Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion

Abstract

Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars1, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse2,3. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel4. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion.

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Figure 1: The operon for galactarate utilization contributes to post-antibiotic expansion of S. Typhimurium.
Figure 2: Post-antibiotic generation of galactarate and glucarate is Nos2-dependent.

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Acknowledgements

This work was supported by Public Health Service grants OD010931 (E.M.V.), AI060555 (S.-P.N.), AI096528 (A.J.B.), AI109799 (R.M.T), AI112241 (C.A.L.), AI112258 (R.M.T), AI112445 (A.J.B.), U24 DK097154 (O.F.), AI112949 (A.J.B.) and AI114922 (A.J.B.).

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F.F. performed bacterial growth assays, most animal experiments and analysed the results. O.F. performed GC/MS measurements. M.X.B. scored histological sections. A.J.B., L.T., C.A.L., E.M.V., T.K. and T.W. assisted with animal experiments. F.F., S.-P.N., R.M.T. and A.J.B. were responsible for the overall study design. F.F. and A.J.B. wrote the manuscript.

Corresponding author

Correspondence to Andreas J. Bäumler.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks D. Bolam, D. Bumann, M. Fischbach & D. M. Monack for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Galactarate/glucarate fermentation by S. enterica.

a, One of the biochemical reactions used in the Salmonella serotyping scheme by Kauffman and White is the ability to ferment galactarate6. We divided 1,367 S. enterica subspecies enterica serovars into two groups: those associated with extraintestinal disease (serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Sendai, Choleraesuis, Typhisuis, Dublin, Bovismorbificans, Abortusovis, Abortusequi, Gallinarum biovar Gallinarum and Galliunarum biovar Pullorum) and those associated with human gastroenteritis (the remaining 1,354 serovars). The bar graph shows the percentages of serovars in each group that are positive, negative, delayed or differing (some isolates within the serovar are positive while others are negative) for this reaction. b, Detection of galactarate in chow for conventional or germ-free mice using GC/MS (n = 4). c, Schematic drawing of the two gene clusters encoding proteins involved in the degradation of glucarate and galactarate in S. Typhimurium (ATCC14028), E. coli (Nissle 1917) and Klebsiella oxytoca (KCTC1686). Arrows indicate genes. The bracket indicates the DNA region deleted in the indicated mutants. d, Minimal medium or mucin broth supplemented with the indicated carbon sources (0.1% w/v) was inoculated with a 1:1 mixture of the wild-type S. Typhimurium and indicated mutants. Competitive index (CI) recovered after 24 h incubation in an anaerobic chamber. e, Streptomycin-treated C57BL6 mice (n = 6) were infected with a 1:1 mixture of the indicated S. Typhimurium strains and the competitive index in colon contents determined 4 days after infection. Bars represent geometric means ± standard errors (d, e). A Student’s t-test was applied to determine statistical significance.

Extended Data Figure 2 Evaluation of caecal inflammation in streptomycin-treated mice 4 days after S. Typhimurium infection.

a, Streptomycin pre-treated mice were infected with the indicated strain mixtures and caecal histopathology was scored four days later for four mice per group. The criteria used for histopathology scoring are listed in Extended Data Table 4. Each bar represents data from an individual animal. b, Representative images of haematoxylin and eosin (H&E)-stained caecal sections scored in a, along with an image from a mock-infected mouse for comparison. All images were taken at the same magnification. m, mucosa; s, submucosa; ml, muscle layer; lu, lumen.

Extended Data Figure 3 Detection of galactaric acid and glucaric acid by GC/MS.

a, Representative GC elution profile of a caecal sample containing galactaric acid and glucaric acid (arrows). b, Representative single ion monitoring scan spectrum of galactaric acid and glucaric acid.

Extended Data Figure 4 Elevated Nos2 expression leads to nitrosyl radical-mediated oxidation of galactose.

a, Expression levels of Nos2 mRNA in RNA isolated from the caecal tip three days after mock-treatment (mock) or treatment of mice with streptomycin (Strep) was determined by quantitative real-time PCR. Bars represent geometric means ± standard errors. A Student’s t-test was applied to determine statistical significance. b, Schematic of the oxidation of galactose to galactarate by TEMPO. 2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPO) is a stable free nitrosyl radical that can oxidize terminal alcohol and aldehyde groups to carboxyl groups15. Consumption of TEMPO during the redox reaction is prevented by addition of a co-oxidant (NaOCl), which regenerates the nitrosyl radical.

Extended Data Figure 5 Galactarate/glucarate fermentation by E. coli.

a, Minimal medium or mucin broth supplemented with the indicated carbon sources was inoculated with a 1:1 mixture of the E. coli wild type (wt) and a garDXP garD mutant. CI, competitive index recovered after 24 h incubation in an anaerobic chamber. Growth was verified with 3 biological replicates. b, Streptomycin-treated C57BL6 mice (n = 6) were infected with a 1:1 mixture of the indicated E. coli strains and received the iNOS inhibitor aminoguanidine (AG) or vehicle control. The competitive index in colon contents was determined four days after infection. Bars represent geometric means ± standard errors (a, b). A Student’s t-test was applied to determine statistical significance.

Extended Data Table 1 Chart indicating scoring criteria for blinded examination of H&E-stained sections from the caecum
Extended Data Table 2 Blinded histopathology scoring scheme
Extended Data Table 3 Bacterial strains used in this study
Extended Data Table 4 Plasmids used in this study
Extended Data Table 5 Primers used in this study

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Faber, F., Tran, L., Byndloss, M. et al. Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion. Nature 534, 697–699 (2016). https://doi.org/10.1038/nature18597

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