Study On Antifungal Effect Of Riccardin D, A Bis(Bibenzyls)Compound, Against Candida Albicans Biofilm

In recent years, the extensive use of efficient and wide-spectrum antimicrobial agents, immunosuppressive agents in organ transplant recipients, antineoplastics in patients with hematologic malignancies, the operation of artificial implantation, such as nearthrosis, heart valve replacement and central venous catheter, contributed to the increasing morbidity associated with fungal infections. The selective pressure of antimicrobials forced microorganisms produced a variety of mechanisms of drug resistance to ensure the continuation of the species, among which biofilms formation is one of the most important drug resistance mechanisms. Fungal biofilms associated infections often occurs when implanting materials are used, such as artificial prosthesis, venous catheters, et al. The susceptibility of microorganisms against antimicrobial agents may change a lot after biofilms formation. For example, Candida albicans biofilms are significantly less susceptible than in planktonic state to antifungals. How to prevent and manage the drug resistance of C. albicans biofilms has become a clinically challenging task.Natural products are still major sources of innovative therapeutic agents in various conditions. Liverworts have been used in some remedies of folk medicine to treat illnesses of the cardiovascular system, bronchitis and skin diseases as well. In the past decades, various types of lipophilic terpenoids and aromatic compounds showing significant biological activities have been isolated from the bryophytes, some of which can be used as good lead compounds for new drug development. Macrocyclic bis(bibenzyls) compounds isolated from liverworts Marchantia were found to have a wide range of biological activities, such as antioxidation, antifungi, antivirus, antibacteria and cytotoxicity, and have a high value for study. Riccardin D (RCD), a macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L.(Marchantiaceae), structurally consisted of two biphenyl bonds linked by diaryl ether bonds and phenolic hydroxyl groups in different numbers. Further investigation found that RCD had the antifungal effect not only against C. albicans, but against C albicans biofilms formed in vitro. Molecular mechanism study revealed that RCD inhibit C. albicans biofilms via retarding hyphae formation. The biofilms clearance effect, susceptibility property, the antifungal efficacy used with other antifungal agents, and the anti-biofilms formed in vivo need to be further studied.The study established in vitro and in vivo C. albicans biofilms-associated infective model, and observed the morphology at different developing stages of biofilms growth. Using this model, Candida biofilms formed in vitro and in vivo were studied, including susceptibility property of C. albicans in biofilms or as planktonic cells against RCD when used alone or in combination with other antifungal agents. Different methods were applied to estimate the antifungal effect. RCD solutions at different concentrations were administered locally by using "antibiotics lock technique" to the infective catheters, using quantitative method of colony culture to assess the inhibition efficacy of experimental agents against C. albicans biofilms in vivo. Scanning electron microscopy and confocol laser scanning microscopy were used to evaluate the morphology of C. albicans biofilms before and after drug challenge. Colony culture and morphology observation were used to assess the effect of RCD used alone or in combination with fluconazole on C. albicans biofilms. Quantitative real-time reverse transcription-PCR (RT-PCR) was used to compare mRNA abundances of the genes related to fungal adherence and hypha growth. The TaqMan MGB probe and primer sets were designed for the target genes and the control gene. The molecular mechanism of the inhibition effect of RCD on C. albicans biofilms was speculated according to the change of genes expression before and after RCD challenge.The study developed in vitro bio-material adherence C. albicans biofilms infective model and CVC-related animal model, and investigated the growth kinetics, impact factors on biofilms development and the micro-morphology properties. During the early phase of biofilms growth, fungal cells increased dramatically. Colony counting was about106CFU when biofilms grew for24h, and afterwards the cell counts increased slowly. Biofilms formation related to the nature of adherence materials and microorganism isolations. Morphology of biofilms changed significantly as time went on, mainly embodied in cell counts, hypha formation and amount of extracellular matrix.The susceptibility of C. albicans isolates ATCC10231、SC5314、YEM30and CA10in both planktonic and biofilms state against experimental agents was also investigated. Results showed that the resistance of C. albicans increased exponentially after biofilms formation. When RCD were used in combination with fluconazole, amphotericin B or caspofungin, it showed synergistic or indifferent effect against various isolates either in planktonic cells or in biofilms. We applied FICI method based on LA theory and△E method based on BI theory to evaluate the antifungal effect when RCD was used with other agents. The accordance ratio of the two methods was75%when used in assessing the antifungal effect on planktonic cells.RCD solutions at different concentrations (8,16,32,64μg/ml) were administered to C. albicans biofilms formed in vitro using antibiotic lock technique for1,3,5or7days continuously to investigate the efficacy of anti-biofilms of RCD. Colony counting proved that RCD showed antifungal effect against24-h-old biofilms. The strength of antifungal effect was related to the drug duration and concentrations. Morphology experiment which aimed to assess cell viability also proved that the increase of drug concentration and duration could decrease the cell viability of the biofilms, even some of them was eliminated totally. The anti-biofilms effect of RCD when combined with fluconazole was studied. Results showed that colony counting in combination group decreased significantly than the that challenged by FLC alone, suggesting RCD had hypersensitivity effect to fluconazole against C. albicans biofilms.Using catheter related C. albicans biofilms infective animal model, the anti-biofilms of experimental drugs. After rabbit model development, RCD solutions were administered at different biofilms formation phase, and evaluated the prevention and therapeutic effect of RCD against C. albicans biofilms. The results were assessed by colony counting and morphology observing. The specimen were taken out after24hours from the initiation of infection. Data showed that RCD could prevent the biofilms formation dose dependently when administered after4h of biofilms growth and maintained in the catheters for8hours. Compared with the control group, colony counting in RCD-challenged group decreased significantly. RCD could increase the prevention effect of fluconazole on C. albicans biofilms according to the results of drug combination experiment. When administered to the24-h-old biofilms at different concentrations and maintained for8hours per day for5days continuously, RCD showed inhibition effect in a dose dependent manner. Micro-morphology observation of scanning electron microscope proved the colony counting results.We designed RT-PCR experiment to investigate the molecular mechanism of the inhibition effect of RCD against C. albicans biofilms. Specimens of C. albicans biofilms formed in vitro and in vivo were collected and genes related to cells adherence and hypha growth ALS1, ALS3, ECE1, EFG1, HWP1and the upstream regulating gene CDC35were selected and amplified. Biofilms not treated by RCD worked as comparison. Results showed that the genes expression were all down-regulated. The expression of HWP1was decreased most significantly in in vitro study, and there was good accordance between in vitro and in vivo experiment. This suggested that RCD inhibited the biofilms growth via fungal cells adherence and hypha formation.