Potential of antimicrobial peptide-fused endolysin LysC02 as therapeutics for infections and disinfectants for food contact surfaces to control Cronobacter sakazakii

Although endolysin (lysin), a muralytic enzyme of bacteriophages, has emerged as a promising antibacterial agent, its use against Gram-negative bacteria is challenging due to the presence of an outer membrane (OM) that acts as a physical barrier. To overcome this limitation, it is crucial to provide lysins with the ability to penetrate or disrupt the OM to access the target peptidoglycan. Here, we fused a lysin LysC02 from phage Phi C02 with nine different amphipathic antimicrobial peptides (AMPs) to enhance its intrinsic bactericidal activity against the notorious opportunistic foodborne Gram-negative pathogen Cronobacter sakazakii. Even without OM permeabilizers, the engineered LysC02 fused at the C-terminus with Cecropin P1, Lactoferricin B, or Thanatin exhibited the most enhanced bactericidal activity against C. sakazakii with similar to 3-log reduction within 2 h. The action of LysC02-AMPs, the physical destruction of bacterial cells facilitated by the destabilization of the OM by fused AMPs and the intracellular turgor pressure of the cell, occurred over a wide range of temperatures up to 55 degrees C and pH from 4 to 10. Not only C. sakazakii but also other Gram-negative pathogens were lysed by LysC02AMPs. Accordingly, LysC02-AMPs were able to significantly rescue Galleria mellonella larvae infected with C. sakazakii in the in vivo treatment model and significantly reduce C. sakazakii cells in food and food contact surface models. Taken together, LysC02-AMPs could be developed as effective alternative disinfectants and to control C. sakazakii.