Research Article: Inhibitory activity of hinokitiol against biofilm formation in fluconazole-resistant Candida species

Date Published: February 2, 2017

Publisher: Public Library of Science

Author(s): Dae Jin Kim, Min Woo Lee, Jeong Su Choi, Seung Gwan Lee, Jee Yoon Park, Suhng Wook Kim, Caroline Mitchell.


The aim of this study was to investigate the ability of hinokitiol to inhibit the formation of Candida biofilms. Biofilm inhibition was evaluated by quantification of the biofilm metabolic activity with XTT assay. Hinokitiol efficiently prevented biofilm formation in both fluconazole-susceptible and fluconazole-resistant strains of Candida species. We determined the expression levels of specific genes previously implicated in biofilm development of C. albicans cells by real-time RT-PCR. The expression levels of genes associated with adhesion process, HWP1 and ALS3, were downregulated by hinokitiol. Transcript levels of UME6 and HGC1, responsible for long-term hyphal maintenance, were also decreased by hinokitiol. The expression level of CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA was suppressed by hinokitiol. Its upstream general regulator RAS1 was also suppressed by hinokitiol. These results indicate that hinokitiol may have therapeutic potential in the treatment and prevention of biofilm-associated Candida infections.

Partial Text

Candida species are opportunistic pathogens that live commensally within the human body. The prevalence of Candida infections, which ranges from superficial to deep-seated invasive candidiasis, is increasing at an alarming rate especially among immunocompromised individuals [1]. The most common isolated Candida species in clinical fungal invasive infection is Candida albicans, followed by C. tropicalis, C. parapsilosis and C. glabrata [2,3]. While C. albicans remains the most frequently isolated species, its incidence is declining and the frequency of other species is increasing. C. tropicalis mostly affects neutropenic patients and individuals with haematological malignancies [4]. C. glabrata affects mostly older patients, diabetics or individuals pre-exposed to azoles or echinocandins, and is rarely isolated from neonates and young children [5]. Candida species are heterogeneous, so understanding their phylogenetic differences may help to explain the reasons underlying variations of prevalence in Candida species. The most closely related species are C. albicans and C. tropicalis whereas C. glabrata is more closely related to Saccharomyces cerevisiae [6]. Most manifestations of candidiasis are associated with biofilm formation occurring on the surfaces of host tissues and medical devices [7,8]. Moreover, the most important feature of Candida biofilms is its role in increasing tolerance to conventional antifungal therapy. Forming biofilms is the answer of microorganisms to hostile environments. The optimal protection of the embedded cells against noxious agents, e.g., antibiotics and the immune system, is the main reason why biofilm infections are so difficult to treat. It has been found that for the eradication of pathogens from biofilms more than 1000 times higher antibiotic concentrations were required than for the same strain living in planktonic form in the serum [9]. The most common agent used in clinic against candidiasis are fluconazole and the candins. However, poor activity of fluconazole against Candida biofilms is a well-known phenomenon, which may decrease efficacy of fluconazole treatment [10]. Also, prolonged exposures to fungistatic fluconazole can result in the emergence of acquired resistant isolates. The spread of drug-resistant pathogens is one of the most serious threats to successful treatment of microbial diseases. Therefore, it has become essential to develop novel and potent antifungal for the treatment of fluconazole-resistant Candida infections.

Cells in biofilms are much better protected against noxious agents than free-living cells. From a medical viewpoint, the most critical feature of biofilm growth is the development of antimicrobial resistance by the microorganisms that constitute the biofilm [29]. The elimination of mature biofilms presents a therapeutic challenge in the management of device-associated Candida infections [30]. In this study, hinokitiol effectively inhibited the generation of Candida biofilms. At higher concentrations, hinokitiol also showed inhibitory activity against mature biofilms. Hinokitiol exhibited inhibitory activity against mature biofilms of C. guiliermondi and C. parapsilosis with MIC values of 12.5 and 25 μg/ml, respectively (Table 3). These MIC values were 2 to 4 times higher for mature biofilms than for planktonic cells. However, hinokitiol exhibited inhibitory activity against mature biofilms of C. albicans, fluconazole-resistant C. albicans, C.glabrata and C. tropicalis with MIC values of 400, 200, 50 and 200 μg/ml, respectively (Table 3). These MIC values were 64 to 250 times higher for mature biofilms than for planktonic cells. Lower activities are observed for other terpenoids, such as thymol, carvacrol and eugenol. These terpenoids showed 2.5-times lower inhibitory activity than that of hinokitiol against C. albicans biofilms [28, 31]. Echinocandins have shown some effectiveness against in vivo mature C. albicans biofilms [32]. Echinocandins were not utilized and this is a limitation of our study. Also, further evaluation is required to determine the antibiofilm activity of hinokitiol in vivo.




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