Screening And Identification Of A Clam Antagonistic Bacteria And Its Antibacterial Substances

Disease outbreak caused by pathogenic bacteria, commonly of the genus Vibrio, is a major mortality in clam farming, with the characteristics of highly and fastly contagious, as well as difficult to be control. Recently, a variety of antibiotics have been used for the prevention of the diseases, but the abuse of antibiotics usually leads to the emergence of resistant bacteria and raises environmental problems. Based on the principle of clam farming heathly and environmentally friendly, the inhibition of the pathogenic bacterium Vibrio alginolyticus was used as a trait to select a candidate probiotic bacterial strain from the breeding habitat of clams. An ideal bacterial strain, SW-1, was isolated from seawater in a clam farm, and could inhibit the growth of Vibrio.MP-1. Thus, SW-1could be used as a probiotic to protect clams from diseases, and meanwhile reduce the effects of antibiotics on aquatic environment. The main results are described as follows:The selected MP-1was identified based on its physiological, morphological and biochemical characteristics, as well as its16S rDNA sequence. The results showed that the strain MP-1had a high similarity to Vibrio, alginolyticus. The mortality of clams was100%after infection with107to109cfu/ml of Vibrio. Alginolyticus.MP-1, indicating the strain MP-1is the clam pathogens. The best growth conditions of the strain MP-1:100mL/250ml of aeration,30℃of temperature, pH8.0and0.5mol/L of salinity. The growth curve of the strain MP-1not only reveals no lag phase, but more resistance to antibiotics, as compared with the strain SW-1. Therefore, disease outbreak caused by pathogenic Vibrio. Alginolyticus. MP-1was the most difficult to be control.The selected SW-1was also identified to have, and showed a high similarity to Pseudoalteromonas. piscicida. It could inhibit the growth of Vibrio, alginolyticus (Vibrio.MP-1) and improve the survival of clams following challenge with the pathogenic Vibrio MP-1. The mortality of clams was100%after infection with108CFU/ml of Vibrio, alginolyticus, whereas mortality was only11%when clams were infected with108CFU/ml of Vibrio. MP-1while simultaneously exposed to the same concentration of Pseudoalteromonas. SW-1. The strain SW-1can inhibit seven strains, which were used in this study. The best growth conditions of strains MP-1:30mL/250ml of aeration,30℃of temperature, pH8.0and0.5mol/L of salinity. The growth curve of strain SW-1not only reveals o lag phase, but more susceptible with antibiotics, as compared with the clams pathogens of Vibrio. MP-1.The inhibition activity of the strain SW-1was the strongest in its highest biomass, and could produce antimicrobial activity for seven strains. The inhitition zone ranged from16to25mm. The best inhibition effect was obtained when the cell-free supernatant (CFS) of the strain SW-1was joined in log phase and stability phase of Vibrio. MP-1. The induction test proved that the strain SW-1could produce antimicrobial substances to inhibit the growth of the clam pathogenic Vibrio. MP-1. The inhibitory activities of the strain SW-1CFS showed differently after treating by heat, acid, alkali, and proteinase K. The CFS of the strain SW-1inhibitory activities was decreased after treating by heat, but the inhibitory activities were still effective after treating by proteinase K in24h. The acid and alkali could increase the inhibitory activities of the strain SW-1CFS, could produce some active compounds to antagonize the pathogenic Vibrio. MP-1.The crude protein substances obtained from the CFS by the means of ammonium sulphate precipitation could produce inhibition activities.The crude protein substance was cut off by10KDa molecular sieves, and the components more than10KDa molecular weight could inhibit the growth of the strain Vibrio. MP-1. Then the crude protein substance was separated with SephadexG-75gel filtration chromatography and eluted three characteristic absorption peaks. Although the first peak and the second peak had higher protein concentration, there were no antibacterial activities. Protein concentration of the third peak was6.1mg/ml and these fractions could inhibit the growth of the strain Vibrio. MP-1. SDS-PAGE gel electrophoresis test could show a single stripe, according to the RF value logging its molecular weight was52.6KDa.The cell-free supernatant of the strain SW-1was collected, and then the CFS was extracted three times with ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure on a rotary evaporator. Antimicrobial activity of the extracts obtained was monitored by the disk diffusion method assay with Vibrio. MP-1as susceptible strain.The inhibition rate reached95%within24hours. According to organic solvent dissolved test, it was found that the crude extracts could be dissolved by Alcohol. The absorbance wave ranged from290nm to440nm. The crude extracts were eluted by silicone column using different proportion Methylene Dichloride and Methanol, then the46tubes were obtained. The methode of thin-layer chromatography couldn’t separate and combine these fractions, so the different wavelength (320nm and440nm) absorbance value was used to separate and combine these fractions. Thus, ten fractions were obtained. The inhibition test by using Microdilution broth assay showed the second and sixth fractions had inhibititon activety, and the inhibition rate reached to83.5%and92.6%, respectively. The HPLC results indicated that the sixth fraction had complex compounds, but the second fraction showed two single peaks, depend on the analysis of HPLC-ESI-MS/MS, the active component of strain SW-1was a bromine compound with701.5Da molecular weight.