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Cited by 259Gut microbiota and healthAntibiotic Resistance in BacteriaEscherichia coli research studies

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Inhibiting antibiotic-resistant Enterobacteriaceae by microbiota-mediated intracellular acidification

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Sorbara, Matthew T., Dubin, Krista, Littmann, Eric R., Moody, Thomas U., Fontana, Emily, Seok, Ruth, Leiner, Ingrid M., Taur, Ying, Peled, Jonathan U., van den Brink, Marcel R.M., Litvak, Yael, Bäumler, Andreas J., Chaubard, Jean-Luc, Pickard, Amanda J., Cross, Justin R., Pamer, Eric G.·2019·Journal of Experimental Medicine
DOI 10.1084/jem.20181639PMC PMC6314524
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Abstract

Klebsiella pneumoniae, Escherichia coli, and other members of the Enterobacteriaceae family are common human pathogens that have acquired broad antibiotic resistance, rendering infection by some strains virtually untreatable. Enterobacteriaceae are intestinal residents, but generally represent <1% of the adult colonic microbiota. Antibiotic-mediated destruction of the microbiota enables Enterobacteriaceae to expand to high densities in the colon, markedly increasing the risk of bloodstream invasion, sepsis, and death. Here, we demonstrate that an antibiotic-naive microbiota suppresses growth of antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis by acidifying the proximal colon and triggering short chain fatty acid (SCFA)–mediated intracellular acidification. High concentrations of SCFAs and the acidic environment counter the competitive edge that O2 and NO3 respiration confer upon Enterobacteriaceae during expansion. Reestablishment of a microbiota that produces SCFAs enhances clearance of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis from the intestinal lumen and represents a potential therapeutic approach to enhance clearance of antibiotic-resistant pathogens.

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Graphical abstract CC BY-NC-SA · PubMed Central
Figure 1. A diverse microbiota blocks enteric expansion and promotes clearance of clinical Enterobacteriaceae isolates. (A) Antibiotic-naive or ampicillin-treated WT mice were challenged with the indicated isolates of Enterobacteriaceae, and colonization after 24 and 72 h was measured by measuring CFUs in fecal pellets (n = 4 mice from two experiments). (B) Colonization of K. pneumoniae MH258 in fecal samples 1–4 d after challenge in gnotobiotic mice reconstituted with an antibiotic-naive microbiota or PBS as indicated in the schematic diagram (n = 3, 4 mice from one representative experiment of two independent experiments). (C–E) Colonization with K. pneumoniae MH258 (C), E. coli MHT18 (D), or P. mirabilis MH42F (E) in fecal pellets from ampicillin-treated WT mice that were challenged with K. pneumoniae MH258, E. coli MHT18, or P. mirabilis MH42F and then given FMT or PBS treatments as indicated. (n = 7–12 mice per group from three to four independent experiments; t test with the Holm-Sidak method to correct for multiple comparisons was used to compare the PBS and FMT groups at each time point). *, P < 0.05; ***, P < 0.001. ns, not significant.
Figure 1. A diverse microbiota blocks enteric expansion and promotes clearance of clinical Enterobacteriaceae isolates. (A) Antibiotic-naive or ampicillin-treated WT mice were challenged with the indicated isolates of Enterobacteriaceae, and colonization after 24 and 72 h was measured by measuring CFUs in fecal pellets (n = 4 mice from two experiments). (B) Colonization of K. pneumoniae MH258 in fecal samples 1–4 d after challenge in gnotobiotic mice reconstituted with an antibiotic-naive microbiota or PBS as indicated in the schematic diagram (n = 3, 4 mice from one representative experiment of two independent experiments). (C–E) Colonization with K. pneumoniae MH258 (C), E. coli MHT18 (D), or P. mirabilis MH42F (E) in fecal pellets from ampicillin-treated WT mice that were challenged with K. pneumoniae MH258, E. coli MHT18, or P. mirabilis MH42F and then given FMT or PBS treatments as indicated. (n = 7–12 mice per group from three to four independent experiments; t test with the Holm-Sidak method to correct for multiple comparisons was used to compare the PBS and FMT groups at each time point). *, P < 0.05; ***, P < 0.001. ns, not significant.CC BY-NC-SA · PubMed Central
Figure 2. An acidified pH is necessary for the microbiota to directly block carbapenem resistant K. pneumoniae expansion. (A) Schematic showing the collection and anaerobic culture of cecal contents from antibiotic-naive or ampicillin-treated mice. (B) Growth of K. pneumoniae MH258 in cecal content cultures from antibiotic-treated or antibiotic-naive mice (n = 7–8 wells from three to four independent experiments; one-way ANOVA followed by Dunnett’s multiple comparison test to PBS control values). (C) Growth of K. pneumoniae MH258 in the supernatant from the indicated cecal content supernatants (n = 6–8 wells from three independent experiments; t test). Data from these conditions is also presented in G. (D) Growth of K. pneumoniae MH258 in diluted supernatants from cultures of antibiotic-naive mice (n = 4 wells from two independent experiments; one-way ANOVA followed by Dunnett’s multiple comparison test to the PBS control values). (E) pH of supernatants from the indicated cecal content cultures (n = 4–5 independent experiments; one-way ANOVA followed by Dunnett’s multiple comparison test to the PBS control values). (F) pH of freshly isolated cecal contents from antibiotic-naive or ampicillin-treated mice (n = 4–5 independent experiments; t test). (G) Growth of K. pneumoniae MH258 in the supernatant from the indicated cecal content cultures, with or without adjusting the pH as indicated (n = 6–8 wells from three independent experiments, one-way ANOVA followed by Sidak’s Multiple comparison test). Control conditions without pH adjustment are also presented in C. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Kp, K. pneumoniae.
Figure 2. An acidified pH is necessary for the microbiota to directly block carbapenem resistant K. pneumoniae expansion. (A) Schematic showing the collection and anaerobic culture of cecal contents from antibiotic-naive or ampicillin-treated mice. (B) Growth of K. pneumoniae MH258 in cecal content cultures from antibiotic-treated or antibiotic-naive mice (n = 7–8 wells from three to four independent experiments; one-way ANOVA followed by Dunnett’s multiple comparison test to PBS control values). (C) Growth of K. pneumoniae MH258 in the supernatant from the indicated cecal content supernatants (n = 6–8 wells from three independent experiments; t test). Data from these conditions is also presented in G. (D) Growth of K. pneumoniae MH258 in diluted supernatants from cultures of antibiotic-naive mice (n = 4 wells from two independent experiments; one-way ANOVA followed by Dunnett’s multiple comparison test to the PBS control values). (E) pH of supernatants from the indicated cecal content cultures (n = 4–5 independent experiments; one-way ANOVA followed by Dunnett’s multiple comparison test to the PBS control values). (F) pH of freshly isolated cecal contents from antibiotic-naive or ampicillin-treated mice (n = 4–5 independent experiments; t test). (G) Growth of K. pneumoniae MH258 in the supernatant from the indicated cecal content cultures, with or without adjusting the pH as indicated (n = 6–8 wells from three independent experiments, one-way ANOVA followed by Sidak’s Multiple comparison test). Control conditions without pH adjustment are also presented in C. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Kp, K. pneumoniae.CC BY-NC-SA · PubMed Central
Figure 3. SCFAs inhibit Enterobacteriaceae isolates. (A) Acetate, propionate, and butyrate concentrations in antibiotic-naive or ampicillin mice. Total SCFA represents the sum of these three most-abundant SCFA (n = 8 mice from two independent experiments; t test). (B) Growth of the indicated isolates of Enterobacteriaceae in LB with or without a mixture of SCFA at the indicated pH was measured after 16 h of incubation (n = 6–8 wells from three to four independent experiments; one-way ANOVA followed by Dunnett’s multiple comparison test to the control LB condition for each isolate). (C) Growth of Enterobacteriaceae isolates in LB at the indicated pH values with a mixture of acetate, propionate, and butyrate was measured after 16 h of incubation (n = 6 wells from two independent experiments). pH values observed directly in cecal contents (Fig. 2 F) are indicated in green (antibiotic naive) or orange (antibiotic treated). Dotted line indicates strain growth in LB alone (n = 6 wells from two independent experiments). (D) The K. pneumoniae, E. coli, or P. mirabilis burdens in the intestinal content (CFU/g) of colonized mice given PBS or FMT treatments or collected before treatment is plotted against SCFA concentration in cecal contents (acetate + propionate + butyrate; n = 3–12 mice per group; Spearman correlation. CFU/g values are also plotted in Fig. 1, C and D).
Figure 3. SCFAs inhibit Enterobacteriaceae isolates. (A) Acetate, propionate, and butyrate concentrations in antibiotic-naive or ampicillin mice. Total SCFA represents the sum of these three most-abundant SCFA (n = 8 mice from two independent experiments; t test). (B) Growth of the indicated isolates of Enterobacteriaceae in LB with or without a mixture of SCFA at the indicated pH was measured after 16 h of incubation (n = 6–8 wells from three to four independent experiments; one-way ANOVA followed by Dunnett’s multiple comparison test to the control LB condition for each isolate). (C) Growth of Enterobacteriaceae isolates in LB at the indicated pH values with a mixture of acetate, propionate, and butyrate was measured after 16 h of incubation (n = 6 wells from two independent experiments). pH values observed directly in cecal contents (Fig. 2 F) are indicated in green (antibiotic naive) or orange (antibiotic treated). Dotted line indicates strain growth in LB alone (n = 6 wells from two independent experiments). (D) The K. pneumoniae, E. coli, or P. mirabilis burdens in the intestinal content (CFU/g) of colonized mice given PBS or FMT treatments or collected before treatment is plotted against SCFA concentration in cecal contents (acetate + propionate + butyrate; n = 3–12 mice per group; Spearman correlation. CFU/g values are also plotted in Fig. 1, C and D).CC BY-NC-SA · PubMed Central
Figure 4. An acidic pH and SCFA reduce the advantage conferred by aerobic or anaerobic respiration. (A) Equal numbers of E. coli Nissle 1917 and the isogenic cydAB/napA/narG/narZ strain were mixed in competitive cultures supplemented with or without SCFAs, NO3−, or oxygen in neutral or acidic pH conditions. Both the total growth of E. coli and the competitive advantage of the WT were measured after 16 h. (B) The total growth of E. coli in the indicated SCFA, NO3, O2, and pH conditions is plotted (n = 3 wells from three independent experiments, mean + SEM). The shading of each bar indicates the mean competitive advantage of respiration-competent WT compared with the mutant strain for that condition. Values and SEM for the CI are provided in Table S2.
Figure 4. An acidic pH and SCFA reduce the advantage conferred by aerobic or anaerobic respiration. (A) Equal numbers of E. coli Nissle 1917 and the isogenic cydAB/napA/narG/narZ strain were mixed in competitive cultures supplemented with or without SCFAs, NO3−, or oxygen in neutral or acidic pH conditions. Both the total growth of E. coli and the competitive advantage of the WT were measured after 16 h. (B) The total growth of E. coli in the indicated SCFA, NO3, O2, and pH conditions is plotted (n = 3 wells from three independent experiments, mean + SEM). The shading of each bar indicates the mean competitive advantage of respiration-competent WT compared with the mutant strain for that condition. Values and SEM for the CI are provided in Table S2.CC BY-NC-SA · PubMed Central
Figure 5. SCFA prevent E. coli and K. pneumoniae replication by decreasing intracellular pH. (A) Representative histograms of FITC and AmCyan fluorescence of pHlourin-expressing K. pneumoniae MH258 at different set pH values in the presence of potassium benzoate and methylamine hydrochloride (left). A representative standard curve depicting the relationship between intracellular pH and AmCyan/FITC intensity ratios (right). (B and C) The intracellular pH of K. pneumoniae MH258 (B) or E. coli MHT18 (C) in the presence of the indicated concentrations of acetate in LB at pH 7.0 or 5.75 (n = 3 from three independent experiments). (D) The intracellular pH of K. pneumoniae MH258 in freshly isolated cecal contents from control or antibiotic-treated mice supplemented with 50 mM of acetate as indicated. External pH was set at 7.5 or 6.0 as indicated (n = 3–7 from three to seven independent experiments; t test). In B–D, values above the upper limit of confidence are indicated with a horizontal line. (E) Anaerobic replication of K. pneumoniae MH258 or E. coli MHT18 in LB with potassium benzoate and methylamine hydrochloride at the indicated pH levels (n = 3–6 wells from three to four independent experiments). Ec, E. coli; Kp, K. pneumoniae; Pm, P. mirabilis.
Figure 5. SCFA prevent E. coli and K. pneumoniae replication by decreasing intracellular pH. (A) Representative histograms of FITC and AmCyan fluorescence of pHlourin-expressing K. pneumoniae MH258 at different set pH values in the presence of potassium benzoate and methylamine hydrochloride (left). A representative standard curve depicting the relationship between intracellular pH and AmCyan/FITC intensity ratios (right). (B and C) The intracellular pH of K. pneumoniae MH258 (B) or E. coli MHT18 (C) in the presence of the indicated concentrations of acetate in LB at pH 7.0 or 5.75 (n = 3 from three independent experiments). (D) The intracellular pH of K. pneumoniae MH258 in freshly isolated cecal contents from control or antibiotic-treated mice supplemented with 50 mM of acetate as indicated. External pH was set at 7.5 or 6.0 as indicated (n = 3–7 from three to seven independent experiments; t test). In B–D, values above the upper limit of confidence are indicated with a horizontal line. (E) Anaerobic replication of K. pneumoniae MH258 or E. coli MHT18 in LB with potassium benzoate and methylamine hydrochloride at the indicated pH levels (n = 3–6 wells from three to four independent experiments). Ec, E. coli; Kp, K. pneumoniae; Pm, P. mirabilis.CC BY-NC-SA · PubMed Central
Figure 6. Loss of SCFA production correlates with E. coli expansion in an allo-HCT patient. (A) Daily changes in the composition of the intestinal microbiota were monitored in a patient undergoing allo-HCT from 9 d before transplant to 5 d after transplant when a BSI with E. coli was detected (dashed red line). Select antibiotics administered to the patient are depicted. (B) The composition of the microbiota in daily fecal samples is plotted. Values are plotted as the percent abundance of each species determined using MetaPhlAn analysis. (C) The inverse Simpson diversity index of the microbiota in daily fecal samples. (D) Levels of the indicated β-lactamase genes in the microbiota from daily fecal samples. (E) Acetate, propionate, and butyrate concentrations in daily fecal samples from the patient. (F) Total SCFA in daily fecal samples from the patient calculated as the sum of acetate, propionate, and butyrate. (G) The relative abundance of E. coli (MetaPhlAn) compared with the total SCFA concentration in the daily fecal sample. At the point highlighted in red, a BSI with E. coli was detected (n = 15 d; Spearman correlation).
Figure 6. Loss of SCFA production correlates with E. coli expansion in an allo-HCT patient. (A) Daily changes in the composition of the intestinal microbiota were monitored in a patient undergoing allo-HCT from 9 d before transplant to 5 d after transplant when a BSI with E. coli was detected (dashed red line). Select antibiotics administered to the patient are depicted. (B) The composition of the microbiota in daily fecal samples is plotted. Values are plotted as the percent abundance of each species determined using MetaPhlAn analysis. (C) The inverse Simpson diversity index of the microbiota in daily fecal samples. (D) Levels of the indicated β-lactamase genes in the microbiota from daily fecal samples. (E) Acetate, propionate, and butyrate concentrations in daily fecal samples from the patient. (F) Total SCFA in daily fecal samples from the patient calculated as the sum of acetate, propionate, and butyrate. (G) The relative abundance of E. coli (MetaPhlAn) compared with the total SCFA concentration in the daily fecal sample. At the point highlighted in red, a BSI with E. coli was detected (n = 15 d; Spearman correlation).CC BY-NC-SA · PubMed Central

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Authors
Sorbara, M. T., Dubin, K., Littmann, E. R., Moody, T. U., Fontana, E., Seok, R., Leiner, I. M., Taur, Y., Peled, J. U., van den Brink, M. R. M., Litvak, Y., Bäumler, A. J., Chaubard, J.-L., Pickard, A. J., Cross, J. R., & Pamer, E. G.
Affiliations
  • Memorial Sloan Kettering Cancer Center (US)
  • Adult Bone Marrow Transplant Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 4
  • University of California, Davis (US)
Corresponding author
  • Sorbara, Matthew T.
  • Pamer, Eric G.
ORCID
Venue
Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2018-12-18
Volume
216
Issue
1
Pages
84-98
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.20181639
PMC
PMC6314524
OpenAlex
W2904430902
Cited by
259
References
46
Citations by year
2026: 36 · 2025: 40 · 2024: 48 · 2023: 34 · 2022: 26 · 2021: 38 · 2020: 25 · 2019: 11
FWCI
6.58
Type
journal article
Language
en
Open access
Licence
Locations
  • The Journal of Experimental Medicine (publishedVersion, cc-by-nc-sa)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by-nc-sa)
Funders
  • Canadian Institute of Health Research (FRN#152527)
  • National Institutes of Health (R01 AI042135, AI095706, U01 AI124275, P30 CA008748)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Health Sciences › Medicine › Microbiology (medical) › Antibiotic Resistance in Bacteria
  • Life Sciences › Immunology and Microbiology › Microbiology › Escherichia coli research studies
Keywords
Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, gut microbiota, short-chain fatty acids, intracellular acidification, antibiotic-induced microbiota depletion, bacterial clearance, sepsis, nitrate respiration, oxygen respiration
MeSH terms
Gastrointestinal Microbiome, Animals, Colon, Enterobacteriaceae, Enterobacteriaceae Infections, Fatty Acids, Female, Humans, Hydrogen-Ion Concentration, Male, Drug Resistance, Bacterial, Mice

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