Novel Burkholderiales 23S rRNA genes identified in ileal biopsy samples from children: preliminary evidence that a subtype is associated with perianal Crohn's disease

Abstract

A novel family of Burkholderiales bacteria was identified in ileal biopsy specimens from children presenting with symptoms of inflammatory bowel disease. A molecular subtyping approach based on sequencing of a variable region of the bacteria's 23S rRNA genes identified three variants. Pilot analysis identified one variant to be significantly associated with perianal Crohn's disease.

FIG. 1.

A schematic representation of the map location of primers used in this study, against a GenBank reference B. petrii 23S rRNA (accession no. AM902716). Primer WSbugF (nt 572 to 591) was designed based on the unique 23S rRNA gene identified previously by subtractive hybridization and amplifies a 1.9-kb region with primer 2490r (nt 2470 to 2488). The typing primer 23S F2 (5′ GGA ACT CGG CAA ATT GAC) binds to the conserved sequence from nt 1657 to nt 1674 and amplifies the variable region from nt 1690 to nt 1810 (in gray). This region contains the 78-bp diagnostic region, nt 1721 to nt 1798.

FIG. 2.

Neighbor-joining phylogenetic tree, based on partial 23S rRNA gene sequences (1.9 kb), showing positions of strains isolated from patient ileal biopsy specimens (CD patients 124, 149, 132, 61, 163, and 138; non-IBD patients 137, 178, 181, and 60) and related Proteobacteria species from GenBank. Four representative reference genes from each of the Burkholderiales families, Alcaligenaceae, Oxalobacteraceae, Burkholderiaceae, and Comamonadaceae, were retrieved from GenBank for this comparison. Additionally, reference genes of the Gammaproteobacteria and Deltaproteobacteria classes were included to serve as outgroups in the phylogenetic tree. GenBank accession numbers are shown beside each reference name. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. Bar, 0.05 number of nucleotide differences.

FIG. 3.

Multiple sequence alignment of 78-bp partial 23S rDNA sequences from 25 patients: 10 CD and 15 non-IBD (N) patients. The patient 43 sequence is taken as the consensus. There are three distinct variants: “124-type,” which includes patients 86, 194, 149, 80, 47, 137, 147, and 124; “var124-type,” which encompasses patients 181, 164, 175, 38, 162, 28, 178, and 100; and “60-type” for patients 170, 153, 193, 132, 61, 60, 138, and 163. Patient 43 is distinct from each of the three variants but has the fewest differences with the var124-type.