Use of pulsed-field gel electrophoresis to determine genomic diversity in strains of Helicobacter hepaticus from geographically distant locations

Abstract

In 1992 a helical microorganism associated with chronic active hepatitis and a high incidence of hepatocellular tumors was identified in the hepatic parenchyma of A/JCr mice. By using biochemical tests, phenotypic characterization, and 16S rRNA gene sequence analysis, the organism was classified as a novel Helicobacter species and named Helicobacter hepaticus. Recent surveys completed in our laboratory indicate that H. hepaticus is widespread in academic and commercial mouse colonies. The aim of this study was to examine the H. hepaticus genome by pulsed-field gel electrophoresis (PFGE) to determine the degree of genomic variation and genomic size. This technique has been used to identify significant genomic diversity among strains of Helicobacter pylori and to demonstrate only slight genomic diversity among strains of Helicobacter mustelae. Genomic DNAs from 11 isolates of H. hepaticus from the United States, Germany, France, and The Netherlands were subjected to PFGE after digestion with SmaI. Isolates from three independent sources within the United States had very similar PFGE patterns, suggesting that the genomic DNAs of these isolates are conserved. Genomic DNA isolated from a fourth source within the United States had a PFGE pattern different from those of the other U.S. isolates. Isolates obtained from Germany, France, and The Netherlands had PFGE patterns that differed markedly from those of the U.S. isolates and from one another. The use of DNA fingerprinting may be useful in subsequent epidemiological studies of H. hepaticus when the source and method of spread of this murine pathogen need to be ascertained. By PFGE, the genomic size of H. hepaticus is estimated to be roughly 1.3 Mb, which compares to 1.67 Mb for H. pylori and 1.7 Mb for H. mustelae.

Genomic characterization of Helicobacter hepaticus: ordered cosmid library and comparative sequence analysis

“…SmaI restriction fragments of H. hepaticus chromosome were separated by PFGE, transferred onto nylon membrane and hybridized with the probes prepared from the terminal clones in each contig. SmaI fragments are numbered as previously described [17]. Approximate sizes of the respective contigs and the overlapping SmaI fragments are indicated in parentheses.…”

“…DNA hybridization was performed with chemiluminescence-labeled probes prepared using an ECL nucleic acid labeling and detection system according to the supplier's recommendations (Amersham Pharmacia Inc., Piscataway, NJ, USA). PFGE separation of SmaIdigested H. hepaticus chromosomal DNA was carried out with the Bio-Rad CHEF-DR III system under the conditions previously described [17]. Cosmid DNA was sequenced using a Thermo Sequenase Cycle Sequencing kit (USB Corporation, Cleveland, OH, USA).…”

“…Initial clones, E1 containing £aA, C43 containing sodB, and C46 containing ureA, were used to identify sets of overlapping clones in order to form contigs. Three hundred and thirty primary cosmid clones were used to construct an ordered genomic library, which should theoretically represent the complete nucleotide content of the H. hepaticus genome [17]. The terminal sequences of the inserts in the cosmid clones containing sodB, ureA and £aA homology were determined using T3 and T7 primers that £ank either end of the insert.…”

“…The H. hepaticus genome is approximately 1.3 Mb as estimated by pulsed-¢eld gel electrophoresis (PFGE) [17] but remains poorly characterized. Only a few genes or gene clusters, including the 16S rRNA gene [5], a gene encoding an 18-kDa immunogenic protein [18], cdtABC encoding a cytolethal distending toxin (CDT) [19,20], and the partial sequence of ureAB encoding the urease structural subunits [21], have been sequenced.…”

Characterization of Helicobacter felis by Pulsed‐Field Gel Electrophoresis, Plasmid Profiling and Ribotyping

“…No animal-passaged strains or follow-up isolates were examined in the present study. Somewhat differently, of the several H. hepaticus strains that have been isolated around the world it has been noted that two different PFGE types appeared within the strains isolated in the USA, but each of the three European strains had unique PFGE patterns [33].…”