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Early E. casseliflavus gut colonization and outcomes of allogeneic hematopoietic cell transplantation

  • Armin Rashidi ,

    Roles Conceptualization, Investigation, Supervision, Writing – original draft

    arashidi@umn.edu

    Affiliation Division of Hematology, Oncology, and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, MN, United States of America

  • Maryam Ebadi,

    Roles Data curation, Visualization, Writing – review & editing

    Affiliation Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Minnesota, Minneapolis, MN, United States of America

  • Robin R. Shields-Cutler,

    Roles Formal analysis, Methodology, Visualization, Writing – review & editing

    Affiliations BioTechnology Institute, College of Biological Sciences, University of Minnesota, MN, United States of America, Department of Biology, Macalester College, Saint Paul, MN, United States of America

  • Kathryn Kruziki,

    Roles Data curation, Formal analysis, Writing – review & editing

    Affiliation Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN, United States of America

  • Dawn A. Manias,

    Roles Data curation, Investigation, Methodology, Writing – review & editing

    Affiliation Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN, United States of America

  • Aaron M. T. Barnes,

    Roles Data curation, Investigation, Methodology, Writing – review & editing

    Affiliations Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN, United States of America, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, United States of America

  • Todd E. DeFor,

    Roles Formal analysis, Methodology, Writing – review & editing

    Affiliation Biostatistics Core, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America

  • Patricia Ferrieri,

    Roles Resources, Writing – review & editing

    Affiliation Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, United States of America

  • Jo-Anne H. Young,

    Roles Methodology, Supervision, Writing – review & editing

    Affiliation Division of Infectious Disease and International Medicine, Department of Medicine, University of Minnesota, Minneapolis, MN, United States of America

  • Dan Knights,

    Roles Formal analysis, Methodology, Software, Supervision, Writing – review & editing

    Affiliation BioTechnology Institute, College of Biological Sciences, University of Minnesota, MN, United States of America

  • Bruce R. Blazar,

    Roles Supervision, Writing – review & editing

    Affiliation Division of Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN, United States of America

  • Daniel J. Weisdorf,

    Roles Resources, Supervision, Writing – review & editing

    Affiliation Division of Hematology, Oncology, and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, MN, United States of America

  • Gary M. Dunny

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Resources, Supervision, Writing – review & editing

    Affiliation Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN, United States of America

Early E. casseliflavus gut colonization and outcomes of allogeneic hematopoietic cell transplantation

  • Armin Rashidi, 
  • Maryam Ebadi, 
  • Robin R. Shields-Cutler, 
  • Kathryn Kruziki, 
  • Dawn A. Manias, 
  • Aaron M. T. Barnes, 
  • Todd E. DeFor, 
  • Patricia Ferrieri, 
  • Jo-Anne H. Young, 
  • Dan Knights
PLOS
x

Abstract

Gut dysbiosis has been associated with worse allogeneic hematopoietic cell transplantation (allo-HCT) outcomes. We reported an association between intrinsically vancomycin-resistant enterococci (iVRE: E. gallinarum and E. casseliflavus) gut colonization and lower post-transplant mortality. In this study, using an expanded cohort, we evaluated whether our previously observed association is species-specific. We included allo-HCT recipients with ≥1 positive rectal swab or stool culture for iVRE between days -14 and +14 of transplant. To investigate whether iVRE modulate the gut microbiota, we performed agar diffusion assays. To investigate whether iVRE differ in their ability to activate the aryl hydrocarbon receptor, we analyzed iVRE genomes for enzymes in the shikimate and tryptophan pathways. Sixty six (23 E. casseliflavus and 43 E. gallinarum) of the 908 allograft recipients (2011–2017) met our inclusion criteria. Overall survival was significantly higher in patients with E. casseliflavus (91% vs. 62% at 3 years, P = 0.04). In multivariable analysis, E. casseliflavus gut colonization was significantly associated with reduced all-cause mortality (hazard ratio 0.20, 95% confidence interval 0.04–0.91, P = 0.04). While agar assays were largely unremarkable, genome mining predicted that E. casseliflavus encodes a larger number of enzymes in the tryptophan metabolism pathway. In conclusion, E. casseliflavus gut colonization is associated with reduced post-HCT morality. Further research is needed to understand the mechanisms for this association.

Introduction

The importance of gut microbiota as a predictor of allogeneic hematopoietic cell transplantation (HCT) outcomes has become apparent in recent years [1]. We recently reported an unexpected association between pre-HCT gut colonization with intrinsically vancomycin-resistant enterococci (iVRE: E. gallinarum and E. casseliflavus) and improved overall survival (OS) due to decreased non-relapse mortality (NRM) [2]. iVRE are characterized by susceptibility to teicoplanin and constitutive low-level vancomycin resistance through the chromosomally encoded vanC genotype [3,4].

The objective of the present study was to determine whether our reported association between pre-HCT iVRE gut colonization and improved outcomes is species-specific (i.e., E. gallinarum vs. E. casseliflavus). In an expanded cohort, now also including patients who were iVRE colonized early after HCT, we demonstrate that this association with improved survival is specific to E. casseliflavus. Using in vitro agar diffusion assays and bioinformatic analysis of iVRE genomes, we explored two potential mechanisms for an E. casseliflavus-mediated effect: (i) a direct effect on the host and (ii) an indirect effect on the host via modulation of the gut microbiota.

Materials and methods

Patients

The University of Minnesota Institutional Review Board approved the study protocol. We retrospectively studied allo-HCT recipients at the University of Minnesota (2011–2017) who had ≥1 positive rectal swab or stool culture for iVRE between days -14 and +14. New admissions for HCT were screened for gut VRE colonization weekly until discharge. SpectraTM VRE chromogenic agar medium (Thermo Fisher Scientific, Waltham, MA) was used for enterococcal culture. Species-level identification was done by Vitek 2 instrument and MALDI-TOF mass spectrometry (both from bioMerieux, Durham, NC) and antibiotic sensitivity were performed by broth microdilution from a subculture of the isolate using the colorimetric Vitek 2 automated system (bioMérieux, Durham, NC). Colonization level, classified as light, moderate, and heavy growth according to our clinical laboratory standards, was extracted from the patients’ electronic medical records. Antimicrobial prophylaxis consisted of levofloxacin, acyclovir, and an azole. Cefepime was our empiric antibiotic for neutropenic fever. OS was estimated using Kaplan-Meier curves. Relapse, grade II-IV acute graft-versus-host-disease (GVHD), chronic GVHD and NRM were estimated using cumulative incidence, treating NRM (for relapse), non-GVHD death (for GVHD), and relapse (for NRM) as competing risks. In the analysis of relapse and NRM, we excluded patients with non-malignant disorders. Fine and Gray regression was used to adjust for potential confounding [5]. All analyses were performed using SAS 9.4 (SAS Institute, Cary, NC).

Agar diffusion assays

Intestinal domination may increase the risk of bacteremia and worsen transplant outcomes [6]. To investigate whether E. casseliflavus produces bacteriocins that create colonization resistance against prototype colonizers of the gut, we screened 16 E. casseliflavus clinical isolates for activity against 6 different E. faecium and E. faecalis isolates via agar diffusion assays. These E. casseliflavus isolates were provided by the clinical laboratory at our institution and retrieved from a repository of samples collected from inpatients (not necessarily HCT patients) over the last 10 years. In addition, a subset of the E. casseliflavus isolates were tested against two E. coli isolates, two Streptococcus agalactiae (group B) isolates, and a Streptococcus gordonii isolate.

A 300 μL aliquot of the indicated growth medium (M9YE [7] or Todd-Hewitt broth) was inoculated with enterococcal producer or indicator strain colonies and incubated for 18 hours at 37°C. The following day, indicator plates were prepared by inoculating 50 mL of warm medium + 1% w/v agar with 50 μL of an overnight culture of the indicator strain. Using a multichannel pipette, 1 μL of producer culture then was stabbed and released into the agar. Plates were then incubated for 18 hours at 37°C prior to imaging. Activity was determined based on the presence or absence of a clear zone of inhibition surrounding the producer strain inoculum.

Bioinformatic analysis of shikimate/tryptophan pathways

Host-microbial crosstalk at the gut barrier is partially regulated by indole, indole-derived metabolites, and other shikimate/tryptophan pathway compounds, some of which are aryl hydrocarbon receptor (AhR) agonists with immunomodulatory and gut barrier protective functions [8,9]. To bioinformatically investigate whether E. casseliflavus, E. gallinarum, E. faecium, and E. faecalis may possess similar ARH agonist pathways, we analyzed the set of shikimate and tryptophan metabolism enzymes present in sequenced genomes from each species. A total of 15 E. casseliflavus (8 human isolates, of which 6 were disease-associated), 8 E. gallinarum (3 human disease-associated isolates), 11 E. faecium (10 human isolates, of which 8 were disease-associated), and 7 E. faecalis genomes (5 human isolates, of which 4 were disease-associated; for all species NCBI RefSeq latest assembly versions were used, accessed 3/19/2018; all classified as full genome representation) were annotated with PROKKA v1.12 [10] using default settings plus the following arguments: “—gcode 11—genus Enterococcus—species [casseliflavus]/[gallinarum]/[faeciuj]/[faecalis]—usegenus—kingdom Bacteria”. The annotations were then manually searched for enzymes present in shikimate and tryptophan metabolism as identified in the KEGG database [11]. The resulting presence/absence data was processed, ordered by unsupervised hierarchical clustering, and plotted using custom scripts in R (version 3.4; R Foundation for Statistical Computing, Vienna, Austria).

Results

Sixty six (23 with E. casseliflavus and 43 with E. gallinarum) of the 842 allograft recipients between 2011–2017 met our inclusion criteria. As expected, since vancomycin resistance in iVRE is constitutive rather than acquired, the groups did not differ in their prior exposure to different classes of antibiotics (Fig 1). The abundance of iVRE in cultures were classified as light in 3 (13%), moderate in 7 (30%), heavy in 10 (43%), and not available in 3 (13%) patients with E. casseliflavus colonization. The abundance of iVRE in cultures were classified as light in 8 (19%), moderate in 9 (21%), and heavy in 26 (60%) in patients with E. gallinarum colonization. Nine (39%) patients colonized with E. casseliflavus and 34 (79%) of those colonized with E. gallinarum had at least one other sample colonized with the same species between days -14 and +14. These findings suggest that E. gallinarum colonization is more stable than E. casseliflavus colonization. Only 2 (9%) patients with E. casseliflavus and none with E. gallinarum colonization had VRE colonization between days -14 and +14. Five patients in each group (22% and 12%) had vancomycin-sensitive enterococcus (VSE) gut colonization between days -14 and +14.

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Fig 1. Antibacterial antibiotic exposure in patients with E. casseliflavus vs. E. gallinarum gut colonization.

There were no significant differences between the groups. We considered antibiotic exposures starting 4 weeks before admission to the hospital until the colonization event. We classified antibiotics to four major groups that are commonly used before or shortly after HCT. Anaerobic: Antibiotics with potent anti-anaerobic activity (piperacillin-tazobactam, carbapenems, metronidazole, clindamycin); FQN: Fluoroquinolone; PCN/Ceph: Penicillins or Cephalosporins.

https://doi.org/10.1371/journal.pone.0220850.g001

There were no significant differences between the groups in patient-, disease-, or transplant-related characteristics, except a higher frequency of sirolimus-based GVHD prophylaxis in the E. casseliflavus group (35% vs. 12%, P = 0.05; Table 1). Fig 2 compares transplant outcomes between the groups with E. casseliflavus gut colonization, E. gallinarum gut colonization, and the 776 control patients who did not have colonization with either species. The median follow up for the first two groups was 30 (interquartile range 22–41) months. No deaths occurred before day +14. Comparing patients with E. casseliflavus vs. E. gallinarum colonization, OS was significantly higher in patients with E. casseliflavus (91% vs. 62% at 3 years, P = 0.04), due to lower NRM (0 vs. 18% at 3 years, P = 0.05). Only two 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 post-HCT: 7 (50%) due to relapse and 7 (50%) from NRM (4 GVHD, 1 infection, 1 graft failure, and 1 veno-occlusive disease). Relapse rates were not significantly different between the groups (47% vs. 30%, P = 0.39). 3-year rates of OS, NRM, relapse, and grade II-IV acute GVHD among controls were 61% (95% confidence interval 57%-64%), 24 (21–27)%, 30 (26–34)%, and 34 (31–37)%, respectively. These rates were similar to the E. gallinarum group.

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Fig 2. Comparison of outcomes between the groups with E. casseliflavus and E. gallinarum gut colonization.

Patients with E. casseliflavus had a significantly higher overall survival (A), due to lower non-relapse mortality (B). The cumulative incidence of relapse (C) and grade II-IV acute GVHD (D) were not significantly different between the groups. Curves for a control group of 776 patients who did not develop E. casseliflavus or E. gallinarum gut colonization are also shown, with outcomes not different from the E. gallinarum group. P values compare patients with E. casseliflavus vs. E. gallinarum. aGVHD: Acute graft-versus-host disease.

https://doi.org/10.1371/journal.pone.0220850.g002

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Table 1. Baseline patient-, disease-, and transplant-related characteristics.

https://doi.org/10.1371/journal.pone.0220850.t001

In multivariable analysis, E. casseliflavus gut colonization was significantly associated with reduced all-cause mortality (hazard ratio 0.20, 95% confidence interval 0.04–0.91, P = 0.04). There were no significant differences between the groups in 180-day grade II-IV acute GVHD (22% in E. casseliflavus vs. 37% in E. gallinarum; P = 0.19), 1-year chronic GVHD (9% vs. 12%, P = 0.56), 100-day bacteremia (22% vs. 28%, P = 0.59), or 100-day Clostridium difficile infection (17% vs. 21%, P = 0.79). The groups did not differ in time to platelet recovery (median 42 vs. 38 days, P = 0.46) but time to neutrophil recovery was longer in the E. casseliflavus group (median 16 vs. 12 days, P = 0.02).

Table 2 shows the results of agar diffusion assays measuring susceptibility of well-characterized other enterococcal species that might be competitors in the gut to potential bacteriocins or other compounds secreted by E. casseliflavus or E. gallinarum. Other than one E. casseliflavus isolate (#10 in Table 2), which had potent activity against both E. faecium and E. faecalis, no other E. casseliflavus isolates showed consistent zones of inhibition. We found no consistent differences between the two growth media tested in these assays. To examine the possibility that E. casseliflavus might produce bacteriocins or other compounds that target potentially-competitive non-enterococcal bacteria strains in the gut, we also tested five additional clinical isolates via the agar diffusion assay—two E. coli isolates, two Streptococcus agalactiae isolates, and a Streptococcus gordonii isolate–against a subset of six of the E. casseliflavus strains noted in Table 2. None of the non-enterococcal isolates were affected by the E. casseliflavus strains tested. These results suggest that a competitive advantage over other bacteria did not contribute to E. casseliflavus dominance in the intestines of patients with improved survival.

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Table 2. Sensitivity of E. faecium and E. faecalis to potential E. casseliflavus bacteriocins as measured by agar diffusion assays.

https://doi.org/10.1371/journal.pone.0220850.t002

Our bioinformatic analysis of 41 publicly available Enterococcus genomes for enzymes in shikimate and tryptophan metabolism pathways predicted that E. casseliflavus encodes a larger number of enzymes in the tryptophan metabolism pathway (Fig 3). This analysis predicted that four enzymes in the chorismate pathway to indole are present only in E. casseliflavus. In addition, tryptophanase, which converts tryptophan to indole, was predicted to be present in four E. casseliflavus genomes but none of the other E. casseliflavus or non-casseliflavus genomes.

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Fig 3. Bioinformatic analysis of 41 publicly available Enterococcus genomes for 14 enzymes in shikimate and tryptophan metabolism pathways.

Predicted presence/absence matrix for each enzyme (shikimate and tryptophan metabolism pathways) found in at least one of the 41 sequenced Enterococcus genomes, with pathways and isolate genomes arranged by unsupervised hierarchical clustering (A). Simplified metabolic pathway demonstrating the two routes to indole production (via chorismate and via tryptophan) (B). 4.2.1.10: 3-dehydroquinate dehydratase; 1.1.1.25: shikimate dehydrogenase; 2.7.1.71: shikimate kinase; 2.5.1.19: 3-phosphoshikimate 1-carboxyvinyltransferase; 4.2.3.5: chorismate synthase; 4.1.327: anthranilate synthase; 2.4.2.18: anthranilate phosphoribosyltransferase; 5.3.1.24: phosphoribosylanthranilate isomerase; 4.1.1.48: indole-3-glycerol-phosphate synthase; 4.2.1.20: tryptophan synthase; 1.2.1.3: aldehyde dehydrogenase (NAD+); 4.1.99.1: tryptophanase; 4.2.3.4: 3-dehydroquinate synthase; 2.5.1.54: 3-deoxy-7-phosphoheptulonate synthase.

https://doi.org/10.1371/journal.pone.0220850.g003

Because of the known role of indole in AhR agonism [12,13], potential involvement of the four casseliflavus-specific enzymes in indole production, and the availability of a sensitive and specific assay for indole production [14], we tested 16 diverse E. casseliflavus strains, and selected isolates of E. gallinarum, E. faecium, and E. faecalis for indole production in vitro (Methods in S1 File). None of the enterococcal isolates produced detectable indole in these assays, while a control Escherichia coli strain produced abundant indole under the same conditions (Fig A in S1 File).

Discussion

The presence of Blautia, butyrate-producing Clostridia spp [15], and Eubacterium limosum [16] in the stool has been associated with reduced mortality after allo-HCT. Our results indicate a similar association for E. casseliflavus. Notably, although both E. casseliflavus and E. gallinarum are motile enterococci with intrinsic vancomycin resistance and have 74% average nucleotide identity [17], our results from this and our previous study show that the association between iVRE and reduced mortality after allo-HCT is specific to E. casseliflavus.

We explored two possibilities to explain the association between E. casseliflavus and reduced post-allo-HCT mortality. First, we hypothesized that E. casseliflavus may have a competitive advantage over pathobionts known to cause clinical infections in transplant recipients. Intestinal domination by pathogenic bacteria such as those with antibiotic resistance genes (e.g., VRE), coupled with a compromised gut barrier due to transplant conditioning and intestinal GVHD, may increase the risk of bloodstream infection [6,18]. Bacteriocin production augments enterococcal niche competition in the mammalian gut [19]. However, our results did not identify E. casseliflavus as a powerful producer of bacteriocins against E. faecium and E. faecalis (including 5 VRE and 1 vancomycin-sensitive strains). Similarly, E. casseliflavus did not have a competitive advantage against several other gut pathobionts in our agar diffusion assays.

Alternatively, E. casseliflavus may produce metabolites that directly benefit the host. One such metabolite is the commensal microbe-derived compound butyrate, which has both local and systemic immunomodulatory effects [20,21], and improves gut barrier integrity [15,22,23]. Another example is indole, a molecule produced from the catabolism of dietary tryptophan by commensal bacteria of the gut [24]. We chose to measure indole because indole and its derivatives have a wide range of immune- and non-immune-mediated effects on the host. Indoleamine 2,3-dioxygenase (an indole derivative) has tolerogenic activity and reduces acute GVHD lethality by causing T-cell anergy [25,26]. In a recent study, indole-3-carboxaldehyde limited gut barrier damage, reduced bacterial translocation, and decreased inflammatory cytokine production, reducing GVHD pathology and its mortality [12]. GVHD and complications of gut barrier injury such as bloodstream infection are the most common causes of NRM in HCT recipients. Our in vitro comparison of E. casseliflavus with other enterococci for indole production was unremarkable. However, indole is only one of the many AhR agonists produced via shikimate and tryptophan pathways. Investigating the other metabolites involved in the shikimate/tryptophan pathway may be informative.

Using a genome mining approach, we predicted the potential presence of more tryptophan pathway metabolites and AhR ligands in E. casseliflavus compared to E. gallinarum. AhR signals augment the gut barrier and modulate the immune system, potentially reducing pathogenic injury and inducing protective immune responses [2730]. Although NRM was lower in E. casseliflavus patients, no specific cause of NRM was identified as the primary driver of this difference. We chose AhR ligands for our bioinformatic analysis because of their broad range of effects on the host and hence potential to explain the overall reduced NRM rates in E. casseliflavus patients. Our genomic prediction needs biochemical validation in future work.

Although iVRE can be isolated from stool in ~5% of hospitalized patients, clinical infections are rare [3135]. Gut colonization with E. casseliflavus may not be in the causal pathway to improved transplant outcomes. Instead, it may be a surrogate for a healthier microbiome permitting colonization by a less pathogenic organism. Future metagenomics studies may identify microbiota signatures of E. casseliflavus-colonized samples and predict metabolic pathways in which these samples are enriched.

Supporting information

S1 File. Methods and Fig A.

Lack of indole in culture supernatants of enterococcal strains.

https://doi.org/10.1371/journal.pone.0220850.s001

(PDF)

References

  1. 1. Andermann T, Peled J, Ho C, Reddy P, Riches M, Storb R, et al. Microbiome-Host Interactions in Hematopoietic Stem Cell Transplant Recipients. Biol Blood Marrow Transplant. 2018; pmid:29471034
  2. 2. Rashidi A, Ebadi M, Shields-Cutler RR, DeFor TE, Al-Ghalith GA, Ferrieri P, et al. Pretransplant Gut Colonization with Intrinsically Vancomycin-Resistant Enterococci (E. gallinarum and E. casseliflavus) and Outcomes of Allogeneic Hematopoietic Cell Transplantation. Biol Blood Marrow Transplant. 2018; pmid:29407252
  3. 3. Vincent S, Knight RG, Green M, Sahm DF, Shlaes DM. Vancomycin susceptibility and identification of motile enterococci. J Clin Microbiol. 1991;29: 2335–7. Available: http://www.ncbi.nlm.nih.gov/pubmed/1939593 pmid:1939593
  4. 4. Leclercq R, Dutka-Malen S, Duval J, Courvalin P. Vancomycin resistance gene vanC is specific to Enterococcus gallinarum. Antimicrob Agents Chemother. 1992;36: 2005–8. Available: http://www.ncbi.nlm.nih.gov/pubmed/1416893 pmid:1416893
  5. 5. Fine JP, Gray RJ. A Proportional Hazards Model for the Subdistribution of a Competing Risk. J Am Stat Assoc. 1999;94: 496.
  6. 6. Taur Y, Xavier JB, Lipuma L, Ubeda C, Goldberg J, Gobourne A, et al. Intestinal Domination and the Risk of Bacteremia in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation. Clin Infect Dis. 2012;55: 905–914. pmid:22718773
  7. 7. Dunny GM, Clewell DB. Transmissible toxin (hemolysin) plasmid in Streptococcus faecalis and its mobilization of a noninfectious drug resistance plasmid. J Bacteriol. 1975;124: 784–90. Available: http://www.ncbi.nlm.nih.gov/pubmed/810482 pmid:810482
  8. 8. Alexeev EE, Lanis JM, Kao DJ, Campbell EL, Kelly CJ, Battista KD, et al. Microbiota-Derived Indole Metabolites Promote Human and Murine Intestinal Homeostasis through Regulation of Interleukin-10 Receptor. Am J Pathol. 2018;188: 1183–1194. pmid:29454749
  9. 9. Whitfield-Cargile CM, Cohen ND, Chapkin RS, Weeks BR, Davidson LA, Goldsby JS, et al. The microbiota-derived metabolite indole decreases mucosal inflammation and injury in a murine model of NSAID enteropathy. Gut Microbes. 2016;7: 246–61. pmid:27007819
  10. 10. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30: 2068–9. pmid:24642063
  11. 11. Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016;44: D457–D462. pmid:26476454
  12. 12. Swimm A, Giver CR, DeFilipp Z, Rangaraju S, Sharma A, Ulezko Antonova A, et al. Indoles derived from intestinal microbiota act via type I interferon signaling to limit graft-versus-host disease. Blood. 2018;132: 2506–2519. pmid:30257880
  13. 13. Zelante T, Iannitti RG, Cunha C, De Luca A, Giovannini G, Pieraccini G, et al. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity. 2013;39: 372–85. pmid:23973224
  14. 14. Darkoh C, Chappell C, Gonzales C, Okhuysen P. A rapid and specific method for the detection of indole in complex biological samples. Schloss PD, editor. Appl Environ Microbiol. 2015;81: 8093–7. pmid:26386049
  15. 15. Mathewson ND, Jenq R, Mathew A V, Koenigsknecht M, Hanash A, Toubai T, et al. Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease. Nat Immunol. 2016;17: 505–513. pmid:26998764
  16. 16. Peled JU, Devlin SM, Staffas A, Lumish M, Khanin R, Littmann ER, et al. Intestinal Microbiota and Relapse After Hematopoietic-Cell Transplantation. J Clin Oncol. 2017;35: 1650–1659. pmid:28296584
  17. 17. Palmer KL, Godfrey P, Griggs A, Kos VN, Zucker J, Desjardins C, et al. Comparative genomics of enterococci: variation in Enterococcus faecalis, clade structure in E. faecium, and defining characteristics of E. gallinarum and E. casseliflavus. MBio. 2012;3: e00318–11. pmid:22354958
  18. 18. Ubeda C, Taur Y, Jenq RR, Equinda MJ, Son T, Samstein M, et al. Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans. J Clin Invest. 2010;120: 4332–4341. pmid:21099116
  19. 19. Kommineni S, Bretl DJ, Lam V, Chakraborty R, Hayward M, Simpson P, et al. Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract. Nature. 2015;526: 719–722. pmid:26479034
  20. 20. Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G, Takahashi D, et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature. 2013;504: 446–450. pmid:24226770
  21. 21. Arpaia N, Campbell C, Fan X, Dikiy S, van der Veeken J, deRoos P, et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature. 2013;504: 451–455. pmid:24226773
  22. 22. Peng L, Li Z-R, Green RS, Holzman IR, Lin J. Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers. J Nutr. 2009;139: 1619–25. pmid:19625695
  23. 23. Kelly CJ, Zheng L, Campbell EL, Saeedi B, Scholz CC, Bayless AJ, et al. Crosstalk between Microbiota-Derived Short-Chain Fatty Acids and Intestinal Epithelial HIF Augments Tissue Barrier Function. Cell Host Microbe. 2015;17: 662–71. pmid:25865369
  24. 24. Li G, Young KD. Indole production by the tryptophanase TnaA in Escherichia coli is determined by the amount of exogenous tryptophan. Microbiology. 2013;159: 402–10. pmid:23397453
  25. 25. Jasperson LK, Bucher C, Panoskaltsis-Mortari A, Taylor PA, Mellor AL, Munn DH, et al. Indoleamine 2,3-dioxygenase is a critical regulator of acute graft-versus-host disease lethality. Blood. 2008;111: 3257–65. pmid:18077788
  26. 26. Jasperson LK, Bucher C, Panoskaltsis-Mortari A, Mellor AL, Munn DH, Blazar BR. Inducing the tryptophan catabolic pathway, indoleamine 2,3-dioxygenase (IDO), for suppression of graft-versus-host disease (GVHD) lethality. Blood. 2009;114: 5062–70. pmid:19828695
  27. 27. Lamas B, Natividad JM, Sokol H. Aryl hydrocarbon receptor and intestinal immunity. Mucosal Immunol. 2018;11: 1024–1038. pmid:29626198
  28. 28. Moura-Alves P, Faé K, Houthuys E, Dorhoi A, Kreuchwig A, Furkert J, et al. AhR sensing of bacterial pigments regulates antibacterial defence. Nature. 2014;512: 387–92. pmid:25119038
  29. 29. Schiering C, Wincent E, Metidji A, Iseppon A, Li Y, Potocnik AJ, et al. Feedback control of AHR signalling regulates intestinal immunity. Nature. 2017;542: 242–245. pmid:28146477
  30. 30. Veldhoen M, Hirota K, Westendorf AM, Buer J, Dumoutier L, Renauld J-C, et al. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature. 2008;453: 106–9. pmid:18362914
  31. 31. Toye B, Shymanski J, Bobrowska M, Woods W, Ramotar K. Clinical and epidemiological significance of enterococci intrinsically resistant to vancomycin (possessing the vanC genotype). J Clin Microbiol. 1997;35: 3166–70. Available: http://www.ncbi.nlm.nih.gov/pubmed/9399514 pmid:9399514
  32. 32. Reid KC, Cockerill FR, Patel R. Clinical and Epidemiological Features of Enterococcus casseliflavus/flavescens and Enterococcus gallinarum Bacteremia: A Report of 20 Cases. Clin Infect Dis. 2001;32: 1540–1546. pmid:11340524
  33. 33. Ruoff KL, de la Maza L, Murtagh MJ, Spargo JD, Ferraro MJ. Species identities of enterococci isolated from clinical specimens. J Clin Microbiol. 1990;28: 435–7. Available: http://www.ncbi.nlm.nih.gov/pubmed/2108992 pmid:2108992
  34. 34. Patterson JE, Sweeney AH, Simms M, Carley N, Mangi R, Sabetta J, et al. An analysis of 110 serious enterococcal infections. Epidemiology, antibiotic susceptibility, and outcome. Medicine (Baltimore). 1995;74: 191–200. Available: http://www.ncbi.nlm.nih.gov/pubmed/7623654
  35. 35. Gordon S, Swenson JM, Hill BC, Pigott NE, Facklam RR, Cooksey RC, et al. Antimicrobial susceptibility patterns of common and unusual species of enterococci causing infections in the United States. Enterococcal Study Group. J Clin Microbiol. 1992;30: 2373–8. Available: http://www.ncbi.nlm.nih.gov/pubmed/1401001 pmid:1401001