Development of novel molecular methods for Campylobacter species and for Helicobacter pylori

Sammanfattning: Background and Aim: Different Campylobacter species and Helicobacter pylori are common causes of gastrointestinal infections that may result in diarrhea and chronic gastritis, respectively. The overall aim of this thesis was to develop novel methods for identification, differentiation and characterization of these bacteria. This included comparison of expression of mRNA encoding virulence associated antigens by H. pylori grown in vitro and in vivo and to study the expression of these antigens on the bacterial surfaces. Results: A specific, PCR-based DNA probe hybridization assay was developed for identification of Campylobacter fetus, a bacterium that has caused several outbreaks of human disease during the last decades. The specificity of the probe was evaluated by DNA sequence homology analyses and highly specific identification of C. fetus could be accomplished using the probe labelled with either 32P or digoxigenin. A highly specific PCR-RFLP method was established that allowed rapid identification of different Campylobacter species and also discrimination between Campylobacter spp. and members of the closely related genera Arcobacter, Wolinella and Helicobacter.A QCRT-PCR method was established that allowed quantification of mRNA in H. pylori grown in vivo and in vitro and also very sensitive enumeration of bacterial cells. By this method mRNA that encodes two putative virulence factors in H. pylori, i.e. urease and the neutrophil activating protein (NAP), was studied in a mouse adapted H. pylori strain (SS1) after 1, 2, 3, 4, 7 and 11 days of growth in broth and after 3 days and 2, 6, 12 and 18 weeks of growth in C57/Bl6 mice. Both ureA and nap mRNA were increased in bacteria grown in vivo as compared to in vitro, suggesting that H. pylori is capable of modulating gene expression in response to environmental stimuli. To enable studies of possible surface expression of different antigens on H. pylori a flow cytometric (FCM) assay was established. By using highly specific monoclonal antibodies (MAbs) it was shown that 100% and 30-70% of in vitro grown H. pylori expressed the LPS and N-acetyl-neuraminyllactose binding hemagglutinin (HpaA), respectively on the bacterial surface during different growth phases. In contrast, NAP was surface-expressed only on few cells at late stage of growth and urease was not expressed at all on the bacterial cell surface at any time point during 11 days of culture. The gene expression and surface localization of HpaA was also analyzed in different reference strains and fresh clinical isolates. Although all strains transcribed the hpa gene, as shown by Northern blotting, and all bacterial colonies of the different strains expressed HpaA in dot blot analyses, the surface expression of this antigen on individual bacteria varied considerably, both between strains and during different growth phases.Conclusion: Novel PCR-based methods have been developed that allow highly specific identification and differentiation of different species of Campylobacter. By using QCRT-PCR H. pylori could be quantified with high sensitivity and the expression of mRNA in H. pylori grown in vivo and in vitro could be compared. A method allowing demonstration of surface localization of putative virulence factors was also developed. The different methods may be useful tools in clinical diagnostics, in epidemiological studies and in efforts to further characterize virulence factors in common enteropathogenic bacteria.