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Lawson, Andrew Jeffrey
(2000).
DOI: https://doi.org/10.21954/ou.ro.0000f9a9
Abstract
A rapid extraction protocol to facilitate the recovery of bacterial DMA from faecal material for use as a template for RCR was developed. This consisted of a basic guanidine-silica extraction technique with an additional PVP/TE wash step to remove PCR inhibitors. The efficacy of the new extraction protocol in combination with Campylobacter species-specific PCR assays was demonstrated using seeded faecal material and compared favourably with detection by existing culture-based methodologies. A series of pilot studies using clinical samples were then performed; firstly a study of 25 samples of known Campylobacter status; and then a comparison of PCR assay with selective culture in a blind study of 200 samples. These experiments confirmed the effectiveness of PCR assays for the detection of Campylobacter species and consequently a major, multi-centre, investigation was undertaken. Over the course of two years, a total of 3,738 faecal samples from seven contributing centres were examined. This is the largest molecular-based survey of Campylobacter species yet undertaken. The detection rates for PCR and culture were similar and in accord in ~ 78% of C. jejuni and C. coli infections. However, a significant proportion were PCR-positive only (12%) or culture-positive only (10%). Detection rates of non-jejuni/non-coli Campylobacter spec\es were significantly higher using PCR assay and C. lari (1), C. upsaliensis (9) and C. hyointestinalis (4) were found in culture-negative faeces. The detection of C. hyointestinalis by PCR assay at an incidence of 0.13% is the highest yet recorded from human gastroenteritis.
In a study of faeces from healthy humans, 16S rDNA amplicons originating from a previously undescribed and initially uncultivable Campylobacter species, termed 'Candidatus C. hominis’, were discovered. Phylogenetic analysis of 16S rDNA suggested a relationship between the novel species and existing anaerobic Campylobacter species. A membrane filtration technique modified for the recovery of anaerobic bacteria with a novel selective dilution and immunomagnetic separation procedure were developed and pure cultures of the bacteria from which the 'Candidatus C. hominis’ 16S rDNA sequences originated were obtained. These strains were characterised and the new species described as C. hominis sp. nov.
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