Antibacterial Activity Of The Lipopeptides Produced By Bacillus Amyloliquefaciens R3Against Multidrug-Resistant Pathogenic E. Coli

Pathogenic E. coli is one of the most common pathogens of clinical infections,including pathogens causing intestinal infections and extraintestinal pathogenic E. coli(ExPEC). The former cause diarrhea in humans and animals and the latter mainlycause urinary tract infections, abscesses, meningitis and septicemia. Traditionally,therapeutic and preventive strategies frequently rely on the use of antibiotics in orderto prevent bacterial growth. However, the overuse of antibiotics has induced theemergence of resistant strains and drastic increase of often multiple antibioticresistances. Therefore, there is an urgent need for alternatives to antibiotics.Bacillus amyloliquefaciens can produce a variety of antimicrobial substancessuch as lipopeptides. Lipopeptides can be generally classified into three families orgroups: surfactin, iturin and fengycin. They have broad-spectrum antimicrobialactivities such as antibacterial, antifungal, antiviral, antimycoplasma and antitumoractivities. Although the cyclic lipopeptides have been found to have many potentialcommercial, therapeutic and environmental applications because of their high yields,easy production，stability and the special antimicrobial mechanisms, it is stillunknown if they have killing activity against the multidrug-resistant pathogenic E.coli.In this study, the R3strain with the highest activity against multidrug-resistantpathogenic E. coli was obtained. By conventional biochemical identification andmolecular biology methods including determination of16S rDNA sequence andalignment, we found that R3strain had the highest similarity to Bacillusamyloliquefaciens. Therefore, R3strain was identified as one strain of B.amyloliquefaciens. R3strain had strong antibacterial activities against six strains ofmultidrug-resistant pathogenic E. coli used in this study. The medium compositions and culture conditions for R3strain to produce thebioactive substances were optimized and the results were as follows: the optimalmedium compositions were2.0%(w/v) of soybean meal and2.0%(w/v) of wheatflour, the optimal cultivation conditions were28oC, pH6.0, inoculation sizes5%andcultivation time27h. Fermentation was carried out using10-L fermenter under theoptimal conditions. The results showed that R3strain produced high level of bioactivesubstances (Diameter of inhibition zone was23.5mm), amylase activity (104.8U/mL), protease activity (88.5U/100ml) and cell mass (1.5×109cells/mL).The bioactive substances produced by R3strain was purified and analysized. TheHPLC-MS analysis results showed that there were five anti-E. coli fractions and themolecular masses ([M+H]+) of them were994.6(F1),1008.6(F2),1022.6(F3),1022.6(F4) and1036.6(F5). These fractions were very close to C12-15surfactins.The purified surfactin (F1) showed the highest anti-E. coli activity under theconditions of28oC and pH7.0.The preliminary study on the mechanism of the lipopeptide against pathogenic E.coli was performed. The results showed that lipopeptide could damage both wholecells and protoplasts of E. coli2#. After treated with the purified surfactin F1andobserved under scanning electronic microscope, the E. coli2#cells were broken, andintegrity of the cell wall disappeared. E. coli cells treated by the surfactin F1werestained by PI, indicating the plasma membrane of the sensitive intact E. coli cells wasdamaged by the surfactin F1. In addition, the lipopeptide can directly damage themembrane of protoplasts no matter whether the cell wall is present or not.