Growth-Inhibitory Activity of Raw and Pasteurized Donkey Milk Against Clinically Relevant Gram-Negative Isolates with Different Antimicrobial Resistance Profiles

Antimicrobial resistance (AMR) is a major global health challenge affecting human and animal health, food safety, and the effectiveness of antimicrobial therapy. Therefore, this study evaluated the antimicrobial activity of raw (RDM) and pasteurized donkey milk (PDM) against clinically relevant Gram-negative bacteria with different AMR profiles. Antimicrobial activity against ten clinical Enterobacterales isolates, including CTX-M-, OXA-48-, KPC-, NDM-, and VIM-producing strains, was assessed by monitoring bacterial growth kinetics in artificially contaminated milk samples during 8 h of incubation at 37 °C. Pasteurized cow milk (CM) and nutrient broth (NB) served as controls. The strongest antimicrobial effects were observed against OXA-48-producing Klebsiella pneumoniae A4 and CTX-M-producing Escherichia coli A1, whereas Citrobacter koseri F5 showed the lowest susceptibility. After 8 h, K. pneumoniae A4 counts remained below 2 log CFU/mL in RDM compared with >6 log CFU/mL in NB. Significant effects of milk type, incubation time, and their interaction on bacterial growth were confirmed (p < 0.001). Donkey milk significantly inhibited bacterial growth compared with NB and CM, while some differences between RDM and PDM were observed depending on the bacterial isolate. These findings support donkey milk as a bioactive matrix with potential antimicrobial applications.