The Study Of Candida Albicans Biofilms In Vitro About Metabolomics By Xianglian Solution

Objective1. To study the antifungal efficacy of Candida albicans biofilms by Xianglian Solution.2. To investigate the probable antifungal mechanism of Candida albicans biofilms in vitro by Xianglian Solution, in the view of metabolomics.Methods1. According to the standard methord of M27-A from CLSI, we tested the MIC of Candida albicans(SC5314) by Xianglian Solution and Fluconazole.2. Refer to the lecture of Ramage GW (Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms), we established C. albicans biofilms in vitro.3. We measured the SMIC50and SMIC8o of the C. albicans biofilms in different maturity (4h,24h,48h) by XTT.4. We measured the SMIC50and SMIC80of the C. albicans biofilms which were cultured in an embedding96-well plate by Xianglian Solution and Fluconazole.5Through the methord of UPLC-Q-TOF-MS, we detected the metabolomics of the planktonic, early (4h), medium term(24h),maturity(48h) biofilms and the biofilms cultured in an embedding96-well plate by Xianglian Solution and Fluconazole.Results1. The MIC of planktonic was1.95mg/ml by the effect of Xianglian Solution. And the value went to4μg/mL by the effect of Fluconazole.2. By the effect of Xianglian Solution, the SMIC50of early (4h),medium term(24h) and maturity(48h) biofilms were7.81,125and500mg/ml respectively, when the SMIC80were up to31.25,250and>1000mg/ml respectively. By the effect of Fluconazole, the SMIC50of early (4h), medium term(24h) and maturity (48h) biofilms were32,64and>1024μg/mL respectively, when the SMIC80were up to64,128and>1024μg/mL respectively.3. The formation of Candida albicans biofilms were inhibited when the emdedding drug concentration of Xianglian Solution up to62.5mg/ml, and of Fluconazole up to64μg/mL in the96-well plate.4The metabolic profiling of planktonic was different from that of biofilms obviously. But the metabolic profiling of24h’s biofilms was quite similar to that of48h’s. There is a big overlap between24h’s biofilms and48h’s. What’s more, the metabolin whose m/z were488.1647,377.1950, maybe the potential discrepant metabolin. The m/z488.1647were identified as Cysteinyl-Threonine. The m/z377.1950were identified as Pentosidine, which had a relationship with pentose phosphate pathway.5. The metabolic profiling of planktonic among Group Xianglian Solution, Group Fluconazole and control group were different from each other. The metabolin whose m/z were247.0964were identified as the potential metabolin. It was supposed to be Lipoamide.6. The metabolic profiling of biofilms (4h,24h,48h) among Group Xianglian Solution, Group Fluconazole and control group were different from each other. The metabolin whose m/z were206.0810were identified as the potential metabolin among4h’s biofilms. It was supposed to be Quinaldic acid, which is a catabolism of L-tryptophan. The metabolin whose m/z were379.2098were identified as the potential metabolin among24h’s biofilms. It was supposed to be tyrosyl-Arginine, which is a tyrosine arginine dipeptide composition.It’s a kind of decomposed products by digestion of the protein or protein decomposition uncompletely. The metabolin whose m/z were387.1754were identified as the potential metabolin among48h’s biofilms. It was supposed to be Indoleacetyl glutamine, which is indolic derivative of tryptophan.7. The metabolic profiling of biofilms (4h,24h,48h) among different concentrations of Xianglian Solution were different from each other. The metabolin whose m/z were201.1121were identified as the potential metabolin. It was supposed to be4-Hydroxynonenal, which is one of the major end products of lipid peroxidation, has been shown to be involved in signal transduction and available evidence suggests that it can affect cell cycle events in a concentration-dependent manner. The metabolic profiling of biofilms (4h,24h,48h) among different concentrations of Fluconazole were different from each other. The metabolin whose m/z were175.0218were identified as the potential metabolin. It was supposed to be3-Dehydro-L-gulonate, which is an intermediate in Ascorbate and aldarate metabolism.3-Dehydro-L-gulonate is the4th to last step in the synthesis of D-Xylulose5-phosphate and is converted from2,3-Diketo-L-gulonate via the enzyme3-dehydro-L-gulonate2-dehydrogenase. It is then converted to3-Dehydro-L-gulonate6-phosphate via the enzyme L-xylulokinase.8. The metabolic profiling of biofilms between an embedding96-well plate by Xianglian Solution and Fluconazole were different from each other. The metabolin whose m/z were144.9225were identified as the potential metabolin. It was supposed to be Superoxide. It is the anionic form02. It is important as the product of the one-electron reduction of dioxygen (oxygen gas). The biological toxicity of superoxide is due to its capacity to inactivate iron-sulfur cluster containing enzymes which are critical in a wide variety of metabolic pathways.Conclusion1. It had been demonstrated that both Xianglian Solution and Fluconazole were effect to C. albiance planktonic in vitro. The MIC of Xianglian Silution was1.95mg/ml, which was equal to that of Fluconazole(4μg/mL). Because different strains were used in different experiments, there would be different sensitivity to drugs. This study provided a reference MIC for the strain used.2. This study had proved that Xianglian Solution was effective to C. albiance biofilms. But the SMIC50、SMIC80of biofilms were obviously increase than planktonic. It’s about10times higher than that of planktonic. Whatever, Xianglian Solution had the exact inhibition for biofilms.3. Xianglian Solution and Fluconazole had the exact inhibition for the C. albicans biofilms which were cultured in an embedding96-well plate by Xianglian Solution and Fluconazole.4. The metabolic model had changed in process of biofilms formation from planktonic. It mainly related to sugar metabolism and amino acid metabolism pathways. For example, the pentose phosphate pathway, D-methionine, phenylalanine, L-tryptophan, tyrosyl-arginine and tryptophan metabolites vary from different drug or different morphology. It is supposed that glucose metabolism and amino acid metabolism may play an important role in the transformation of morphology, such as the transformation of yeast and hypha.5. What’s more important, Superoxide would be the key pathway of oxidative stress during the biofilms formation.