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Background Clostridium difficile is the main cause of antibiotic associated diarrhea. In the past decade, the number of C. difficile patients has increased dramatically, coinciding with the emergence of two PCR ribotypes 027 and 078. PCR ribotype 078 is also frequently found during C. difficile outbreaks in pigfarms. Previously, the genome of the PCR ribotype 078 strain M120, a human isolate, was described to contain a unique insert of 100 kilobases. Results Analysis of this insert revealed over 90 open reading frames, encoding proteins originating from transposons, phages and plasmids. The insert was shown to be a transposon (Tn6164), as evidenced by the presence of an excised and circularised molecule, containing the ligated 5’and 3’ends of the insert. Transfer of the element could not be shown through filter-mating experiments. Whole genome sequencing of PCR ribotype 078 strain 31618, isolated from a diarrheic piglet, showed that Tn6164 was not present in this strain. To test the prevalence of Tn6164, a collection of 231 Clostridium difficile PCR ribotype 078 isolates from human (n = 173) and porcine (n = 58) origin was tested for the presence of this element by PCR. The transposon was present in 9 human, tetracycline resistant isolates, originating from various countries in Europe, and none of the pig strains. Nine other strains, also tetracycline resistant human isolates, contained half of the transposon, suggesting multiple insertion steps yielding the full Tn6164. Other PCR ribotypes (n = 66) were all negative for the presence of the transposon. Multi locus variable tandem repeat analysis revealed genetic relatedness among transposon containing isolates. Although the element contained several potential antibiotic resistance genes, it did not yield a readily distinguishable phenotype. Conclusions Tn6164 is a newly described transposon, occurring sporadically in C. difficile PCR ribotype 078 strains. Although no transfer of the element could be shown, we hypothesize that the element could serve as a reservoir of antibiotic resistance genes for other bacteria. Further research is needed to investigate the transfer capabilities of the element and to substantiate the possible role of Tn6164 as a source of antibiotic resistance genes for other gut pathogens.

Antimicrobial resistance (AMR) is a growing problem worldwide. This is mainly the result of the presence of resistance genes in bacteria. When these genes are located in small pieces of DNA, so called plasmids, they can easily spread between bacteria. This thesis gives an overview the different types of plasmids, all with their different resistance genes, and their presence around the globe. Bacteria may contain a number of different plasmids in a single cell. whether any of these plasmids can be stably maintained depends on several factors. One of these factors is called incompatibility, which is the inability of plasmids to be maintained in one cell. Based on this incompatibility plasmids can be categorized in 40 different “Inc” groups, a biological phenomenon that was long used as a typing system to study epidemiology. This thesis focusses on the members of the “I-complex” incompatibility group, found in Enterobacteriaceae and frequently associated with important resistance gene classes such as the so-called Extended Spectrum Beta-lactamases (ESBLs). The I-complex includes IncI1α, IncI1γ, IncI2, IncK, IncB/O and IncZ plasmids. In this thesis the basis of their incompatibility is analysed and the results show that a single mutation (change in DNA) can determine if two plasmids are compatible or not. It concludes that IncB/O and IncZ plasmids, which were previously considered compatible and thus different plasmids, were actually incompatible and should be considered as highly similar. It also shows that the IncK plasmid group, which is an important carrier of resistance genes in human and poultry bacteria, consists of two compatible plasmid lineages, designated IncK1 and IncK2. Studies into the molecular effects of these plasmid types on the physiology of their host cell showed that IncK2 plasmids provide a fitness advantage at a higher environmental temperature which may have resulted in their predominance in poultry over IncK1, which is mostly found in mammals. Overall, the results presented in this thesis highlight the importance of plasmids in the spread of AMR, and it underlines the importance of understanding the plasmid compatibility and its adaptation to the bacterial or animal host.

Antimicrobial resistance (AMR) is a growing problem worldwide. This is mainly the result of the presence of resistance genes in bacteria. When these genes are located in small pieces of DNA, so called plasmids, they can easily spread between bacteria. This thesis gives an overview the different types of plasmids, all with their different resistance genes, and their presence around the globe. Bacteria may contain a number of different plasmids in a single cell. whether any of these plasmids can be stably maintained depends on several factors. One of these factors is called incompatibility, which is the inability of plasmids to be maintained in one cell. Based on this incompatibility plasmids can be categorized in 40 different “Inc” groups, a biological phenomenon that was long used as a typing system to study epidemiology. This thesis focusses on the members of the “I-complex” incompatibility group, found in Enterobacteriaceae and frequently associated with important resistance gene classes such as the so-called Extended Spectrum Beta-lactamases (ESBLs). The I-complex includes IncI1α, IncI1γ, IncI2, IncK, IncB/O and IncZ plasmids. In this thesis the basis of their incompatibility is analysed and the results show that a single mutation (change in DNA) can determine if two plasmids are compatible or not. It concludes that IncB/O and IncZ plasmids, which were previously considered compatible and thus different plasmids, were actually incompatible and should be considered as highly similar. It also shows that the IncK plasmid group, which is an important carrier of resistance genes in human and poultry bacteria, consists of two compatible plasmid lineages, designated IncK1 and IncK2. Studies into the molecular effects of these plasmid types on the physiology of their host cell showed that IncK2 plasmids provide a fitness advantage at a higher environmental temperature which may have resulted in their predominance in poultry over IncK1, which is mostly found in mammals. Overall, the results presented in this thesis highlight the importance of plasmids in the spread of AMR, and it underlines the importance of understanding the plasmid compatibility and its adaptation to the bacterial or animal host.