Human-derived probiotic Lactobacillus reuteri demonstrate antimicrobial activities targeting diverse enteric bacterial pathogens

Abstract

Lactobacillus reuteri is a commensal-derived anaerobic probiotic that resides in the human gastrointestinal tract. L. reuteri converts glycerol into a potent broad-spectrum antimicrobial compound, reuterin, which inhibits the growth of gram-positive and gram-negative bacteria. In this study, we compared four human-derived L. reuteri isolates (ATCC 55730, ATCC PTA 6475, ATCC PTA 4659 and ATCC PTA 5289) in their ability to produce reuterin and to inhibit the growth of different enteric pathogens in vitro. Reuterin was produced by each of the four L. reuteri strains and assessed for biological activity. The minimum inhibitory concentration (MIC) of reuterin derived from each strain was determined for the following enteric pathogens: enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, Salmonella enterica, Shigella sonnei and Vibrio cholerae. We also analyzed the relative abilities of L. reuteri to inhibit enteric pathogens in a pathogen overlay assay. The magnitude of reuterin production did not directly correlate with the relative ability of L. reuteri to suppress the proliferation of enteric pathogens. Additional antimicrobial factors may be produced by L. reuteri, and multiple factors may act synergistically with reuterin to inhibit enteric pathogens.

Figure 1. Comparison of reuterin production among probiotic L. reuteri strains

Reuterin was produced from L. reuteri strains 55730, 6475, 4659 and 5289 in glycerol solution three times in triplicate using a two-step fermentation process. Reuterin concentrations were determined using a modified Trp-HCl assay and HPLC-quantified reuterin as the standard. Reuterin concentrations were compared against concentrations of glycerol remaining in solution to yield a percentage of glycerol converted to reuterin. An asterisk (*) indicates that the amount of reuterin produced by 55730 is significantly different (P < 0.05) than the amounts produced by the other strains.

Figure 2. Reuterin inhibits proliferation of enteric pathogens

Reuterin was produced by L. reuteri strains 55730, 6475, 4659 and 5289 and tested for inhibitory effects on various enteric pathogens in an MIC assay. The growth of each pathogen was determined by measuring the A₆₀₀ after 18–24 hr incubation in the presence or absence of reuterin. Pathogen growth at 18–24 hr in the absence of reuterin was normalized to 100% for each replicate. The concentration of reuterin that consistently resulted in > 95% growth inhibition for each of the pathogens tested was 10 mM for each L. reuteri strain. EHEC, enterohemorrhagic E. coli; ETEC, enterotoxigenic E. coli.