Bacillus phage endolysin, lys46, bactericidal properties against Gram-negative bacteria

BACKGROUND AND OBJECTIVES: The great potential of bacteriophage for removing pathogen bacteria via targeting the cell wall is highly concerned. With a priority for overcoming drug-resistance, we screened against endolysins targeting Gram-negative bacteria to introduce a new antibacterial agent. This study was aimed to identify endolysins from the lysogenic phage of the Siphoviridea family in Bacillus subtilis. MATERIALS AND METHODS: The Bacillus subtilis strain DDBCC46 was isolated from a preliminary antibacterial screening program. The endolysin (s) was extracted, concentrated with ammonium sulfate saturation, and their activity evaluated against the indicator bacteria. The phage particles were extracted from the bacteria using the minimum inhibition concentration of mitomycin C, followed by testing the phage inhibitory effect on the growth of indicator bacteria. The NCBI, Virus-Host DB, and EXPASY databases were used to obtain and confirm the sequences of the genes encoding PG hydrolases in Siphoviridea phages hosted in B. subtilis. RESULTS: An 816 bp gene encoding an endolysin enzyme, was approved in the B. subtilis DDBCC 46, with specific primers of Bacillus phage SPP1. The purified-endolysin indicated antibacterial activity against Klebsiella pneumoniae, Salmonella typhimurium, Proteus (sp), and Escherichia coli. SDS-PAGE profiling followed by silica gel purification, led to introduce Lys46(30) as a therapeutic product and food preservative. CONCLUSION: lys46(30) showed antibacterial effects on the common Gram-negative pathogens in clinics and food industries; E. coli, P. aeruginosa and Salmonella (sp).