Rationally-defined microbial consortia suppress multidrug-resistant proinflammatory Enterobacteriaceae via ecological control

Abstract

Persistent colonization and outgrowth of pathogenic organisms in the intestine may occur due to long-term antibiotic usage or inflammatory conditions, which perpetuate dysregulated immunity and tissue damage^1,2. Gram-negative Enterobacteriaceae gut pathobionts are particularly recalcitrant to conventional antibiotic treatment^3,4, though an emerging body of evidence suggests that manipulation of the commensal microbiota may be a practical alternative therapeutic strategy^5-7. In this study, we rationally isolated and down-selected commensal bacterial consortia from healthy human stool samples capable of strongly and specifically suppressing intestinal Enterobacteriaceae. One of the elaborated consortia, consisting of 18 commensal strains, effectively controlled ecological niches by regulating gluconate availability, thereby reestablishing colonization resistance and alleviating antibiotic-resistant Klebsiella-driven intestinal inflammation in mice. Harnessing these microbial activities in the form of live bacterial therapeutics may represent a promising solution to combat the growing threat of proinflammatory, antimicrobial-resistant bacterial infection.

Figure 1. Elaboration of an 18 strain-consortium capable of decolonizing Klebsiella.

a-c, e, f, Germ-free (GF) C57BL/6 mice were monocolonized with Kp-2H7, followed by oral administration of stool samples from one of five healthy human donors (A, F, I, J, and K) (a) or the indicated mixture of bacterial isolates (b, c, e, f). Faecal CFU of Kp-2H7 throughout the experiment (a, c, e, f). The combination of strains administered and faecal CFU of Kp-2H7 on day 28 are shown in b. d, GF mice (n = 5) were monocolonized with Kp-2H7, followed by oral administration of F31-mix. Ampicillin (200 mg/L) was given in the drinking water from day 32 to 63. Relative abundance of each of the 31 strains was quantified by qPCR in two technical replicates and average data are shown. In a-c, e, and f, median ± IQR (interquartile range) are shown for representative data from two independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; by Kruskal-Wallis test using the Benjamini-Hochberg correction for multiple comparisons at day 28.

Figure 2. Control of intestinal pathogens and colitis by F18-mix.

a, GF mice (n = 3–10 per group) were monocolonized with the indicated pathogenic or antibiotic-resistant (pathobiont) strain, and then treated with the indicated bacterial mixture. Faecal pathobiont load was examined by counting CFUs or by qPCR of bacterial DNA (for C. difficile). b-e, GF Il10^−/− mice were monocolonized with Kp-2H7, followed by oral administration with the indicated bacterial mix. Representative haematoxylin and eosin staining of the colon (b), histological colitis scores (c), faecal lipocalin-2 and calprotectin levels (d), and frequencies of IFN-γ⁺ cells among colonic lamina propria CD4⁺TCRβ⁺ T-cells (e) are shown. In a and c-e, median ± IQR are shown for representative data from two independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; by Kruskal-Wallis test using the Benjamini and Hochberg correction for multiple comparisons (c-e).

Figure 3. F18-mix competes with Kp-2H7 for gluconate.

a, Using random mutagenesis with the Tn5-based transposon EZ-Tn5, 8 × 10⁵ kanamycin resistant (KmR) Kp-2H7 mutants (Kp-TPs) were obtained. Kp-TPs were pooled together and administered to GF mice, followed by oral administration of F18-mix or F13-mix. Faecal samples were collected and sequenced at day 0, 4, 10, and 28. b, Heatmap shows 194 Kp-2H7 genes that were significantly downregulated on day 10 post-F18-mix administration. c, Relative abundance of Kp-TP mutants in each mouse (4 mice per group). Mutants representing >15% of the total reads in any samples are noted in the legend. d, Gluconate metabolic pathway in K. pneumoniae. GntR suppresses expression of genes encoding gluconate transporter (gntU), gluconate kinase (gntK), and Entner-Doudoroff pathway enzymes (edd and eda). e, GF mice were colonized with a 1:1 mixture of wild-type (WT) and D gntK Kp-2H7, followed by oral administration of F18-mix or F13-mix. Faecal Kp-2H7 CFUs are shown. Data are expressed as the median ± IQR, representative from two independent experiments. *P < 0.05; by Mann-Whitney U test at last day. f, LC-MS/MS analysis of the indicated carbon source in faeces of GF mice fed a nutrient-rich diet (CL-2). g, Faecal gluconate levels in uncolonized GF mice or GF mice colonized with Kp-2H7, F18-mix, or F13-mix. Data are expressed as the median ± IQR. *P < 0.05; by Kruskal-Wallis test using the Benjamini-Hochberg for multiple comparisons. h, GF mice were monocolonized with Kp-2H7, followed by oral administration of F18-mix. Diet was switched from CL-2 to AIN93G supplemented with or without gluconate at day 21. Faecal Kp-2H7 CFUs are shown as median ± IQR, representative from two independent experiments. *P < 0.05; ns, not significant; by Kruskal-Wallis test using the Benjamini-Hochberg correction for multiple comparisons at day 28. i, Pathogenic strains were incubated with 300 mM gluconic acid in mGAM broth for 48 hours. Gluconate concentration in the culture supernatant was measured by LC-MS/MS in n = 3 biological replicates.

Figure 4. Association of gluconate pathway genes with IBD.

a, In vitro gluconate consumption capabilities of the F18 strains are shown in the middle bar graph (n = 3 biological replicates). Genome neighbourhood of putative gluconate metabolism genes identified in the F18 strains are shown in the right. Asterisk indicates a non-functional frameshift mutation. b, GF mice were monocolonized with Kp-2H7 and treated with F8- or F18-mix. Faecal Kp-2H7 CFUs are shown. Data are expressed as the median ± IQR, representative from two independent experiments. *P < 0.05; by Mann-Whitney U test at day 28. c, Classical and alternative gluconate metabolism pathways typically carried by Klebsiella and Blautia species. d, Comparative analysis of the abundance of species containing gluconate-related genes among paediatric patients with ulcerative colitis (UC), classified as moderate/severe versus inactive disease (maroon) or mild versus inactive disease (grey). Species were grouped based on the combination of gluconate-related genes in their MSP bins. e, Overview of the sequence identity and sequence ID for gluconate-related genes per MSP. f, MSP prevalence across the cohort, shown as a percentage. g, Disease ratio of MSP prevalence between either moderate/severe vs. inactive disease (maroon) or mild vs. inactive disease (grey).