| Summary: | Infections caused by antibiotic-resistant bacteria pose a significant global challenge. This study explores the antibacterial effects of a bacteriophage-derived endolysin, LysAB1245, against important pathogens, including <i>Acinetobacter baumannii</i>, <i>Escherichia coli</i>, <i>Klebsiella pneumoniae</i>, <i>Pseudomonas aeruginosa,</i> and <i>Staphylococcus aureus.</i> We determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for all tested isolates. A time–kill study was conducted to evaluate the reduction in bacterial survival following treatment with LysAB1245. Additionally, the effects of LysAB1245 on <i>P. aeruginosa</i> K1455 and methicillin-resistant <i>S. aureus</i> (MRSA) NPRC 001R-formed biofilms were investigated. The MIC and MBC of LysAB1245 against all the tested isolates ranged from 4.68 to 9.36 µg/mL and 4.68 to 18.72 µg/mL, respectively. The time–kill study demonstrated more than a 4 log CFU/mL (99.99%) reduction in bacterial survival within 6 h of LysAB1245 treatment at 2MIC. LysAB1245 (1/8–1/2MIC) treatment significantly reduced biofilms formed by <i>P. aeruginosa</i> and MRSA in a concentration-dependent manner. Furthermore, scanning electron and confocal laser scanning microscopy confirmed the potential inhibition effects on 3-day established biofilms formed on abiotic surfaces upon treatment with LysAB1245 at 2MIC. The findings indicate that endolysin LysAB1245 could be employed as a new alternative therapeutic antibacterial and anti-biofilm agent for combating biofilm-related infections.
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