Bacteriophage Endolysins As Promising Tools for Detection and Control of Foodborne Pathogens

Endolyins are bacteriophage-encoded cell wall-lytic enzymes (peptidoglycan hydrolases) that have recently gained attention as potential antimicrobial agents. These enzymes rapidly and specifically kill Gram-positive bacteria and are refractory to resistance development. Endolysins feature a modular architecture, consisting of enzymatically active domains (EAD), which cleave certain bonds within the peptidogylcan and cell wall binding domains (CBD), which direct the enzyme to its cell wall substrate with high affinity and specificity. These properties make CBDs ideal tools for diagnostics. CBDs, from Listeria phages, fused to fluorescent proteins allowed simultaneous detection and differentiation of Listeria cells from different serovars in mixed bacterial cultures. Furthermore, L. monocytogenes cells could be recovered from contaminated food samples via paramagnetic beads coated with these high-affinity binding molecules. This CBD-based magnetic separation procedure was demonstrated to be superior to established, standard detection protocols in terms of sensitivity and time requirement.

Besides diagnostic applications, endolysins hold promise for control of foodborne pathogens. A collection of unique staphylococcal peptidoglycan hydrolases exhibited lytic activity against S. aureus planktonic cells and biofilms, as demonstrated in static and dynamic models. In addition to these parental enzymes, we have compiled a large library of engineered endolysin constructs (> 400 constructs) featuring versatile enzymatic and antimicrobial properties. Using a newly developed screening protocol, we identified enzymes with high activity in cow milk from this library. The most promising candidates eradicated S. aureus in milk, within minutes, at nanomolar concentrations, acted synergistically when applied in combination, and retained their activity in raw milk. Overall, our results demonstrate the high potential of bacteriophage endolysins as tools for detection and control of bacterial pathogens in food production.