Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis

Abstract

A polymerase chain reaction (PCR) amplification of 16S rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name 'Anguillina coli'. The PCR incorporated a universal eubacterial 16S rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for 'A. coli'. The PCR was used to correctly identify DNA extracted from 43 isolates of 'A. coli' from humans and pigs, whilst no product was produced from Escherichia coli, or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi. The amplification provided a rapid and simple means of identifying DNA from isolates of 'A. coli', and could be used on boiled whole 'A. coli' cells, with a detection limit equivalent to 2.5 x 10(2) cells. The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs.

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons

“…This seqience was not present in any other 16s rRNA gene sequence determined in this study. Other investigators have used this sequence to design a primer for a PCR-based diagnostic test for specifically identifying these organisms [22]. As deduced from 16s rRNA comparisons and MEE analyses, P43/6/78, HRM-7, and Wes-B are likely to be related at the species level.…”

Brachyspira (Serpulina) pilosicoli Spirochetemia in an Immunocompromised Patient

“…Subculturing of the culture fluid under aerobic and anaerobic conditions in blood agar base (Oxoid) and 5% human blood and with MacConkey agar led to the develop- ment under anaerobic conditions of multiple, tiny, white colonies that were again gram-negative spirochetes. The latter were identified as B. pilosicoli by PCR according to the method described by Park et al [4]. Briefly, the colonies taken from the culture fluid were washed with phosphate buffer solution and centrifuged.…”

“…Brachyspira pilosicoli (formerly known as Serpulina pilosicoli or Anguillina coli) [1], a 4-8 µm spiral, weakly coiled flagellated spirochete, causes porcine intestinal spirochetosis [2] and has been isolated from the gastrointestinal tract of many species including humans [3][4][5]. However, its role in human disease outside the gastrointestinal tract remains controversial.…”

Tsukamurella tyrosinosolvens - An unusual case report of bacteremic pneumonia after lung transplantation

“…Concerning the genotypic identification, a 16S rRNA gene sequencing was first performed with the universal forward 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and reverse 1492R (5'-TACGGYTACCTTGTTACGACTT-3') primers [7,8] generating amplicons of 1485 bp. PCR was performed with the Taq DNA polymerase (Promega, Charbonnières-les-Bains, France).…”