Robert J. Owen

Rapid extraction of bacterial genomic DNA with guanidium thiocyanate

A method is described for the rapid isolation and purification of bacterial genomic DNA. A total of 215 bacterial strains representing species of Campylobacter, Corynebacterium, Escherichia, Legionella, Neisseria, Staphylococcus and Streptococcus, were lysed with guanidium thiocyanate. DNA was prepared using just three other reagents and one high‐speed centrifugation step. The method, which was applicable to both Gram‐positive and Gram‐negative bacteria, eliminated endogenous nuclease activity and avoided the need for phenol, RNase and protease treatments. The DNA was of high purity, high molecular mass and double‐stranded.

Evaluation of the polymerase chain reaction for detecting the urease C gene of Helicobacter pylori in gastric biopsy samples and dental plaque.

A polymerase chain reaction (PCR) assay with oligonucleotide primers homologous to a portion of the urease C gene of Helicobacter pylori was evaluated for specificity with pure DNA and biopsy material. The assay was used to test for the presence of the organism in dental plaque. The species specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from H. cinaedi, H. felis, H. fennelliae, H. mustelae and H. nemestrinae. Sixty-two gastric biopsy samples collected from 14 patients (antrum, body and duodenal sites) were cultured and PCR was performed on the samples after culture. Primer sites were conserved in genomically diverse strains. Samples prepared by single-step heat lysis of bacterial cells and biopsy material did not inhibit PCR. The overall specificity was 96% irrespective of genotype. H. pylori was not cultured from dental plaque (15 patients), neither was H. pylori DNA detected by PCR in either urea breath test-positive or -negative individuals. The results showed that primer pair sequences within the urease C gene are conserved in most strains and provide an accurate basis for detecting H. pylori. As the PCR assay was not inhibited and did not yield false positive results with crude extracts from organisms or in the presence of biopsy material, its value as a diagnostic test was confirmed.

Description ofCampylobacter laridis, a new species comprising the nalidixic acid resistant thermophilicCampylobacter (NARTC) group

Ten strains of the nalidixic acid-resistant thermophilicCampylobacter (NARTC) group, of which 2 were isolated from human feces, were compared with 12 reference strains representing various species ofCampylobacter. The NARTC strains were a homogeneous group with respect to their cell morphology and 28 physiological and biochemical characters. All were microaerophilic, motile (amphitrichate), gram-negative, curved, S-shaped or helical rods, and representative strains had mean DNA base compositions of 31 to 32 mol % G+C. Distinctive features of the 10 strains were resistance to nalidixic acid and anaerobic growth in the presence of trimethylamine N-oxide hydrochloride (TMAO). The latter feature may account for the common occurrence of NARTC strains in the fecal contents of seagulls. DNA-DNA hybridizations indicated high (≥76%) base sequence relatedness within the group and low (≤15%) relatedness to other species ofCampylobacter. The 10 strains were classified in the genusCampylobacter but they could not be assigned to any previously defined species. Therefore, a new species, with the nameCampylobacter laridis, is proposed for these 10 strains; the type strain is NCTC 11352.

DNA patterns of Helicobacter pylori isolated from gastric antrum, body, and suodenum

Biopsy specimens for culture of Helicobacter pylori were obtained from two different sites in the antrum, gastric body, and duodenal cap in 20 patients during endoscopic investigation of dyspepsia. H. pylori was identified in 64 isolates obtained from 15 of the 20 patients. Analysis of chromosomal DNA from these isolates of H. pylori showed that 13 of 15 patients harbored a single strain of H. pylori throughout their stomach and duodenum. Two differing H. pylori types were found in two patients. Unique DNA patterns were shown in each of the 15 patients. The genetic heterogeneity of H. pylori is unexplained but it could be of considerable value for epidemiological studies.

Classification of Campylobacter sputorum and allied campylobacters based on numerical analysis of electrophoretic protein patterns

Summary A total of 28 strains of Campylobacter comprising representatives of C. sputorum subsp. sputorum , C. sputorum subsp. bubulus , C. sputorum subsp. mucosalis , C. concisus , “ C. fecalis ”, “ C. hyointestinalis ” and the CNW Campylobacter group have been characterized by SDS-PAGE of cellular proteins. The protein patterns, which contained 40 to 50 discrete bands, were highly reproducible and were used as the basis of a numerical taxonomic analysis. Eight clusters were obtained at the 55% similarity level and each corresponded to a recognized species or group. The analysis demonstrated unequivocally that C. sputorum subsp. mucosalis constituted a distinct species although the serogroup A strains differed slightly from the serogroup B and C strains. Also, it was shown that C. sputorum subsp. bubulus and “ C. fecalis ” were closely related and should be classified in the same species. As that taxon contained catalase-positive and catalase-negative strains, over-reliance on that test could lead to incorrect identification. Each of the species clusters had distinctive protein band patterns and the main bands provide useful diagnostic markers for the rapid identification of human and animal isolates.

Lineages within Campylobacter jejuni defined by numerical analysis of pulsed-field gel electrophoretic DNA profiles

Forty-seven Penner heat-stable (HS) serotype reference strains for Campylobacter jejuni and 47 serologically non-typable strains were examined by pulsed field gel electrophoresis (PFGE) DNA restriction analysis. The SmaI and KpnI digest profiles were compared by numerical analysis. Most strains grouped differently in the two analyses but strain lineages were inferred where the two agreed. Genetic relationships between reference strains in the cross-reacting HS4 complex were examined. Three clonal lines were evident and comprised: (i) HS4, HS13 and HS16; (ii) HS50 and HS65; (iii) HS43. The majority of those C. jejuni expressing HS antigens not recognised by currently available antisera had > 50% PFGE DNA digest similarity to one or more Penner scheme reference strain(s) and so did not necessarily represent distinct genetic lineages. PFGE analysis provided a high level of discrimination amongst strains of C. jejuni but overall similarity estimates for defining types must be based on the analysis of more than one restriction pattern.

Genomic Variation in Helicobacter pylori: Application to Identification of Strains

DNA digest analysis, ribopatterns, and plasmid profiling were used to determine genomic variation in 55 strains of Helicobacter pylori from patients with gastritis in the USA, Peru, Australia, and the U.K. HaeIII-ribopatterns and total DNA digest patterns showed a high degree of heterogeneity, with at least 33 different genomic types among strains, including some sequential isolates. Plasmids, present in 51% of strains, were less useful as epidemiologic markers. Investigation of 14 multiple isolate sets showed that genotypic variants were present in pre- and post-treatment gastric mucosa, that relapse in some patients was due to reinfection by a genotypically different strain, and that the same strain persisted in most treatment failures. We conclude that molecular methods were excellent for precise identification of H. pylori, but ribopatterns had the advantages of reproducibility, high discrimination, and visual simplicity.

Restriction fragment length polymorphism analysis shows that the hippuricase gene of Campylobacter jejuni is highly conserved

A 1151‐bp amplicon containing the hippuricase (hipO) gene was obtained from 118strains of Campylobacter jejuni and double‐digested with AluI and DdeI togive five different PCR‐RFLP patterns. Most strains had the six‐banded profile predicted fromsequence data. Lack of polymorphisms within the hipO gene indicated it was highlyconserved amongst strains of Camp. jejuni, and that RFLP analysis provided only lowdiscrimination as an epidemiological typing method. Detection of hipO by PCR provided auseful test for confirmatory identification of Camp. jejuni.

Use of DNA restriction endonuclease digest and ribosomal RNA gene probe patterns to fingerprint Helicobacter pylori and Helicobacter mustelae isolated from human and animal hosts.

Variation amongst strains of Helicobacter pylori and Helicobacter mustelae was examined by DNA restriction endonuclease digestion and rRNA gene patterns generated using a non-radioactive probe. Chromosomal DNA was extracted from 30 cultures of H. pylori from human, Rhesus monkey and pig gastric mucosa, and from three H. mustelae isolates from ferret gastric mucosa. DNA fingerprinting with Hae III and Hind III showed H. mustelae was relatively homogeneous but revealed genomic heterogeneity within H. pylori with at least 18 different DNA patterns identifiable amongst the 30 isolates. Five sets of strains other than duplicates with matching DNA fingerprints were identified within H. pylori. The Peruvian isolates were the largest identical set and comprised eight isolates from four different patients with five consecutive isolates from one patient. The Rhesus monkey strains were a relatively homogeneous set as were several Australian human isolates. The study demonstrates that rRNA gene restriction patterns provide a simple but highly discriminatory electrophoretic fingerprint for H. pylori with potential for use as a novel epidemiological marker in addition to total DNA digest analysis.

Strain variation in Campylobacter pylori detected by numerical analysis of one-dimensional electrophoretic protein patterns

A total of 21 clinical isolates of Campylobacter pylori from Peru and the United Kingdom and two reference strains (from Australia), including the type strain (NCTC 11637T), were characterized by high resolution one-dimensional SDS-polyacrylamide gel electrophoresis of cellular proteins. The protein patterns contained more than 40 discrete bands and the approximate molecular weights of the major bands were 22, 27, 46, 57, 60, 65 and 93 kD. The total patterns were used as the basis of numerical analysis. Most strains were clustered in four phenons at 91% similarity with the exception of six ungrouped strains. Overall similarity was high with all strains linked in the phenogram at ≥81%. Variation among strains was attributable principally to qualitative and quantitative band differences in the 47 to 56 kD (hypervariable) region of the C. pylori protein profile. From the analysis, ten different electropherotypes (EP-types) were identified. We demonstrated that differences were detectable among isolates from widely separated geographical locations as well as from the same location, although multiple isolates from two Peruvian patients had the same electropherotype. Our results indicate that determination of protein profiles provides the basis of a reproducible method for characterization of C. pylori isolates.