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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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Methods for characterization of persistent state after exposure to selected antibiotics in Staphylococcus aureus
Valtová, Aneta ; Lichá, Irena (advisor) ; Tkadlec, Jan (referee)
Staphylococcus aureus is a opportunistic pathogen that can cause severe and chronic infections. The reason of the infections relapse is often the persistence. It is about adapting to stressful conditions by inducing a dormant state, which would allow bacteria to survive exposure to antibiotics and grow again after their elimination. Bacteria that persist in the patient acquire various adaptive mutations, which are transmited creating subpopulations that have a better ability to persist. The aim of this diploma thesis was to compare individual methods of persistent study that could be used in clinical practice in the future, and at the same time to try a closer molecular characterization of the persistent state with using methods for calculating gene expression. I had chronological isolates of Staphylococcus aureus at my disposal, the initial one being the primoisolate, an isolate taken at the diagnostics of cystic fibrosis before the start of antibiotic treatment. Another was taken at a distance of three-quarters of a year and the last with a half-year interval from the previous one. Following whole genome sequencing, genes in which adaptive mutations occurred were identified. The first method determines the degree of persistence by calculating CFU (Colony Forming Units) after antibiotic treatment....
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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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Effect of knock out of yxkO gene on environmental stress adaptation in genus Bacillus
Tkadlec, Jan ; Lichá, Irena (advisor) ; Krásný, Libor (referee)
We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to...
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Analysis of methicillin resistant Staphylococcus aureus strains from Czech Republic and Slovakia
Brajerová, Marie ; Tkadlec, Jan (advisor) ; Karpíšková, Renáta (referee)
Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen that has spread into the community since the 90s. In general, the community-associated (CA- MRSA) strains are more virulent, but less resistant to antimicrobials than the hospital- acquired strains (HA-MRSA). Some lineages of MRSA such as sequence type 8 have been transmitted more successfully around the world than others and there are situations where a dominant lineage has been replaced by a new one. The factors that are crucial for the selection of dominant lineage are often not clear. As part of this thesis, a longitudinal study of MRSA epidemiology in the Motol University Hospital during the period of 2018-2020 was performed and the occurrence of MRSA clonal complexes was characterized. A multicenter study of the epidemiological situation of MRSA in Slovakia during the period of January - April 2020 was also performed concomitantly. Moreover, several isolates from dominant lineages were further characterized by their phenotype. In the Motol University Hospital study, it was found that the HA-MRSA of the clonal complex 5 dominates and is represented mainly by spa-type t586; a finding that concurs with recent studies from the Czech Republic. In Slovakia, similar to the Czech Republic, MRSA lineages from clonal...
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Changes in the ability to form persisters in chronological isolates of Staphylococcus aureus
Kotková, Hana ; Lichá, Irena (advisor) ; Tkadlec, Jan (referee)
In immunodeficient patients, for example with cystic fibrosis (CF), the opportunistic pathogen Staphylococcus aureus causes chronic infections of respiratory tract that are treated with antibiotics (ATB) in the long term. However, exposure to antibiotics can lead to persistence, thereby result a recurrence of infection. The aim of this work was to examine in selected pairs of S. aureus chronological isolates from the respiratory tract of CF patients how their ability to form persisters is changing in time. I have found that the ability to persist within the clonal pair does not change significantly after two years of survival in the host, and that the ability to persist depends on the adaptative mutations of the isolates. Persister formation may depend on mutations in operon of the alternative sigma B factor (sigB) and the major virulence gene regulator (agr). By dual staining with DioC2(3) and To-pro-3, I was able to determine the changes in membrane potential and membrane permeability during the killing curve with ATBs. The distribution into subpopulations according to these parameters depends primarily on the antibiotic used. I conclude that various antibiotics can induce different mechanisms causing a persistent state. Futhermore, I have constructed plasmids with a labeled promoter to determine...
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Colistin resistance in clinically important Enterobacteriaceae
Smělíková, Eva ; Tkadlec, Jan (advisor) ; Ježek, Petr (referee)
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...
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Methods for characterization of persistent state after exposure to selected antibiotics in Staphylococcus aureus
Valtová, Aneta ; Lichá, Irena (advisor) ; Tkadlec, Jan (referee)
Staphylococcus aureus is a opportunistic pathogen that can cause severe and chronic infections. The reason of the infections relapse is often the persistence. It is about adapting to stressful conditions by inducing a dormant state, which would allow bacteria to survive exposure to antibiotics and grow again after their elimination. Bacteria that persist in the patient acquire various adaptive mutations, which are transmited creating subpopulations that have a better ability to persist. The aim of this diploma thesis was to compare individual methods of persistent study that could be used in clinical practice in the future, and at the same time to try a closer molecular characterization of the persistent state with using methods for calculating gene expression. I had chronological isolates of Staphylococcus aureus at my disposal, the initial one being the primoisolate, an isolate taken at the diagnostics of cystic fibrosis before the start of antibiotic treatment. Another was taken at a distance of three-quarters of a year and the last with a half-year interval from the previous one. Following whole genome sequencing, genes in which adaptive mutations occurred were identified. The first method determines the degree of persistence by calculating CFU (Colony Forming Units) after antibiotic treatment....
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Changes in the ability to form persisters in chronological isolates of Staphylococcus aureus
Kotková, Hana ; Lichá, Irena (advisor) ; Tkadlec, Jan (referee)
In immunodeficient patients, for example with cystic fibrosis (CF), the opportunistic pathogen Staphylococcus aureus causes chronic infections of respiratory tract that are treated with antibiotics (ATB) in the long term. However, exposure to antibiotics can lead to persistence, thereby result a recurrence of infection. The aim of this work was to examine in selected pairs of S. aureus chronological isolates from the respiratory tract of CF patients how their ability to form persisters is changing in time. I have found that the ability to persist within the clonal pair does not change significantly after two years of survival in the host, and that the ability to persist depends on the adaptative mutations of the isolates. Persister formation may depend on mutations in operon of the alternative sigma B factor (sigB) and the major virulence gene regulator (agr). By dual staining with DioC2(3) and To-pro-3, I was able to determine the changes in membrane potential and membrane permeability during the killing curve with ATBs. The distribution into subpopulations according to these parameters depends primarily on the antibiotic used. I conclude that various antibiotics can induce different mechanisms causing a persistent state. Futhermore, I have constructed plasmids with a labeled promoter to determine...
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