An antibiotic-free antimicrobial combination of bacteriocins and a peptidoglycan hydrolase: <i>in vitro</i> and <i>in vivo</i> assessment of its efficacy

Mastitis is an inflammatory disease of the mammary gland usually caused by bacterial pathogens that gain physical access to the glandular epithelium through the teat canal. In bovines, common mastitis-causing agents are environmental or pathogenic bacterial species, including staphylococci, streptococci, enterococci, and gram-negative bacteria such as <i>Escherichia coli</i>. Current therapeutic strategies for bovine mastitis typically involve the administration of antibiotic formulations into the infected udder, which can result in increased selection for antibiotic resistance and the accumulation of antibiotic residues in milk. In this study, we sought to develop an antibiotic-free antimicrobial formulation for the treatment of bovine mastitis based on bacterial antimicrobial peptides (bacteriocins) and proteins (peptidoglycan hydrolases). Using a combination of <i>in vitro</i> assays with a range of bacteriocins, we show that the combination of the thiopeptide micrococcin P1 (MP1) and the lantibiotic nisin A (NisA) is a robust antimicrobial formulation that effectively inhibits the growth of bovine mastitis-derived bacteria, both in planktonic and biofilm-associated growth modes. The addition of AuresinePlus (Aur, a staphylococcus-specific peptidoglycan hydrolase) further increased the antimicrobial potency against <i>S. aureus</i>. Furthermore, using two mouse models, a skin infection model and a mastitis model, we show that the combination MP1-NisA-Aur effectively inhibits methicillin-resistant <i>S. aureus in vivo</i>. We discuss the potential and challenges of using antibiotic-free antimicrobial combinations in the treatment of bacterial infections.IMPORTANCEThe spread of antibiotic resistance is a major global concern. This is reflected in the One Health concept, which is based on the premise that the spread of antibiotic resistance can only be addressed through coordinated efforts to promote "healthy people, healthy environments, and healthy animals." It is therefore of great importance to reduce the use of medically important antibiotics in agriculture, where treatment of bovine mastitis is one of the major drivers of antibiotics use. In this work, we investigate the use of antimicrobial peptides and proteins as an alternative treatment for bovine mastitis pathogens.