Development of engineered endolysins with in vitro intracellular activity against streptococcal bovine mastitis‐causing pathogens

Bacteriophage‐derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic‐resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae, also targeting intracellular survival and biofilm formation. For this purpose, high‐throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram‐positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time‐kill assays in phosphate‐buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis, also showing similar activity against S. agalactiae and S. dysgalactiae. This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta‐lactam penicillin used to intramammarily treat bovine Gram‐positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add‐on in the treatment of bovine streptococcal mastitis.