Antibacterial Activity Of The Lipopetides Produced By Bacillus Amyloliquefaciens M1against Multidrug-resistant Vibrio Spp. Isolated From Diseased Marine Animals

Bacillus amyloliquefaciens could produce a variety of lipopeptides, which havesignificant antibacterial, antifungal and antimycoplasma activity. The lipopeptide iscomposed of lipophilic hydroxyl-fatty acid chain and hydrophilic peptide chain.Antibacterial lipopeptide has great potential in the fields of biocontrol in agriculture,therapeutic, and environmental management. But it is still unknown if they have killingactivity against the pathogenic Vibrio spp. isolated from diseased marine animals. Vibriosiscould cause significant economic losses in mariculture. Different kinds of antibiotics,which would encourage the natural emergence of bacterial resistance, are used to preventor cure the bacterial diseases in marine animals. So it is urgent need to find a natural andsafe antibacterial substance that will kill the pathogenic Vibrio spp. withmultidrug-resistant profiles.In this work, the M1strain with the strong antibacterial activity was isolated fromMangrove and examined against9species of pathogenic Vibrio spp. isolated from diseasedmarine animals. By using conventional biochemical identification and molecular biologymethod, the M1strain was identified to be one strain of B. amyloliquefaciens. It was foundthat all the Vibrio spp. showed resistance to several different antibiotics, suggesting thatthey were the multidrug resistance.In order to produce the maximal lipopeptides by the bacterial strain M1, a new andcheap medium which contained1.5%wheat flour and1.0%soybean powder wasdeveloped. The optimal cultivation conditions were28℃, pH7.0and cultivation time24h.Then, fermentation was carried out in10-L fermenter. It was found that the cell density ofstrain M1reached about1.6×109cells/mL after21h of the fermentation, and the diameterof the clear zone against V. anguillarum on the plate was34.0mm at the21st hour of the fermentation, and a high proteinase activity of2.3U/mL and amylase activity of48.7U/mL were detected during the fermentation. The antibacterial lipopeptides produced by B.amyloliquefaciens M1were purified by acid precipitation, thin-layer chromatography(TLC) and high pressure liquid chromatography (HPLC). The mass spectrometry analysisshowed that the lipopeptide NQ was the main product and the surfactin isoforms withamino acids (Glu-Leu-Leu-Val-Asp-Leu-Leu) and hydroxy fatty acids (of12–15carbons inlength). The antibacterial activity of the lipopeptide NQ was examined against9species ofVibrio spp. and all the Vibrio spp. strains were sensitive to the surfactin NQ, in particular V.anguillarum. The results indicated that the lipopeptides produced by B. amyloliquefaciensM1had a broad spectrum of action, including antibacterial activity against the pathogenicVibrio spp. with multidrug-resistant profiles.The lipopeptide NQ was able to kill not only the whole V. anguillarum cells, but alsothe protoplast of V. anguillarum. The antibacterial lipopeptide NQ probably killed bacteriaby disrupting cell membranes because the treated cells could be stained by PI, while thenormal cells could not. This confirmed that both the cell membranes of V. anguillarum andthe whole cells of the bacteria were damaged after the treatment with the lipopeptide NQ.