Microbiota dynamics in a randomized trial of gut decontamination during allogeneic hematopoietic cell transplantation

Abstract

BACKGROUNDGut decontamination (GD) can decrease the incidence and severity of acute graft-versus-host disease (aGVHD) in murine models of allogeneic hematopoietic cell transplantation (HCT). In this pilot study, we examined the impact of GD on gut microbiome composition and the incidence of aGVHD in HCT patients.METHODSWe randomized 20 patients undergoing allogeneic HCT to receive (GD) or not receive (no-GD) oral vancomycin-polymyxin B from day -5 through neutrophil engraftment. We evaluated shotgun metagenomic sequencing of serial stool samples to compare the composition and diversity of the gut microbiome between study arms. We assessed clinical outcomes in the 2 arms and performed strain-specific analyses of pathogens that caused bloodstream infections (BSI).RESULTSThe 2 arms did not differ in the predefined primary outcome of Shannon diversity of the gut microbiome at 2 weeks post-HCT (genus, P = 0.8; species, P = 0.44) or aGVHD incidence (P = 0.58). Immune reconstitution of T cell and B cell subsets was similar between groups. Five patients in the no-GD arm had 8 BSI episodes versus 1 episode in the GD arm (P = 0.09). The BSI-causing pathogens were traceable to the gut in 7 of 8 BSI episodes in the no-GD arm, including Staphylococcus species.CONCLUSIONWhile GD did not differentially affect Shannon diversity or clinical outcomes, our findings suggest that GD may protect against gut-derived BSI in HCT patients by decreasing the prevalence or abundance of gut pathogens.TRIAL REGISTRATIONClinicalTrials.gov NCT02641236.FUNDINGNIH, Damon Runyon Cancer Research Foundation, V Foundation, Sloan Foundation, Emerson Collective, and Stanford Maternal & Child Health Research Institute.

Keywords: Bacterial infections; Hematology; Infectious disease; Molecular genetics; Stem cell transplantation.

Figures

Figure 1. Study flow diagram.

ClinicalTrials.gov Identifier NCT02641236. Self-reported racial and ethnic categories in Supplemental Table 11. ANC, absolute neutrophil count.

Figure 2. Study design.

Twenty patients undergoing allo-HCT were randomized to 2 arms, 10 patients with GD and 10 patients with no GD. The GD arm received vancomycin-polymyxin B starting day –5 through engraftment (median neutrophil engraftment day +25, see Supplemental Figure 1) and was analyzed as intention-to-treat (Supplemental Table 1). The no-GD arm had the same stool and blood collection time points and did not receive oral vancomycin-polymyxin B. Black circles show time of stool collections, including pretransplant, weekly until engraftment, and monthly until day +100. An additional cohort of 2 healthy sibling donors serve as a stool control comparison group (Supplemental Figure 8). For immune reconstitution studies, blood samples (red circles) were collected at pretransplant, at 2 weeks, monthly for the first 3 months, and then at months 6, 9, and 12.

Figure 3. Shannon diversity is similar between the GD and no-GD groups based on intention-to-treat analysis at the species taxonomic level.

Samples from patients undergoing GD (red) and no GD (blue). (A) Shannon diversity over time analyzed at the species level using local polynomial regression fitting (LOESS, locally estimated scatterplot smoothing of the mean Shannon diversity) showing similarity between the 2 groups. n = 48 samples from 10 patients in GD arm, n = 51 samples from 10 patients in no-GD arm. (B) Shannon diversity of individual patients from pretransplant (before GD antibiotics) to 2 weeks after HCT connected with a line. Boxes shown are the median with hinges at the 25% and 75%. All comparisons not significant (see Supplemental Table 4 for details) using Wilcoxon rank-sum test. n = 10 GD arm, n = 10 no-GD arm.

Figure 4. Immune reconstitution.

Peripheral blood samples at pretransplant, monthly for the first 3 months, and then months 6, 9, and 12. Shown are the median values ± interquartile range, along with the number of patients sampled at each time point below each graph. (A) CD4+ T helper cells, (B) Treg/Tcon, (C) CD8+ cytotoxic T cells, (D) CD4+ Tcon naive cells, (E) CD19+ B cells, (F) CD56+CD3– natural killer cells. For CD19+ B cells at 12 months, an uncorrected P = 0.016 with a Wilcoxon rank-sum test was not significant when tested against a stringent Bonferroni-adjusted α level of 0.0045 (0.05/11 biomarkers tested).

Figure 5. Cumulative incidence of BSI during the first 100 days of transplant.

Patients are separated by treatment group with GD (dashed red line, n = 10 patients) and no-GD (solid blue line, n = 10 patients). In the 6 patients with a BSI, 5 BSIs occurred within the first 31 days; 1 patient in the no-GD arm had BSI on day +85 relative to the first transplant (on day +6 of the second transplant). P = 0.0483, Gray’s test.

Figure 6. Bacterial abundance in the gut around the time of BSI.

Multiple pathogens are present in the gut around the time of the BSI. Days relative to the course of the transplant shown on the x axis (from top to bottom on the y axis), antibiotic administration, α (Shannon) diversity, relative abundance of microbes in the stool samples at the genus taxonomic level (with organisms listed by color according to the key at the lower left), and relative abundance in the gut of the BSI-causing organism with the date of the BSI shown as an asterisk (*). Note: y axis is a different scale between abundance plots for focused organisms found in the BSI. (A) Patient C04 had 2 Staphylococcus aureus BSIs 89 days apart (day +5 and day +94) and a Klebsiella oxytoca BSI on day +18. (B) E. coli BSI on day +8 in patient C10. (C) Staphylococcus epidermidis BSI on day +23 in patient C20. (D) Patient C22 received 2 transplants and had low abundance of Rothia dentrocariosa in the gut during the first transplant; Rothia was not detectable in the gut after day +15 of the first transplant, with a Rothia BSI on day +6 of the second transplant (day +85 relative to the first transplant). An Enterococcus faecium BSI occurred on day +20 of the second transplant (day +99 relative to the first transplant). Antifungal and antiviral medications are shown in Supplemental Figure 13. Information on patients C03 and C11 may be found in Supplemental Figure 11. Azithro, azithromycin; Cipro, ciprofloxacin; Clinda, clindamycin; Levo, levofloxacin; Mero, meropenem; PipTazo, piperacillin/tazobactam; TMP/SMX, trimethoprim/sulfamethoxazole (cotrimoxazole).