Rapid and Accurate Diagnosis of Human Intestinal Spirochetosis by Fluorescence In Situ Hybridization

Abstract

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.

Colonic mucosa-associated microbiota is influenced by an interaction of Crohn disease and FUT2 ( Secretor ) genotype

“…S1). This individual was most likely displaying asymptomatic human intestinal spirochaetosis at the time of sampling and was removed because of its unclear clinical significance (28) and severe effect on bacterial community structure. All other individuals displayed communities dominated by the Firmicutes, Bacteroidetes, and Proteobacteria along with other less abundant phyla (Fig.…”

FISH analysis of Lactobacillus biofilms in the gastrointestinal tract of different hosts

“…FISH is increasingly applied for the diagnosis of bacterial biofilm‐related infections (e.g. (Schmiedel et al. 2009)), but is not commonly used for the detection of probiotic bacteria that are intentionally administered to the GIT.…”

“…Thick biofilms are sometimes observed in disease conditions, such as severe inflammatory bowel diseases (Swidsinski et al. 2005) and human intestinal spirochaetosis (Schmiedel et al. 2009).…”

The Spirochete Brachyspira pilosicoli, Enteric Pathogen of Animals and Humans

“…Another adjunct to diagnosis is the use of a fluorescent in situ hybridization technique which uses fluorescent oligonucleotide probes to visualize and localize spirochetes associated with the mucosa in formalin-fixed tissues. This approach has been used with colonic samples from animals (198,199) and with colorectal biopsy specimens and fecal material from humans (164,200). It has been combined with DNA extraction from samples, followed by PCR amplification of the 16S rRNA gene and DNA sequencing to identity the spirochetes involved (164).…”