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Despite efforts to discover novel therapeutic options, few antibiotics targeting MDR have been approved by the Food and Drug Administration (FDA). Lactic acid bacteria have emerged as a promising therapeutic alternative due to their demonstrated ability to combat MDR pathogens <jats:italic>in vitro<\/jats:italic>. Our previous co-culture studies showed <jats:italic>Lacticaseibacillus rhamnosus<\/jats:italic> CRL 2244 as having a potent killing effect against carbapenem-resistant <jats:italic>Acinetobacter baumannii<\/jats:italic> (CRAB) strains. Here we report that cell-free conditioned media (CFCM) samples obtained from <jats:italic>Lcb<\/jats:italic>. <jats:italic>rhamnosus<\/jats:italic> CRL 2244 cultures incubated at different times display antimicrobial activity against 43 different pathogens, including CRAB, methicillin-resistant <jats:italic>Staphylococcus aureus<\/jats:italic> (MRSA) and carbapenemase <jats:italic>Klebsiella pneumoniae<\/jats:italic> (KPC)-positive strains. Furthermore, transwell and ultrafiltration analyses together with physical and chemical\/biochemical tests showed that <jats:italic>Lcb<\/jats:italic>. <jats:italic>rhamnosus<\/jats:italic> CRL 2244 secretes a &lt;3 kDa metabolite(s) whose antimicrobial activity is not significantly impaired by mild changes in pH, temperature and various enzymatic treatments. Furthermore, sensitivity and time-kill assays showed that the bactericidal activity of the <jats:italic>Lcb<\/jats:italic>. <jats:italic>rhamnosus<\/jats:italic> CRL 2244 metabolite(s) enhances the activity of some current FDA approved antibiotics. We hypothesize that this observation could be due to the effects of <jats:italic>Lcb<\/jats:italic>. <jats:italic>rhamnosus<\/jats:italic> CRL 2244 metabolite(s) on cell morphology and the enhanced transcriptional expression of genes coding for the phenylacetate (PAA) and histidine catabolic Hut pathways, metal acquisition and biofilm formation, all of which are associated with bacterial virulence. Interestingly, the extracellular presence of <jats:italic>Lcb<\/jats:italic>. <jats:italic>rhamnosus<\/jats:italic> CRL 2244 induced the transcription of the gene coding for the CidA\/LgrA protein, which is involved in programmed cell death in some bacteria. Overall, the findings presented in this report underscore the promising potential of the compound(s) released by <jats:italic>Lcb<\/jats:italic>. <jats:italic>rhamnosus<\/jats:italic> CRL2244 as an alternative and\/or complementary option to treat infections caused by <jats:italic>A<\/jats:italic>. <jats:italic>baumannii<\/jats:italic> as well as other MDR bacterial pathogens.<\/jats:p>","DOI":"10.1371\/journal.pone.0306273","type":"journal-article","created":{"date-parts":[[2024,6,28]],"date-time":"2024-06-28T18:11:23Z","timestamp":1719598283000},"page":"e0306273","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":3,"title":["Lacticaseibacillus rhamnosus CRL 2244 secreted metabolites display killing and antibiotic synergistic activity against multi-drug resistant 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