Article Figures & Data
Figures
- FIG 1
Reuterin synthesis pathway. Microbial fermentation of glycerol by L. reuteri results in the synthesis of reuterin (3-HPA) and the corresponding by-product, 1,3-propanediol.
- FIG 2
Zones of inhibition show that L. reuteri inhibits C. difficile in a strain-specific and reuterin-dependent manner in vitro. A vancomycin (Vanc) disc (5 μg) was placed and L. reuteri strains were spotted and developed on BHI medium with 20 mM glucose. C. difficile strains were overlaid in BHI soft agar containing 2% glycerol and incubated for growth. Clear zones of inhibition (in millimeters) were measured. (A) Representative images of pathogen overlays showing clear zones where the growth of C. difficile R20291 was inhibited. Wild-type L. reuteri strains 17938 and 6475, isogenic pocR insertion mutants (17938::pocR and 6475::pocR), and complemented strains (pJKS100 is the vector control, pJKS102 is the vector expressing the wild-type strain 6475 pocR gene) are shown. (B) Bar graph representing zone of inhibition measurements for L. reuteri strains tested against C. difficile. Results represent the means ± SEMs (n = 3). Inhibitory zones significantly larger (unpaired, 2-tailed t test with equal variances) than the zones for the corresponding vancomycin control, the 6475 wild type, or the 6475::pocR strain are indicated (*, P < 0.05).
- FIG 3
Precursor-directed reuterin production by L. reuteri suppresses the growth of C. difficile in a human fecal microbial community. (A) Time line depicting the experimental design of the MBRA experiments. (B) Quantities of 16S rRNA gene copies of C. difficile relative to the total number of 16S rRNA gene copies in bioreactor samples over time. Data are represented as means ± SDs. Significant differences in relative C. difficile 16S rRNA gene copy numbers over seven time points between the Ctrl and Lreu-Glyc groups are indicated (repeated-measures ANOVA).
- FIG 4
Reuterin actively targets C. difficile with minimal consequences to the overall microbial community structure. (A) Temporal analysis of microbial community composition and structure using the 16S rRNA gene sequence data generated from bioreactor samples. Pairwise relationships between samples were determined using the Bray-Curtis dissimilarity measure and plotted with nonmetric multidimensional scaling (NMDS). (B) Distribution of the abundance of MBRA OTUs represented as a heatmap, with family-level classifications being represented by the colored bars on the right. OTUs significantly differing in abundance between groups at each time point are labeled on the left.
- FIG 5
Global metabolomic changes correlate with glycerol fermentation and associated by-products. (Top) Analysis of metabolic profiles from MBRAs using the normalized metabolite concentrations generated by Metabolon. Pairwise relationships between samples were determined using the Bray-Curtis dissimilarity measure and plotted with NMDS. (Bottom) Glycerol-associated metabolite concentrations that positively correlated (Spearman rho [rs] > 0.6, P < 0.05) with NMDS axis 1 loadings.
Tables
- TABLE 1
MICs of Lactobacillus spp. indicate resistance to antibiotics associated with an increased risk of development of CDI at clinically relevant concentrations
Strain Source MICa (μg/ml) Vancomycin Metronidazole Fidaxomicin C. difficile R20291b CDI patient stool 4 2 0.5 L. casei LC-39 Infant feces 256 >256 2 L. gasseri LG-3 Infant feces 1 >256 2 L. rhamnosus LR-34 Infant feces 256 >256 2 L. reuteri 17938 Breast milk 256 >256 >32 L. reuteri 6475 Breast milk 256 128 >32 - TABLE 2
Spearman correlations of reuterin- and glycerol-associated metabolites to global metabolite NMDS axis 1 loadings
Biochemicala Subpathway rs P valueb 1,3-Propanediol in reuterin pathway Chemical 0.765 1.271E−02 Biochemicals in glycerol metabolism 1-Palmitoyl-2-oleoyl-GPC Phospholipid metabolism 0.952 7.892E−13 1-Palmitoleoyl-2-linoleoyl-GPE Phospholipid metabolism 0.897 2.167E−09 1-Palmitoylglycerol Monoacylglycerol 0.783 9.006E−06 Glycerophosphorylcholine Phospholipid metabolism 0.770 1.167E−04 1-Stearoyl-2-linoleoyl-GPC Phospholipid metabolism 0.801 1.327E−04 Palmitoyl-oleoyl-glycerol Diacylglycerol 0.675 4.877E−04 Trimethylamine N-oxide Phospholipid metabolism 0.668 5.937E−04 Choline phosphate Phospholipid metabolism 0.674 6.665E−04 Glycerol-3-phosphate Glycerolipid metabolism 0.643 1.079E−03 Choline Phospholipid metabolism 0.642 1.104E−03 Glycerophosphoethanolamine Phospholipid metabolism 0.831 3.872E−03 Glycerol Glycerolipid metabolism 0.736 5.007E−03 1-Palmitoyl-GPC Lysolipid 0.812 5.560E−03 1-Palmitoyl-2-linoleoyl-GPC Phospholipid metabolism 0.605 2.793E−02 1-Stearoyl-2-oleoyl-GPC Phospholipid metabolism 0.618 4.629E−02
Supplemental material
- Supplemental file 1 -
Table S1. Bacterial strains and primers included in this study. Table S2. Sample identifiers and barcode sequences. Fig. S1. Reuterin-dependent C. difficile inhibition requires the glycerol dehydratase (gdh) gene. Fig. S2. Ex vivo analysis of glycerol-dependent L. reuteri-mediated growth inhibition of C. difficile. Fig. S3. Antibiotic treatment of human fecal MBRAs results in community dynamics mimicking CDI.
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- Supplemental file 1 -
