Next-Generation Probiotics Targeting Clostridium difficile through Precursor-Directed Antimicrobial Biosynthesis

Abstract

Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. <i>Clostridium difficile</i> infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified <i>Lactobacillus reuteri</i> to be a promising candidate for adjunct therapy. Human-derived <i>L. reuteri</i> bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the <i>pocR</i> gene locus were potent reuterin producers, with <i>L. reuteri</i> 17938 inhibiting <i>C. difficile</i> growth at a level on par with the level of growth inhibition by vancomycin. Targeted <i>pocR</i> mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of <i>L. reuteri</i> with glycerol was effective against <i>C. difficile</i> colonization in complex human fecal microbial communities, whereas treatment with either glycerol or <i>L. reuteri</i> alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with <i>L. reuteri</i> elicited changes in the composition and function of the human microbial community that preferentially targets <i>C. difficile</i> outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of <i>C. difficile</i>, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials.