| ObjectiveIn recent years, the antibiotic resistance crisis has received more and more attention in the world. Saccharomyces albicans (also called Candida albicans) is a commensal of human microflora, residing at the oral cavity, the vaginal and the urinary environments, that acts as an opportunistic human fungal pathogen and one of the pathogens of nosocomial infection. When the host becomes debilitated or immunocompromised, S. albicans can behave as a pathogen causing both superficial and systemic infections, the latter with possible infections of internal organs. Bloodstream infections are associated with considerable attributable mortality rates varying from 29 to 76%. It is the fourth most leading cause of death in nosocomial infections. Many patients didn’t die of primary disease but died of fungal infection eventually. Like other microorganisms, under the natural condition, S.albicans adhered to the surface of object, and then formed the so-called biofilm structure to survive. Biofilms are surface-associated structured microbial communities surrounded by a matrix of extracellular polymeric substances produced by the microorganism cells. Saccharomyces biofilms carries important clinical repercussions because of their increased resistance to antifungal therapy and the ability of cells within biofilms to withstand host immune defenses. Numerous studies have demonstrated the vast majority of chronic infection is closely associated with microbial biofilm formation in the host body. It was proved, however, biofilms-associated cells show resistance against antimicrobials therapy as well as against host defense agents such as phagocytes and antibodies. For this reason, biofilms are the main cause of the recurrence of chronic infectious diseases. However, there are few types of antifungal agents which have little choice. Systemic use is limited because antifungal agents generally have a strong side effects. And now the fungus resistance to antifungal drugs are becoming more and more common, research and development of new antifungal difficulty is much higher than anti-bacterial drugs. These difficulties make it more serious to control fungal infection in clinic.For this reason, measures need to be taken to treat with biofilms-associated infection and high levels of drug resistance, and new antifungal agent development is necessary and feasible. In recent years, it was found reasonable compatibility with an small molecule compounds can achieve two goals:to improve the therapeutic effect of antibiotics; to reduce the emergence of resistant strains. Accordingly, we are trying to depend on our natural enrich resource products and high-throughput screening technology to find small molecule compounds that can enhance the effect of amphatericin B against S. albicans biofilms. So as to provide new ideas for clinical treatment of refractory recurrent Saccharomyces infections.Methods1. Reviewing the relevant literature, We find out the small molecular compounds which have been proved to be a part of antifungal potentiators against S. albicans biofilm. Then we send this part small molecule compounds to the Chinese National Compound Library, and the similar structure of small molecule compounds can be matched.2. Determine the concentration of antifungal amphotericin B. S. albicans biofilm(48h) formation was determined using preseterilized polystyrene 96-well microplates and adding amphotericin B with 2-fold dilution. Following incubation for 24 hours, the plates were detected by Alamarblue reagents.3. High-throughput screening:Biofilm(48h) growth, in individual wells of 384-well microplates, was impacted by amphotericin B with small molecule compounds. After 24 hours, the biofilm was measured using the metabolic indicator Alamarblue reagent. Wells were scored as hits if compounds that enhanced the activity of amphotericin B percentage inhibition was>30%.4. We further rescreen the small molecule compounds which enhanced the activity of amphotericin B by CFU(colony counting) method and Alamarblue reagent method.5. We synthesis the small molecular compounds that have been screened and further analysis the antifungal susceptibility to S. albicans biofilms in vitro by LIVE/DEAD(?) FungaLightTM yeast viability kit.Results1. By matching, we finally decided to carry out high-throughput screening for 51520 small molecular compounds.2. Measured by Alamarblue reagent, we found that amphotericin B sessile minimal inhibitory concentration 50%(SMIC50) is about 2μg/mL, and then based on this concentration to test small molecule compounds if have synergistic effect with amphotericin B.3. By high-throughput screening for 51520 small molecular compounds, we found that only 10 small molecule compounds can enhances the effect of amphatericin B against Saccharomyces albicans biofilms4. By re-screening, We further determined these 10 small molecule compounds can assist amphotericin B drug to eliminate S. albicans biofilms.5. The results showed that the small molecule compounds have no effect on S. albicans biofilms almostly. However, compared with the group that without small molecular compound, the group which small molecular compound in conjunction with amphotericin B drug have higher inhibition rate to S. albicans biofilms (P<0.05)ConclusionWe have validated a novel approach to identify antifungal potentiators and completed a high-throughput screen to identify small molecules with activity against S. albicans biofilms. These small molecule compounds maybe have no obvious antifungal effect on S. albicans biofilms, but they make currently available antifungals more effective. This will provide new ideas and methods for clinical treatment of refractory recurrent Saccharomyces infections. |
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