Isolates from Colonic Spirochetosis in Humans Show High Genomic Divergence and Potential Pathogenic Features but Are Not Detected Using Standard Primers for the Human Microbiota

Abstract

Colonic spirochetosis, diagnosed based on the striking appearance in histological sections, still has an obscure clinical relevance, and only a few bacterial isolates from this condition have been characterized to date. In a randomized, population-based study in Stockholm, Sweden, 745 healthy individuals underwent colonoscopy with biopsy sampling. Of these individuals, 17 (2.3%) had colonic spirochetosis, which was associated with eosinophilic infiltration and a 3-fold-increased risk for irritable bowel syndrome (IBS). We aimed to culture the bacteria and perform whole-genome sequencing of the isolates from this unique representative population sample. From 14 out of 17 individuals with spirochetosis we successfully isolated, cultured, and performed whole-genome sequencing of in total 17 isolates, including the Brachyspira aalborgi type strain, 513A. Also, 16S analysis of the mucosa-associated microbiota was performed in the cases and nonspirochetosis controls. We found one isolate to be of the species Brachyspira pilosicoli; all remaining isolates were of the species Brachyspira aalborgi Besides displaying extensive genetic heterogeneity, the isolates harbored several mucin-degrading enzymes and other virulence-associated genes that could confer a pathogenic potential in the human colon. We also showed that 16S amplicon sequencing using standard primers for human microbiota studies failed to detect Brachyspira due to primer incompatibility.IMPORTANCE This is the first report of whole-genome analysis of clinical isolates from individuals with colonic spirochetosis. This characterization provides new opportunities in understanding the physiology and potentials of these bacteria that densely colonize the gut in the individuals infected. The observation that standard 16S amplicon primers fail to detect colonic spirochetosis may have major implications for studies searching for associations between members of the microbiota and clinical conditions such as irritable bowel syndrome (IBS) and should be taken into consideration in project design and interpretation of gastrointestinal tract microbiota in population-based and clinical settings.

Keywords: Brachyspira; comparative genomics; spirochetosis; whole-genome sequencing.

FIG 1

Histological sections showing, in the same formalin-fixed, paraffin-embedded biopsy specimen, representative pictures of spirochetosis using hematoxylin and eosin staining (a), immunohistochemistry using polyclonal spirochete-specific antibodies (b), and Warthin-Starry silver staining (c).

FIG 2

Phylogenetic trees based on the 16S rRNA sequences (a) and the NADH oxidase (NOX) protein sequence (b). The isolates marked in red were sequenced within this study. The sequences marked in green come from the whole-genome sequences described in Table S1. The taxa in bold are the type strains for their respective species. In panel b, clades containing only sequences from one species are collapsed for better readability.

FIG 3

Maximum likelihood tree of 16S rRNA sequences from human intestinal spirochetosis. Isolates in red were sequenced in this study, the ones in blue are from the work of Pettersson et al. (8), isolates in green are from the work of Mikosza et al. (9), and those in orange are from the work of Westerman et al. (10). The phylogenetic clusters are annotated according to the terminology used in the three respective papers.

FIG 4

Heat map showing hierarchical clustering of the accessory genome in Brachyspira aalborgi, i.e., genes present in <99% of the genomes, together with the results from the biochemical characterization of isolates. The presence or absence of an orthologous group is shown in blue or white, respectively. Included genomes are those of the 16 newly sequenced B. aalborgi isolates together with Brachyspira sp. CAG-700.