To understand transmissible human diseases, disciplines such as epidemiology and the surveillance of affected cases are as essential as the knowledge about the pathogenesis and the course of a disease. Epidemiologists categorize and estimate factors for public health risks by taking metadata into account including geographic aspects, health and social states to study a disease transmission and prevent further cases. In addition, a focus on the causative agents itself is necessary in order to understand their ecology and hence their virulence traits. The causative agents for a severe pneumonia named Legionnaires’ disease (LD) are bacteria of the genus Legionella. The putative sources of LD infection are any aerosol-generating natural or man-made fresh water systems. Due to this ubiquitous distribution of legionellae, it is difficult to find the source of infection. Therefore, it is necessary to isolate the bacterium from the suffering patients to further characterize it in the laboratory and to compare the clinical isolates with isolates obtained from probable environmental sources.
The predominant species isolated from LD patients is Legionella pneumophila serogroup (Sg) 1. Intensive genotyping of L. pneumophila Sg1 isolates by using the current gold standard method, the sequence-based typing scheme (SBT), revealed limitations in the discrimination of several sequence types (ST) which could not be compensated for by additional phenotypic typing scheme. In practical terms, this means that several clones or STs are disproportional frequently found in both, patients and water systems, and cannot be distinguished by current methods. Therefore, a distorted picture of endemic and globally-spread clones is generated and current typing methods cannot add substantial information during the identification of the infectious source. The aim of this thesis is to develop and implement new typing methods for L. pneumophila isolates with a higher resolution than the gold standard methods.
A DNA-DNA hybridization based microarray was designed and equipped with probes that target specifically L. pneumophila virulence factors and genes that are involved in the biosynthesis of lipopolysaccharide structures. Legionellae can be subgrouped on the basis of their lipopolysaccharide structures. Here, the usually phenotypic characterization of L. pneumophila Sg1 is successfully transmitted to a DNA-based genotypic method. Furthermore, the detailed validation of the DNA-microarray revealed a higher discriminatory power in comparison to the gold standard methods. It enables previously indistinguishable clones to be subdivided, providing valuable information about probable sources of infection.
The second new tool for typing of L. pneumophila is based on the core genome of the bacteria. An extended SBT-scheme was extracted from the core genome and accordingly named core genome multilocus sequence typing (cgMLST). This genome wide gene-by-gene typing approach allows a high genomic resolution of L. pneumophila isolates by retaining epidemiological concordance. A major advantage of this genome-based method is the detection of large recombination events within the analysed genomes, which is, so far, reserved for whole genome sequencing. The population structure of legionellae is largely driven by recombination and horizontal gene transfer rather than by spontaneous mutations. Therefore, the detection of recombination events is essential for typing of L. pneumophila isolates. In addition, the cgMLST-scheme assigns a core genome sequence type to the analysed isolate and allows backwards compatibility with the current SBT-scheme.
Both methods proved to be fast, reliable and robust typing methods through their application during outbreak investigations. Furthermore, both systems are particularly suited as routine molecular typing tools for the surveillance of single cases. The raw data are verified and translated into uniform portable codes, which enables the easy transfer and comparison of results. The standardized and portable quality of the results of both methods enables the establishment of a curated global database. This qualifies both methods as potential new gold standard methods for the genotyping of L. pneumophila isolates.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:bsz:14-qucosa-233546 |
| Date | 06 March 2018 |
| Creators | Petzold, Markus |
| Contributors | Technische Universität Dresden, Medizinische Fakultät Carl Gustav Carus, Dr. med. Christian Lück, Prof. Dr. med. Enno Jacobs, Prof. Dr. rer. nat. Michael Göttfert |
| Publisher | Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
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