Kee Peng Ng

Comparison between allicin and fluconazole in Candida albicans biofilm inhibition and in suppression of HWP1 gene expression

Candida albicans is an opportunistic human pathogen with the ability to differentiate and grow in filamentous forms and exist as biofilms. The biofilms are a barrier to treatment as they are often resistant to the antifungal drugs. In this study, we investigated the antifungal activity of allicin, an active compound of garlic on various isolates of C. albicans. The effect of allicin on biofilm production in C. albicans as compared to fluconazole, an antifungal drug, was investigated using the tetrazolium (XTT) reduction-dependent growth and crystal violet assays as well as scanning electron microscopy (SEM). Allicin-treated cells exhibited significant reduction in biofilm growth (p<0.05) compared to fluconazole-treated and also growth control cells. Moreover, observation by SEM of allicin and fluconazole-treated cells confirmed a dose-dependent membrane disruption and decreased production of organisms. Finally, the expression of selected genes involved in biofilm formation such as HWP1 was evaluated by semi-quantitative RT-PCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of HWP1.

The mycological and molecular study of Hortaea werneckii isolated from blood and splenic abscess

Hortaea werneckii is an environmental dematiaceous fungus found in the halophilic environment. It causes tinea nigra. We report the isolation of H. werneckii from blood and splenic abscess of two patients with acute myelomonocytic leukaemia. H. werneckii grew at room temperature but not at 37 °C, it was identified by biochemical tests, growth characteristics and the presence of conspicuous collarette intercalary on dividing yeast cells. The use of specific oligonucleotide primer Hor-F (5′-TGGACACCTTCA TAACTCTTG-3′) and Hor-R (5′-TCACAACGCTTAGAGACGG-3′) confirmed the two isolates were H. werneckii. The sequence for 281 nucleotide of HW299 and HW403 were 99% identical but differed only in one nucleotide. In vitro anti-fungal susceptibility testing showed that the isolates were resistant to amphotericin B and flucytosine.

Immunoproteomic analysis of antibody response to cell wall‐associated proteins of Candida tropicalis

This study was conducted to identify antigenic proteins of Candida tropicalis that are targeted by the host immune system.

Identification of immunogenic proteins of Candida parapsilosis by serological proteome analysis

Systemic candidiasis is the leading fungal bloodstream infection, and its incidence has been on the rise. Recently, Candida parapsilosis has emerged as an increasingly prevalent fungal pathogen, but little is known about its antigenic profile. Hence, the current work was performed to discover immunogenic proteins of C. parapsilosis using serological proteome analysis.

2-dodecanol (decyl methyl carbinol) inhibits hyphal formation and SIR2 expression in C. albicans.

Candida albicans is capable of undergoing yeast‐hypha transition to attain pathogenicity in humans. In this study, we investigated the differential expression of CaSIR2 via quantitative real‐time PCR (qPCR), during yeast‐hypha transition with and without the presence of 2‐dodecanol. SIR2 transcript levels were found to be significantly enhanced after hyphal induction as compared to the yeast form. This study found that 2‐dodecanol is able to inhibit hyphal development and block SIR2 up‐regulation, even in hyphal‐inducing growth conditions. We suggest that SIR2 may be involved in Candida albicans quorum‐sensing and serum‐induced yeast‐hyphae transition via the Ras1‐cAMP‐Efg1 signalling cascade. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Detection of 10 medically important Candida species by seminested polymerase chain reaction

A seminested PCR detecting ten medically important Candida species were achieved. Analytical sensitivity and specificity were not compromised.

Detection of medically important Candida species by absolute quantitation real-time polymerase chain reaction

Background: The number of invasive candidiasis cases has risen especially with an increase in the number of immunosuppressed and immunocom promised patients. The early detection of Candida species which is specific and sensitive is important in determining the correct administration of antifungal drugs to patients. Objectives: This study aims to develop a method for the detection, identification and quantitation of medically important Candida species through quantitative polymerase chain reaction (qPCR). Materials and Methods: The isocitrate lyase (ICL) gene which is not found in mammals was chosen as the target gene of real-time PCR. Absolute quantitation of the gene copy number was achieved by constructing the plasmid containing the ICL gene which is used to generate standard curve. Twenty fungal species, two bacterial species and human DNA were tested to check the specificity of the detection method. Results: All eight Candida species were successfully detected, identified and quantitated based on the ICL gene. A seven-log range of the gene copy number and a minimum detection limit of 103 copies were achieved. Conclusions: A one-tube absolute quantification real-time PCR that differentiates medically important Candida species via individual unique melting temperature was achieved. Analytical sensitivity and specificity were not compromised.

Comparative Study of the Effects of Fluconazole and Voriconazole on Candida glabrata , Candida parapsilosis and Candida rugosa Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC50 of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.