Research ArticleGraft-Versus-Host Disease

Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice

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Science Translational Medicine  18 May 2016:
Vol. 8, Issue 339, pp. 339ra71
DOI: 10.1126/scitranslmed.aaf2311
  • Fig. 1. The clinical use of imipenem-cilastatin and piperacillin-tazobactam is associated with increased GVHD-related mortality.

    (A to D) A retrospective cohort of 857 adult patients was identified as recipients of non–T cell–depleted allo-HSCT at our center from 1992 to 2015. (A) GVHD-related mortality in patients exposed to aztreonam, cefepime, imipenem-cilastatin, or piperacillin-tazobactam. ABX, antibiotics. (B) Analyses of overall grades 2 to 4 GVHD are shown. (C) Analyses of upper GI grade 1 GVHD are shown. (D) Analyses of lower GI grades 1 to 4 GVHD are shown. (E) A subset of patients who had been treated for neutropenic fever was stratified according to whether they received first-line treatment with imipenem-cilastatin or piperacillin-tazobactam, versus aztreonam or cefepime. Outcomes indicated were depicted by Kaplan-Meier plots and curves compared by the log-rank test. *P < 0.05, **P < 0.01 in (A) to (E). (F) Intestinal microbiota composition analysis using 16S rRNA sequencing before and after beginning treatment with the indicated antibiotics in allo-HSCT recipients. *P < 0.05 after multiple comparisons with Holm-Sidak correction.

  • Fig. 2. Imipenem-cilastatin treatment, compared to aztreonam treatment, suppresses anaerobic commensals and elevates GVHD severity in mice.

    (A) Intestinal microbiota composition analysis using 16S rRNA sequencing before and after treatment with the indicated antibiotics in healthy C57BL/6 mice. Mice were treated with subcutaneous injections of each antibiotic twice a day for 2 days (100 mg/kg), and stool specimens were collected the following day. Values represent means ± SEM (n = 6 to 7). *P < 0.05, **P < 0.01 by multiple comparisons corrected by Holm-Sidak test. Data are combined from two independent experiments. (B to D) Lethally irradiated 129S1 recipients were transplanted with MHC-matched C57BL/6 T cell–depleted bone marrow cells (BM) and 1 × 106 C57BL/6 T cells (T). Recipients were treated with aztreonam, cefepime, imipenem-cilastatin, or piperacillin-tazobactam (pip-tazo) (100 mg/kg, sc, three times a week from days 10 to 24 after allo-HSCT). (B) Comparison of overall survival, with combined data from two independent experiments (n = 17 to 18). *P < 0.05, **P < 0.01 by Mantel-Cox log-rank test. (C) Comparison with control mice with T cell–depleted BM only (no GVHD). Overall survival, with combined data from three independent experiments (n = 15 to 43). ****P < 0.0001 by Mantel-Cox log-rank test. Tx indicates the period of antibiotic treatment in (B) and (C). (D) Mice with GVHD treated with antibiotics were sacrificed on day 21, and GVHD histology scores in target organs were quantified by a blinded pathologist. Data are combined from three independent experiments (n = 5 to 20). *P < 0.05 by Mann-Whitney U test.

  • Fig. 3. Treatment with imipenem-cilastatin in GVHD mice results in elevated numbers of donor CD4+ T cells and increased production of IL-23 in the colon.

    (A to F) Lethally irradiated 129S1 recipients were transplanted with C57BL/6 T cell–depleted bone marrow cells with 1× 106 C57BL/6 T cells. Recipients were treated with imipenem-cilastatin or aztreonam as described in Fig. 2. (A) Colonic lamina propria–infiltrating leukocytes from recipients were analyzed on day 21 by flow cytometry. Data are combined from two independent experiments. Values represent means ± SEM (n = 10 to 14). *P < 0.05, **P < 0.01 by Mann-Whitney U test. QOD, every other day. (B) Concentration of IL-23 in serum, whole–small intestine homogenate, and whole-colon homogenate. Data are combined from two independent experiments. Values represent means ± SEM (n = 10 to 12). *P < 0.05 by Mann-Whitney U test. The individual plots in the graphs indicate individual animals sacrificed at the time of the analysis in (A) and (B). (C) Lamina propria–infiltrating leukocytes from the colon on day 21 were enriched for CD11b and CD11c simultaneously using a mixture of magnetic beads, and IL-23 transcripts were quantified by real-time PCR (IL-23 expression relative to β-actin control). Data are representative of two independent experiments. Values represent means ± SEM (n = 3). *P < 0.05 by Mann-Whitney U test. (D) Immunofluorescence staining was used to quantify phosphorylated STAT3 (pSTAT3)–, CD3-, and DAPI (4′,6-diamidino-2-phenylindole)–positive cells in colonic tissue collected on day 21. (E) RNA sequencing analysis of the distal colon on day 16 after allo-HSCT. The top 50 regulated genes are shown in the heatmap panel. IMI/AZT, imipenem/aztreonam. (F) Immunofluorescence staining was used to quantify CD11b-, B220-, and DAPI-positive cells in colonic tissue collected on day 21. Data are representative of two independent experiments. Values represent means ± SEM (n = 7 to 8). **P < 0.01, ***P < 0.001, ****P < 0.0001 in (D) and (F) by Mann-Whitney U test.

  • Fig. 4. Mice treated with imipenem-cilastatin in the setting of GVHD show increased abundance of Akkermansia.

    (A) Stool specimens obtained from mice treated with imipenem-cilastatin or aztreonam were collected on day 21 and analyzed by 16S rRNA gene sequencing (as in Fig. 2), followed by principal components (PC) analysis of weighted and normalized UniFrac distances. Proportion of variance accounted for by each principal component is indicated. (B and C) Differential taxonomic abundance between aztreonam- and imipenem-cilastatin–treated recipients was analyzed by linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) (B) and by LEfSe projected as a cladogram (C). Data are representative of more than five independent experiments in (A) to (C). (D and E) Comparisons of bacterial abundance at the phylogenetic levels of order (D) and genus (E). Data are combined from six independent experiments (n = 32 to 36). ***P < 0.001 by multiple comparisons, corrected by Holm-Sidak test. (F to H) Stool specimens collected from mice with GVHD treated with antibiotics were collected on day 21 and evaluated by metagenomic shotgun sequence analysis. (F) Comparison of bacterial species abundance determined by taxonomy. Numbers 1 through 6 along the x axis represent the individual subjects. (G) Principal component analysis of quantification of sequence reads from KEGG (Kyoto Encyclopedia of Genes and Genomes) gene orthologs comparing specimens from mice treated with aztreonam and imipenem-cilastatin. (H) Quantification of gene sequences by homology was performed on stool specimens collected on day 21. Amuc_0953 (a sulfatase) and Amuc_2164 (a glycosyl hydrolase) are two predicted secreted mucolytic genes found in the genome of A. muciniphila ATCC BAA-835, isolated from human feces. **P < 0.01 by Mann-Whitney U test.

  • Fig. 5. Mice treated with imipenem-cilastatin in the setting of GVHD result in the loss of the colonic mucus layer and impaired intestinal barrier function.

    (A to C) Colon tissues from mouse recipients were fixed by water-free methanol-Carnoy’s fixative on day 21, stained with periodic acid–Schiff stain, and visualized by light microscopy. Orange triangles in (A) indicate the location of the inner mucus layer. Quantification of mucus layer thickness is shown in (B). Number of goblet cells are shown in (C). Data are representative of two independent experiments. Values represent means ± SEM (n = 10). **P < 0.01 by Mann-Whitney U test. (D) Immunostaining of colon sections for MUC2 (green) with general bacterial 16S rRNA gene in situ hybridization (FISH) probe EUB338 (EUB) (red) counterstained with Hoechst (blue). Data are representative of two independent experiments (n = 10). Orange arrowheads indicate inner mucus layer; red arrowheads indicate bacteria penetrating beyond the mucus layer and colonic epithelium. (E) Allo-HSCT recipients of bone marrow and T cells treated with aztreonam or imipenen-cilastatin were challenged with oral gavage of FITC-dextran on day 15 after transplant. The graph shows plasma FITC-dextran concentrations. Data are representative of two independent experiments. Values represent means ± SEM (n = 6 to 8). *P < 0.05 by Mann-Whitney U test.

  • Table 1. Clinical characteristics of 857 allo-HSCT patients transplanted at Memorial Sloan Kettering Cancer Center.

    NHL, non-Hodgkin’s lymphoma; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; MDS, myelodysplastic syndrome; MPD, myeloproliferative disorder; ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; HLA, human lymphocyte antigen.

    Dates of transplantMay 1992–July 2015
    Age (years)18 to 77; median, 49
    GenderFemale, 341 (40%); male, 516 (60%)
    Primary malignancyNHL, 242 (28%); AML, 277 (32%); CML, 48 (5.6%); MDS/MPD, 71 (8.3%); ALL, 74 (8.6%);
    CLL, 41 (4.8%); Hodgkin’s disease, 70 (8.2%); multiple myeloma, 24 (2.8%); other hematological
    malignancies, 16 (1.9%); other malignancies, 8 (0.9%); nonmalignant hematological disorders,
    22 (2.6%)
    Graft sourcePeripheral blood, 497 (58%); cord blood, 197 (23%); bone marrow, 163 (19%)
    Conditioning intensityStandard intensity myeloablative, 305 (36%); reduced intensity myeloablative, 283 (33%);
    nonmyeloablative, 268 (31%)
    HLA matchingHLA identical, 604 (70%); HLA mismatch, 253 (30%)
    Antibiotics for neutropenic feverNo antibiotics for neutropenic fever, 474 (55%)
    Imipenem-cilastatin or piperacillin-tazobactam first-line, 306 (36%)
      Received imipenem-cilastatin only, 6 (0.7%)
      Received piperacillin-tazobactam only, 183 (21%)
      Received both, 117 (14%)
      Also received aztreonam or cefepime, 106 (12%)
      Aztreonam or cefepime first-line, 77 (9%)
      Received aztreonam only, 18 (2.1%)
      Received cefepime only, 40 (4.7%)
      Received both, 19 (2.2%)
      Also received imipenem-cilastatin, 23 (2.7%)
  • Table 2. Effects of antibiotic exposure on increased 5-year GVHD-related mortality.

    PO, per os; IV, intravenous.

    AntibioticUntreated 5-year
    GVHD-related
    mortality (n)
    Treated 5-year
    GVHD-related
    mortality (n)
    P
    value
    Atovaquone (PO)14.1% (772)19.1% (85)0.496
    Aztreonam (IV)14.2% (793)17.5% (64)0.777
    Cefepime (IV)14.6% (705)13.8% (152)0.980
    Ciprofloxacin (IV)14.4% (535)14.7% (322)0.862
    Imipenem-cilastatin
    (IV)
    13.1% (709)21.5% (148)0.025
    Metronidazole (IV)14.0% (779)18.6% (78)0.197
    Metronidazole (PO)14.0% (801)20.8% (56)0.206
    Piperacillin-
    tazobactam (IV)
    11.9% (557)19.8% (300)0.007
    Sulfamethoxazole-
    trimethoprim (IV)
    14.8% (769)12.9% (88)0.625
    Sulfamethoxazole-
    trimethoprim (PO)
    14.6% (727)12.8% (130)0.522
    Vancomycin (IV)13.4% (408)15.6% (449)0.579
    Vancomycin (PO)14.4% (796)17.3% (61)0.942
  • Table 3. Association between antibiotic treatment and increased GVHD in target organs.
    AntibioticGVHD target organ (P value)
    Skin
    (stages 1–4)
    Liver
    (stages 1–4)
    Upper
    GI
    (stage 1)
    Lower
    GI
    (stages 1–4)
    Aztreonam0.1530.1070.6010.702
    Cefepime0.1330.1830.3320.514
    Imipenem-cilastatin0.1880.2000.0450.036
    Piperacillin-tazobactam0.1800.1650.0020.019
  • Table 4. Univariate and multivariate analyses of factors for GVHD-related mortality.

    HR, hazard ratio; CI, confidence interval; PBSC, peripheral blood stem cells; BM, bone marrow; TBI, total body irradiation.

    UnivariateMultivariate
    HR
    (95% CI)
    P
    value
    HR
    (95% CI)
    P
    value
    Antibiotic treatment group
      Imipenem/piperacillin-
    tazobactam
    ReferenceReference
      Aztreonam/cefepime0.38
    (0.15–0.94)
    0.0370.4
    (0.16–0.99)
    0.048
    Age >401.35
    (0.69–2.64)
    0.38
    Race
      White/BlackReference
      Asian/Hispanic1.19
    (0.56–2.53)
    0.65
      Other1.89
    (0.64–5.57)
    0.25
    Source
      PBSCReference
      BM1.27
    (0.44–3.72)
    0.66
      Cord1.21
    (0.69–2.11)
    0.51
    Donor
      Sibling identicalReference
      Unrelated identical1.47
    (0.64–3.35)
    0.36
      Nonidentical2.08
    (0.96–4.47)
    0.062
    Intensity
      AblativeReferenceReference
      Reduced1.03
    (0.49–2.19)
    0.931.04
    (0.49–2.2)
    0.920
      Nonablative2.37
    (1.15–4.91)
    0.0202.3
    (1.11–4.76)
    0.026
    TBI (any)1.72
    (0.92–3.22)
    0.087

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/339/339ra71/DC1

    Fig. S1. Six representative clinical cases with time courses depicting fecal microbiota composition and administration of antibiotic treatments during the course of allo-HSCT.

    Fig. S2. Recipients treated with imipenem-cilastatin exhibit enhanced histological GVHD on day 21.

    Fig. S3. Flow cytometric analysis of colon tissues in recipients on day 21.

    Fig. S4. Recipients treated with imipenem-cilastatin show increased production of IL-23 in the colon on day 21.

    Fig. S5. Abundance of bacterial OTUs, clustered by 16S rRNA sequence similarity, in recipients treated with imipenem-cilastatin or aztreonam.

    Fig. S6. Analysis of SCFAs in cecum stool specimens.

    Fig. S7. LEfSe analysis of relative numbers of sequences assigned to KEGG ortholog gene pathways after metagenomic shotgun sequencing.

    Table S1. Summary of day 21 analyses after allo-HSCT.

  • Supplementary Material for:

    Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice

    Yusuke Shono,* Melissa D. Docampo, Jonathan U. Peled, Suelen M. Perobelli, Enrico Velardi, Jennifer J. Tsai, Ann E. Slingerland, Odette M. Smith, Lauren F. Young, Jyotsna Gupta, Sophia R. Lieberman, Hillary V. Jay, Katya F. Ahr, Kori A. Porosnicu Rodriguez, Ke Xu, Marco Calarfiore, Hendrik Poeck, Silvia Caballero, Sean M. Devlin, Franck Rapaport, Jarrod A. Dudakov, Alan M. Hanash, Boglarka Gyurkocza, George F. Murphy, Camilla Gomes, Chen Liu, Eli L. Moss, Shannon B. Falconer, Ami S. Bhatt, Ying Taur, Eric G. Pamer, Marcel R. M. van den Brink,* Robert R. Jenq*

    *Corresponding author. Email: jenqr{at}mskcc.org (R.R.J.); shonoy{at}mskcc.org (Y.S.); vandenbm{at}mskcc.org (M.R.M.v.d.B)

    Published 18 May 2016, Sci. Transl. Med. 8, 339ra71 (2016)
    DOI: 10.1126/scitranslmed.aaf2311

    This PDF file includes:

    • Fig. S1. Six representative clinical cases with time courses depicting fecal microbiota composition and administration of antibiotic treatments during the course of allo-HSCT.
    • Fig. S2. Recipients treated with imipenem-cilastatin exhibit enhanced histological GVHD on day 21.
    • Fig. S3. Flow cytometric analysis of colon tissues in recipients on day 21.
    • Fig. S4. Recipients treated with imipenem-cilastatin show increased production of IL-23 in the colon on day 21.
    • Fig. S5. Abundance of bacterial OTUs, clustered by 16S rRNA sequence similarity, in recipients treated with imipenem-cilastatin or aztreonam.
    • Fig. S6. Analysis of SCFAs in cecum stool specimens.
    • Fig. S7. LEfSe analysis of relative numbers of sequences assigned to KEGG ortholog gene pathways after metagenomic shotgun sequencing.
    • Table S1. Summary of day 21 analyses after allo-HSCT.

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