Metabolomics and proteomics of L. rhamnosus GG and E. coli Nissle probiotic supernatants identify distinct pathways that mediate growth suppression of antimicrobial-resistant pathogens

Probiotics merit testing as alternatives to conventional antibiotics and are receiving increased attention for efficacy against multi-drug resistant pathogen infections. This study hypothesis was that the Gram-positive probiotic, L. rhamnosus GG (LGG) and Gram-negative E. coli Nissle (ECN) secrete distinct proteins and metabolites to suppress pathogen growth. LGG and ECN cell free supernatants were tested in a dose-dependent manner for differential growth suppression of Salmonella Typhimurium, Escherichia coli, and Klebsiella oxytoca that harbor antimicrobial resistance (AMR). Across supernatant doses, LGG was 6.27% to 20.55% more effective than ECN at suppressing AMR pathogen growth. Proteomics and metabolomics were performed to identify pathways that distinguished LGG and ECN for antimicrobial functions. From the 667 detected metabolites in probiotic cell free supernatants, 304 metabolites had significantly different relative abundance between LGG and ECN, and only 5 and 6 unique metabolites were identified for LGG and ECN respectively. LGG and ECN differences involved amino acid, energy and nucleotide metabolism. Proteomics analysis of ECN and LGG cell free supernatants identified distinctions in 87 proteins, where many were related to carbohydrate and energy metabolism. Integration of genome-proteome-metabolome signatures from LGG and ECN with predictive metabolic modeling supported differential use of substrates by these two probiotics as drivers of antimicrobial actions. ECN metabolized a range of carbon sources, largely purines, whereas LGG consumed primarily carbohydrates. Understanding functional biosynthesis, utilization and secretion of bioactive metabolites and proteins from genetically distinct probiotics will guide strategic approaches for developing antibiotic alternatives and for controlling spread of multi-drug resistant pathogens. ImportanceProbiotics are practical alternatives for protection against antimicrobial resistant pathogens. Bioactive probiotics molecules merit further investigation using high throughput - omic approaches. This study identified functional differences between Gram-positive L. rhamnosus GG (LGG) and Gram-negative E. coli Nissle (ECN) probiotics that suppressed the growth of antimicrobial resistant S. Typhimurium, K. oxytoca, and E. coli. Proteomes and metabolomes of the probiotic cell free supernatants showed metabolic differences between LGG and ECN for mediating pathogen growth suppression. Metabolites distinguishing LGG versus ECN growth suppression included carbohydrates, lipids, amino acids, and nucleic acids. The metabolic flux differences between ECN and LGG, which coincided with observed separations in the proteomes and metabolomes, was hypothesized to explain the differential suppression of AMR pathogens. Integrated metabolite and protein signatures produced by each probiotic merit attention as adjuvant therapeutics for antimicrobial resistant infections.