Background/Objectives: Multidrug-resistant (MDR) pathogens pose a critical challenge in infection treatment. Pediococcus pentosaceus (P. pentosaceus) is known for its antimicrobial activity; however, studies on its effects against MDR pathogens remain limited. This study aimed to evaluate the antimicrobial and biological activities of P. pentosaceus PMY2, isolated from fermented porcine colostrum yogurt, against MDR pathogens, including Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli). Methods: The antimicrobial, anti-inflammatory, and cytotoxic effects of P. pentosaceus PMY2 were evaluated in vitro. In addition, IL-6 and TNF-α levels were analyzed using an ELISA kit. Results: The MIC value against S. aureus KCTC 3881 and MRSA (CCARM 3089) was 0.31 mg/mL, while the MBC values were 0.63 mg/mL and 2.5 mg/mL, respectively. At MIC, biofilm formation was inhibited by 62.2% in S. aureus KCTC 3881 and by 51.5% in MRSA. CFS exhibited low cytotoxicity in RAW 264.7 macrophages and significantly reduced NO production, IL-6, and TNF-α levels, indicating strong anti-inflammatory effects. Conclusions: These findings suggest that P. pentosaceus PMY2 exhibited excellent antimicrobial and anti-inflammatory activity against MDR pathogens, demonstrating its potential as a natural antimicrobial agent. These results indicate that PMY2 CFS could be a promising candidate for addressing antibiotic resistance issues.
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