The overall aim of this study was to establish molecular techniques for the detection and identification of "difficult to grow" - bacteria in mixed bacterial populations in clinical samples without the need for culturing procedures.
Material and Methods: Thirty-nine strains of Mobiluncus were used as a model system for phylogenetic classification of fastidious bacteria based on 16S ribosomal DNA (rDNA) sequences. To test the application of the 16S rDNA broad range PCR concept for detecting bacteria clinically, urine samples spiked with Chlamydia trachomatis elementary bodies and real urine test samples from 12 C. trachomatis positive and negative male volunteers were tested in a semiblind manner against routine procedures. Furthermore, gastric biopsy samples from 22 individuals (13 defined as having Helicobacter pylori-associated gastritis, and 9 defined as normal controls) were evaluated for the presence of Helicobacter 16S rDNA and the virulence genes cagA, vacA, and ureA. PCR products were also applied to temporal temperature gel electrophoresis (TTGE) gels for profiling the microbial flora. Finally, intestinal biopsies from 22 patients (11 diagnosed as Crohn's disease (CD), and 11 non-CD patients) were investigated using probes targeting potential pathogens that have been suggested to be involved in CD.
Results: We were able to confirm the current species designation of Mobiluncus, although the results did not support the division of M. curtisii into subspecies. For the detection of C. trachomatis in urine, the in-house system was shown to be as sensitive as a commercially available PCR system. The search for Helicobacter pylori in gastric biopsies revealed the presence of Helicobacter DNA in 20 of 22 individuals. The molecular techniques were apparently too sensitive compared with routinely used techniques. TTGE revealed a complex microbial flora both in the normal control group and in the gastritis group, with dominance of Helicobacter in the gastritis group. The present results might lend support to the hypothesis that Helicobacter are indigenous biota of the human stomach. cagA was amplified in all Helicobacter positive specimens. None of the specimens in the control group carried a H. pylori type strain identical vacA genotype. ureA negative mutants were also found in this group. The 16S rDNA sequence data might also indicate phylogenetic heterogeneity. The mixed bacterial flora found in CD inflammatory lesions is consistent with the idea that the enteric microflora enters primary lesions, where secondary invaders may elicit chronic inflannnatory response.
Conclusion: This thesis has demonstrated the usefulness of 16S rDNA based techniques for detection, identification, and characterisation of individual bacterial pathogens as well as for profiling of mixed bacterial flora in clinical specimens.
Linköping: Linköpings universitet , 2000. , 112 p.