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

Mastitis is an inflammatory disease of the mammary gland commonly brought about by bac-terial pathogens that gain physical access to the glandular epithelium through the teat canal. In bovines, common mastitis-causing agents are environmental or pathogenic bacterial spe-cies, including staphylococci, streptococci, enterococci, and Gram-negative bacteria such as Escherichia coli. Current therapeutic strategies for bovine mastitis typically involve the ad-ministration of antibiotic formulations within the infected udder, possibly resulting in in-creased selection of antibiotic resistance and the accumulation of antibiotic residues within the milk. In this study, we sought to design an antibiotic-free antimicrobial formulation to treat bovine mastitis based on bacterial antimicrobial peptides (bacteriocins) and proteins (pepti-doglycan hydrolases). Using a combination of in vitro 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 bo-vine mastitis-derived bacteria, both in planktonic and biofilm-associated growth modes. The addition of AuresinePlus (Aur, a staphylococcus-specific PGH) further increased the antimi-crobial potency against S. aureus. 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 S. aureus (MRSA) in vivo. We discuss the potential and challenges of using antibiotic-free antimicrobial combinations in the treatment of bacterial infections.