A Novel Protein Demonstrating Antibacterial Activity Against Multidrug-Resistant Escherichia coli Purified from Bacillus velezensis CB6

In recent years, multidrug resistance in pathogenic bacteria has become increasingly serious, causing serious harm to the livestock and poultry breeding industries and posing severe challenges to its clinical prevention and treatment; therefore, the development of new antibacterial agents is urgently needed. We previously isolated <i>Bacillus velezensis</i> CB6, which exhibits broad-spectrum antibacterial activity, from Changbaishan in China. In this study, multidrug-resistant <i>Escherichia coli</i> B2(MDR <i>E. coli</i> B2) was used as an indicator bacterium. Ammonium sulfate precipitation, dextran gel chromatography, and Diethylaminoethyl Bestarose High Performance was used to isolate antibacterial protein with strong activity against MDR <i>E. coli</i> B2. SDS-PAGE combined with liquid chromatography-mass spectrometry was used to obtain the antibacterial protein CB6-E, which has a molecular weight of 54.537 kDa. Our study found that CB6-E has a strong inhibitory effect on Gram-negative bacteria such as <i>Pseudomonas aeruginosa</i> Z1, <i>Salmonella</i> H9812, and <i>Shigella castellani</i> Z1; among them, the minimum inhibitory concentration for MDR <i>E. coli</i> B2 was 32 µg/mL. In addition, CB6-E is stable under various conditions including exposure to various temperatures, organic reagents, pH values, and proteolytic enzymes. The hemolytic activity test and cytotoxicity test also showed that CB6-E is safe. Research on antibacterial mechanisms showed that CB6-E destroys cell membranes in a dose-dependent manner and can inhibit the growth of MDR <i>E. coli</i> B2 by targeting lipopolysaccharides on the cell membrane, showing good therapeutic effects in model animals. In summary, CB6-E is a newly discovered antibacterial protein with a high therapeutic index that is safe, nontoxic, and stabile, and is expected to be an effective antibacterial agent.