Antibacterial activity and mechanism of novel phage endolysin lysSEP21 against dual-species biofilm of Salmonella and Escherichia coli and its application in food preservation.

Biofilms of Salmonella and Escherichia coli promote drug resistance and pathogenicity, as their multi-lipid structures hinder the eradication of these bacteria. Phage lytic proteins provide viable treatment strategies to eradicate biofilms. In this context, the antibiofilm efficacy of phage endolysin lysSEP21 was investigated against both species. Results of antibacterial activity showed the potent minimum inhibitory concentration (MIC) of ≤0.025 mg/mL, resulting in substantial 80 % lytic effects against Gram-negative and Gram-positive strains. The membrane disruption mechanism was further confirmed with increased release of β-lactamase and β-galactosidase from periplasm and cytosol, indicating effective degradation of outer and inner membranes (OM, IM), respectively. Furthermore, larger reductions up to 3.68 log CFU/mL were quantified in 1 h treated groups, leading to a ≥ 90 % reduction in biofilm-mass after 6 h. The viability of 36-42 h mature biofilm eradication was assessed by confocal laser scanning microscopy (CLSM) in a dead/live cell staining. Importantly, quantitative real-time PCR (qRT-PCR) analysis demonstrated that lysSEP21 significantly suppressed the relative gene expressions of key biofilm-regulating and virulence genes in Salmonella and E. coli. Moreover, this endolysin exhibited robust MDR Salmonella inhibition across food matrices, with reductions between 0.93 and 3.12 log CFU/mL. Altogether, lysSEP21 efficiently degraded mature biofilms and decontaminated food surfaces. Its application represents a remarkable advancement in food safety interventions and provides an exceptional strategy to mitigate public health risks associated with Salmonella and E. coli biofilms.