Antifungal activity and mechanism of action of dichloromethane extract fraction A from Streptomyces libani against Aspergillus fumigatus.

AIMS: This study aimed to investigate the mechanism of antifungal action of Streptomyces libani dichloromethane extract fraction A (DCEFA) against Aspergillus fumigatus and the host cytotoxicity. METHODS AND RESULTS: DCEFA was purified from S. libani by autobiography and showed strong antifungal activity against A. fumigatus. A combination of electron microscopy, cell permeability assays, total oxidant status (TOS) assay, cell cytotoxicity assay and haemolysis activity was carried out to determine the target site of DCEFA. Exposure of A. fumigatus to DCEFA caused the damage to membranous cellular structures and increased release of cellular materials, potassium ions and TOS production. DCEFA was bound to ergosterol but did not affect fungal cell wall and ergosterol content. DCEFA did not show any obvious haemolytic activity for RBCs and toxicity against HEK-293 cell line. CONCLUSIONS: DCEFA may inhibit A. fumigatus growth by targeting fungal cell membrane which results in the leakage of potassium ions and other cellular components, TOS production and final cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: DCEFA of S. libani could be considered as a potential source of novel antifungals which may be useful for drug development against A. fumigatus as a life-threatening human pathogen.

Mycotoxin-producing ability and chemotype diversity of Aspergillus section flavi from soils of peanut-growing regions in iran.

Invasion of crops with Aspergillus flavus may result in contamination of food and feed with carcinogenic mycotoxins such as aflatoxins (AF) and cyclopiazonic acid (CPA). In the present study, distribution and toxigenicity of Aspergillus flavus and A. parasiticus in soils of five peanut fields located in Guilan province, Northern Iran was investigated. From a total of 30 soil samples, 53 strains were isolated which all of them were finally identified as A. flavus by a combination of colony morphology, microscopic criteria and mycotoxin profiles. Chromatographic analysis of fungal cultures on yeast extract sucrose broth by tip culture method showed that 45 of the 53 A. flavus isolates (84.9 %) were able to produce either CPA or AFB1, while eight of the isolates (15.1 %) were non-toxigenic. The amounts of CPA and AFB1 produced by the isolates were reported in the range of 18.2-403.8 µg/g and 53.3-7446.3 µg/g fungal dry weights, respectively. Chemotype classification of A. flavus isolates based on the ability for producing mycotoxins and sclerotia showed that 43.4 % were producers of CPA, AFB1 and sclerotia (group I), 13.2 % of CPA and AFB1 (group II), 9.4 % of AFB1 and sclerotia (group III), 13.2 % of AFB1 (group IV), 5.7 % of CPA and sclerotia (group V) and 15.1 % were non-toxigenic with no sclerotia (group VI). No strain was found as producer of only CPA or sclerotia. These results indicate different populations of mycotoxigenic A. flavus strains enable to produce hazardous amounts of AFB1 and CPA are present in peanuts field soils which can be quite important regard to their potential to contaminate peanuts as a main crop consumed in human and animal nutrition.

Genotyping of Candidaalbicans isolates from oropharyngeal candidiasis in head and neck cancer patients in Iran: Molecular epidemiology and SAP2 gene expression.

OBJECTIVE: Multilocus sequence typing is a powerful method for genotyping of clinical isolates of Candidaalbicans. Cross-contamination between the patients is an important reason for nosocomial infections. Oropharyngeal candidiasis (OPC) caused by C. albicans is an important problem in patients with head and neck cancer, in Cancer Institute of Tehran. Here we studied the endemic genotypes of C. albicans isolates and the relationship between geographic distributions, potential cross-contaminations and the expression of SAP2 gene, an important gene in oral candidiasis, with MLST groups. MATERIAL AND METHODS: A total of 32 clinical strains of C. albicans isolated from head and neck cancer patients with oropharyngeal candidiasis were subjected to MLST analysis and SAP2 gene expression was analyzed by Real-time PCR. RESULTS: We identified 75 polymorphic sites in 7 loci of C. albicans isolates and 30 diploid sequence types which 27 of them were found as new. After eBURST analysis, our results determined that CC 124 was the most prevalent group among all CCs. SAP2 gene showed high expression in almost all OPC patients' isolates, compared to the control. CONCLUSION: We found few genetically-related as well as identical isolates among the 32 Candida strains which indicated low cross-contaminations among the patients. There was no relationship between C. albicans MLST profiles and their geographic distribution, cancer type and SAP2 gene expression. This lack of correlation was possibly due to the small understudy population; hence, finding more relevance requires studies with a higher number of samples.

Comparative analysis of proteinase, phospholipase, hydrophobicity and biofilm forming ability in Candida species isolated from clinical specimens.

Candida species are the commensal organisms of human and animal mucosa that cause a wide range of debilitating diseases in immunocompromised patients and other susceptible individuals. The present study aimed to investigate the ability of clinical isolates of various Candida species to produce proteinase and phospholipase, hydrophobicity and biofilm forming ability that assumed to have a vital role in Candida pathogenicity. Eighty-four Candida strains belonged to Candida albicans (44.1%), C. glabrata (5.9%), C. guilliermondii (5.9%), C. krusei (10.8%), C. parapsilosis (26.2%), and C. tropicalis (7.1%) were examined for proteinase and phospholipase production, cell surface hydrophobicity and biofilm forming ability. The production of proteinase and phospholipase was detected in 81 (96.4%) and 79 (94.1%) of the strains, respectively. C. albicans showed the highest proteinase and phospholipase activity (mean Pz values of 0.42±0.25 and 0.72±0.28) and biofilm formation ability (0.66±0.22). C. parapsilosis had the highest hydrophobicity (42.97±16.1), which showed a good correlation with biofilm formation ability. A considerable percentage of non-albicans Candida strains produced significant amounts of proteinase and phospholipase with a good ability of biofilm formation in vitro. Taken together, our results further substantiated that enzymatic activity, hydrophobicity and the ability for biofilm formation are important virulence factors which may be account for pathogenicity of various Candida species distributed in albicans and non-albicans groups.

Molecular characterization of Aspergilli isolated from outdoor air.

Ubiquitous airborne conidia of the genus Aspergillus are responsible for a diverse group of human disorders from allergy to life treating invasive aspergillosis and mycotoxicoses. The aim of this study was to determine the population structure of Aspergillus isolated from outdoor air in Tehran by comparing the nucleotide sequences of ITS region and the PCR-RFLP molecular method. Internal transcribed spacer domains of 47 Aspergillus spp. were amplified and sequenced and PCR products were digested individually with restriction enzymes TaqI and EcoRI. For all species the PCR reaction produced a fragment of approximately 600bp in length. All of the nucleotide sequences were highly similar with the corresponding reference sequences registered at the gene bank. The all isolates displayed same banding pattern on the basis EcoR1 cleavage. While Taq1 enzyme profiling provided 5 different banding pattern. The results show that the A. niger section has the highest frequency with 27 isolates (57.4%). Of these, 23 isolates (48.9%) belonged to the A. niger complex and 4 isolates (8.5%) to the A. aculeatus complex. The A. flavus complex was also placed in the next ranking with 9 isolates (19.1%). These results strongly support the need for using molecular markers as an auxiliary tool in differentiating Aspergillus species.

Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile.

OBJECTIVE: Non-Candida albicans (NCA) species now account for a significant part of clinical candidiasis worldwide. In the present study, epidemiology and antifungal susceptibility profile of NCA isolated from various forms of candidiasis were studied with special focus on their phylogenetic relationship by ITS sequencing. PATIENTS AND METHODS: Seventy-nine NCA isolates were isolated from skin and nail scrapings (67.0%), vaginal discharges (8.8%), blood (8.8%), sputa (5.0%), urine (5.0%), oral swabs (2.6%), biopsy and eye tumor, each (1.4%). These isolates were identified by morphological, biochemical and molecular (ITS sequencing) techniques. In vitro antifungal susceptibility of the isolates to fluconazole (FCZ) was tested according to the CLSI method (M27-S4). RESULTS: Among a total number of 79 cases of proven NCA infections, C. parapsilosis (36.8%) was the most prevalent species followed by C. glabrata (32.9%), C. orthopsilosis (11.4%), C. tropicalis (8.9%), C. krusei (5.0%) and C. guilliermondii (5.0%). The susceptibility to FCZ was assessed for C. parapsilosis (96.5%), C. orthopsilosis (88.9%), C. tropicalis (85.7%) and C. guilliermondii (50.0%). C. glabrata and C. krusei isolates were not susceptible to FCZ. NCA species were distributed in various phylogenetic clades including C. glabrata (1), C. tropicalis (3), C. parapsilosis (6) and C. orthopsilosis, C. krusei and C. guilliermondii (each 2). CONCLUSION: C. parapsilosis and C. glabrata were the most predominant NCA species involve in the etiology of candidiasis. C. orthopsilosis was reported from superficial candidiasis. Taken together, our results further substantiate the increasing importance of the involvement of NCA species in the etiology of candidiasis.

Oropharyngeal candidiasis in head and neck cancer patients in Iran: Species identification, antifungal susceptibility and pathogenic characterization.

OBJECTIVE: Oropharyngeal candidiasis (OPC) is the most frequent opportunistic fungal infection in head and neck cancer patients. This study was done to identify the Candida species, which cause OPC, and to evaluate their antifungal susceptibility pattern and pathogenic characteristics in Iranian head and neck cancer patients treated by radiotherapy. MATERIAL AND METHODS: The oral clinical samples were determined by culturing on CHROMagar, carbohydrate assimilation and ITS sequencing methods. Biofilm formation, phospholipase and proteinase activity and antifungal susceptibility were examined too. RESULTS: Among 54 patients with confirmed OPC, 39 (72.22%) patients were male and 15 (27.77%) were female. The most frequently Candida species from a total of 60 isolates was C. albicans (53.3%), followed by C. tropicalis (21.66%), C. glabrata (15%), C. kefyr (5%) and C. dubliniensis (1.66%). All the isolates were high-producers of biofilm. All of Candida isolates were proteinase positive and 47 isolates (81.04%) represented phospholipase activity. The maximum and minimum rates of antifungal resistance belonged to ketoconazole (93.75% of C. albicans and 89.28% of Candida non-albicans) and fluconazole (62.50% and 42.85% of C. albicans and Candida non-albicans), respectively. The most effective antifungal against all candida isolates was fluconazole. CONCLUSION: Our data can estimate abundance of OPC in male and female head and neck cancer patients and is helpful to use effective strategies for antifungal treatment, prophylaxis, and preventive therapies in these patients.

Epidemiological trends of dermatophytosis in Tehran, Iran: A five-year retrospective study.

OBJECTIVE: Dermatophytosis is the most frequent fungal infection all over the world and its frequency is constantly increasing. The aim of this study was to evaluate clinical features and epidemiological trends of dermatophytosis over the years 2010 to 2014 in Tehran, Iran. PATIENTS AND METHODS: A total of 13,312 patients clinically suspected of cutaneous fungal infections were examined. Skin scales, plucked hairs, nail clippings and sub-ungual debris were examined by direct microscopy and culture. Dermatophyte species were identified at the species level by a combination of morphological and physiological criteria. RESULTS: Direct microscopy confirmed a contamination rate of 19.7% (2622/13,312 cases) of which 1535 cases (58.5%) were culture positive distributed in male (1022 cases) and female (513 cases). The most commonly infected age group was the 30-39 years old. Tinea pedis (30.4%) was the most prevalent type of dermatophytosis followed by tinea cruris (29.8%) and tinea corporis (15.8%). Epidermophyton floccosum (31%) was the most prevalent causative agent, followed by Trichophyton rubrum (26.2%) and Trichophyton mentagrophytes (20.3%). CONCLUSION: Our results showed considerable distribution of dermatophytosis from zoophilic, anthropophilic and geophilic species among population with diverse age groups. Although anthropophilic fungi such as T. mentagrophytes, E. floccosum, and T. rubrum were the main etiologic agents of dermatophytosis, the prevalence of T. verrucosum showed a meaningful increase over the years, which highlights the importance of rural dermatophytosis mainly transmitted from large animals. This noticeable information improves our current knowledge about dermatophytosis and assists to establish effective prevention and therapeutic strategies to overcome the disease.

The predatory capability of Arthrobotrys cladodes var. macroides in the control of Haemonchus contortus infective larvae.

One hundred compost samples were examined for the presence of nematophagous fungi on the sheep farms of Mazanderan, province, Iran. Arthrobotrys cladodes var. macroides (IRAN 677C=CBS 143565) was isolated from 3% of the samples examined. Nematophagous activity of this fungus which was shown for the first time in this study, revealed the addition of 1000, 8000, 20000 and 100000 conidia per gram of feces of sheep reduced significantly (P<0.001) the number of Haemonchus contortus infective larvae in the feces by 41.71%, 63, 27%, 73.49% and 94.96%, respectively. These results show that A. cladodes var. macroides is a promising candidate for biological control of H. contortus.

Antifungal activity of a soil isolate of Pseudomonas chlororaphis against medically important dermatophytes and identification of a phenazine-like compound as its bioactive metabolite.

OBJECTIVE: The increasing importance of dermatophytoses and emerging resistance of dermatophytes to current synthetic antifungals have stimulated the search for safer and more effective alternative drugs from natural sources. The present study was carried out to identify antagonistic bacteria of soil origin with strong inhibitory activities on the growth of major human pathogenic dermatophytes. MATERIALS AND METHODS: Antifungal activity of isolated soil bacteria was screened against the dermatophytes from three genera Microsporum (M. canis, M. gypseum), Epidermophyton (E. floccosum) and Trichophyton (T. mentagrophytes, T. rubrum, T. violaceum, T. tonsurans) by using visual plate agar assay method. A Pseudomonas chlororaphis isolate S105, identified at the species level by 16S ribosomal RNA sequence analysis, was reported as the strongest antagonistic bacterium. P. chlororaphis S105 culture supernatant (PCCS) was examined against tested dermatophytes by GY (glucose-yeast extract) broth bioassay in 6-well microplates. Antifungal compound of the bacterium was partially purified from the culture supernatant through a purification scheme of methanol extraction, Diaion HP20 ion-exchange chromatography and preparative thin layer chromatography. RESULTS: P. chlororaphis S105 was the most potent inhibitor of fungal growth for all tested dermatophytes with a percent inhibition ranged from 57.1% to 99.8%. The PCCS suppressed the growth of all fungi tested in the range of 18.5% to 84.8%. Partially purified antifungal compound of the bacterium was identified as a phenazine-like compound with an Rf value of 0.51. The compound inhibited fungal growth by 73.6% to 97.9% on GY broth. Fungal growth inhibition was significant for all dermatophytes tested in comparison with the controls (Anova, P<0.05). CONCLUSION: With respect to the strong inhibitory activity of P. chlororaphis against pathogenic dermatophytes reported here, it may be considered as a rich source of useful metabolites with potential application in antifungal drug discovery.

Species distribution and antifungal susceptibility of Candida spp. isolated from superficial candidiasis in outpatients in Iran.

OBJECTIVE: Candidiasis is the most prevalent fungal infection affecting human and animals all over the world. This study represents the epidemiological aspects of superficial candidiasis in outpatients and in vitro antifungal susceptibility of etiologic Candida species. PATIENTS AND METHODS: Clinical samples were taken from 173 patients including skin and nail scrapings (107; 61.8%), vaginal discharge (28; 16.2%), sputum (20; 11.6%), oral swabs (7; 4.0%), bronchoalveolar lavage (6; 3.5%) and 1 specimen (0.6%) of each eye tumor, gastric juice, urine, biopsy and urinary catheter and confirmed as candidiasis by direct microscopy, culture and histopathology. Susceptibility patterns of the isolated Candida species were determined using the disk diffusion and broth microdilution methods. RESULTS: Among 173 Candida isolates, C. albicans (72.3%) was the most prevalent species followed by C. parapsilosis (11.5%). Other identified species were C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C. intermedia and C. sake. Majority of the Candida isolates were susceptible to fluconazole (95.4%) followed by 5-flucytosine (89.6%), voriconazole (78.6%) itraconazole (48.0%) and ketoconazole (42.8%). Caspofungin was the most potent antifungal drug against C. albicans (MICs; 0.062-1 µg/mL), ketoconazole for C. parapsilosis and C. tropicalis (MICs; 0.031-0.25 µg/mL) and itraconazole for C. krusei, C. glabrata and C. guilliermondii (MICs; 0.031-1 µg/mL). CONCLUSION: This study reinforces the significance of superficial candidiasis as an important fungal infection with multiple clinical presentations. Our results further indicate that susceptibility testing to commonly used antifungals is crucial in order to select the appropriate therapeutic strategies which minimize complications while improving patients' life.

Rhinocerebral mucormycosis due to Rhizopus oryzae in a diabetic patient: a case report.

Mucormycosis is an opportunistic fulminant fungal infection, which affects nose and paranasal sinuses by direct invasion or through the blood vessels mainly in diabetic and immunocompromised patients. In the present study, a rhino-orbito-cerebral mucormycosis in a 24-year-old female with diabetes mellitus as underlying disease was reported. Computed tomography (CT scan) demonstrated an increase in the soft tissue densities in the left nasal cavity, maxillary and ethmoid sinuses and destruction of the antral wall with soft tissue densities in the pterygopalatine fossa and retromaxillary fissure. In histopathology, wide ribbon-shape non-septate hyphae were seen in tissue sections stained with hematoxylin-eosin (H&E). The etiologic fungus isolated from tissue biopsy on mycological media was identified by the amplification and sequencing of the 5.8S RNA gene and of the adjacent internal transcriber spacer domains, ITS1/ITS4, as Rhizopous oryzae. Recognition of the unique patterns of this high mortality rhinocerebral fungal infection in patients with diabetes is a key to early diagnosis and successful treatment.

Effect of Curcumin on Aspergillus parasiticus Growth and Expression of Major Genes Involved in the Early and Late Stages of Aflatoxin Biosynthesis.

BACKGROUND: The effect of curcumin as a natural safe compound with different biological activities was examined on fungal growth and aflatoxin production in Aspergillus parasiticus NRRL 2999. METHODS: The fungus was cultured in presence of serial two-fold concentrations of curcumin (125-2000 µg/ml) in yeast extract sucrose broth for 3 days at 28°C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography (HPLC). The expression of ver-1, nor-1, pksA, omtA and aflR genes in aflatoxin biosynthetic pathway was evaluated by real time PCR. RESULTS: Curcumin strongly inhibited aflatoxin B(1) production in the range of 26.6 to 94.9% by serial two-fold concentrations from 125 to 2000 µg/ml. Fungal growth was also inhibited by the compound in the range of 34.0 to 60.8%. Analysis of the expression of aflatoxin pathway genes by real time PCR showed that curcumin inhibited the expression of ver-1, nor-1, pksA, omtA and aflR genes at concentrations of 250 and 1000 µg/ml. In concentration of 1000 µg/ml, gene expression was reduced by 31.3%, 44.6%, 57.1% 110.9% and 286.7% accordingly. Reduction in the expression of aflatoxin biosynthesis genes was significant only for aflR. In ferric reducing ability of plasma (FRAP) assay, curcumin showed strong antioxidant activity at all concentrations tested. CONCLUSION: Curcumin may be employed successfully as a good candidate in controlling of toxigenic fungal growth on food and feed and subsequent contamination with aflatoxins in practice.

Chemical composition and antifungal activity of Matricaria recutita flower essential oil against medically important dermatophytes and soil-borne pathogens.

OBJECTIVE: Fungal infections are potential public health threats all over the world. In the present study, effect of Matricaria recutita flower essential oil (EO) was evaluated against medically important dermatophytes and opportunistic saprophytes using microbioassay technique. MATERIALS AND METHODS: Flower essential oil (EO) of M. recutita prepared by hydrodistillation was analyzed by gas chromatography/mass spectrometry (GC/MS). The effect of plant EO on the growth of pathogenic dermatophytes and opportunistic saprophytes was assessed using microbioassay technique. In the bioassay, fungi were cultured in 6-well flat-bottom microplates in presence of various concentrations of plant EO (2.5-1000µg/mL) for 4-10days at 28°C. RESULTS: A total of 14 compounds were identified in the plant oil by GC/MS accounting for 97.5% of the oil composition. The main compound identified was chamazulene (61.3%) followed by isopropyl hexadecanoate (12.7%), trans-trans-farnesol (6.9%) and E-ß-farnesol (5.2%). Growth inhibition for the dermatophytes exposed to serial two-fold concentrations of plant EO (2.5 to 80µg/mL) was reported in the range of 3.24 to 68.15% for Microsporum gypseum, 24.48 to 100% for M. canis, 11.40 to 96.65% for Trichophyton mentagrophytes, 27.79 to 100% for T. rubrum and 45.73 to 100% for T. tonsurans. M. recutita EO inhibited the growth of opportunistic saprophytes by 3.98 to 64.29% for Aspergillus flavus, 6.38 to 93.62% for A. fumigatus, 3.52 to 89.45% for A. niger, 6.38 to 77.66% for Trichoderma harzianum and 17.41 to 89.41% for Fusarium oxysporum in serial two-fold concentrations of 15.62 to 1000µg/mL. CONCLUSION: Results of the present study indicate that M. recutita could be considered as a potential candidate for designing effective antifungal formulations suitable for treatment of dermatophytosis and other fungal infections.

Molecular identification of antagonistic bacteria from Tehran soils and evaluation of their inhibitory activities toward pathogenic fungi.

BACKGROUND AND OBJECTIVES: To find antagonistic bacteria with potential antifungal activity against some pathogenic fungi, including Aspergillus niger, A. flavus, Fusarium moniliforme and Penicillium marneffei, a total of 148 agricultural soil samples from different sites of Tehran were examined. MATERIALS AND METHODS: Antagonistic soils were selected by screening against A. niger on glucose-yeast extract (GY) agar using a visual agar plate assay method. All growing bacteria were examined for antifungal activity, and antagonistic bacteria identified based on 16S rRNA sequence analysis. Among a total number of 97 bacteria isolated form inhibitory soils (36 samples), 16 bacteria were reported as strong growth inhibitors in co-cultures on GY agar with all tested fungi at variable degrees. Fungal growth inhibitory bacteria were cultured against all fungi and growth inhibition was measured and analyzed between test and control groups by statistical analysis (ANOVA). RESULTS: Molecular identification of antagonistic bacteria indicated that most bacterial isolates belonged to the genus Bacillus (81.25%), including B. subtilis (5 isolates), B. amyloliquefaciens (6 isolates) and B. valismortis (2 isolates), followed by one isolate (6.25%) from each Streptomyces sp., Pseudomonas chlororaphis and Acinetobacter baumannii. Based on the visual plate assay results, total fungal growth inhibition of all bacteria was reported in the range of 13.2 to 68.3%. P. chlororaphis S105 was reported as the most potent antagonistic bacterium which inhibited the growth of A. niger by 68.3%, followed by F. moniliforme (66.4%), A. flavus (64.7%) and P. marneffei (57.1%). CONCLUSION: P. chlororaphis and some other inhibitory bacteria reported in the present study, they may be considered not only as a rich source of useful metabolites with potential application in antifungal drug discovery, but also as potential candidates for biological control programs.