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Development and genetic basis of glycopeptide resistance in coagulase-negative staphylococci
Prášilová, Jana ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
Glycopeptides are the so-called last-resort antibiotics in clinical practice used to treat heavier, predominantly nosocomial infections caused by multi-resistant coagulase-negative staphylococci. The origin and genetic basis of resistance to glycopeptide antibiotics has not yet been elucidated within coagulase-negative staphylococci. Research on Staphylococcus aureus has shown, that intermediate resistance to glycopeptide antibiotics is associated with the presence of one or more mutations, rather than being conditioned by the support of a particular genetic element, such as in enterococci. By using various types of in vitro resistant mutant selection, we were able to obtain isogenic pairs of glycopeptide sensitive and resistant strains of Staphylococcus epidermidis and Staphylococcus haemolyticus. By sequencing the genomes of these pairs, one nucleotide polymorphisms were identified and predominantly found in metabolic and cell wall control systems. Phenotypic analysis did not reveal a direct association of glycopeptide resistance with increased biofilm formation. In clinical practice, the cross-resistance of glycopeptides and other antibiotics is problematic. For the non-glycopeptide antibiotics imipenem and rifampicin, the incidence of cross-resistance with glycopeptide antibiotics in S. aureus...
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Mode of daptomycin action and mechanism of resistance
Helusová, Michaela ; Mikušová, Gabriela (advisor) ; Lichá, Irena (referee)
Daptomycin is one of antimicrobial peptides. These molecules are part of immune system of all organisms. Daptomycin consists of a cyclic peptide core and a lipophilic tail. Daptomycin is produced by Streptomyces roseosporus and is used to treat serious gram-positive infections. Daptomycin is active also against methicillin-resistant Staphylococcus aureus. Its antimicrobial effect depends on the presence of calcium ions and phosphatidylglycerol. Daptomycin targets bacterial cytoplasmic membrane, where it forms oligomers. Mode of action of daptomycin probably includes pore-forming effect leading to membrane damage. This disturbance causes ion efflux from cytoplasm which leads to membrane potential disruption, which results in inhibition of macromolecular synthesis and cell death. Daptomycin also causes changes in cell morphology. Despite its unusual mode of action, several mechanisms of resistance have emerged in some pathogenic strains. These are for example decrease in the amount of phosphatidylglycerol in the membrane, increase in the amount of lysylphosphatidylglycerol, release of membrane phospholipids or mutations in genes which control peptidoglycan synthesis.
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The effect of vanZTei and vanZg expression on resistance to glycopeptide antibiotics in Staphylococcus aureus
Zieglerová, Leona ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
A membrane protein VanZTei which is encoded by the gene vanZ from the vanA glycopeptide resistance gene cluster is a part of the large family of VanZ proteins. VanZTei confers resistance to teicoplanin in Enterococcus faecalis without the presence of other proteins encoded by the cluster. The aim of my work was to compare the ability of two orthologous proteins VanZTei and VanZg (from the genome of Enterococcus faecium) to confer resistance to glycopeptides in Staphylococcus aureus RN4220 and Enterococcus faecium. We have shown that VanZg increases resistance to teicoplanin (Tei) 8 to 16 times the and also to dalbavancin (Dalb) 8 times. VanZTei also confers resistance to Tei and Dalb, but the increase is only twofold. Conversely VanZTei confers resistance to newly synthetized glycopeptides more effectively than VanZg (fourfold increase of resistance confered by VanZTei and two to fourfold increase of resistance confered by VanZg). It suggests that both proteins have different specificity to antibiotics. In despite the mutants of S. aureus RN4220 VanZTei pRMC2 with increased resistance to teicoplanin (MICTei> 8 µg/ml) in which the resistance is dependent on vanZTei expression were selected. These resistant mutants do not carry mutation in a gene vanZTei or in its ribosomal binding site. Neither of the...
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Study of gene expression profile of ellipticine - resistant neuroblastoma cell line UKF-NB-4
Pinkas, Michael ; Poljaková, Jitka (advisor) ; Hinďoš Hřebačková, Jana (referee)
Neuroblastoma (NB) is a malignant embryonal tumor of the peripheral sympathetic nervous system derived from neural crest and is the most common tumor in infants. If the protooncogen MYCN is amplified in the case of high risk neuroblastoma, the current therapy fails. The biggest issue is the development of resistance. Ellipticine (ELLI) is a potential antineoplastic drug, whose cytotoxic effect is mainly based on the inhibition of topoisomerase II, its intercalation into the double helix structure of DNA and formation of adducts with DNA after enzymatic activation by cytochromes P450, peroxidases, sulfotransferases and N,O-acetyltransferases. Long-term cultivation of NB cell line UKF-NB-4 with ELLI leads to resistance, which is multifactorial. (i) It appears that ELLI is not effluxed from cells of the line UKF-NB-4ELLI as in the case of doxorubicin resistance in UKF-NB-4, but is transported from the nucleus and sequestrated in intracellular compartments. Cytotoxicity of ELLI is reduced also by (ii) low intracellular pH and (iii) decreased expression of topoisomerase II. (iv) Expression of enzymes activating ELLI is unchanged on the mRNA level detected by DNA microarray. However, enhanced expression of enzymes activating ELLI (cytochrome P450 3A4 and cyclooxygenase-1) is detected by qRT-PCR. Moreover...
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