Inhibitory Effect of Carvacrol on the Expression of Candida (albicans Hyphae-Specific Gene (HWP1

BACKGROUND AND OBJECTIVE: Candida albicans infection is a problem of growing clinical importance, particularly in immunocompromised populations. This study aimed to investigate the inhibitory effect of carvacrol on the hyphae formation and expression of C. albicans HWP1. METHODS: This cross-sectional study was done over a 6-month period in 2016-2017. Vaginal, mouth and skin surface swabs were obtained from immunocompromised patients. Colonizing clinical isolates of C. albicans were identified and drug susceptible isolates detected using WHONET software. The susceptibility test for carvacrol (range 6.25–300 μg/ml) were carried out with a broth microdilution according to the CLSI guidelines against drug susceptible C. albicans. The time kill assay of carvacrol (range 2 × MIC to ¼ × MIC) was determined. Hyphae inhibition was evaluated by light microscopy. We determined the expression levels of HWP1 implicated in hyphae formation of C. albicans ATCC 14053 cells by quantitative RT-PCR. FINDINGS: Ten colonizing clinical isolates of drug susceptible C. albicans were identified. Carvacrol was inhibited the growth of all drugs susceptible isolates of C. albicans (MIC range, 25-200 µg/ml). Time kill curve assay demonstrated that carvacrol could significantly inhibit the growth of C. albicans (p≤0.05). Carvacrol efficiently prevented hyphae formation in drug susceptible C. albicans. The expression levels of HWP1 gene were down-regulated by 1.82- and 1.62-fold at concentrations of 2 × MIC and 1×MIC of carvacrol, respectively. CONCLUSION: These results suggest that carvacrol could provide an improved and safe clinical approach in treating Candida infections by prevention of hyphae formatio

Simplex and triplex polymerase chain reaction (PCR) for identification of three medically important Candida species

Candida species are a major cause of invasive infections in both critically ill and immunocompromised patients. Hence, rapid identification of these pathogens may facilitate specific therapy and patient management. The development of rapid and specific diagnostic methods remains a challenge. Herein, we developed the simplex and triplex polymerase chain reaction (PCR) for the identification of three medically important Candida species namely C. albicans, C. parapsilosis and C. tropicalis. The developed methods target the phospholipase B gene (PLB). The primers designed achieved highly specific identification of the selected species using both the simplex PCR and the triplex PCR formats, which were confirmed by DNA sequencing. The primers did not show any non-specific amplification when tested with DNA from other Candida species and other fungal species such as Aspergillus and Cryptococcus. These results showed that the PLB gene provides a novel target that could be used for the detection of medically important Candida species from clinical specimens.Key words: Candida species, primers, phospholipase B gene (PLB), polymerase chain reaction (PCR)

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 (XXT) 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. (C) 2011 Elsevier GmbH. All rights reserved

Identification and differentiation of Candida species using specific polymerase chain reaction (PCR) amplification of the phospholipase B gene

Candida species are the major cause of mortality in both immunocompromised and critically ill patients. Therefore, the early diagnosis and differentiation of Candida species isolated from clinical samples is principally important due to their inherently variable antifungal resistance pattern. Herein, rapid and species-specific polymerase chain reaction (PCR)-based molecular method was developed for the identification of the four species of Candida most commonly isolated from clinical specimens, namely Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis. The developed method targeted the phospholipase B gene (PLB) as a novel target. We determined the sequences of this gene in previous work. The primers designed achieved highly specific identification of the selected species using simplex PCR assay, which were confirmed by sequencing. There was no cross-amplification of other Candida species nor other fungal organisms tested. The simplex PCR assay yielded detection limits of 1 to 10 cells/ml and 10 fg/µl DNA. These results showed that the PLB gene provides a novel target that could be used for the identification and detection of medically important Candida species from the clinical samples