Virulence Factors and Azole-Resistant Mechanism of Candida Tropicalis Isolated from Candidemia.

BACKGROUND: Limited knowledge exists on the virulence factors of Candida tropicalis and the mechanisms of azole resistance that lead to an intensified pathogenicity and treatment failure. We aimed to evaluate the virulence factors and molecular mechanisms of azole resistance among C. tropicalis isolated from patients with candidemia. MATERIALS AND METHODS: Several virulence factors, including extracellular enzymatic activities, cell surface hydrophobicity (CSH), and biofilm formation, were evaluated. Antifungal susceptibility pattern and expression level of ERG11, UPC2, MDR1, and CDR1 genes of eight (4 fluconazole resistance and 4 fluconazole susceptible) clinical C. tropicalis isolates were assessed. The correlation between the virulence factors and antifungal susceptibility patterns was analyzed. RESULTS: During a 4 year study, forty-five C. tropicalis isolates were recovered from candidemia patients. The isolates expressed different frequencies of virulence determinants as follows: coagulase 4 (8.9%), phospholipase 5 (11.1%), proteinase 31 (68.9%), esterase 43 (95.6%), hemolysin 44 (97.8%), biofilm formation 45 (100%) and CSH 45(100%). All the isolates were susceptible to amphotericin B and showed the highest resistance to voriconazole. There was a significant positive correlation between micafungin minimum inhibitory concentrations (MICs) and hemolysin production (rs = 0.316). However, we found a negative correlation between fluconazole MICs and esterase production (rs = -0.383). We observed the high expression of ERG11 and UPC2 genes in fluconazole-resistant C. tropicalis isolates. CONCLUSION: C. tropicalis isolated from candidemia patients extensively displayed capacities for biofilm formation, hemolysis, esterase activity, and hydrophobicity. In addition, the overexpression of ERG11 and UPC2 genes was considered one of the possible mechanisms of azole resistance.

The relationship between biofilm formation and mortality in patients with Candida tropicalis candidemia.

BACKGROUND: Biofilm formation by Candida species is an influential virulence factor in candidemia pathogenesis. We investigated the relationship between biofilm formation of Candida tropicalis isolates with the clinical characteristics and mortality outcomes in patients with candidemia. MATERIALS AND METHODS: Thirty-nine C. tropicalis isolates were recovered from patients with candidemia admitted to two university hospitals in Tehran, Iran. Biofilm mass and metabolic activity of C. tropicalis biofilms were assessed in vitro with two colorimetric methods. The sessile minimum inhibitory concentrations (SMICs) were evaluated in vitro by treating preformed biofilms with diluted concentrations of azoles according to CLSI-M27 A3/S4 protocol, followed by metabolic activity quantification. The expressions of ERG11, UPC2, MDR1, and CDR1 genes were also evaluated. RESULTS: All C. tropicalis isolates produced biofilm. Respectively, higher <7-day and ≥7-day mortality rates were found among cases with high metabolic activity (46.7% vs. 13%, P = 0.03) and high biofilm mass (31.8% vs. 0, P = 0.029). Sessile cells had high resistance to fluconazole, voriconazole, and itraconazole. The azole minimum inhibitory concentrations (MICs) of C. tropicalis sessile were significantly greater than the planktonic minimum inhibitory concentrations (PMICs). In fluconazole-treated biofilms, the expression of ERG11 and UPC2 genes was increased. CONCLUSION: Our findings highlight the importance of C. tropicalis biofilm formation as an important factor in candidemia pathogenesis and the clinical outcome of patients with candidemia.

The effect of released new synthetic peptide from nanofibrous scaffold of peptide/Poly (Vinyl Alcohol)/Poly l-Lactic Acid on expression of Secretory aspartyl proteinases 4 to 6 genes of Candida albicans.

Secreted aspartyl proteinases (Saps), are gene products that have been shown to directly contribute to Candida albicans pathogenicity. Despite the clear difficulties of systemic C. albicans infections control, Antimicrobial peptides (AMPs) are regarded as one of the most promising alternatives in this regard. Recently, drug-loaded electrospun nanofibers have attracted a great deal of attention. In this study we have established the nanofibrous scaffold of new synthetic peptide/Poly (Vinyl Alcohol)/Poly l-Lactic Acid on expression of Secretory aspartyl proteinases 4 to 6 genes of C. albicans in comparison with free peptide. We designed new synthetic Antimicrobial peptide (AMPs) used bioinformatics tools to predict structure and stabilities. The electrospinning method was used to produce the polymeric nanofibers. Scanning Electron Microscopy (SEM) was used to measure the nanofibers diameters and morphology. To analyze the expression of SAP4, SAP5 and SAP6 genes, RNA was extracted from clinical isolates of C. albicans before and after treatment with subinhibitory concentrations of new synthetic peptide on nanofibrous scaffold of new synthetic peptide/PVA/PLLA and then the cDNA was synthesized and used for Real-time PCR assay. Scanning electron microscopy (SEM) images showed that the morphology, the diameter, are affected from peptide. Reletive quantitative Real-time PCR results revealed that the mRNA levels of SAP4, SAP5 and SAP6 genes significantly decreased after treatment with nanofibrous scaffold of new synthetic peptide/PVA/PLLA (P < .05). Electrospun nanofibrous of new Synthetic Peptide/PVA/PLLA is effective in downregulating of expression SAP4, SAP5 and SAP6 genes of C. albicans strains.

Inhibitory effects of carvacrol on the expression of secreted aspartyl proteinases 1-3 in fluconazole-resistant Candida albicans isolates.

BACKGROUND AND OBJECTIVES: Secreted Aspartyl Proteinase (SAP) is one of the main virulence factors in the pathogenesis of Candida. This enzyme is encoded by a family of at least ten genes. Among these genes, the role of SAP1-3 in mucosal infections is evident. This study aimed to investigate the expression of SAP1-3 genes of Candida albicans isolates after treatment with Echinophora platyloba extract, carvacrol and caspofungin drug. MATERIALS AND METHODS: Vaginal samples of 68 women with suspected vaginitis were obtained and cultured. Canida albicans species were identified using phenotypic and genotyping methods. Spectrophotometry was used to investigate the presence of SAP protein in the vaginal samples, and SDS-PAGE was used to confirm its protein composition. Real-time PCR was performed to ascertain the effects of subinhibitory concentrations of Echinophora platyloba extract, carvacrol and caspofungin on the expression of SAP1-3 genes before and after treatment. RESULTS: C. albicans was found as the abundant species (59.6%), and different amounts of SAP were present in all vaginal samples, which were higher than Candida krusei strain. The protein composition of SAP in C. albicans samples was estimated with the approximate molecular weight of 45 kDa. mRNA levels of total SAP in FLU-resistant isolates (P=0.01) were more than those of FLU-susceptible isolates (P=0.07). The findings indicated that carvacrol is effective in reduction of SAP1-3 expression with a particular effect against FLU-resistant isolates. CONCLUSION: Carvacrol contains an essential oil (carvacrol); therefore, it can be considered as an alternative effective antifungal compound.

Fluconazole Resistance Candida albicans in Females With Recurrent Vaginitis and Pir1 Overexpression.

BACKGROUND: Some genes may be associated with Candida albicans resistance to azoles. Pir1 gene is described as responsible to induce resistance in C. albicans. OBJECTIVES: The current study aimed to find the relationship between fluconazole resistance and Pir1 protein (Pir1p) overexpression in the females with recurrent C. albicans vaginitis requiring longer fluconazole therapy. PATIENTS AND METHODS: A total of 52l vaginal samples were obtained from the females with C. albicans vaginitis. The azole susceptibility phenotype was determined according to the Clinical Laboratory for Standards Institute (CLSI) protocol for disk diffusion method and inhibition zone for fluconazole. Expression of pir1 gene and fluconazole -resistance were evaluated using polymerase chain reaction (PCR) in C. albicans. RESULTS: In the 52 isolates, 49 (94%) were resistant to fluconazole. Overexpression of Pir1 gene was detected in 47 (96%) fluconazole-resistant C. albicans isolates. CONCLUSIONS: The findings show fluconazole -resistance in C. albicans isolates with overexpression of Pir1p.

In Vitro Evaluation of Enzymatic and Antifungal Activities of Soil-Actinomycetes Isolates and Their Molecular Identification by PCR.

BACKGROUND: Human cutaneous infection caused by a homogeneous group of keratinophilic fungi called dermatophytes. These fungi are the most common infectious agents in humans that are free of any population and geographic area. Microsporum canis is a cause of dermatophytosis (Tinea) in recent years in Iran and atypical strain has been isolated in Iran. Its cases occur sporadically due to M. canis transmission from puppies and cats to humans. Since this pathogenic dermatophyte is eukaryotes, chemical treatment with antifungal drugs may also affect host tissue cells. OBJECTIVES: The aim of the current study was to find a new antifungal agent of soil-Actinomycetes from Kerman province against M. canis and Actinomycete isolates were identified by PCR. MATERIALS AND METHODS: A number of hundred Actinomycete isolated strains were evaluated from soil of Kerman province, for their antagonistic activity against the M. canis. M. canis of the Persian Type Culture Collection (PTCC) was obtained from the Iranian Research Organization for Science and Technology (IROST). Electron microscope studies of these isolates were performed based on the physiological properties of these antagonists including lipase, amylase, protease and chitinase activities according to the relevant protocols and were identified using gene 16SrDNA. RESULTS: In this study the most antagonist of Actinomycete isolates with antifungal activity against M. canis isolates of L1, D5, Ks1m, Km2, Kn1, Ks8 and Ks1 were shown in vitro. Electron microscopic studies showed that some fungal strains form spores, mycelia and spore chain. Nucleotide analysis showed that Ks8 had maximum homology (98%) to Streptomyces zaomyceticus strain xsd08149 and L1 displayed 100% homology to Streptomyces sp. HVG6 using 16SrDNA studies. CONCLUSIONS: Our findings showed that Streptomyces has antifungal effects against M. canis.

Functionalized nanoscale ß-1,3-glucan to improve Her2+ breast cancer therapy: In vitro and in vivo study.

We fabricated a targeted delivery system for doxorubicin (Dox) using ß-1,3-glucan (Glu) as a carrier and decorated by trastuzumab antibody having the status of targeting agent against Her2+ breast tumors. Glu-Dox conjugates were also functionalized with polyethylenimine (PEI) intended for increasing specific cellular uptake of prepared nanoparticles. The self-assembled nanoparticles were prepared through conjugation of Dox- [Glu-Dox-] using succinic anhydride (Sa) in place of a linker. Nanoparticles had spherical morphology with positive zeta potential. In-vitro cell viability assay on two breast cancer cell lines demonstrated acceptable toxicity against tested cell lines. Confocal microscopic images demonstrated the remarkable cytoplasmic uptake of the nanoparticles in Her2-overexpressing 4T1 cells. A controlled release of Dox from Glu-Dox nanoparticles was investigated. In-vivo studies were performed on female Balb/C mice. The volume of the induced tumors was calculated following intravenous administration of nanoparticles. The tumor volume diminished efficiently and more rapidly after administration of nanoparticles containing Dox. Based on survival results, the formulation of Dox targeted nanoparticles appeared very promising for the treatment of tumors. It could be concluded that Glu-Dox targeted nanoparticles have potential advantages for delivering anticancer drugs to the target tissue.

Allergens from Fusarium solani identified by immunoblotting in asthma patients In Iran.

We extracted Fusarium solani antigens to evaluate specific anti-F. solani IgE in fifty-one patients with asthma (33 men and 18 women) and in 22 non-atopic healthy subjects (15 men and 7 women). F. solani strains were cultured in Sabouraud glucose agar and subjected to cell disruption using the freeze-and-thaw method. The obtained cytoplasmic extracts were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Sensitisation to F. solani antigens has been evaluated in asthmatic patients using the immunoblotting assay. The SDS-PAGE identified 29 protein bands in the cytoplasmic extracts of F. solani isolates, with molecular weights ranging from 24 kDa to 112 kDa. Immunoblotting detected specific anti-F. solani IgE antibody in all asthma patients, but not in the control group. The predominant reactive allergens in patients corresponded to the bands with molecular weights of 24 kDa, 58.5 kDa, 64.5 kDa, 69 kDa, 72 kDa, and 97 kDa. Our results suggest that various allergenic components of F. solani may produce symptoms of asthma in susceptible individuals and they call for further research.

Candida albicans structural and secreted proteins modulate CD4/CD8 ratio in tumor infiltrating lymphocytes of spontaneous adenocarcinoma bearing mice.

BACKGROUND: Candida albicans is one of the most important opportunistic pathogens that suppress immunologic mechanisms of the host. It is speculated that structural and secretory proteins of C. albicans have immunomodulatory effects in cancer. OBJECTIVE: To evaluate the effects of C. albicans structural and secreted proteins on intratumoral CD4/CD8 ratio as well as the survival rate in BALB/c tumor model. METHODS: Structural and secretory proteins from C. albicans were isolated and examined for their effects on tumor growth and survival of adenocarcinoma bearing mice. RESULTS: The results indicated that in mice treated with C. albicans structural protein, the survival rate significantly decreased compared with the control groups. Also, mice treated with secretory proteins showed a decrease in survival rate but it was not statistically significant (p>0.05). Investigating the frequency of tumor infiltrated CD4+ and CD8+ T lymphocytes indicated that the percentages of tumor infiltrated CD4+ T lymphocytes in response to structural and secreted proteins were higher compared to the control groups. CONCLUSION: Our study suggests that C. albicans structural and secreted proteins modulate intratumor T lymphocyte infiltration.

Comparison of glutathione S-transferase activity and concentration in aflatoxin-producing and their non-toxigenic counterpart isolates.

In this study, two techniques were used to compare the specific activity and total concentration of mycelial glutathione S-transferase (GST) in fungal strains isolated from natural sources. The fungi identified as Aspergillus parasiticus and Aspergillus flavus have been divided into two groups based on their ability to produce aflatoxins. Altogether 26 fungi were isolated, among which 12 were capable of producing varying levels of aflatoxin and 14 were proved to be non-toxigenic. GST specific activity in mycelial preparation was measured spectrophotometrically using 2,1-chloro-2,4-dinitrobenzene as the substrate. The results showed that the mean GST activity in toxigenic isolates was 25.06 +/- 9.8 micromol/mg protein/min which was 2.8-fold greater than that measured in non-toxigenic isolates (8.84 +/- 5.5 micromol/mg protein/min). Moreover, the GST concentration was compared in toxigenic and non-toxigenic isolates using an Enzyme Linked Immunosorbent Assay based on antigen (fungal preparation) and antibody (antibody produced against fungal GST in rabbit). The results of ELISA showed that the mean GST level in toxigenic and non-toxigenic fungi was 1.17 +/- 0.55 and 0.40 +/- 0.24, respectively. These results further confirm that the aflatoxin production in the fungal strains is correlated with GST expression and using ELISA, it is possible to discriminate aflatoxin-producing fungi from their non-toxigenic counterparts.

Evaluation the anti proliferative activity of structural proteins and fraction of supernatant from culture of Candida albicans.

In this study, anti proliferative activity of structural proteins and fraction of supernatant from culture of Candida albicans on proliferation responses of lymph node cells in Balb/c mice have been evaluated. For this reason Candida albicans was cultured in RPMI medium supplemented with 10% FBS at 37 in 5% CO2 until reaching a confluent state for 2 weeks. The culture supernatant was obtained by centrifugation and purified by gel filtration chromatography and structural proteins of C. albicans were obtained from breakage of cell wall by vortexing with glass beads in suspension of PBS and 1 mM PMSF and then cultured with lymph node cells and evaluated by MTT assay. The results in this study demonstrated that both of structural proteins and fraction of supernatant from culture of Candida albicans suppress immune responses compared with Control group. Our study provides evidence that proteins of C. albicans have anti proliferative activity.