dc.description.abstract
Thermophilic Campylobacter spp. are zoonotic bacteria leading to the most often reported gastroenteritis in the European Union. Campylobacter jejuni and Campylobacter coli are the major species relevant for human campylobacteriosis, which manifests with watery or bloody diarrhea, abdominal cramps, fever and in few cases leads to autoimmune sequelae, such as reactive arthritis, irritable bowel disease and Guillain-Barré syndrome. Even though the pathogens are fastidious in vitro and only grow at reduced oxygen levels, they are very successful in colonizing different warm-blooded hosts and in quickly spreading within a chicken flock. This PhD theses was focused on genomic diversity of C. jejuni and C. coli and the underlying mechanisms, in particular, natural transformation, which is the capacity to take up free DNA from the environment and adapt to changing conditions. We identified strains with ambiguous results in species differentiation by real-time PCR targeting genes, usually distinct for C. coli and C. jejuni. Using next generation sequencing and k-mer analysis the strains were identified as C. coli, harboring a substantial amount of recombined C. jejuni sequences, leading to gene variants. Interestingly, these so-called hybrid strains shared a common set of genes with C. jejuni sequence introgression, which may have a potential role in stress defense. Since the hybrid strains were preferentially isolated from egg shells, which is a dry and harmful environment for thermophilic Campylobacter spp., this may hint at selection for strains which survived harsh environmental conditions. It also demonstrated that Campylobacter is able to undergo extensive genetic exchange. The question arose, under which conditions the pathogen acquires genetic material by natural transformation, leading to the introduction of such an amount of new genetic material. Factors regulating natural transformation and competence development are only poorly understood in C. jejuni. Therefore, we developed a single cell-based uptake assay to monitor competence development. As a fresh field isolate we used C. jejuni strain BfR-CA-14430 which had recently been isolated from chicken meat and belongs to clonal complex 21 (ST44), which is also commonly found in human isolates. For better comparability this strain was distributed for common use in the PAC-CAMPY consortium. Since no sequence data was available we sequenced this strain using short-read Illumina and single molecule real-time (SMRT) long-read sequence analysis. BfR-CA-14430 had a genome size of 1.6 Mb and harbored a 41 kb plasmid. For visualization of DNA uptake, we covalently labelled C. jejuni genomic DNA with a fluorophore and added this labelled DNA to C. jejuni cells under different growth conditions. The results obtained by the single cell assay correlated well with the results obtained by a classical assay approach based on the transformation and subsequent selection for a resistance marker. This showed that the assay is a powerful tool to analyze competence development in C. jejuni. Increase from pH 6.5 to 7.5 resulted in a higher fraction of competent cells. Below pH 5 neither competence development nor DNA uptake was observed. Furthermore, aerobic conditions abolished competence development but not DNA uptake in already competent C. jejuni. These findings suggested a strong regulation of competence development in C. jejuni. We further showed that competence development in C. jejuni did not depend on growth temperatures or carbon dioxide concentrations. Since C. jejuni often resides in the slightly alkaline intestine of poultry, the results implicate extensive genetic exchange in the host. This might be beneficial to switch hosts and/or to survive in the environment. In a cooperation study, the single cell assay was used to check the effect of an immune-stimulative substance, curcumin, which was suggested as a potential treatment option for campylobacteriosis, on natural transformation. The DNA uptake assay did not identify either stimulating or inhibiting effects of curcumin on natural transformation in C. jejuni, which corroborates its application during campylobacteriosis without triggering the adaptive potential of the pathogen. In conclusion, extensive genetic exchange occurs in Campylobacter, potentially enlarging the adaptive potential of the food-borne pathogen. This might support the pathogen as successful colonizer and survivor. Besides, high genetic plasticity may hinder correct diagnostics and has to be taken into account for continuous monitoring of Campylobacter variants. It remains important to study natural transformation in detail in order to reduce the adaptability of thermophilic Campylobacter spp. in the future.
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