Study On Isolation,Identification And Screening Antagonistic Bacteria Of Fish Pathogenic Strains Aeromonas Hydrophila

Aeromonas hydrophila is a gram-negative,facultative anaerobic organism and an autochthonous inhabitant of fresh water environments.It causes a variety of diseases in different animal species,from aquatic to terrestrial animals,including humans.In fish,it is responsible for fatal aeromonad septicemia,which causes significant economic losses to aquaculture worldwide each year.Lesion tissue of fish was collected from a farm in Hunan Province,then the isolated stains were identified as Aeromonas hydrophila,and screening its antagonistic bacteria.The main results are as follows.1.Isolation and identification of A.hydrophia AhX040 strain.In this study,we isolated and purified thirty suspected strains of Aeromonas hydrophila from lesion tissue of fish,collected from a farm in Hunan Province.We selected a strain which has the typical characteristic to carry on the appraisal.According to physiological&biochemical characteristics,and the homologous analysis of 16S rDNA sequences of nucleotides,the isolated fish pathogenic bacterium was identified as Aeromonas hydrophila and named as AhX040(CCTCC NO:M 2014157).This bacteria can make grass carp pathogenic by artificial infection test,and study its Antibiotic susceptibility.2.Optimization of culture conditions of Aeromonas hydrophila AhX040.The most suitable culture temperature of AhX040 strain is 30 degrees Celsius;the optimum pH is 6.5;the optimum NaCl concentration is 0.2%(w/v).The optimal medium for bacteria were determined by orthogonal experiment is peptone 40g/L,yeast powder 20 g/L,K2HP0410g/L.The growth curve of this strain was determined:0-2 h is the lag phase;2-14 h is logarithmic phase;14-24 h is stable phase and decline phase after 24 h.3.Screening antagonistic bacteria of Aeromonas hydrophila and antagonistic substance was studied.The study found that Pseudomonas aeruginosa has a broad-spectrum antagonistic activity against fish pathogenic bacteria and having obvious antagonistic activity against Aeromonas spp.Animal protection tests showed that Pseudomonas aeruginosa has a 90%immune protection rate for grass carp.The toxicity of Pseudomonas aeruginosa to animal is very low.Subsequently,the antibacterial activity of Pseudomonas aeruginosa was identified by physical and chemical properties.The results showed that the antibacterial substances has heat resistance,acid and alkali resistance,and can be extracted by organic solvent.It is speculated that the antimicrobial activity is a non polar molecule.4.Isolation and identification of antibacterial substances of Pseudomonas aeruginosa.The antibacterial substances of Pseudomonas aeruginosa were isolated and purified by UHPLC and identified by LTQ-XL mass spectrometry.It was found that the antimicrobial substance was a kind of pale yellow substance;the maximum UV absorption peak was 310 nm;easily soluble in organic solvents,slightly soluble in water and the molecular weight was 272.07.According to the previous studies,we speculate that the antimicrobial active substance is likely to be a aromatic polyketide material,Pyoluteorin,molecular formula being C11H7O3NC12.It is expected that the antimicrobial substance is developed as a biological control agent and applied to the prevention and treatment of the disease in aquaculture system.