Comparative genome and phenotypic analysis of Clostridioides difficile strains
Dissertation
Datum der mündl. Prüfung:2023-11-14
Erschienen:2024-01-16
Betreuer:Prof. Dr. Rolf Daniel
Gutachter:Prof. Dr. Rolf Daniel
Gutachter:PD Dr. Michael Hoppert
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Zusammenfassung
Englisch
The anaerobic bacterium Clostridioides difficile, renamed from Clostridium difficile, substantially accounts for nosocomial, antibiotic-associated infections worldwide. The complex dynamics of various traits shape the bacterial virulence, such as toxin production and gene sequence variants, multidrug resistances, and sporulation. C. difficile is also known for its mobile genome that comprises diverse mobile genetic elements (MGE), thereby enabling the spread of advantageous genes. The pathogen is extensively investigated to enlighten its virulence and dissemination. Strain isolation is mainly done in clinical context, although C. difficile also inhabits human and mammalian intestines without disease manifestation or is present in environmental surroundings. Further, these isolations always employ antibiotic based cultivation, which might lead to an isolation bias. Within this thesis, non-clinical strains were isolated from environmental samples. Five strains in total were obtained from which two were isolated without antibiotic treatment. An effective protocol for antibiotic-free isolation could not be established but offers potential for further efforts. Isolation approaches were supported by the established detection PCR that targets the C. difficile hpdBCA-operon and detects even low abundances of C. difficile in environmental DNA. Specificity of the detection PCR was verified by next generation sequencing of detection PCR amplicons from environmental samples to ensure that amplified sequences only originated from C. difficile DNA. These environmental samples were also analyzed via 16S rRNA gene sequencing, in which C. difficile could not be detected, what clearly demonstrated their low abundance. Besides validation of environmental sources for promising C. difficile isolation, amplicons from the established detection PCR further allow phylogenetic estimation, which was determined by average nucleotide identity analyses of representative sequences. Analysis of PCR amplicons also supported the assumed isolation bias due to antibiotic treatment, as detected sequences from environmental samples did not accord with sequences from strains obtained by antibiotic-based protocols. This indicated an abundance shift of the present C. difficile strains and selective enrichment of the un-detected strains upon antibiotic treatment. The genomes of the non-clinical strains isolated within this thesis were compared to clinical reference strains to identify clinical-related features with influence on strain virulence. Besides analysis of crucial virulence factors, the genome analyses addressed MGEs, and further included antibiotic resistances and accessory genes, which were all incorporated in pairwise genome alignments. An overall trend of more ARGs, MGEs and accessory genes in the clinical strains was observed. The accessory genes mainly comprised specific functions such as transcription, which are correlated to higher strain virulence. This observation might further indicate the ability of the clinical strains to adapt more rapidly to environmental fluctuations. It is therefore advisable to increasingly implement non-clinical strains in comparative analyses to unravel C. difficile virulence progression. ARG, MGE and pan genome analyses were put into genomic context to identify conjunctions between specific elements. This revealed that accessory genes, which were identified to correlate with higher strain virulence, resided within MGEs. This connection highlights the relevance of MGEs in C. difficile virulence. All non-clinical isolates and the clinical reference strains were subjected to phenotypic investigations on spontaneous and induced prophage activity. Active prophages were determined by isolation and sequencing of particle-protected DNA and mapping of sequencing reads onto the host genomes. Phages were either not induced, or induced with the secondary bile salt deoxycholate, which is a stressful component in the natural C. difficile habitat. Corresponding clinical and non-clinical strains showed no difference in prophage carriage or tolerance to deoxycholate, as determined by relative growth in the presence of varying concentrations. The sequencing data verified spontaneous prophage activity in all strains with one exception and revealed multiple active phages co-existing within the same host. The inducing effect of deoxycholate was also confirmed and observed for almost all phages, which underlines the relevance of natural conditions in investigations of C. difficile phage activity. Differences in increased phage activity upon induction could be observed between corresponding strains, but the data could not ensure a clinical relation. One corresponding strain pair however indicated a connection between the higher induction in the clinical strain and its higher reactivity determined in the comprehensive genome analyses. Fourteen novel phages in total were identified. Further, several regions with clear activity but missing phage identity were apparent in the sequencing read mapping. Different mechanisms might be responsible for the envelopment of these mobile DNA elements, such as phage-mediated transduction. One of these active DNA elements could be assigned to lateral transduction, which is the first description of this mechanism in C. difficile. These findings point to so far unraveled HGT strategies in C. difficile and encourage further investigations.
Keywords: Clostridioides difficile; mobile genetic element; genome comparison; virulence; bacteriophage; detection; environmental sample; antibiotic-free isolation; clinical isolate; non-clinical isolate; pan genome