Next-Generation Sequencing for the Identification of Transplantation-Associated Pathogens

Hematopoietic stem cell transplantation (HSCT), while a powerful tool in the clinician’s armamentarium against refractory hematological diseases, is associated with high non-relapse mortality. Infectious, immunological and idiopathic syndromes all contribute to the nearly 30% treatment related mortality in the first year post-transplantation. Microbiological triggers have been suggested in many of the idiopathic post-transplantation syndromes that occur, but actual pathogens have been discovered in only a small subset.

Recently, Herrera and colleagues described an antibiotic-responsive colitis unique to patients who have undergone umbilical cord stem cell transplantion.¹ This “cord colitis syndrome” (CCS) is clinically and histopathologically distinct from other known causes of colitis in transplantation patients, such as viral colitis, bacterial colitis and intestinal graft versus-host disease (GVHD). Contemporary microbiological tools did not reveal a causative organism in CCS. We therefore sought to apply an unbiased, “deep sequencing” approach for the discovery of a potential CCS-associated pathogen.

Of the 11 patients described in the original CCS cohort, five patients had undergone colonic biopsies in the time frame just before and just after diagnosis with and treatment for CCS. We selected these formalin fixed, paraffin embedded biopsies, which measured approximately 2mm × 2mm × 1mm, and extracted DNA. Four samples (two temporally separated biopsies from two patients) had adequate DNA to perform whole genome sequencing (WGS). Following sequencing at two separate facilities (to control for potential sources of contamination), we performed taxonomic classification of all resultant sequencing “reads” using the PathSeq software.² Computational assembly of unclassifiable “reads” was performed for the discovery of novel microbial organisms. An attempt was then made to investigate for the presence of candidate microbes identified by this method in the three additional patients for whom colonic biopsies were available and in various controls.

Using this approach, a large proportion of non-human reads (over 2.5 million high-quality reads) from all four sequenced samples were unclassifiable, suggesting the presence of a yet-undiscovered microbial organism within the tissue specimens. In order to infer the genomic sequence of this organism, computational assembly was performed with reads from a single patient using two different DNA assembly programs (VELVET and AllPaths-LG).^3,4  Both assembly methods independently resulted in the generation of a nearly 7.65 Mb draft genome. Ninety-eight of 99 contiguous overlapping sequences (“contigs”) demonstrated homology to Bradyrhizobium species. This novel organism was provisionally named Bradyrhizobium entericabased on the results of a rooted phylogenetic analysis. PCR confirmed the presence of B. enterica in three additional CCS patients and demonstrated absence of B. enterica in normal colon, colon cancer and GVHD controls.

Using an unbiased, sequencing-based method for pathogen discovery, we report the discovery of Bradyrhizobium enterica, a novel bacterium, from colon biopsies of patients affected with CCS. This is the first demonstration, to our knowledge, of the discovery of a novel bacterium through sequencing of a diseased human tissue. We showed that B. enterica is present in additional cord colitis patients and is absent in normal colon, colon cancer and GVHD controls. Given the ease, rapidity and culture-independent method of discovery of a disease-associated bacterium that was demonstrated in this previously idiopathic syndrome, we anticipate that this methodology will be more widely applied in the field of stem cell transplantation for the identification of potential disease-associated pathogens.

Marty:Chimerix: Consultancy, Research Funding. Meyerson:Foundation Medicine: Consultancy, Equity Ownership; Microgenomica: Equity Ownership; Novartis: Consultancy.