Summary Objectives and hypothesis: This thesis concerns the study of plasmids of vancomycin- resistant enterococci isolated from feces of American crows in the years 2012 - 2013 period. The hypothesis is that, in various environments, there is one or more types of epidemiologically significant vanA gene-carrying plasmids that are capable of horizontally spread. Methods: Based on PFGE method the number and size of plasmids were detected in selected isolates of vancomycin-resistant E. faecium. Using PCR method the isolates were subjected to detection of genes of replicases, relaxases and toxin-antitoxin system of plasmid-bound resistance genes. Using 19 primers were characterized types of Tn1546. Results: Of the 12 tested vancomycin-resistant isolates of E. faecium the following number and size of plasmids was proven using PFGE method: 2 isolates contained two plasmids (17%), 3 isolates contained three plasmids (25 %), 5 isolates contained four plasmids (42 %) and 2 isolates contained five plasmids (17 %). All isolates (n = 12) were then subjected to the detection of genes of replicases, relaxases and toxin-antitoxin system for typing of plasmids from each plasmid families. RepA_N family of plasmids: genes characterizing plasmids related to pRUM: rep17 in 11 isolates (92 %), gene Axe-Txe was detected in 5 isolates (42 %) genes characterizing plasmids related to pLG1: rep20 in 7 isolates (58 %) genes characterizing plasmids related to pAD1: relpAD1 gene was detected in one isolate (8 %) Inc18 family of plasmids: genes characterizing plasmids related to pIL501: rep1 gene detected in one case (8 %) genes characterizing plasmids related to pRES25: rep2 gene in 2 isolates (17 %) genes characterizing plasmids related to pEF1: relpEF1 detected in 11 isolates (92 %) pHTB family of plasmids: genes characterizing plasmids related to pHTB: rep22 gene was detected in 4 isolates (33%) and in 2 isolates gene relpHTB was detected (17%) RCR family of plasmids: genes characterizing plasmids related to pRI: positive detection of Rep14 gene in 8 isolates (67%) and in 4 isolates relpRI gene was detected Small theta-replicating plasmids: genes characterizing plasmids related to pEF418 plasmids: rep18a gene in 2 isolates (17%) genes characterizing plasmids related to pB82: rep18b gene was detected in one isolate (8%) genes characterizing plasmids related to pCIZ2: relpCIZ2 gene was detected in 9 isolates tested (75%) Types of transposon Tn1546 Using the PCR method types of Tn1546 were characterized. In 4 isolates (n = 12; 33 %) Tn1546 was characterized as a F3 type. In one isolate (8 %) Tn1546 was characterized as a type F5, in one isolate (8 %) as a type PP-16. In 6 isolates Tn1546 was untypeable. Most likely these are new, yet unknown types. Conclusion: This is the first study of plasmids of vancomycin-resistant isolates E. faecium isolated from feces of American crows. These results emphasize not only a high proportion of plasmids in individual isolates, but also a high proportion of genes with horizontal transfer.

Colistin is a last-resort antibiotic used to treat serious infections caused by Enterobacteriaceae and other multidrug resistant gram-negative bacteria. Recently discovered plasmid-borne colistin resistance, mediated by the mcr genes, poses a serious risk to colistin therapy. The aim of this diploma thesis was to map the occurrence of Enterobacteriaceae carrying the mcr-1 to 8 genes in hospitalized patients, travellers, prospective colistin-resistant clinical isolates and in a retrospective collection of Enterobacteriaceae using a combination of selective cultivation and qPCR. Isolates with a detected mcr gene were characterized by Whole-Genome Sequencing. The localization of mcr genes was determined and other resistance genes and plasmids were identified. Furthermore, the physiological profile of selected colistin- resistant Escherichia coli isolates was characterized. In the presence of a subinhibitory amount of colistin, a strain carrying the mcr-1 gene may be favored. Later, the mcr-9 gene was described and its occurence was subsequently tested retrospectively. Enterobacter spp. isolates carrying the mcr-9 gene were mostly colistin-sensitive but, in some cases, resistance was induced after exposure to sublethal doses of colistin. The results of the study show that the incidence of plasmid-mediated...

Bacillus subtilis strain 3610 is an ancestral undomesticated strain. It diers from the laboratory strain 168 in many aspects. One dierence in strain 3610 is the presence of plasmid pBS32 encoding the sigma factor N (σN). This σ factor is activated when DNA damage occurs and induces the bacteria's cell death. The aim of the Thesis was a systematic characterisation of σN-dependent transcription. First, I showed that plasmid-borne but not chromosome-borne predicted σN-dependent promoters were ac- tive in transcription in vitro. Next, the anities of RNAP with σN for DNA, initiating NTP (iNTP) were determined for both relaxed and supercoiled DNA templates. Sur- prisingly, the activity of RNAP on relaxed σN-dependent promoters was higher than on their supercoiled versions, an opposite trend than displayed by RNAP associated with other σ factors. This property of σN-dependent promoters was not encoded by the core promoter sequence. In summary, this Thesis contributed to our understanding of the bacterial transcription apparatus. 1

This diploma thesis is focused on topic of genetically modified organisms and their use in the practical sectors of human life. Theoretical part of the thesis defines general terms GMO, plasmid, genetic engineering, biotechnology. It also records historical milestones relating to the problematic, deals with individual techniques of genetic engineering and briefly states legislative procedures in context of dealing with GMO. It gives examples of transgenic organisms and summarizes advantages and disadvantages of their use.Practical part of the thesis contains educational program called "Genetically modified organisms", which was conceived by the author and includes a draft of a lesson inclusive of teaching materials - powerpoint presentations, worksheets, interactive worksheets, auxiliary text for teacher and written preparation. Research part deals with high school students change of view about using GMOs after completing the educational program. Due to analysis was proven that most of the attitudes and knowledge about GMO was changed after completing the educational program (for example in issues of willingness to consume GM food and animal products, perception of advantages and disadvantages etc.) Data was still unchanged in questions which cannot be affected by the program (control of food packaging or...