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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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Evaluation of TD test for analysis of persistence or tolerance in clinical isolates of Staphylococcus aureus
Kotková, Hana ; Lichá, Irena (advisor) ; Fišer, Radovan (referee)
Persistence is the ability of bacteria to survive the impact of antibiotics even when the bacteria do not encode resistance genes. This is a very complex process, which is probably consequence of a reduction physiological process in subpopulation of bacteria. The aim of this study was to verify the suitability of the newly developed "Tolerance Disk Test" (TD test, Gefen et al, 2017) for detection of persistent or tolerant subpopulations of bacterial cells in clinical isolates of Staphylococcus aureus. We performed TD test for this kind of bacteria, which is a significant opportunistic pathogenic organism in humans, and compared its results with the killing curves. We have found that the ability to persist can be monitored semi-quantitatively also in this case and we consider this test suitable for introduction into clinical practice. In addition, we suggest that TD test could distinguish between persistent and tolerant subpopulations. Key words: Staphylococcus aureus, persistence, antibiotics, tolerance
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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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General stress response in Staphylococcus aureus and its role in adaptation to hyperosmotic stress
Kotková, Hana ; Lichá, Irena (advisor) ; Seydlová, Gabriela (referee)
Hyperosmotic stress induces a stress reponse in a halotolerant bacteria Staphylococcus aureus that helps it to survive even the osmotic conditions that are inhibiting for most of the bacteria. Stress responses of S. aureus include a general stress response that activates rapidly on a wide range of stresses and is ensured by expression of genes induced by an alternative sigma B subunit of RNA polymerase (SigB), and more specific responses to hyperosmotic stress associated with transporters of potassium and transporters of compatible solutes. Also, some virulence factors (such as the adhesive Ebh protein) allow S. aureus to survive hyperosmotic stress within the host, thereby helping to colonize it. It is currently known that the general stress response directly and indirectly participates in the specific stress adaptation through the Kdp transport system for potassium (Kdp transporter) and transporters of compatible solutes (PutP and OpuD). Expression of the Kdp transporter and the large S. aureus Ebh protein is SigB dependent, via Agr or ArlRS virulence regulator systems. The formation of a biofilm, in which SigB is significantly involved, may also enable to colonize host niches with low water activity. Hyperosmotic stress by triggering SigB initiates a complex response that is associated not only...
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