| 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.Human intestinal spirochetosis (HIS) is a histologically defined condition of the human distal intestinal tract characterized by helical microorganisms attached at one end to the surface epithelium of the colonic mucosa. This association forms a so-called “false brush border” (13). While certain Brachyspira spp. are recognized as the causative agents of swine dysentery and porcine intestinal spirochetosis (3, 11), their clinical significance and pathogenic potential for humans remain unclear. Various studies have reported on the association of these bacteria with intestinal disorders such as chronic watery diarrhea (8, 12) and on clinical improvement following antimicrobial therapy (29). In contrast, others have suggested that intestinal spirochetes are harmless commensals in humans (7). The prevalence of HIS ranges from 1.2% (23) to >40% (17, 34), depending on presumable patient risk factors, such as origin from developing countries, immunodeficiency, or homosexuality. Recently, Peruzzi et al. (30) discovered a prevalence of 12% in a selected population, indicating that HIS is an important differential diagnosis for patients with chronic gastrointestinal disorders and risk factors.Two intestinal spirochetes have been identified in humans so far: Brachyspira aalborgi (15) and Brachyspira pilosicoli (36). Both species require selective media, and B. aalborgi is an extremely slow growing, fastidious microorganism that requires anaerobic incubation for as long as 4 weeks (4, 34). For this reason, HIS is primarily diagnosed histopathologically. The fuzzy basophilic fringe, 4 to 7 μm thick, on the epithelial layer of the colonic mucosa is visible in hematoxylin-and-eosin (HE)-stained histological sections and is considered pathognomonic for HIS. Tissue morphology usually remains unaltered, and no inflammatory reaction is observed (20).However, diagnosis of HIS on the basis of HE staining requires experienced laboratory personnel and accurate interpretation, and silver staining is often needed to confirm the diagnosis (10). Therefore, a significant portion of cases may be missed, especially since B. pilosicoli might also colonize the epithelium without the characteristic end-on attachment, impeding identification by light microscopy at low magnification (24). Furthermore, histopathology does not provide information about the identity of the microorganisms, thereby precluding epidemiological studies. More importantly, the inability to identify the organism also hampers accurate therapy, since the intestinal spirochetes are suspected to differ in virulence, and therefore some cases of HIS may require antibiotic therapy more urgently than others (5, 29).The genus Brachyspira currently comprises seven established species and several proposed species. Among some Brachyspira species, the high level of 16S rRNA gene conservation precludes interspecies differentiation by 16S rRNA gene methods and necessitates further molecular analyses. However, all known species isolated from humans can be identified and differentiated via their 16S rRNA genes. In line with the genetic variation discovered in Brachyspira species, such as Brachyspira hyodysenteriae (2) and Brachyspira innocens (9), recent molecular studies have also identified human Brachyspira strains genetically distinct from B. aalborgi and B. pilosicoli. This heterogeneity was confirmed by sequencing of 16S rRNA (14, 21) or NADH oxidase (25) genes, fluorescence in situ hybridization (FISH) (16, 17), or multilocus enzyme electrophoresis (33). Pettersson et al. (31) analyzed biopsy samples from two adults by 16S rRNA gene sequencing and consequently proposed to divide the B. aalborgi lineage into three phylogenetic clusters, including the type strain, B. aalborgi 513A, in the first cluster.The extent of intraspecies genetic variation in human intestinal spirochetes is unclear and difficult to estimate, because few complete 16S rRNA gene sequences are available. Further epidemiologic and phylogenetic investigations are needed to elucidate spirochete genetic diversity and to facilitate the evaluation of the molecular diagnostic tools that are presently available.FISH is a microscopic method that allows simultaneous visualization and identification of microorganisms. Jensen and colleagues (3, 16, 17) designed several genus- or species-specific oligonucleotide probes targeting the 16S or 23S rRNA of Brachyspira spp. and applied them successfully to porcine and human intestinal biopsy specimens. However, no genus-specific 16S rRNA-directed probe for diagnostic use targeting all Brachyspira spp. known so far has been developed.In the present study, intestinal biopsy specimens from five patients with possible HIS were analyzed histopathologically and by culture, FISH, PCR amplification, and 16S rRNA gene sequencing. Biopsy specimens from a healthy control group were analyzed retrospectively by histopathology and FISH. The purpose was (i) to acquire further information about the phylogenetic structure of the Brachyspira spp. associated with HIS, (ii) to design a FISH probe covering all Brachyspira spp. based on the currently available sequence data, and (iii) to evaluate FISH as a fast and robust diagnostic screening tool for HIS. |
