Abstract

Background/Objectives: Severe and recurrent infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa necessitate alternative antimicrobial strategies. Fermented cacao beans represent a niche microbial ecosystem with the potential to harbor beneficial lactic acid bacteria (LAB). This study aimed to isolate and characterize LAB strains from fermented cacao beans in southern Thailand and to evaluate their probiotic potential and antimicrobial activity against MDR P. aeruginosa. Methods and Results: Five Lactiplantibacillus plantarum isolates were identified via MALDI-TOF MS and whole-genome sequencing (WGS). All strains demonstrated antimicrobial activity against 17 clinical MDR P. aeruginosa isolates and CR14 exhibited the largest inhibition zone. The isolates displayed robust probiotic traits, including survival under simulated gastrointestinal conditions. Acid tolerance (pH 2.0) reached 61.15 ± 7.75%, while resistance to pepsin, pancreatin, and bile salts exceeded 88%, 91%, and 92%, respectively. Strong adhesion was confirmed via auto-aggregation (55.02 ± 1.75%), hydrophobicity (45.58 ± 0.96%) and Caco-2 cell attachment (up to 98.11 ± 3.28%). WGS revealed multiple plantaricin-encoding clusters. Coarse-grained molecular dynamic simulations showed that two-peptide plantaricins (plnJ/K and plnNC8-αβ) self-assembled and formed stable pores in bacterial membrane models, confirming a pore-forming antimicrobial mechanism. The strains lacked acquired resistance genes and virulence factors, confirmed by in silico safety assessments. Conclusions: Thus, these L. plantarum strains are promising probiotics for managing MDR P. aeruginosa via functional foods or adjunct therapies.