Interaction of carvacroland voriconazole against drug - resistant Candida strains isolated from patients with candidiasis.

OBJECTIVE: Terpenoid phenols, especially 5-isopropyl-2-methylphenol (carvacrol), are components of plant essential oils that exhibit potent antifungal activity against a wide range of pathogens, including Candida species. The purpose of this study was to determine the interaction activity of carvacrol in combination with voriconazole against Candida albicans (C. albicans), C. glabrata and C. krusei isolates. MATERIALS AND METHODS: The minimum inhibitory and fungicidal concentrations (MICs and MFCs) of carvacrol and voriconazole were determined against various Candida species isolated from patients with candidiasis using the Clinical Laboratory Standards Institute (CLSI) M27-A2 broth microdilution method. The nature of the interaction was studied from fractional inhibitory concentration indices (FICIs) for carvacrol plus voriconazole combination calculated from checkerboard microdilution assay. RESULTS: Carvacrol presented an antifungal effect, with mean MICs of 66.87µg/mL for C. albicans, 75µg/mL for C. glabrata and 95µg/mL for C. krusei isolates. The mean MICs of voriconazole against C. albicans, C. glabrata and C. krusei isolates were 0.087, 1.25 and 0.35µg/mL, respectively. Carvacrol in combination with voriconazole exhibited the synergistic anti-Candida effects against all species of Candida tested. FICI values for carvacrol plus voriconazole combination ranged from 0.370 to 0.853 for C. albicans isolates, 0.412 to 0.625 for C. glabrata isolates, and 0.474 to 0.748 for C. krusei isolates. No antagonistic activity was seen in the strains tested. CONCLUSION: From these results we suggest that carvacrol has great potential as antifungal, and that voriconazole can be supplemented with carvacrol to inhibit clinical Candida isolates.

Potential effect of 2-isopropyl-5-methylphenol (thymol) alone and in combination with fluconazole against clinical isolates of Candida albicans, C. glabrata and C. krusei.

Limitations of antifungals used in the treatment of candidiasis, as the development of resistant strains, are known by the scientific community. In this context, the aim of this study was to investigate the activity of 2-isopropyl-5-methylphenol (thymol) in combination with fluconazole (FLZ) against clinical Candida strains. The antifungal activity of thymol along with FLZ was evaluated by the Clinical Laboratory Standards Institute (CLSI) M27-A2 broth microdilution method. In addition, synergism was observed for clinical strains of Candida spp. with combination of thymol-FLZ evaluated by the chequerboard microdilution method. The mean of minimum inhibitory concentration (MIC) values of thymol and FLZ were 49.37 and 0.475µg/ml for C. albicans, 51.25 and 18.80µg/ml for C. glabrata and 70 and 179.20µg/ml for C. krusei strains, respectively. Thymol in combination with FLZ exhibited the synergistic effects against all species of Candida tested. FICI values for thymol plus FLZ ranged from 0.366 to 0.607 for C. albicans strains, 0.367 to 0.482 for C. glabrata strains, and 0.375 to 0.563 for C. krusei strains. No antagonistic activity was seen in the strains tested. Thymol was found to have a fungicidal effect on Candida species and a synergistic effect when combined with FLZ.

Chemical composition, antioxidant activity and antifungal effects of five Iranian essential oils against Candida strains isolated from urine samples.

Systemic candidiasis has become an emerging fungal infection in recent years. Anti-Candida resistance to conventional antifungal agents has subsequently increased. This study reported the chemical composition, antioxidant and anti-Candida activity of Origanum majorana, Artemisia dracunculus, Cymbopogon citrate, Cinnamomum verum and Caryophyllus aromaticus essential oils. Different Candida species, from urine tracts of hospitalized patients, were included to be challenged with understudied essential oils. Chemical compositions were determined using gas chromatography/mass spectroscopy (GC/MS) analysis and antioxidant activity was measured using DDPH assay. MIC of these essential oils was evaluated using broth micro-dilution test. Caryophyllus aromaticus had the highest antioxidant activity while the lowest antioxidant activity was for Artemisia dracunculus. MICs of Cinnamomum verum, Caryophillium aromaticus, Artemisia dracunculus, Origanum vulgare and Cymbopogon citratus essential oils ranged from 125 to 175µg/mL (mean value: 147.7±25.5µg/mL), 700 to 1000µg/mL (mean value: 740.9±105.4µg/mL), 1000 to 2000µg/mL (mean value: 1454.5±509.6µg/mL), 173 to 350µg/mL (mean value: 208±55.8µg/mL) and 125 to 175µg/mL (mean value: 156.8±24.6µg/mL) for different Candida species, respectively. In general, natural compounds are suitable to be used as anti-Candida and antioxidant agents. However in this stage, these compounds could be applied as supplementary agents along with conventional antifungal drugs.

Fungicidal efficacy of various honeys against fluconazole-resistant Candida species isolated from HIV+ patients with candidiasis.

OBJECTIVE: Honey is well known to possess a broad spectrum of activity against medically important organisms. The purpose of this study was to assess the antifungal activity of different honeys against 40 fluconazole (FLU) resistant Candida species, including Candida albicans (C. albicans), Candida glabrata, Candida krusei and Candida tropicalis. MATERIALS AND METHODS: Three honey samples were collected from northern (Mazandaran, A), southern (Hormozgan, B) and central (Lorestan, C) regions of Iran. A microdilution technique based on the CLSI, M27-A2 protocol was employed to compare the susceptibility of honeys "A", "B" and "C" against different pathogenic Candida isolates. RESULTS: The results showed that different Candida isolates were resistant to FLU, ranging from 64µg/mL to 512µg/mL. All of the honeys tested had antifungal activities against FLU-resistant Candida species, ranging from 20% to 56.25% (v/v) and 25% to 56.25% (v/v) for minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs), respectively. Honey "A" (MIC: 31.59%, v/v) showed higher anti-Candida activity than honey "B" (MIC: 35.99%, v/v) and honey "C" (MIC: 39.2%, v/v). No statistically significant differences were observed among the mean MIC values of the honey samples (P>0.05). The order of overall susceptibility of Candida species to honey samples were; C. krusei>C. glabrata>C. tropicalis>C. albicans (P>0.05). In addition, the mean MICs of Candida strains isolated from the nail, vagina and oral cavity were 33.68%, 36.44% and 39.89%, respectively, and were not significantly different (P>0.05). CONCLUSION: Overall, varying susceptibilities to the anti-Candida properties of different honeys were observed with four FLU-resistant species of Candida. Further research is needed to assess the efficacy of honey as an inhibitor of candidal growth in clinical trials.

Presence and distribution of yeasts in the reproductive tract in healthy female horses.

BACKGROUND: Yeasts are commensal organisms found in the reproductive and gastrointestinal tracts, and on the skin and other mucosa in mammals. OBJECTIVES: The purpose of this study was to isolate and identify yeast flora in the caudal reproductive tract in healthy female horses. STUDY DESIGN: Longitudinal study. METHODS: A total of 453 samples were collected using double-guarded swabs from the vestibule, clitoral fossa and vagina in 151 horses. All samples were cultured on Sabouraud 4% dextrose agar and incubated at 35°C for 7-10 days. Isolates were identified according to their morphological characteristics and biochemical profiles. RESULTS: Yeast colonies were isolated from 60 (39.7%) of the 151 horses. The isolated yeasts belonged to nine genera, and included Candida spp. (53.2%), Cryptococcus spp. (12.2%), Saccharomyces spp. (10.5%), Geotrichum spp. (8.0%), Rhodotorula spp. (7.1%), Malassezia spp. (3.7%), Trichosporon spp. (2.6%), Kluyveromyces spp. (2.6%) and Sporothrix spp. (0.2%). Candida krusei (43.1%) was the most frequent Candida species isolated. There was a significant difference in prevalence between C. krusei and other Candida species (P<0.05). The vestibule contained more yeast isolates (48.0%) than the vagina (18.3%). The isolation of yeast colonies from multiparous females (76.8%) was significantly higher than from maiden mares (P<0.05). MAIN LIMITATIONS: The study was limited by the difficulty of distinguishing between normal flora and potential pathogens. CONCLUSIONS: Candida spp., in particular C. krusei, represent important flora resident in the caudal reproductive tract in healthy female horses. This is particularly important in contexts that require the initiation of empirical treatment prior to the completion of culture results.

Antifungal activity and influence of propolis against germ tube formation as a critical virulence attribute by clinical isolates of Candida albicans.

OBJECTIVE: The potentiality to switch between yeast and hyphal morphologies plays an important role in the virulence of Candida albicans (C. albicans). The objective of the present study was to evaluate the chemical composition, antifungal activity and influence of propolis upon germ tube formation of C. albicans clinical isolates. METHODS: The major compounds of propolis ethanolic extract (PEE) was revealed by gas chromatography-mass spectrometry (GC/MS) analysis. Broth microdilution method was used for determination of minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of this extract. C. albicans isolates were exposed to sub-MIC concentrations of propolis and germ tube reduction percent (GRP) and morphological transition from yeast to filamentous form were monitored microscopically. RESULTS: Phenolic compounds, aromatic acids, aliphatic acids, sugars and polycyclic aromatic hydrocarbons were the major compound classes in PEE. Mean of MIC and MFC values of PEE against C. albicans isolates were 360.6µg·mL-1 and 1250.1µg·mL-1, respectively. The increase in concentration led to a significant reduction in germ tube formation, so mean of GRP was 36.7±11.1 and 22±10.1 at concentration of ½ MIC and » MIC of PEE, respectively. CONCLUSIONS: In conclusion, the present study indicated that PEE has interesting potential as a therapeutic agent that targets germ tube formation by C. albicans as a critical virulence factor.

Evaluation of antioxidant and antifungal properties of the traditional plants against foodborne fungal pathogens.

OBJECTIVE: To determine the antioxidant and antifungal activities of the essential oils from five aromatic herbs, including Thymus vulgaris, Chamaemelum nobile, Ziziphora clinopodioides, Zingiber officinale and Cuminum cyminum, against different Aspergillus and Penicillium species. METHODS: The oils were subjected to screening for their possible antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The susceptibility test for the oils was carried out in terms of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) using microdilution method. RESULTS: The values of the essential oils in DPPH assay were as follows: T. vulgaris (450.11±5.23 µg/mL), Ch. nobile (602.73±4.8 µg/mL), Ziz. clinopodioides (1238.82±9.3 µg/mL), Cu. cyminum (1255.52±8.92 µg/mL) and Zin. officinale (5595.06±8.24 µg/mL). Our findings also indicated a strong activity against tested fungi for the oil of T. vulgaris (1250 µg/mL), followed by Cu. cyminum (1416 µg/mL), Zin. officinale (1833 µg/mL), Ziz. clinopodioides (2166 µg/mL) and Ch. nobile (3750 µg/mL). This study confirmed the excellent antifungal and antioxidant properties of the essential oils, especially T. vulgaris, against foodborne pathogenic fungi. CONCLUSION: Owing to their strong protective features, these oils could be used in ethno-medicine as preventers of lipid peroxidation and cellular damage, and in food industries as preservers of foodstuffs against spoilage fungi. Also, they could be the candidates to develop new antibiotics and disinfectants to control infective agents.

Lactic acid bacteria as functional probiotic isolates for inhibiting the growth of Aspergillus flavus, A. parasiticus, A. niger and Penicillium chrysogenum.

OBJECTIVE: The aim of this study was to assess the potential of lactic acid bacteria (LAB) such as Lactobacillus acidophilus, L. rhamnosus, L. casei, L. paracasei and Bifidobacterium bifidum to inhibit the outgrowth of some common food-spoiling fungi including Aspergillus niger, A. flavus, A. parasiticus and Penicillium chrysogenum. METHODS: Bacterial isolates were cultured on Mann Rogosa Sharpe (MRS) broth and liquid cultures and supernatants were prepared. The antifungal activity was tested using the agar well diffusion method. RESULTS: Both liquid culture and supernatant of L. casei isolate exhibited high antifungal activity, followed by L. acidophilus and L. paracasei isolates. The least activity was recorded for the isolates B. bifidum, while the isolate L. rhamnosus was moderately active against tested fungi. The antifungal activity of the supernatants obtained from all probiotic isolates against fungi was significantly less than that of liquid cultures (P<0.05). Antifungal activity evaluation showed that A. flavus was the most inhibited fungus by probiotic bacteria, followed by P. chrysogenum, A. niger and A. parasiticus. CONCLUSION: These results suggest that probiotic bacteria strains have the ability to prevent the growth of pathogenic and mycotoxigenic fungi as antifungal agents for various biomedical applications.

A study of onychomycosis in patients attending a dermatology center in Tehran, Iran.

OBJECTIVE: To determine the incidence of onychomycosis based on age and sex, morphological pattern of the disease, predisposing factors and identification of fungus by direct microscopy and culture methods. METHODS: A prospective study was conducted on 140 patients with nail disorders. A detailed history and thorough examination was done in all patients. The samples were taken from patients clinically suspected of fingernails and toenails infections attending a dermatology center in Tehran, Iran. The nails were subjected to potassium hydroxide (KOH) examination and fungal culture on Sabouraud's dextrose agar (SDA) medium. RESULTS: Specimens from 79 patients (56.4%) were positive for onychomycosis. The mycological observations showing positive fining with KOH were observed in 79 (56.4%) and culture positive in 35 (25%) cases. Females were more infected than males. The most common age group infected was 41-60 years (40.7%). Toenails were affected more frequently than fingernails and dystrophic onychomycosis was the most common clinical type seen in 39.2% patients. From the culture-positive samples, yeasts were the most common pathogens isolated from 25 (71.4%) patients, followed by non-dermatophytic moulds in 6 (17.1%) and dermatophytes in 4 (11.5%) patients. CONCLUSION: This study demonstrated that Candida species were the main agents causing onychomycosis in our region and accurate diagnosis of onychomycosis was based on direct microscopy and fungal culture.

Antifungal effect of Trachyspermum ammi against susceptible and fluconazole-resistant strains of Candida albicans.

OBJECTIVE: Trachyspermum ammi (T. ammi) has been known as having many therapeutic properties and its antimicrobial activity has currently received a renewed interest. This study aimed to verify the effectiveness of T. ammi essential oil to inhibit the growth of Candida albicans (C. albicans) strains isolated from HIV(+) patients with oropharyngeal candidiasis (OPC). MATERIALS AND METHODS: The essential oil was obtained by hydrodistillation in a Clevenger apparatus and analyzed by gas chromatography. Susceptibility tests were expressed as inhibition zone by the disk diffusion method and minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) by the broth microdilution method. RESULTS: Thymol (63.4%), p-cymene (19%) and γ-terpinen (16.9%) were found as the most abundant constituents. The disk diffusion results revealed that 67% of oral C. albicans isolates were susceptible, 9% susceptible-dose dependent and 24% resistant to fluconazole. In the broth microdilution method, 68% of isolates were susceptible, 5% susceptible-dose dependent and 27% resistant to fluconazole. The increase in concentration led to a significant reduction in yeasts that were growing in exponential phase. In addition, with increasing in T. ammi oil concentration, the time of remaining cells in lag phase was significantly increased. CONCLUSION: This study showed that all clinical C. albicans isolates were susceptible to T. ammi essential oil, indicating a significant reduction in the yeast growth in exponential phase.

Antifungal efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth of food-relevant fungi.

OBJECTIVE: This work is an attempt to examine the antifungal activity of thymol, carvacrol, eugenol and menthol against 11 food-decaying fungi. METHODS: The susceptibility test for the compounds was carried out in terms of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) using microdilution method in 96 multi-well microtiter plates. RESULTS: Results indicated that all compounds were effective to varying extents against various fungal isolates, with the highest efficacy displayed by carvacrol (mean MIC value: 154.5 µg/mL) (P<0.05). The incorporation of increased concentrations of all compounds to the media led to progressive and significant reduction in growth for all fungi. The most potent inhibitory activity of thymol, carvacrol, eugenol and menthol was found for Cladosporium spp. (MIC: 100 µg/mL), Aspergillus spp. (MIC: 100 µg/mL), Cladosporium spp. (MIC: 350 µg/mL), and Aspergillus spp. and Cladosporium spp. (MIC: 125 µg/mL), respectively. CONCLUSION: Thus, the application of these herbal components could be considered as a good alternatives to inhibit fungal growth and to reduce the use of synthetic fungicides.

Ocular fungal flora from healthy horses in Iran.

OBJECTIVE: This study was carried out in order to isolate and identify the normal conjunctival fungal flora from Caspian miniature, Thoroughbred, Turkmen and Persian Arab breeds in Tehran, Iran. MATERIALS AND METHODS: A total of seventy-two adult healthy horses were studied. Ocular samples were collected from right and left eyes by using sterile cotton swabs; samples were cultured on Sabouraud dextrose agar and incubated at 30°C for 7-10 days. Molds and yeasts were identified using macro and micro-morphological and physiological characteristics. RESULTS AND CONCLUSION: Number of fungal colonies per eye varied between 0 and 123 colony forming units (CFUs). The most predominant fungal isolates were Aspergillus (19.9%), Rhizopus (15.9%) and Penicillium (15.1%). No significant differences were observed between types of eye fungal floras in different breeds. Caspian miniature horses had significantly the highest number of fungal isolates in compare with other breeds (P<0.001), however no significant difference was observed among other breeds under study. The fungal isolates were almost the same as with studies performed in other countries, although differences in species isolated could be related to geographic and climate difference.

Oral microflora and their relation to risk factors in HIV+ patients with oropharyngeal candidiasis.

OBJECTIVE: The purpose of this study was to determine the prevalence of oral microflora and association of oral candidiasis and multiple risk factors in HIV(+) patients. PATIENTS AND METHODS: The present study included 100 HIV-infected patients participated in Imam Khomeini Hospital, Tehran, Iran for Oropharyngeal candidiasis (OPC) and HIV. We assessed the presence or absence of OPC, and samples were obtained from the oral cavity and direct microscopic examination, gram staining and culture on standard microbiological media were performed in all patients. CD4(+) cell count/CD4(+) percentage were also calculated. RESULTS: The demographic characteristics showed that the patients had a mean age of 32.3 years old, 78% male and 22% female. Patients belonging to 'O(+)' blood group (27%) were more prone to develop OPC. A total of 460 bacterial colonies were obtained and Streptococcus mutans (15.4%) was the most frequently isolated species in the HIV(+) patients, followed by Staphylococcus epidermidis (12.8%) and Corynebacterium (8.7%). In addition, 254 yeasts (from four different genera) were isolated from the patient under study. Candida species (94.4%) were the most frequently obtained genera, followed by Saccharomyces (2.4%), Kluyveromyces and Cryptococcus (1.6% for both) species. Candida albicans (37.2%) was the most common species isolated from HIV(+) patients with OPC and its frequency was significantly higher than that of other Candida species (P<0.05). Candida glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, C. lusitaniae, C. guilliermondii and C. norvegensis were also identified. Forty percent of the patients had angular cheilitis as the most frequent clinical variant. The mean CD4(+) cell counts were 154.5 cells/µL, with a range of 8 to 611 cells/µL. Thirty percent patients had a CD4(+) cell count between 101 and 200 cells/µL (28.7% of total yeasts isolated). Yeast and bacteria counts did not differ statistically among HIV(+) patients' subgroups with different levels of CD4(+) cells counts. CONCLUSION: Our results showed that yeasts of the genus Candida were isolated at a comparable rate from the oral cavity of HIV(+) patients and there was no significant difference of the variables CD4(+) cell count and yeast counts. The findings of this study would be helpful in any further study, which, if done prospectively on a large cohort, can be confirmatory.

Severe tinea corporis due to Trichophyton verrucosum mimicking discoid lupus erythematosus.

OBJECTIVE: This study was carried out to show possible similarities between discoid lupus erythematosus clinical signs and histopathological findings and dermatophytosis. PATIENT AND METHODS: A 43-year-old woman with diffused erythematous scaling lesions on her neck was studied. Skin biopsy was performed using H&E and PAS staining. Direct microscopic examination and mycological culture of specimens were done as well. RESULTS: Histopathological examination suggested she was suffering from discoid lupus erythematosus; however mycological findings confirmed Trichophyton verrucosum as the etiologic agent of lesions. She was successfully treated with oral terbinafine and clotrimazole cream. CONCLUSION: Regarding to the results of this study, there are some similarities between dermatophytosis and skin infections such as discoid lupus erythematosus.

Anti-Candida zeylanoides activity of some Iranian plants used in traditional medicine.

OBJECTIVE: To assess anti-Candida zeylanoides activity of the essential oils (EOs) of five Iranian medicinal plants and to determine the different components of the EOs. METHODS: Anti-C. zeylanoides effects of the EOs and reference drugs were determined by disc diffusion method. The EOs from Trachyspermum copticum, Zataria multiflora, Nigella sativa, Ziziphora clinopodiodes and Heracleum persicum were analyzed by gas chromatography/mass spectroscopy (GC/MS). RESULTS: The mean values of inhibition zones were found to be more than 60mm for T. copticum, 56.7 mm for Z. multiflora, 40.8mm for N. sativa, 33.7 mm for Z. clinopodiodes and 18.7 mm for H. persicum. In GC/MS analysis, thymol (63.4%), carvacrol (61%), trans anthol (39%), pulegone (37%) and hexyl butyrate (30.2%) were found to be the major components of T. copticum, Z. multiflora, N. sativa, Z. clinopodiodes and H. persicum, respectively. CONCLUSION: The EOs showed strong anti-C. zeylanoides activities, which strengthen the potential use of these substances for the treatment of candidiasis.

Acidic residue modifications restore chaperone activity of ß-casein interacting with lysozyme.

An important factor in medicine and related industries is the use of chaperones to reduce protein aggregation. Here we show that chaperone ability is induced in ß-casein by modification of its acidic residues using Woodward's Reagent K (WRK). Lysozyme at pH 7.2 was used as a target protein to study ß-casein chaperone activities. The mechanism for chaperone activity of the modified ß-casein was determined using UV-vis absorbencies, fluorescence spectroscopy, differential scanning calorimetry and theoretical calculations. Our results indicated that the ß-casein destabilizes the lysozyme and increases its aggregation rate. However, WRK-ring sulfonate anion modifications enhanced the hydrophobicity of ß-casein resulting in its altered net negative charge upon interactions with lysozyme. The reversible stability of lysozyme increased in the presence of WRK-modified ß-casein, and hence its aggregation rate decreased. These results demonstrate the enhanced chaperone activity of modified ß-casein and its protective effects on lysozyme refolding.