On-site Detection Of Aeromonas Hydrophila And Mining Of Related Antibacterial Secondary Metabolites From Marine Fungus ZZ1657

Aeromonas hydrophila is the main pathogen of motile aeromonas septicemia in freshwater fish.In addition,Aeromonas hydrophila is a human opportunistic pathogen.People will be infected by wounds and eating food contaminated by Aeromonas hydrophila,resulting in gastroenteritis,bacteremia and soft tissue infection.As a common pathogen of human and fish,Aeromonas hydrophila spreads widely and causes great economic losses.In order to prevent the above hazards,early detection and antibacterial treatment of Aeromonas hydrophila infected subjects should be performed.Recombinase polymerase amplification(RPA)is an isothermal amplification technology.The detection personnel can complete the amplification reaction by hand or simple constant temperature equipment.The detection results can be visualized by lateral flow strips(LFS).The establishment of the recombinase polymerase amplification combined with lateral flow test strip(RPA-LFS)detection method of Aeromonas hydrophila is conducive to the timely discovery of the bacteria,and provides guidance for the selection of the bacteria’s treatment plan.At present,the treatment of Aeromonas hydrophila infection mainly uses antibiotics.However,due to the abuse of antibiotics,Aeromonas hydrophila has shown multidrug resistance.Therefore,to develop new anti Aeromonas hydrophila drugs is an urgent task to alleviate the multidrug resistance of it.Marine microorganisms have many kinds,fast reproduction,and can metabolize unique compounds with antibacterial activity to obtain extremely limited resources in the ocean,which is the basis of the development of new antibiotics.Rare drimane sesquiterpenes containing N-acetyl-L-valine group have been reported in the conventional fermentation products of marine fungus Penicillium minioluteum ZZ1657.The median inhibition concentration of these compounds against three common pathogens including MRSA、E.coli and C.albicansare is less than 15μg/m L,with strong bacteriostatic effect.Therefore,the marine fungus Penicillium minioluteum ZZ1657 shows great advantages in mining compounds against Aeromonas hydrophila.Chemical epigenetic modifiers can activate the biosynthetic gene cluster of marine fungi and stimulate the strains to produce compounds with novel structure and good antibacterial activity that cannot be produced in conventional culture.Using Penicillium minioluteum ZZ1657 as biological resource and culturing it by chemical epigenetic modification is a new way to find antibacterial substances against Aeromonas hydrophila.This test is mainly divided into two parts:part 1:Establishment of RPA-LFS detection method for Aeromonas hydrophila.This study established a rapid detection method based on recombinant polymerase amplification combined with lateral flow strip(RPA-LFS)for Aeromonas hydrophila.Specific primers and probes were designed targeting the aerolysin gene(aer A)of Aeromonas hydrophila.The result showed that the RPA-LFS detection method could be completed in 20 min with 37°C incubation.No cross reaction with other aquaculture and food safety pathogenic bacteria was observed.The detection limit was 10 CFU for a single reaction.The method of RPA-LFS for the detection of Aeromonas hydrophila has the advantages of rapidity,specificity and sensitivity.This method does not rely on sophisticated equipment and is suitable for the on-site detection of Aeromonas hydrophila with limited experimental resources.Part 2:Mining of antibacterial secondary metabolites related to marine fungus zz1657In this study,chemical epigenetic modification was used to explore the new natural products of marine fungus Penicillium minioluteum ZZ1657 for the treatment of Aeromonas hydrophila infection.The fungus was cultured by adding histone deacetylase inhibitor vorinostat or sodium butyrate.The Et OAc extract from the strain fermentation products were separated and purified by the traditional chromatography methods(e.g.,TLC,silica gel column chromatography,sephadex LH-20)combined with the modern ones(e.g.,HPLC);the structures of isolated compounds were determined by NMR and HRMS,as well as the literature data comparison;the antimicrobial activities of the compounds were evaluated MIC methods.The result showed that six compounds:N~1-Phenyloctanediamide(1),Tyrosol(2),?-Hydroxybenzoic acid(3),2,4-Dihydroxy-6-(4-hydroxy-2-oxopentyl)-3-methylbenzaldehyde(4),Orcinol(5),Orsellinic acid(6),Purpuresters A(7)were isolated and identified from the its fermentation products.The epigenetic reagent SAHA or Na Bu was able to affect the secondary metabolites of ZZ1657 significantly then increased the varieties of its products.Compounds 2-6 were isolated from the marine fungus ZZ1657 for the first time.The results of MIC experiment showed that compounds 2-7 had no inhibitory activity against Aeromonas hydrophila.Aiming at the control requirements of Aeromonas hydrophila,this study established an RPA-LFS method for its rapid detection and mining of related antibacterial secondary metabolites from marine fungus ZZ1657.The established RPA-LFS detection technology provides an effective monitoring method for the timely discovery of Aeromonas hydrophila.However,no compounds that can effectively treat Aeromonas hydrophila infection have been found from the marine fungus ZZ1657.In the future,it is necessary to expand the types of marine fungi for more extensive excavation of antibacterial compounds.