genotype C were predominant in blood and vaginal samples, respectively. In both groups, YPD agar medium demonstrated the most variety of phenotypes that was related to genotypes A and C. The variety of phenotypes in both groups was the same in genotypes A and C on the Spider medium.

The efficacy of complementary treatment with marshmallow (Althaea officinalis L.) on vulvovaginal candidiasis: A randomized double-blinded controlled clinical trial.

BACKGROUND: Vulvovaginal candidiasis is a common gynecologic infection, and recurring cases are yet incurable. This trial was based on Persian medicine to compare how effective marshmallow aqueous extract 4% plus clotrimazole 1% (CLOT-M) is compared to clotrimazole 1% vaginal creams on VVC. METHODS: This study randomly assigned 100 women with VVC into two groups. The target group (n = 50) was treated with CLOT-M while controls (n = 50) with clotrimazole vaginal creams for seven consecutive nights. Different VVC symptoms and signs, and yeast culture from vaginal discharge were evaluated as the outcome measures before the intervention and 7 and 30 days after. RESULTS: The efficacy of CLOT-M vaginal cream was assessed during the 1st and 2nd follow-ups, indicating a significant decrease in mean itching (P = 0.001 for both comparisons) and dyspareunia score (P = 0.001 and P = 0.04, respectively) as compared to treatment with clotrimazole vaginal cream. Moreover, after 7 days of the intervention, patients in the CLOT-M group experienced significant improvement in mean dysuria score compared to those in the control group (P = 0.001). Neither cream caused any significant adverse events. CONCLUSION: It seems that CLOT-M vaginal cream had a significant effect on the VVC symptoms improvement, without any significant side effects. However, larger sample-sized trials are needed for more evidence-based judgment.

Synthesis and structure-activity relationship studies of benzimidazole-thioquinoline derivatives as α-glucosidase inhibitors.

In this article, different s-substituted benzimidazole-thioquinoline derivatives were designed, synthesized, and evaluated for their possible α-glucosidase inhibitory activities. The most active compound in this series, 6j (X = 4-bromobenzyl) exhibited significant potency with an IC50 value of 28.0 ± 0.6 µM compared to acarbose as the positive control with an IC50 value of 750.0 µM. The kinetic study showed a competitive inhibition pattern against α-glucosidase for the 6j derivative. Also, the molecular dynamic simulations were performed to determine key interactions between compounds and the targeted enzyme. The in silico pharmacodynamics and ADMET properties were executed to illustrate the druggability of the novel derivatives. In general, it can be concluded that these derivatives can serve as promising leads to the design of potential α-glucosidase inhibitors.

New thioxothiazolidinyl-acetamides derivatives as potent urease inhibitors: design, synthesis, in vitro inhibition, and molecular dynamic simulation.

To identify potent urease inhibitors, in the current study, a series of thioxothiazolidinyl-acetamides were designed and synthesized. The prepared compounds were characterized by spectroscopic techniques, including FTIR, 1HNMR, 13CNMR, and elemental analysis. In the enzymatic assessments, it was demonstrated that all derivatives had significant urease inhibition with IC50 values in the range of 1.473-9.274 µM in comparison with the positive control hydroxyurea (IC50 = 100.21 ± 2.5 µM) and thiourea (IC50 = 23.62 ± 0.84 µM). Compound 6i (N-benzyl-3-butyl-4-oxo-2-thioxothiazolidine-5-carboxamide) was the most active agent with an IC50 value of 1.473 µM. Additionally, kinetic investigation and in silico assessments of 6i was carried out to understand the type of inhibition and behavior of the most potent derivative within the binding site of the enzyme. Noteworthy, the anti-urease assay against P. vulgaris revealed 6e and 6i as the most active agents with IC50 values of 15.27 ± 2.40 and 17.78 ± 3.75 µg/mL, respectively. Antimicrobial evaluations of all compounds reveal that compounds 6n and 6o were the most potent antimicrobial agents against the standard and resistant S. aureus. 6n and 6o also showed 37 and 27% inhibition in the development of biofilm by S. aureus at 512 µg/ml. Furthermore, the MTT test showed no toxicity up to 100 µM. Taken together, the study suggests that the synthesized thioxothiazolidinyl-acetamides bases derivatives may serve as potential hits as urease inhibitors.

Quantitative analysis of in vitro biofilm formation by clinical isolates of dermatophyte and antibiofilm activity of common antifungal drugs.

BACKGROUND: The ability of dermatophytes to develop biofilm, as one of the virulence factors in fungal infections which contribute to antifungal resistance, is an outstanding aspect of dermatophytosis that has been noted recently. Because of the paucity of data about the biofilm formation by dermatophytes and their susceptibility to antifungal drugs, this study evaluated the biofilm formation by clinical isolates of dermatophytes and antibiofilm activity of common antifungals widely used to manage dermatophytosis. METHODS: The ribosomal DNA internal transcribed spacer (ITS) regions sequencing for species identification of 50 clinical dermatophyte isolates was performed. The ability of isolates to form biofilm and inhibitory activity of itraconazole, terbinafine, and griseofulvin against biofilm formation was assayed by the crystal violet staining method. Optical microscopy and scanning electron microscopy (SEM) were applied for the visualization of the biofilm structures. RESULTS: Trichophyton (T.) mentagrophytes (n: 14; 28%) and T. rubrum (n: 13;26%) were included in more than half of the dermatophyte isolates. Biofilm formation was observed in 37 out of 50 (74%) isolates that were classified as follows: nonproducers (n: 13; 26%), weak producers (n: 4; 8%), moderate producers (n: 16; 32%), and strong producers (n: 17; 34%) by comparison of the absorbance of biofilms produced by clinical strains with control. The mean IC50 values for terbinafine, griseofulvin, and itraconazole were 2.42, 3.18, and 3.78 µg/ml, respectively. CONCLUSIONS: The results demonstrated that most of the clinical dermatophyte isolates are capable to form biofilm in vitro with variable strength. Moreover, terbinafine can be suggested as the first-line choice for the treatment of biofilm-formed dermatophytosis.

Time and cost-efficient identification of Trichophyton indotineae.

BACKGROUND: During the past 5 years, an outbreak of recalcitrant dermatophytosis due to a novel Trichophyton species generally resistant to terbinafine, T. indotineae, has spread out from South Asia to many countries around the World. These isolates cannot be reliably differentiated from other Trichophyton spp. on the basis of morphological traits and the current laboratory diagnostics relies on sequencing of ribosomal DNA ITS region. OBJECTIVES: In this study, we aimed to introduce two inexpensive and rapid PCR-based assays for differentiation between T. indotineae and other dermatophytes. METHODS: The first introduced assay is based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, involving the amplification of TOP2 sequences and the digestion of PCR products by Cfr13I restriction enzyme. The second assay is proposed as conventional endpoint species-specific PCR amplification of the C120-287 intergenic locus. To validate the assays, a total of 191 Trichophyton spp. and 2 Microsporum canis strains with known ITS region sequences were used. From the T. mentagrophytes / T. interdigitale species complex (TMTISC), strains with 18 different ITS genotypes were tested. The sample of TMTISC isolates included 41 T. indotineae strains. RESULTS: TOP2 PCR-RFLP and T. indotineae-specific PCR were positive with testing on DNA of all 41 T. indotineae isolates and two strains of T. mentagrophytes belonging to ITS Types XIII and XVI, but negative with other species and other TMTISC ITS genotypes (n = 152). Therefore, the specificity of both new assays was 99%. CONCLUSION: The two developed diagnostic assays provide accurate and cost-effective means of identifying cultured T. indotineae isolates.