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Global epidemiology of Staphylococcus aureus circulating in healthcare system and community
Mrázková, Pavlína ; Tkadlec, Jan (advisor) ; Balíková Novotná, Gabriela (referee)
Staphylococcus aureus is an important human pathogen responsible for a wide range of infections occurring among patients in the healthcare setting and in the community. Much attention is focused on methicillin-resistant strains (MRSA), but methicillin-sensitive strains (MSSA) also remain the main cause of infections. The epidemiology of S. aureus, especially among MRSA strains, has shown a rapid development in recent years, characterized by the appearance of epidemic lines that dominate in certain geographical areas. Molecular typing techniques have made it possible to identify the main successful lineages of MRSA and MSSA and to track their spread and report the prevalence of certain clones in the area at the same time. To understand the evolution and spread of S. aureus, as well as to deploy effective preventive measures against the spread of this dangerous pathogen, the knowledge of the S. aureus strains epidemiology is absolutely necessary. As part of the bachelor's thesis, a recent overview of the methods used for the clonal analysis of Staphylococcus aureus will first be presented. Furthermore, a literature search will be conducted with the aim of creating an overview of the dominant strains of S. aureus and MRSA circulating in the hospitals and in the community with an emphasis on the...
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Cellular factors influencing the antibiotic resistance by ABC-F proteins
Kýr, Jan ; Balíková Novotná, Gabriela (advisor) ; Dolejšová, Tereza (referee)
Antibiotic resistance is one of the main problems modern medicine has to face. In order to control it, it is important first to understand the mechanisms by which resistant pathogens bypass antibiotic treatment. One of the important protein families conferring resistance to 50S binding antibiotics is the ARE ABC-F protein family. A member of this protein family is the MsrA protein, which confers resistance to 14- and 15-membered macrolides. Loss-of-function mutations in the non-essential chaperone ClpX were found to a significant ly enhance the action of the MsrA protein. This leads to the significant increase in the resistance conferred by this protein. The exact mechanism by which ClpX affects MsrA function is still unknown. In this the diploma thesis was demonstrated that chaperone protein ClpX affects the resistance to erythromycin conferred by MsrA protein, due to the interaction with an unknown essential protein, which is mediated by a functional N-terminal zinc-binding domain of the chaperone. Furthermore, it was demonstrated in this work that loss of GluTR function influence the MsrA ability to confer resistance. The results of this work will bet he basis for further reasearch, which will lead to a more detailed understanding of the mechanism of resistance conferred by these proteins and...
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Glycopeptide resistance in staphylococci
Rozsypálková, Adéla ; Tkadlec, Jan (advisor) ; Balíková Novotná, Gabriela (referee)
The aim of this thesis is to describe the mechanism of the resistance to glycopeptide antibiotics in a genus Staphylococcus, especially in a species Staphylococcus aureus which is common cause of nosocomial infections resulting frequently in expensive and long-term treatment. This pathogen is dangerous due to its ability to acquire resistance to most antibiotics used in a clinical practice. The resistance of these microorganisms can develop very easily due to inappropriate treatment (administration, drug concentration, duration), which, if not detected, could ultimately results in treatment failure and the death of the patient. The vancomycin resistance of S. aureus could be divide into groups according to their values of vancomycin MIC: VSSA, VISA, hVISA and VRSA. Vancomycin intermediate resistance is associated with mutation, e.g., which affect cell wall synthesis. In contrast, VRSA is associated with the transfer of the mobile genetic element with the vanA or vanB operon from genus Enterococcus. This transmission is due to co-infection with both pathogens. Glycopeptide resistance has also been shown to be very common in coagulase-negative staphylococci (CNS), such as S. capitis, which cause infection in preterm infants. Glycopeptide resistance in CNS and intermediate resistance of S. aureus is...
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Subcellular localization of resistant proteins Vga(A)LC and Msr(A) using fluorescence microscopy
Nguyen Thi Ngoc, Bich ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
Vga(A)LC and Msr(A) are clinically significant resistant proteins in staphylococci that confer resistance to translational inhibitors. They belong to ARE ABC-F protein subfamily, which is part of ABC transporters. Unlike typical ABC transporters, ABC-F proteins do not have transmembrane domains that are responsible for the transport of substances through the membrane. Therefore, they do not have characteristic transport function but regulatory or resistance function. Their mechanism of action on the ribosome has been described only recently, where these proteins displace the antibiotic from the ribosome. However, some aspects of their function are still unclear. For example, what is the function of the Vga(A) location on a membrane that has been detected in the membrane fraction but not in the ribosomal. In this work, using fluorescence microscopy, I observed subcellular localization of the Vga(A)LC-mEos2, Vga(A)LC-GFP and Msr(A)-eqFP650 resistant fusion proteins in live cells of S. aureus under different culture conditions . It has been shown that Vga(A)LC-GFP and Msr(A)-eqFP650 occur in a foci near the membrane. Depending on ATPase activity or the presence of an antibiotic, the localization of Msr(A)-eqFP650 in the cell changes from focal to diffuse, presumably on ribosomes, suggesting a...
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Antibiotic resistance conferred by members of ARE subfamily of ABC proteins
Veselá, Ludmila ; Balíková Novotná, Gabriela (advisor) ; Borčin, Kateřina (referee)
The main topic of this thesis is the ARE subfamily of ABC transporters. The importance of the proteins of this subfamily lies in the fact that they confer resistance to several classes of clinically important antibiotics: macrolides, lincosamides, streptogramines and pleuromutilines and they do it in significant pathogens, as for example Staphylococcus aureus. Compared to canonical ABC transporters, the structure of ABC proteins lacks the transmembrane domain (TMD) and so far, there where not even found an integrating transmembrane protein. Due to these facts, the mechanism of resistance conferred by these proteins remains unclear. In the thesis, both suggested hypotheses of the mechanism of how these proteins work are discussed. The first hypothesis presumes the active efflux of antibiotics out of the bacteria. The second hypothesis suggests release of antibiotic from its binding site initiated by ARE proteins, followed by its passive diffusion out of the cell. Keywords: ABC proteins, ARE proteins, resistance, MLS, Vga
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The effect of aminoacid variability on the resistance phenotype in ARE subfamily of ABC proteins
Lenart, Jakub ; Balíková Novotná, Gabriela (advisor) ; Fišer, Radovan (referee)
ARE subfamily proteins belonging to ABC transporters confers a different degree of resistance to macrolides, linkosamides and streptogramins antibiotics. Among the most clinically ARE subfamily proteins in staphylococci is Vga(A) protein lead to the award resistance to streptogtramins A. In 2006, discovered the new variant called the Vga(A)LC, which in addition to streptogramins A resistance also confers linkosamides. Vga(A) and Vga(A)LC differ in only 7 amino acids, yet confer different resistance phenotypes. In previous experiments it was found that the central role in determining substrate specificity play a 4 amino acid differences that accumulate in the section of 15 amino acids within the linker connecting the two ABC domains (positions 212, 219, 220 and 226). The combination of amino acids LGAG Vga(A) increases resistance to streptogramins A while present in combination SVTS Vga(A)LC increased resistance to linkosamides. Although in this subfamily includes a large number of resistance proteins, the mechanism of resistance has not yet been established with certainty. The aim was to create a new Vga(A) variants that contain specific combinations of amino acids for Vga(A) and Vga(A)LC protein at positions 212, 219, 220 and 226 and compared their ability to grant resistance to linkosamides. We also...
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Critical sites determining the resistance phenotype of ABC proteins from the ARE subfamily and the molecular mechanism of their function
Lenart, Jakub ; Balíková Novotná, Gabriela (advisor) ; Melter, Oto (referee) ; Branny, Pavel (referee)
Vga(A) and Msr(A) are resistance proteins belonging to the ARE subfamily of ABC -F proteins. They confer resistance to inhibitors of the peptidyltransferase center. It has been proposed that the mechanism of resistance is based on interaction with a transmembrane partner that forms the functional transporter. Their ribosomal function has been described by cryoelectron microscopy of ribosome complexes with ABCF mutants unable to hydrolyze ATP. However, the exact mechanism of resistance is not yet known. We have produced the mutant proteins combining the four amino acid residues in Vga(A) and Vga(A)LC at the linker tip, and we were the first to describe the effects of substrate specificity of the single mutants. Amino acid positions 212 and 220 are important for resistance to lincosamides and pleuromutilins, respectively, while position 219 is responsible for resistance to streptogramin A. Each amino acid property plays a critical role in conferring antibiotic specificity, as confirmed by the fact that amino acid substitution at position K218T in the Vga(A) protein causes the shift in resistance from streptogramins to lincosamides and pleuromutilins. The mechanism of resistance conferred by Vga(A) is ribosomal protection. This is supported by the fact that the rate of [3H]-lincomycin accumulation in...
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Mechanism of inducible gene expression of resistance protein Vga(A)LC from Staphylococcus haemolyticus.
Novotná, Michaela ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
The staphylococcal protein VgaA belongs to ARE ABCF family, which confers resistance to ribosome binding antibiotics by the target protection mechanism. VgaA confers resistance to lincosamides, streptogramins A and pleuromutilins and thus provides the so-called LSAP resistance phenotype. The expression of resistance genes often reduces fitness in the absence of an antibiotic, therefore the expression of resistance genes is often tightly controlled and triggered only in response to the presence of an antibiotic to which the protein confers resistance. The inducible expression has also been observed for the vgaA gene, nevertheless, its mechanism has not been elucidated. In the diploma thesis, it was shown that the vgaALC gene from Staphylococcus haemolyticus is regulated by ribosome-mediated attenuation. The mechanism is based on the detection of translation inhibitors via a ribosome translating a special regulatory open reading frame (uORF), which is part of an attenuator located in the 5' untranslated region of the mRNA. The vgaALC gene is regulated at the transcriptional level in response to LSAP antibiotics. Antibiotic specificity of induction is affected not only by the nature of the peptide encoded by uORF but also by the antibiotic specificity of the resistance protein. Fluorescence microscopy...
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Enterococcus spp. as a reservoir of resistance genes.
Zajíčková, Adéla ; Lichá, Irena (advisor) ; Balíková Novotná, Gabriela (referee)
Bacteria of the genus Enterococcus are categorised among common nosocomial pathogens. They are a significant reservoir of resistance genes to a majority of antibiotics and exhibit an intrinsic resistance to low levels of beta-lactams, glycopeptides, aminoglycosides, streptogramines and lincosamides. The aim of this paper is to review the main resistance genes and other mechanisms involved in the resistance of bacteria of this genus to antibiotics. The paper is mainly focused on the resistance to beta-lactam antibiotics, which is provided by the expression and mutations of low-affinity PBPs, the individual van resistance types mediating resistance to vancomycin, and the expression of enzymes capable of modifying the functional groups of aminoglycoside antibiotics. The paper also describes the resistance to newer antibiotics that are used to treat vancomycin-resistant isolates. The resistance to individual antibiotics can arise from the coding of their own chromosomal genes or entire signaling pathways leading to a reduction in the effect of antibiotics, the acquisition of genetic mutations, and especially the spread of new resistance genes by horizontal transfer.
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Characterization of the ABC-F protein Sco0636 in Streptomyces coelicolor
Pinďáková, Nikola ; Balíková Novotná, Gabriela (advisor) ; Mikušová, Gabriela (referee)
The main topic of this diploma thesis is ARE (resistance) proteins from the ABC-F family of the second class of ABC proteins. ARE proteins confer resistance to antibiotics that bind to a large ribosomal subunit and therefore inhibit proteosynthesis. One of the ARE proteins is the Lmr (C) protein, which is part of the linkomycin biosynthesis cluster of Streptomyces lincolnensis, and according to new results, Lmr (C) does not have to be just resistant protein but may have also regulatory function. We decided to study Sco0636, the closest homologue to Lmr (C) in Streptomyces coelicolor, which is a model organism in the study of secondary metabolism. Thanks to the production of color pigments, it is possible to monitor the effect of ARE proteins on secondary metabolism directly on the plates. I prepared the deletion mutant and the strain with constitutive expression of sco0636, and observed the effect on the phenotype. I followed the production of a blue asset and set a minimum inhibitory concentration to selected antibiotics, which bind to the ribosome. I have found that Sco0636 gives high resistance to tiamulin and so it has been named TiaA. The deletion of gene sco0636 accelerated production of actinorodine, and constitutive expression of this gene slowed down production. Keywords: ABC proteins,...
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