Plant glycan-conditioned probiotic cell-free supernatants reduce growth and biofilm formation in methicillin-resistant Staphylococcus aureus

Abstract Managing Methicillin-resistant Staphylococcus aureus (MRSA) remains challenging due to its resistance to β-lactam antibiotics and its capacity to form biofilms. This study employed carbohydrate-active enzyme (CAZyme) profiling, an independent hypothesis-generating molecular modeling analysis, and in vitro fermentation to evaluate Arabic gum (AG) and baobab (BB) as fermentable substrates for Lactiplantibacillus plantarum and Bifidobacterium longum . Additionally, the activity of the resulting cell-free supernatants (CFS) against MRSA was assessed. The consensus CAZyme profiling revealed the presence of 52 and 55 genes annotated as glycoside hydrolases (GH) in the reference genomes of L. plantarum and B. longum , respectively. In contrast, only 13 such genes were identified in the S. aureus genome. These findings correspond to 19, 25, and six distinct GH families, respectively. In the computational analysis, digalacturonic acid and D-glucuronic acid exhibited predicted docking scores of − 7.0 and − 6.8 kcal/mol against PBP2a, with MM/GBSA ΔGbind values of − 24.22 and − 4.53 kcal/mol, respectively, compared to − 47.27 kcal/mol for the co-crystallized reference ligand. During 300-ns molecular dynamics simulations, D-glucuronic acid maintained repeated predicted contacts with Lys597, Gly599, Thr600, His583, and Ser598, whereas digalacturonic acid demonstrated greater conformational mobility. These in silico findings represent predicted ligand–PBP2a interactions and do not demonstrate experimental PBP2a inhibition. Experimentally, substrate supplementation significantly affected probiotic OD₆₀₀, viable cell counts, and culture pH (all ANOVA, P ≤ 0.0004), with 2% AG producing the strongest overall growth and fermentation response. Selected AG pH-matched preparations reduced MRSA growth by approximately 47–100% ( P < 0.0001), while selected BB-derived preparations resulted in approximately 39–94% growth reduction. AG-derived CFS at 1% and 2% significantly reduced MRSA biofilm biomass, corresponding to calculated reductions of 65.94% and 71.70%, respectively (adjusted P = 0.0401 and 0.0135). Overall, AG, and to a lesser extent BB, supported probiotic growth and fermentation and generated CFS preparations with measurable effects on MRSA growth and biofilm biomass, while the computational PBP2a findings remain hypothesis-generating and require direct biochemical validation.