Mastitis, an infectious disease of the mammary gland in cows, is mainly caused by Staphylococcus aureus and Streptococcus uberis. These udder infections are difficult to cure due to 1. the intracellular persistence of pathogens, 2. the increasing prevalence of antibiotic-resistant strains and 3. biofilm formation, three factors that lead to a low cure rate of intramammary antibiotics. Consequently, there is an urgent need for alternative antimicrobial agents to treat mastitis more effectively. In this proposal, bacterial virus-encoded endolysins are put forward as a novel and sustainable type of antimicrobials with high killing efficacy, specific binding capacity and low chance of resistance development. This project will apply new protein engineering approaches to generate unique fusion enzymes ("enzy-biotics") that eliminate intramammary infections caused by streptococci and staphylococci. These chimeric endolysins are known to act intracellularly and kill dormant bacteria in biofilms. Our group's biotechnological expertise will be combined with an in-house developed mouse model, to not only assess the in vitro but also the in vivo efficacy and safety of our novel antimicrobial endolysins. This proposal therefore combines fundamental insights in the most recent protein engineering technology with relevant animal models and points towards the possible veterinary application in dairy agriculture, in line with the breakthrough of designer endolysins in human medicine.