HTS By Resazurin Assay And Dxr Inhibitors For Anti-Vibrio Agents From Marine Fungi

Vibriosis is one of the major diseases in shrimp aquaculture, causing devastating mortality in the farm as well as in hatchery stage. The inappropriate use of antibiotics in aquaculture is one of the causes for the high incidence of antimicrobial resistant bacteria. It’s necessary to screen new microbial natural extracts for antibacterial activity, but the testing methods present many challenges, standard broth microdilution is more suitable for pure compounds. In this work, we developed and validated a low cost, convenient, reliable, simple colorimetric microtiter assay using the resazurin viability dye. The parameters of the resazurin method for high-throughput screening (HTS) using microbial natural extracts against vibrios were optimized and set up. An HTS of microbial extracts of cultivated marine fungal was carried out against three pathogenic strains Vibrio rotiferianus, Vibrio Vulnificus and Vibrio campbellii, and2.2%of the extracts displayed antibacterial activity. According to the screening results and related literatures, we selected Engyodontium album, Fusarium oxysporum and Aspergillus flavus to be fermented again. The secondary extracts of the fungi were purified by organic solvent extraction, column chromatography, reverse phase chromatography combined with TLC, HPLC. We used resazurin screening method for active tracking, separating a new compound EngyodontiuminA from the products of Engyodontium album.1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) is involved in the systhesis of isoprenoids by the methylerythritol phosphate pathway (MEP). DXR is essential in vibrios, absent in human beings and animals which use only MVA pathway. Therefore,1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) is the rate-limiting enzyme of MEP pathway and can be employed as ideal targets to screen antibiotics.We established a reaction system which was used for quickly detecting DXR activity. We obtained2potential inhibitory activity extracts on DXR from20extracts which inhibit vibrios. However, the compounds which inhibit the activity of DXR need further separating and detecting, laying the foundation for the discovering the new antibacterial drug or lead compounds.