Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease

Abstract

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

Keywords: Bacteroides; Bacteroides thetaiotaomicron; allogeneic hematopoietic stem cell transplantation; broad-spectrum antibiotics; carbapenem; graft-versus-host disease; intestinal microbiome; mucus layer; mucus-degrading bacteria; xylose.

Figure 1.. Meropenem increased the incidence of intestinal GVHD in allo-HSCT patients and mice.

(A) Incidence of intestinal GVHD in 295 allo-HSCT patients. Patients were stratified by antibiotic treatment from days -10 to 30 relative to allo-HSCT. (B) Experimental schema of murine GVHD model. Mice were treated with meropenem from days 3 to 15. TBI, total body irradiation. (C) Overall survival. Data are combined from three independent experiments. (D) H&E staining of histological sections of GVHD target organs collected on day 18. Bar, 100 μm. (E) GVHD histology scores of GVHD target organs harvested on day 18. All GVHD histology scores were quantified by a blinded pathologist. Combined data from two independent experiments are shown as means ± SEM. (F) Experimental schema of murine GVHD model. Mice were decontaminated with piperacillin/tazobactam plus nystatin from days 5 to 15. (G) Bacterial density of fecal samples collected from mice treated as indicated. Data are shown from one representative experiment. (H) Overall survival. Data are combined from three independent experiments.

Figure 2.. The effects of meropenem treatment on the composition of the intestinal microbiome in both patients and mice are characterized by expansion of Bacteroides.

(A - C) Composition of fecal samples from 44 allo-HSCT patients collected pre-HSCT and on day 14. (A) Bacterial genera composition of fecal samples collected pre-HSCT (top) and on day 14 (bottom). (B - C) Differentially abundant bacterial genera comparing pre-HSCT and day 14 samples collected from (B) meropenem-treated or (C) meropenem-untreated patients, analyzed by the paired-Wilcoxon test and adjusted for false discovery. (D - E) Paired-Wilcoxon test of the genus Bacteroides between at pre-HSCT and on day 14 in (D) meropenem-treated or (E) meropenem-untreated patients. (F) Experimental schema of murine GVHD model. The composition of the intestinal microbiome of fecal samples collected from day 21 was evaluated. (G) Bacterial density was measured using qPCR of 16S rRNA. (H) Alpha diversity, measured by the inverse Simpson index, was quantified in fecal samples. (I) Principle coordinate analysis (PCoA) of fecal samples. (J) Bacterial genera composition of fecal samples. (K) Differentially abundant bacterial genera comparing fecal samples. (L) Relative abundance of Bacteroides in fecal samples. (G - L) Data are combined from three independent experiments.

Figure 3.. Murine BT associated with meropenem-induced colonic GVHD.

(A) Relative abundance of distinguishable Bacteroides sequence variants on day 21. (B) Longitudinal relative abundance of BT. (C) Meropenem-treated mice (Figure 1C) were stratified by median abundance of BT into a high abundant BT arm (relative abundance > 0.2) and low abundant BT arm (relative abundance < 0.2). (D) Experimental schema of murine GVHD model using decontamination therapy followed by oral introduction of BT. Mice were decontaminated as in Figure 1F followed by oral gavage of 2×10⁷ CFUs of BT (MDA-JAX BT001) daily for 3 days. (E) Overall survival. (A - C, and E) Data are combined from three independent experiments. (F) H&E staining of histological sections of the colon collected on day 28. Bar, 100 μm. (G) GVHD histology scores of the colon collected on day 28. Data from one representative experiment are shown.

Figure 4.. Meropenem treatment results in increased localization of BT to the colonic mucosa in allogeneic mice.

(A) Experimental schema to analyzed paired fecal and mucosal microbiome by 16S rRNA sequencing. Paired stool and mucus were collected in distal colon. (B) PCoA of stool and mucus in distal colon from normal B6D2F1 mice. (C) Ratio of relative abundance of Bacteroidia/Clostridia in stool and mucus from normal B6D2F1 mice. (D) PCoA of stool and mucus in distal colon from allogeneic mice (left) and meropenem-treated allogeneic mice (right). Numbers shown in each PCoA depict samples collected from the same individual mouse. (E) Beta diversity distances within individual mouse. (F) Ratio of relative abundance of Bacteroidia/Clostridia in stool and mucus from allogeneic mice treated or untreated with meropenem. (B - F) Data from one representative experiment are shown.

Figure 5.. Meropenem-induced compromise of the colonic mucus layer in allogeneic mice.

(A) PAS staining of histological colon sections collected on day 18. Bar, 100 μm. The areas inside dotted lines indicate the inner dense colonic mucus layer. (B) Mucus thickness on day 18. Combined data from two independent experiments are shown as means ± SEM. (C) Immunofluorescent staining of colon sections for MUC2 (green) with universal bacterial 16S rRNA gene in situ hybridization probe EUB338 (red) counterstained with DAPI. Bar, 100 μm. Arrowheads indicate infiltrating bacteria. Areas in white squares are magnified and shown below original images. Areas inside dotted lines indicate the inner dense colonic mucus layer. (D) Numbers of bacterial CFUs cultivated from MLNs on day 18. Combined data from three independent experiments are shown. (E) Identified translocated bacteria in MLNs by MALDI Biotyper. Number indicates bacterial CFUs. (F) Immunohistochemistry staining of CD11b in histological colon sections. Bar, 100 μm. (G) Numbers of dendritic cells and neutrophils in the colon were determined by flow cytometric analysis. Combined data from two independent experiments are shown as means ± SEM.

Figure 6.. Mucolytic activity of BT is suppressed by ambient xylose

(A) Relative expression levels of PULs in BT RNA transcripts sequenced from stool collected from allogeneic mice treated or untreated with meropenem on day 18. Left: PUL identification numbers. Right: enzymatic functional annotations. (B) Relative abundances of monosaccharides of supernatants from stool collected from normal mice and allogeneic mice treated or untreated with meropenem on day 18 measured by IC-MS. (C) Experimental schema of murine GVHD model. (D) PAS staining of histological colon sections collected on day 20. Bar, 100 μm. (E) Mucus thickness on day 20. Data is shown as means ± SEM. (F) Relative abundance of BT on day 20. (G) Correlation analysis of the relative expression levels of PULs in BT RNA transcripts sequenced from stool collected from meropenem-treated allogeneic mice or those with xylose supplementation on day 21. X axis indicates calculated expression levels of PULs in BT RNA transcripts as mean expression levels of meropenem-treated allogeneic mice to allogeneic mice and Y axis indicates calculated expression levels of PULs in BT RNA transcripts as mean expression levels of meropenem-treated allogeneic mice to those with xylose supplementation. (H) Relative expression levels of PULs in BT RNA transcripts. (I) Overall survival. (E-F, I) Data are combined from two independent experiments.