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Influence of Staphylococcus aureus Quorum sensing system to antibiotic tolerance
Šaňková, Michaela ; Lichá, Irena (advisor) ; Buriánková, Karolina (referee)
Staphylococcus aureus is a gram-positive opportunistic pathogenic bacterium that causes a range of chronic infections especially to weakened individuals. Recently, the most serious problem in clinical medicine has been infections caused by so-called persisters. They form a subpopulation of cells that, thanks to adaptations to stressful conditions, are able to withstand lethal doses of antibiotics and then cause an infection again. Agr system, which is responsible for the mechanism of quorum-sensing, plays the main role in switching between the active virulent form and the persistent form associated with biofilm formation. During chronic infections, adaptive mutations accumulate in this Agr system and affect the ability to persist. For the purpose of this thesis, clinical isolates were obtained from patients diagnosed with cystic fibrosis in chronological order within the first three years of their lives. The aim of this thesis was to confirm the influence of adaptive mutations on the persistence ability of clinical isolates and also to observe how the membrane potential and metabolic activity change depending on the on the action of selected antibiotics. Additional aim was to create a deletion of the response regulator AgrA gene using the CRISPR/Cas9 system. In order to characterize the persistence...
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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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Regulation of virulence factors of Staphylococcus aureus
Šaňková, Michaela ; Lichá, Irena (advisor) ; Černý, Ondřej (referee)
Staphylococcus aureus is a gram-positive pathogenic bacterium that regulates virulence factors production in response to changing environmental conditions. S. aureus cells evolved a complex regulatory network, including a number of regulatory proteins, transcriptional factors and two- component systems. One of the most important S. aureus regulatory systems is the Agr system (Accessory gene regulator) that perceives its own population density by sensing a "quorum-sensing" signal in a form of autoinducing peptid (AIP). Agr system encodes a global regulatory RNAIII that regulates the expression of target virulence factors, which includes surface proteins as well as extracellular toxins and enzymes. The family of global protein regulators SarA and transcriptional sigma factor B also play a significant role in the regulation of S. aureus virulence. The production of virulence factors is also regulated in response to specific signals from extracellular environment by two- component-systems, which includes the regulator of exoprotein production SaeRS, the regulator of autolysis ArlRS and the regulator of respiratory response SrrAB. Key words: Staphylococcus aureus, virulence factors, Agr, quorum-sensing, RNAIII, SarA, SigB
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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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