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Abstract

Carbapenemase-producing bacteria are distributed worldwide in healthcare facilities with continuously increasing numbers and are drastically limiting treatment options for severe bacterial infections. Hygienic measures and infection control are essential to counteract their distribution. Hence, sensitive, and reliable detection methods are needed to identify patients, colonized with carbapenem-resistant bacteria, and environmental contaminations, which are often involved in nosocomial infections and outbreaks. One of the major aims of this thesis was to evaluate a newly developed multiplex qPCR as a primary method for carbapenemase detection directly from rectal swab specimens and environmental samples. The qPCR showed excellent performances for the current local carbapenemase epidemiology and revealed the value of initial genotypic detection when compared to phenotypic methods. Besides the temporal advantages to initiate dissemination preventions strategies, the selection of growing isolates for further characterization by cultural screening is avoided. For wastewater samples, the direct-qPCR was less accurate, yet environmental reservoirs with critical levels of carbapenemase-producers could be identified, which may serve as potential sources for hospital-acquired infections and outbreaks. Carbapenemase genes are usually found on plasmids that facilitate their transmission within the bacterial community through horizontal gene transfer. Selection pressure under antibiotic therapy is supposed to contribute to the transmission of resistance genes. Therefore, the investigation of transferability of blaVIM-1-encoding plasmids between Enterobacterales and the impact of antibiotic therapy on conjugation was the second aim of this dissertation. The efficient transfer could be mainly attributed to the plasmid Inc-type for the tested Enterobacterales. An IncN2-type plasmid and its two homologs emerged as highly transmissible plasmids in this thesis. Furthermore, regulatory genes and transposons or insertion sequences, present on the plasmid play an important role in transmission, even though a certain gene could not be pointed out. Minor contributions could be ascribed to co-existing plasmids that usually have an impeding effect and to the negative correlation of donor and recipient relatedness to conjugative transfer. Sublethal concentrations of the fluoroquinolone antibiotic ciprofloxacin were able to induce conjugation of the IncN2-type plasmids and an IncN1-type plasmid, indicating that this effect is not widely distributed among the tested blaVIM-1-encoding plasmids (14.3 %). Different mechanisms were assessed that potentially contribute to elevated IncN2 plasmid transfer of C. amalonaticus KE3510. Overexpression of conjugation-related tra genes could not be confirmed at the tested time. Since other DNA-interfering substances were able to induce the IncN2 plasmid transfer, the contribution of the DNA damage-inducible SOS response was likely. Expression of SOS response-related genes was slightly increased, suggesting a mutagenic effect of ciprofloxacin in an SOS-dependent manner. Deletions of two genes, regulating the plasmid copy number, were observed in the mutant. An increase of plasmid copy numbers under ciprofloxacin treatment, however, could not be confirmed for C. amalonaticus KE3510, therefore, the exact mechanism of how antibiotics contribute to higher plasmid transfer rates could not be uncovered. Altogether, data presented in this dissertation point out that highly efficient transfer is not widely distributed among carbapenemase-positive bacteria and only few plasmids can be positively influenced by antibiotic therapy. A small number of bacteria harboring highly transmissible plasmids, however, is sufficient to successfully distribute resistance genes throughout the bacterial community and contribute to the development of new multidrug-resistant bacteria.