Genetic diversity and population structure of the intestinal spirochaete Brachyspira pilosicoli

The work described in this thesis relates to the fastidious anaerobic intestinal spirochaete Brachyspira pilosicoli, a bacterium that colonises the large intestine of a variety of species. The spirochaete can attach to the colorectal epithelium of the host, forming a ‘false brush border’. Colonised individuals may develop gastrointestinal disorders including chronic diarrhoea, abdominal pain and rectal bleeding. Despite the widespread occurrance of the infection, and the fact that it causes disease in important production animal species (pigs and chickens), as well as in human beings, the spirochaete has not been extensively studied. The aim of the work was to analyse and improve understanding of the genetic diversity and population structure of B. pilosicoli, including gaining insights into how this species has been shaped, and how it is continuing to evolve. To achieve this overarching aim, three objectives were developed: 1. To assemble a large collection of isolates of B. pilosicoli from different host species and locations. 2. To develop multilocus sequence typing (MLST) and multiple locus variable number tandem repeat analysis (MLVA) as new molecular methods for analyzing the B. pilosicoli population, and applying them to the collection. 3. To examine the occurrence of the beta-lactamase gene blaOXA-63 in B. pilosicoli strains from different origins, to assess variations in the sequence of the gene to deduce its molecular evolution, and to compare it to that of other housekeeping genes and repeat sequences that were studied. MLST and MLVA were both successfully developed for the first time, and the application of both methods identified extensive variation amongst the 131 isolates that were studied. A total of 127 sequence types (STs) were found with MLST, whilst using MLVA 102 variable number tandem repeats (VTs) were identified from a subset of 119 isolates. In MLST a large number of alleles were identified at each locus, with all loci being highly polymorphic. The calculated Standardized Index of Association value and the results of a Shimodaira-Hasegawa (SH) test both indicated that there was extensive recombination within the population. When using eBURST on the MLST data, five small clonal complexes (Cc) were identified, indicating that limited clonality occurs in the species. This recombinant population structure of B. pilosicoli was in distinct contrast to those of the related species Brachyspira hyodysenteriae and Brachyspira intermedia, where previous MLST studies have indicated that both species are essentially clonal. Recombinant species are less stable and more rapidly evolving than clonal species; indeed, some of the clonal Brachyspira species may have arisen from the generation of stable variants of a recombinant species such as B. pilosicoli, with these new variants being selected for because they were particularly suited to a specialised ecological niche. To complement these data, the distribution and sequence variation of the blaOXA-63 gene, encoding a group IV class D beta-lactamase, was analysed in the B. pilosicoli population. Unlike the housekeeping genes used in MLST, this gene should only be under positive selection pressure in situations where the spirochaete is regularly exposed to beta-lactam antibiotics. Amongst the 119 isolates tested, 15 apparently lacked the gene, 16 had the full gene, and the rest only had portions of the gene amplifiable. Of the 16 full genes, nine showed less than 20 nucleotides difference from the original blaOXA-63 gene, but seven showed differences of 25 to 207 nucleotides in the expected 804 base pairs. When the blaOXA-63 sequence and the sequences of the seven housekeeping genes used in MLST in these 16 strains were compared with those in control strain 95/1000, the esterase fragment showed the greatest difference (19.5%); the other six housekeeping loci varied by 1.3% to 5.0%, and blaOXA-63 differed by 9.1%. These results suggest that MLST analysis based on housekeeping loci may actually underestimate the overall extent of nucleotide variation between B. pilosicoli strains.