Unveiling the antibacterial effects of endolysin POE and holin Hol8 from Pseudomonas otitidis phage vB_PotS-PotUPM1

The global rise of antibiotic resistance poses a major threat to human health, driving the urgent search for novel antimicrobial strategies. Phage-derived proteins, such as endolysins and holins, represent promising alternatives to conventional antibiotics. Endolysins enzymatically degrade bacterial peptidoglycan but are often ineffective against Gram-negative bacteria due to the outer membrane. Holins, by disrupting cytoplasmic membranes, may bypass this barrier and provide a distinct antibacterial mechanism. In this study, a phage-derived endolysin (POE) and holin (Hol8) from Pseudomonas phage vB_PotS-PotUPM1 were cloned and expressed in Escherichia coli. Antibacterial activity against Pseudomonas otitidis NK1, an emerging zoonotic pathogen, was evaluated using well diffusion, plate lysis, turbidity reduction, and time-kill assays, with structural properties predicted through in silico analysis. POE, a globular protein with a single lysozyme-like domain, exhibited lytic activity only when the outer membrane was permeabilized, achieving a 40.4 ± 1.3% OD₅₉₅ reduction after 3 h at 500 µg/mL. In contrast, Hol8, a holin with four transmembrane domains, exhibited significant antibacterial activity. Crude Hol8 preparations showed bacteriostatic (MIC of 31.25 ± 0 µg/mL) and bactericidal (2× MIC of 62.50 µg/mL) activities against P. otitidis NK1, along with a broader host range activity against multiple Gram-negative and Gram-positive bacteria. The combination of POE and Hol8 showed no synergistic antibacterial effects under the tested conditions, with Hol8 showing superior efficacy. These results highlight Hol8 as a promising broad-spectrum antibacterial candidate capable of overcoming the outer membrane barrier that limits endolysin activity. The findings provide a strong foundation for further investigation of phage-derived holins as alternative therapeutics to combat multidrug-resistant bacterial infections.