Metabolite profiling and bioactivity guided fractionation of Lactobacillaceae and rice bran postbiotics for antimicrobial-resistant Salmonella Typhimurium growth suppression.

Probiotic-fermented supplements (postbiotics) are becoming increasingly explored for their activity against antibiotic-resistant enteropathogens. Prebiotics are often incorporated into postbiotics to enhance their efficacy, but due to strain differences in probiotic activity, postbiotic antimicrobial effects are poorly understood. To improve postbiotic antimicrobial efficacy, we investigated and compared metabolite profiles of postbiotics prepared with three lactic acid bacteria strains (L. fermentum, L. paracasei, and L. rhamnosus) cultured with and without rice bran, a globally abundant, rich source of prebiotics. At their minimum inhibitory dose, L. fermentum and L. paracasei postbiotics + rice bran suppressed S. Typhimurium growth 42-55% more versus their respective probiotic-alone postbiotics. The global, non-targeted metabolome of these postbiotics identified 109 metabolites increased in L. fermentum and L. paracasei rice bran postbiotics, including 49 amino acids, 20 lipids, and 12 phytochemicals metabolites. To identify key metabolite contributors to postbiotic antimicrobial activity, bioactivity-guided fractionation was applied to L. fermentum and L. paracasei rice bran-fermented postbiotics. Fractionation resulted in four L. fermentum and seven L. paracasei fractions capable of suppressing S. Typhimurium growth more effectively versus the negative control. These fractions were enriched in 15 metabolites that were significantly increased in the global metabolome of postbiotics prepared with rice bran versus postbiotic alone. These metabolites included imidazole propionate (enriched in L. fermentum + rice bran, 1.61-fold increase; L. paracasei + rice bran 1.28-fold increase), dihydroferulate (L. fermentum + rice bran, 5.18-fold increase), and linoleate (L. fermentum + rice bran, 1.82-fold increase; L. paracasei + rice bran, 3.19-fold increase), suggesting that they may be key metabolite drivers of S. Typhimurium growth suppression. Here, we show distinct mechanisms by which postbiotics prepared with lactic acid bacteria and rice bran produce metabolites with antimicrobial activity capable of suppressing S. Typhimurium growth. Probiotic strain differences contributing to postbiotic antimicrobial activity attract attention as adjunctive treatments against pathogens.

Integrated Profiling of Gram-Positive and Gram-Negative Probiotic Genomes, Proteomes and Metabolomes Revealed Small Molecules with Differential Growth Inhibition of Antimicrobial-Resistant Pathogens.

Probiotics produce small molecules that may serve as alternatives to conventional antibiotics by suppressing growth of antimicrobial resistant (AMR) pathogens. The objective of this study was to identify and examine antimicrobials produced and secreted by probiotics using 'omics' profiling with computer-based metabolic flux analyses. The cell-free supernatant of Gram-positive Lacticaseibacillus rhamnosus GG (LGG) and Gram-negative Escherichia coli Nissle (ECN) probiotics inhibited growth of AMR Salmonella Typhimurium, Escherichia coli, and Klebsiella oxytoca ranging between 28.85 - 41.20% (LGG) and 11.48 - 29.45% (ECN). A dose dependent analysis of probiotic supernatants showed LGG was 6.27% to 20.55% more effective at reducing AMR pathogen growth when compared to ECN. Principal component analysis showed clear separation of ECN and LGG cell free supernatant metabolomes. Among 667 metabolites in the supernatant, 304 were differentially abundant between LGG and ECN probiotics. Proteomics identified 87 proteins, whereby 67 (ECN) and 14 (LGG) showed differential expression as enzymes related to carbohydrate and energy metabolic pathways. The whole genomes and metabolomes were next used for in-silico metabolic network analysis. The model predicted the production of 166 metabolites by LGG and ECN probiotics across amino acid, carbohydrate/energy, and nucleotide metabolism with antimicrobial functions. The predictive accuracy of the metabolic flux analysis highlights the novel utility for profiling probiotic supplements as dietary-based antimicrobial alternatives in the control of AMR pathogen growth.

Antimicrobial-Resistant Escherichia coli from Environmental Waters in Northern Colorado.

Waterborne Escherichia coli are a major reservoir of antimicrobial resistance (AMR), including but not limited to extended-spectrum beta-lactamase (ESBL) and Klebsiella pneumoniae carbapenemase (KPC) mechanisms. This study quantified and described ESBL- and KPC-producing E. coli in Northern Colorado from sewer water, surface water, and influent and effluent wastewater treatment sources. Total detected bacteria and E. coli abundances, and the percentages that contain ESBL and/or KPC, were compared between water sources. Seventy E. coli isolates from the various waters had drug resistance validated with a panel of 17 antibiotics using a broth microdilution assay. The diverse drug resistance observed across E. coli isolates was further documented by polymerase chain reaction of common ESBL genes and functional relatedness by PhenePlate assay-generated dendrograms (n=70). The total E. coli abundance decreased through the water treatment process as expected, yet the percentages of E. coli harboring ESBL resistance were increased (1.70%) in surface water. Whole-genome sequencing analysis was completed for 185 AMR genes in wastewater E. coli isolates and confirmed the presence of diverse AMR gene classes (e.g., beta-lactams and efflux pumps) in isolate genomes. This study completed surveillance of AMR patterns in E. coli that reside in environmental water systems and suggests a role for integrating both phenotypic and genotypic profiling beyond ESBL and KPC mechanisms. AMR screening via multiple approaches may assist in the prevention of drug-resistant E. coli spread from waters to animals and humans.