Development of a Real-Time PCR for Identification of Brachyspira Species in Human Colonic Biopsies

Background: Brachyspira species are fastidious anaerobic microorganisms, that infect the colon of various animals. The genus contains both important pathogens of livestock as well as commensals. Two species are known to infect humans: B. aalborgi and B. pilosicoli. There is some evidence suggesting that the veterinary pathogenic B. pilosicoli is a potential zoonotic agent, however, since diagnosis in humans is based on histopathology of colon biopsies, species identification is not routinely performed in human materials. Methods: The study population comprised 57 patients with microscopic evidence of Brachyspira infection and 26 patients with no histopathological evidence of Brachyspira infection. Concomitant faecal samples were available from three infected patients. Based on publically available 16S rDNA gene sequences of all Brachyspira species, species-specific primer sets were designed. DNA was extracted and tested by real-time PCR and 16S rDNA was sequenced. Results: Sensitivity and specificity for identification of Brachyspira species in colon biopsies was 100% and 87.7% respectively. Sequencing revealed B. pilosicoli in 15.4% of patients, B. aalborgi in 76.9% and a third species, tentatively named ‘‘Brachyspira hominis’’, in 26.2%. Ten patients (12.3%) had a double and two (3.1%) a triple infection. The presence of Brachyspira pilosicoli was significantly associated with inflammatory changes in the colon-biopsy (p = 0.028). Conclusions: This newly designed PCR allows for sub-differentiation of Brachyspira species in patient material and thus allows large-scaled surveillance studies to elucidate the pathogenicity of human Brachyspira infections. One-third of affected patients appeared to be infected with a novel species

Intestinal Spirochetosis mimicking inflammatory bowel disease in children.

ABSTRACT: BACKGROUND: Intestinal spirochetosis is an unusual infection in children and its clinical significance in humans is uncertain. The presence of these microorganisms in humans is well-known since the late 1800's and was first described in 1967 by Harland and Lee by electron microscopy. CASE PRESENTATION: This article reports the findings of one pediatric case, review of the current literature, and an overview of therapeutic options. CONCLUSION: A high degree of suspicion is required in cases presenting with abdominal pain, chronic diarrhoea and/or hematochezia associated with a normal endoscopic examination, thus emphasizing the importance of multiple biopsies throughout the colon

Colonic Spirochetes: What Has Genomics Taught Us?

The ‘colonic’ spirochetes assigned to the genus Brachyspira are slow-growing anaerobic bacteria. The genus includes both pathogenic and non-pathogenic species, and these variously colonise the large intestines of different species of birds and animals, including humans. Scientific understanding of the physiology and molecular biology of Brachyspira spp. remains very limited compared with that of other pathogenic spirochetes, and there are few descriptions of successful genetic manipulations undertaken to investigate gene function. An important boost to knowledge occurred in 2009 when, for the first time, the whole genome sequence of a Brachyspira strain (Brachyspira hyodysenteriae strain WA1) was obtained. The genomics analysis provided a significant increase in knowledge: for example, a previously unknown ~36 Kb plasmid was discovered and metabolic pathways were constructed. The study also revealed likely acquisition of genes involved in transport and central metabolic functions from other enteric bacterial species. Four subsequent publications have provided a similarly detailed analysis of other Brachyspira genomes, but of these only two included more than one strain of a species (20 strains of B. hyodysenteriae in one and three strains of B. pilosicoli in the other). Since then, more Brachyspira genomes have been made publicly available, with the sequences of at least one representative of each of the nine officially recognised species deposited at public genome repositories. All species have a single circular chromosome varying in size from ~2.5 to 3.3 Mb, with a C + G content of around 27%. In this chapter, we summarise the current knowledge and present a preliminary comparative genomic analysis conducted on 56 strains covering the official Brachyspira species. Besides providing detailed genetic maps of the bacteria, this analysis has revealed gene island rearrangements, putative phenotypes (including antimicrobial drug resistance) and genetic mutation mechanisms that enable brachyspires to evolve and respond to stress. The application of Next-Generation Sequencing (NGS) to generate genomic data from many more Brachyspira species and strains increasing will improve our understanding of these enigmatic spirochetes