D. Linton

Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals

The nucleotide sequences for the 16S ribosomal RNA gene were compared for 33 species comprising the epsilon subdivision of the Proteobacteria (genera: Campylobacter, Helicobacter, Arcobacter and Wolinella). Regions specific for the genus Campylobacter and for five Campylobacter species important in human and/or veterinary medicine were identified. From these regions, PCR primer pairs were designed for use in species-specific identification. Primer pairs were validated against strains representing all taxa of campylobacter-like organisms. They did not amplify products other than from their five target species (C. upsaliensis, C. helveticus, C. fetus, C. hyointestinalis and C. lari), and they generated amplicons of defined size from large numbers of strains of those species. A primer pair suitable for identification of the genus Campylobacter was also identified and validated. This generated amplicons from all species of Campylobacter as well as from unnamed groups known to be within the genus, but not from any species or unnamed strains of Helicobacter, Arcobacter or Wolinella.

Polymerase chain reaction detection and speciation of Campylobacter upsaliensis and C. helveticus in human faeces and comparison with culture techniques

A polymerase chain reaction (PCR) assay based on the 16S rRNA gene and an improved DNA extraction procedure were developed for the direct detection and differentiation of Campylobacter upsaliensis and C. helveticus in seeded human faeces. The PCR assay was compared with culture detection by a membrane filter (MF) technique and on selective agar (SA) containing 8 mg l−1 cefoperazone. Both MF culture and the PCR assay detected 105 colony‐forming units (cfu) g−1 faeces. Selective agar culture of some strains could detect as few as 103 cfu g−1 faeces. However, some strains were susceptible to cefoperazone and either failed to grow or were detected only with reduced sensitivity in the presence of the antibiotic. Detection by MF and SA both required 48–96 h incubation in a microaerobic atmosphere and did not specifically identify the isolate. By contrast, the PCR assay could be completed within 8 h and accurately identified the two phenotypically similar species, C. upsaliensis and C. helveticus.

Molecular typing of Helicobacter pylori isolates from asymptomatic, ulcer and gastritis patients by urease gene polymorphism

The gastric-adapted bacterium Helicobacter pylori plays an important role in gastritis and ulcer disease, but no phenotypic typing scheme presently exists for this organism. With a view to the development of genotypic typing, we have compared isolates of H. pylori from gastritis or ulcer patients with those from subjects exhibiting no disease. Variation was analysed at the urease genes, ureA and ureCD, by employing PCR-generated probes in genomic Southern blot hybridizations. Whilst ureA restriction fragments provided a fourfold subgrouping of strains, ureCD fragments were considerably more discriminatory. Twenty-four combined ureACD profiles were generated with Hind III, subdividing the 64 strains into 11 types and 13 single profiles. The most prevalent profile (UI) was found in 33% of strains, almost all from gastritis or ulcer patients. On the other hand strains isolated from asymptomatic individuals had the most diverse ureACD profiles. A key finding from this set of isolates was that strains of H. pylori associated with general gastroduodenal disease were genetically more homogeneous than strains carried by people without disease symptoms.

Campylobacter coli strains with enlarged flagellin genes isolated from river water

A group of campylobacters isolated from river water were found to possess unusually large flagellin genes. Both phenotype and serology were consistent with identification as Campylobacter coli. Phylogenetic analysis of small (16S, rrs) and large subunit (23S, rrl) rRNA genes of a representative strain, NCTC 13006, demonstrated high levels of relatedness with C. jejuni and C. coli (99.1 and 98.3% similarity for 16S; 99.3 and 99.4% similarity for 23S). Large flagellin proteins were demonstrated by SDS-PAGE analysis. The flaA and flaB genes were sequenced and aligned with known campylobacter flagellin amino acid sequences. The encoded FlaA protein of the new group exhibited a high degree of divergence from other Campylobacter species. Within the central variable region of FlaA, a further hypervariable domain was identified containing characteristic repeated motifs. Separate pairwise alignments performed for the variable regions of the polypeptide indicated these large fla genes were more closely related to those of C. upsaliensis than to those of C. coli or C. jejuni.