Pre-Engraftment Gut Colonization with Enterococcus Casseliflavus Improves Survival after Allogeneic Hematopoietic Cell Transplantation

Gut bacteria modulate the immune system and influence outcomes of allogeneic hematopoietic cell transplantation (HCT). We reported an unexpected association between pre-HCT gut colonization with intrinsically vancomycin-resistant enterococci (iVRE: E. gallinarum and E. casseliflavus) and better overall survival (OS). In an expanded cohort, now also including patients who were iVRE-colonized early post-HCT (before day +14), we demonstrate that the iVRE-OS association is specific to E. casseliflavus. We studied allo-HCT recipients who had ≥1 positive rectal swab or stool culture for iVRE (days -14 to +14). SpectraTM VRE chromogenic agar medium (ThermoFisher Scientific) was used for species-level identification. 66 (E. casseliflavus: 23, E. gallinarum: 43) of the 873 allograft recipients (2011-2017) met our inclusion criteria. As expected from the constitutive vancomycin resistance of iVRE, the groups did not differ in their prior exposure to different antibiotic classes. The groups were not different in patient, disease, or transplant characteristics, except a marginally higher frequency of sirolimus-based GVHD prophylaxis in the E. casseliflavus group (35% vs. 12%, P = 0.05). With a median follow up of 30 months, 3-year OS was better in patients with E. casseliflavus (91% vs. 62%, P = 0.04), due to lower 3-year non-relapse mortality (NRM: 0 vs. 18%, P = 0.05) (Fig. 1A-B). Only 2 patients with E. casseliflavus died within 3 years post-HCT, both due to relapse. In contrast, 14 patients with E. gallinarum died within 3 years: 7 (50%) due to relapse, and 7 (50%) from NRM (4 GVHD, 1 infection, 1 graft failure, 1 VOD). In multivariable analysis, E. casseliflavus gut colonization was associated with reduced all-cause mortality (HR 0.20, 95%CI 0.04-0.91, P = 0.04). There were no differences between the groups in 180-day acute grade II-IV GVHD (P = 0.19), 1-year chronic GVHD (P = 0.56), 100-day bacteremia (P = 0.59), or 100-day Clostridium difficile infection (P = 0.79). To probe the mechanism of this protection against mortality, first we mined 15 E. casseliflavus and 8 E. gallinarum genomes for 14 shikimate and tryptophan metabolism enzymes. This analysis predicted that E. casseliflavus encodes a larger number of enzymes in the tryptophan metabolism pathway (Fig. 1C). Several compounds in this pathway are ligands for the aryl hydrocarbon receptor (AhR). Signals downstream of AhR augment the gut barrier and modulate the immune system, potentially reducing injury and inducing protective responses. Next, we performed agar diffusion assays. We did not find significant bacteriocin production by E. casseliflavus, arguing against direct modulation of the gut microbiota (Fig. 2). Pre-engraftment gut colonization with E. casseliflavus, but not E. gallinarum, improves survival. Mechanistic studies of E. casseliflavus-host interactions can guide the development of novel microbiota therapeutics.