Sequence-based discovery of Bradyrhizobium enterica in cord colitis syndrome

Abstract

Background: Immunosuppression is associated with a variety of idiopathic clinical syndromes that may have infectious causes. It has been hypothesized that the cord colitis syndrome, a complication of umbilical-cord hematopoietic stem-cell transplantation, is infectious in origin.

Methods: We performed shotgun DNA sequencing on four archived, paraffin-embedded endoscopic colon-biopsy specimens obtained from two patients with cord colitis. Computational subtraction of human and known microbial sequences and assembly of residual sequences into a bacterial draft genome were performed. We used polymerase-chain-reaction (PCR) assays and fluorescence in situ hybridization to determine whether the corresponding bacterium was present in additional patients and controls.

Results: DNA sequencing of the biopsy specimens revealed more than 2.5 million sequencing reads that did not match known organisms. These sequences were computationally assembled into a 7.65-Mb draft genome showing a high degree of homology with genomes of bacteria in the bradyrhizobium genus. The corresponding newly discovered bacterium was provisionally named Bradyrhizobium enterica. PCR identified B. enterica nucleotide sequences in biopsy specimens from all three additional patients with cord colitis whose samples were tested, whereas B. enterica sequences were absent in samples obtained from healthy controls and patients with colon cancer or graft-versus-host disease.

Conclusions: We assembled a novel bacterial draft genome from the direct sequencing of tissue specimens from patients with cord colitis. Association of these sequences with cord colitis suggests that B. enterica may be an opportunistic human pathogen. (Funded by the National Cancer Institute and others.)

Figure 1. Sample Selection and Analyses

Formalin-fixed, paraffin-embedded samples obtained from patients with cord colitis were selected for molecular analysis on the basis of clinical criteria. Patients for whom colon-biopsy samples were available for the period ranging from 120 days before antibiotic therapy to 200 days after such therapy were selected for inclusion in the initial cohort. DNA extraction and sequencing were followed by PathSeq analysis, in which computational subtraction was applied for the removal of human and known microbial sequences. The remaining unmapped sequencing reads and the reads with a high degree of homology with known microbial sequences were then computationally assembled into longer contiguous overlapping sequences (contigs) representing genomic fragments of a novel organism. Candidate pathogens, which were predicted by PathSeq analysis of the discovery cohort, were detected by targeted methods, such as polymerase-chain-reaction (PCR) assay, in the validation cohort.

Figure 2. Phylogenetic Analysis and Genomic Assembly of the Organism Associated with Cord Colitis and Comparison of the Provisional Genome for Bradyrhizobium enterica with the Genome of B. japonicum USDA 110

A rooted phylogenetic tree showing the predicted evolutionary relationship between B. enterica and related species was constructed by means of multisequence alignment of 400 core, protein-coding genes (Panel A). Bootstrap analysis was performed showing more than 99% consensus at all branch points except the circled one, where the bootstrap value was 18.1%. Shown is a Circos plot of the draft B. enterica genome, which was assembled with the use of unmappable reads from shotgun whole-genome sequencing of samples of cord colitis (Panel B). The draft genome, which is composed of linear contigs, is represented circularly in the middle track in order of descending contig size. A circular contig that probably represents a plasmid similar to that found in bradyrhizobium species BTAi1 was excluded from this representation. On the inner track, dark blue lines that are perpendicular to the circular genome plot indicate genes present in B. enterica that are not present in B. japonicum USDA 110. On the outer track, the amino acid sequence identity of each B. enterica protein to its closest B. japonicum homologue is represented. In the middle track, each contig is represented by a dark green block, with its borders outlined in light green.

Figure 3. Abundance of Bradyrhizobium enterica in Samples Obtained from Patients with Cord Colitis, as Compared with Control Samples

B. enterica was more abundant in samples obtained from patients with the cord colitis syndrome (CCS) than in samples obtained from healthy controls, patients with colon cancer, or patients with graft-versus-host disease (GVHD). In addition, B. enterica was present in colon-biopsy samples from three additional patients with cord colitis. In Panels A through F, the top lanes show amplification of a B. enterica target after 35 cycles of a polymerase-chain-reaction (PCR) assay, and the bottom lanes show amplification of a human actin target after 35 PCR cycles. A lane containing reagents but no DNA (no-template control) is indicated by 0. Shown are results for five healthy control specimens (p1 through p5) (Panel A), five colon-cancer specimens (c1 through c5) (Panel B), three colon-biopsy specimens from patients with pathologically confirmed GVHD (g1 through g3) (Panel C), and DNA from temporally distinct colon-biopsy samples from Patient 4 (4a, 4b, 4c, and 4e) (Panel D), from Patient 9 (9b through 9f) (Panel E), and from Patient 6 (6a and 6b) (Panel F). Samples are displayed chronologically. Milestones in therapy for the three patients with cord colitis are indicated by colored arrowheads. Microscopical images of colon tissue obtained from a patient with cord colitis are shown, including a section stained with hematoxylin and eosin (Panel G) and a corresponding section (Panels H through K), along with colon tissue from healthy controls (Panels L and M) stained with either a universal eubacterial probe (EUB, yellow) or a bradyrhizobium-specific probe (Brady) and counterstained with 4',6-diamidino-2-phenylindole (DAPI, orange).