R.J. Owen

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.

Comparison of PFGE, ribotyping and phage-typing in the epidemiological analysis of Campylobacter jejuni serotype HS2 infections

In this study we have evaluated the ability of three typing methods, pulsed field gel electrophoresis (PFGE), phage-typing and ribotyping, to discriminate not only between strains of differing serotypes but also between strains within a single serotype, heat stable serotype 2 (HS2). Forty-five isolates derived from cases of campylobacter enteritis occurring in the Cardiff area were examined. These included 18, mostly HS2, strains associated with an outbreak. The typing results for these and a further 39 epidemiologically unrelated strains of serotype HS2 were compared. This is the first report documenting the use of PFGE in an epidemiological investigation of Campylobacter jejuni in the UK. The results presented suggest that this technique is the most discriminatory of the three subtyping methods examined.

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.

Genetic diversity in Helicobacter pullorum from human and poultry sources identified by an amplified fragment length polymorphism technique and pulsed-field gel electrophoresis.

Helicobacter pullorum was first isolated from the faeces and carcasses of poultry and has been associated with human gastroenteritis. The aim of this study was to examine interstrain genetic diversity within H. pullorum. Two fingerprinting techniques were used: amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoretic (PFGE) analysis. The 20 strains examined were from four countries and comprised 13 human isolates and seven poultry isolates. Their identity was confirmed by a species‐specific PCR assay. The human and poultry isolates had distinct genotypes and most strains showed a high degree of genetic diversity. Genotyping also indicated a clonal origin for two strains from the same poultry flock, and established a close relatedness between three chicken carcass isolates from a processing plant. It is concluded that these two genotyping techniques will provide a useful basis for future epidemiological investigations of H. pullorum in poultry, and may provide a link with its possible causal role in human gastrointestinal infections.

DNA restriction digest and ribosomal RNA gene patterns of Campylobacter jejuni : a comparison with bio-, sero-, and bacteriophage-types of united Kingdom outbreak strains

DNA restriction endonuclease (Hae III and Hind III) total digest and 16S and 23S ribosomal (r)RNA gene patterns (ribopatterns) were determined for 18 isolates of Campylobacter jejuni from three separate outbreaks of diarrhoea in the north of England. Strains were also characterized by biotyping, serotyping and phage typing. Comparisons of the DNA patterns by visual and numerical methods revealed five distinct strain groupings with clear differences between isolates from different outbreaks as well as some heterogeneity between strains within the community outbreak and one of the school outbreaks. An excellent correlation was observed between the genomic DNA fingerprints data and the Preston bacteriophage group, both of which gave better discrimination than biotyping and serotyping alone or in combination. Only one phage group (PG 37) was not confirmed by the DNA data. DNA fingerprints therefore provide additional information of value in studying the epidemiology of outbreaks of C. jejuni.

Flagellin gene polymorphism analysis of Campylobacter jejuni infecting man and other hosts and comparison with biotyping and somatic antigen serotyping

Flagellin gene sequence polymorphisms were used to discriminate amongst 77 strains of Campylobacter jejuni from sporadic and outbreak-associated human enteric infections, and from chickens, sheep and calves. The results were assessed in relation to Lior biotyping and serotyping (Penner somatic antigens). Eight DNA PCR-RFLP patterns (genotypes) were identified by analysis of HinfI fragment length polymorphisms in flagellin gene (flaA) polymerase chain reaction (PCR) products. One genotype (F-1) was a feature of 55% of strains. Strains within the genotypes were heterogeneous with respect to somatic antigens with 12 serogroups represented amongst the C. jejuni isolates of flaA type F-1. Serogroups Pen 1, 2 and 23 were the commonest (45%) amongst the 20 different serogroups represented. Several unique clusters of isolates with diverse biotypes were defined, and one cluster (F-7/Pen 23) contained epidemiologically implicated outbreak strains as well as sheep and calf isolates. We conclude that HinfI flaA typing is reproducible and offers high typability, and its combination with serogrouping provides a novel approach to characterizing isolates of C. jejuni with improved discrimination.

Comparisons between degree of histological gastritis and DNA fingerprints, cytotoxicity and adhesivity of Helicobacter pylori from different gastric sites.

Thirty-six isolates of H. pylori from up to three gastric biopsy sites (antrum, corpus and fundus) from 13 patients in Italy with different degrees of histological gastritis were investigated. All strains were tested for motility, cytotoxicity and degree of adhesion, and were typed by analysis of ribosomal RNA gene patterns (ribopatterns). Seventeen different DNA types (ribotypes) were identified, with each patient possessing H. pylori of one or more unique types. Only two patients had identical H. pylori at three sites. Most patients had H. pylori with different ribotypes or subtypes, but nine strains were not typable. Five patients had the same strain colonizing two of the three sites and atypical strains were mostly from the antrum. A complex pattern of H. pylori colonization in the stomach of some individuals was evident and suggested multiple sources of infection. No consistent associations were detected between degree of gastritis and adherence, cytotoxicity and motility but a 2.56Kb rRNA gene fragment that had a higher frequency in strains associated with severe gastritis than mild gastritis, may provide a useful molecular marker for future pathogenicity studies.

Subtypes of Campylobacter jejuni from sporadic cases of diarrhoeal disease at different locations in England are highly diverse

Serotyping (heat stable antigens) was performed on 398 strains of Campylobacter jejuni from faeces of human enteritis cases in England. Strains isolated over 12 months at three locations were heterogeneous with 33 HS serotypes represented. HS1 and HS4 complex were the predominant types (34% of all strains). The monthly strain frequency distributions were similar at the three locations. The late spring peak appeared to be associated with a rise in miscellaneous serotypes rather than with the emergence of any characteristic predominant serotype. PFGE DNA restriction profiling provided evidence of a high degree of genomic diversity within the common HS1 and HS4 complex serotypes, irrespective of the geographical source, yet some subtypes were common to more than one location. The study showed that C. jejuni strain subtypes from human enteric infections in England were highly diverse, and that HS serotyping must be combined with a more discriminatory subtyping method such as PFGE DNA profiling to provide an accurate basis for epidemiological surveillance.

Molecular subtyping by genome and plasmid analysis of Campylobacter jejuni serogroups O1 and O2 (Penner) from sporadic and outbreak cases of human diarrhoea

Ribosomal RNA gene patterns, randomly amplified polymorphic genomic DNA (RAPD) profiles and plasmid profiles were used to discriminate between 28 strains of Campylobacter jejuni serogroups O1 and O2 (Penner). Most isolates were biotype I (Lior). The strains were representative isolates from a UK school outbreak of enteritis (7 cases) and from 21 sporadic human cases of enteritis in 4 countries. The molecular techniques discriminated to various degrees between strains in each of the serogroups. The outbreak strains were homogeneous in most molecular features but a variety of types was detected amongst the isolates from the sporadic cases. Five groups of two or more strains with identical ribopatterns were identified and within each, strains from different patients were homogenous with respect to serogroup. RAPD profile typing based on numerical analysis generally matched ribotyping. Plasmid profiling overall gave least discrimination but was useful in separating some strains similar in other features. We concluded that optimal discrimination of C. jejuni could best be achieved using a combination of phenotypic and genotypic properties. Hae III ribotyping was the single most discriminatory and reproducible technique investigated. Several strains of C. jejuni from sporadic infections had similar molecular profiles which have potential for general typing purposes.

Flagellin gene profiling of Campylobacter jejuni heat-stable serotype 1 and 4 complex

Flagellin gene (flaA) sequence polymorphisms were used to discriminate amongst 167 strains of Campylobacter jejuni serotype HS1 and the HS4 complex. Direct PCR of cell suspensions provided a rapid method for analysing DNase-negative strains, whereas purified DNA was necessary for the DNase-positive strains. Nine different PCR-RFLP patterns (genotypes) were identified by analysis with Hinfl and 12 with Ddel, giving a total of 19 combined flaA profile types. The most common combined fla types were H1D1 (35%) and H1D2 (20%) for serotype HS1, and H1D2 (23%) and H4D7 (43%) for serotype HS4. Comparison of flaA typing with other key subtyping methods for C. jejuni showed it to be less discriminatory than pulsed field gel electrophoretic (PFGE) profiling, but more so than ribotyping. Fla types provided a useful indication of strain diversity, but as some were conserved across different serotypes, ribotypes and PFGE types, the same fla type could not be used as the sole basis for grouping strains. We provide evidence for several distinct subgroups based on conserved multiple genomic criteria within the HS1 and HS4 strains, and conclude that monitoring of such subgroups could provide a novel basis for future epidemiological surveillance of C. jejuni.