Exometabolite-Based Antimicrobial Formulations from Lactic Acid Bacteria as a Multi-Target Strategy Against Multidrug-Resistant Escherichia coli

<b>Background/Objectives</b>: The global increase in multidrug-resistant (MDR) bacterial infections underscores the urgent need for effective and sustainable antimicrobial alternatives. This study investigates the antimicrobial activity of exometabolite-based formulations (ExAFs), derived from the cell-free supernatants (CFS) of native lactic acid bacteria (LAB) applied individually or in combination thereof, against MDR-<i>Escherichia coli</i> strain L1PEag1. <b>Methods</b>: Fourteen ExAFs were screened for inhibitory activity using time-kill assays, and structural damage to bacterial cells was assessed via scanning and transmission electron microscopy (SEM/TEM). The most potent formulation was further characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) employing a Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH) approach for untargeted metabolite profiling. <b>Results</b>: Among the tested formulations, E10, comprising CFS from <i>Weissella cibaria</i> UTNGt21O, exhibited the strongest inhibitory activity (zone of inhibition: 17.12 ± 0.22 mm), followed by E1 (CFS from <i>Lactiplantibacillus plantarum</i> Gt28L and <i>Lactiplantibacillus plantarum</i> Gt2, 3:1 <i>v</i>/<i>v</i>) and E2 (Gt28L CFS + EPS from Gt2, 3:1 <i>v</i>/<i>v</i>). Time-kill assays demonstrated rapid, dose-dependent bactericidal activity: E1 and E10 achieved >98% reduction in viable counts within 2-3 h, at 1× MIC, while E2 sustained 98.24% inhibition over 18 h, at 0.25× MIC. SEM and TEM revealed pronounced ultrastructural damage, including membrane disruption, cytoplasmic condensation, and intracellular disintegration, consistent with a membrane-targeting mode of action. Metabolomic profiling of E10 identified 22 bioactive metabolites, including lincomycin, the proline-rich peptide Val-Leu-Pro-Val-Pro-Gln, multiple flavonoids, and loperamide. Several compounds shared structural similarity with ribosomally synthesized and post-translationally modified peptides (RiPPs), including lanthipeptides and lassopeptides, suggesting a multifaceted antimicrobial mechanism. <b>Conclusions</b>: These findings position ExAFs, particularly E10, as promising, peptide-rich, bio-based antimicrobial candidates for food safety or therapeutic applications. The co-occurrence of RiPP analogs and secondary metabolites in the formulation suggests the potential for complementary or multi-modal bactericidal effects, positioning these compounds as promising eco-friendly alternatives for combating MDR pathogens.