Investigation of potential genetic instability within the Helicobacter pylori genome

The human gastric pathogen Helicobacter pylori has been reported to be genetically
diverse. However, initial research in this study indicated that the development of
resistance to non-therapeutic antibiotics in vitro is comparable in frequency to other
species of bacteria. Both spontaneous and induced mutation frequencies were found
to be no higher than those observed in Escherichia coli.
Conjugation experiments were performed between two type strains of H. pylori; one
containing plasmid DNA of a conjugative size and the other being a plasmid-free
antibiotic-resistant mutant, developed from earlier mutation studies. Following
conjugation and antibiotic selection the isolates were screened for the presence of
plasmid DNA. In initial studies, screening for plasmid DNA was performed using a
non-radioactively labelled probe derived from plasmid DNA, in connection with
Southern blotting / DNA hybridisation experiments. Due to problems with
reproducibility, a simpler method was developed, using a PCR specific for H. pylori
plasmid DNA, delivering consistent, accurate results. It was shown that conjugation
occurred between these two H. pylori strains.
Analysis of the 16S rDNA of a number of H. pylori strains was undertaken. Little
genetic variation between isolates was observed in this study, using both nucleotide
sequence data and restriction digest analysis. In a comparative study, the variation in
16S rDNA sequence observed between H. pylori isolates was found to be no greater
than that observed between E.coli isolates.
Despite all previous reports of genetic variability within the species, H. pylori did
not display high mutation frequencies or a large amount of inherent genetic
variability in this study. The discovery of conjugative DNA transfer does, however,
suggest one possible mechanism for the generation of genetic variation and transfer
of antibiotic resistance between H. pylori strains.

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