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Motility and virulence of bacterial pathogens in patients with cystic fibrosis
Benešová, Nikol ; Melter, Oto (advisor) ; Smělíková, Eva (referee)
Cystic fibrosis is an autosomal recessive disease that mainly effects the Europoid race. The disease is caused by a mutation in the cystic fibrosis transmembrane conductance regulator gene. This gene encodes the formation of chloride channel proteins. The disease leads to the formation of highly viscous secretions from the exocrine glands. The most commonly affected organ is the lung, which accumulates mucus and bacteria. Bacteria that colonize the lungs of patients with cystic fibrosis are exposed to many stress factors such as large amounts of mucus, oxidative stress, antibiotics and immune system of the host. That leads to the selection of mutants that are better adapted to the environment of the lungs. In acute infection, one of the important virulence factors for bacteria is their motility. Motility is provided by flagella and allows the bacterium to move to sites with higher nutrient content and to colonise different parts of the lung. In chronic infection, we can see changes in the genotype and phenotype of bacteria. Nonmotile mutants are selected because they activate the immune system less than motile bacteria, that helps in the persistence of bacteria in the lungs. Chronic infections lead to a higher mortality rate. The aim of this bachelor's thesis is to compare the motility of bacteria...
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Study of biofilm Pseudomonas aeruginosa and introduction of new method for antibiotics susceptibility
Mošničková, Markéta ; Melter, Oto (advisor) ; Chrenková, Vanda (referee)
Biofilm is a complex structure produced by bacterial cells which contain highly hydratated filamentous exopolysacharides. Biofilm exists everywhere in our surroundings or nature. It creates for instance a sticky viscous structure covering the surface of stones or other solid objects underwater. The technology of sewage cleaning remediation is essentially dependent on the presence and function of biofilm producing bacteria. Adhered biofilm in medicine has usually negative consequences for patient health, hence, reduction of its role is a goal of researches in the field. Here biofilm first of all adheres and colonizes catheters or artificial joints or other implants and is a source of a chronic infection which limits the healing process or normal function of the implants. In cytic fibrosis patient biofilm production is also responsible for the fact that acute infection caused by P. aeruginosa could change within a few months in chronic infection when mucoid strain could be cultivated from the patient. In my bachelor thesis I have analyzed clinical material and bacterial isolate of P. aeruginosa of a cystic fibrosis patient which dissociated into mucoid and smooth phenotype using light-, electron- and immunofluorescence microscopy. I have applied an original method using resuspended bacterial culture in solid...
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Molecular epidemiology and characteristics of bacterial pathogens on lung infection in patients with cystic fibrosis
Vošahlíková, Šárka ; Nemec, Alexandr (advisor) ; Melter, Oto (referee) ; Osička, Radim (referee)
Cystic fibrosis is the most abundant inherited autosomal recessive disease in Caucasian population. Cystic fibrosis is caused by a dysfunction of a transport channel which is responsible for the transport of chloride ions on the apical side of the plasma membrane. Despite the fact that the dysfunction of the transport channel is present in several organs, the most severely affected one is the respiratory system. Because of the ion imbalance, thick sticky mucus is produced on the surface of the airways which then prevents the removal of dust particles and bacteria. The main complications of cystic fibrosis are the bacterial infections of the respiratory system which become chronic during the patient's life and thus are the most common causes of the respiratory failure and premature death. The most important agents causing these infections are Pseudomonas aeruginosa and Burkholderia cepacia (Bcc). Infections caused by those bacteria are practically untreatable and serious complications arise from the existence of epidemic strains which can be transfered from patient to patient. Precise and fast diagnostics of pathogenic strains is a critical step to avoid spreading bacterial infections as well as strictly followed anti- epidemic strategies mainly based on isolation of cystic fibrosis patients according to...
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Molecular characterization of resistance to MLSb antibiotics in Staphylococcus aureus and SCV Staphylococcus aureus strains of cystic fibrosis patients
Vařeková, Eva ; Melter, Oto (advisor) ; Zikánová, Blanka (referee)
Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder in Caucasians. Lower respiratory tract of CF patients is colonized by specific bacteria, often leading to chronic infection and lung tissue damage. In this thesis we characterized 338 isolates of S. aureus from 92 Czech CF patients isolated in 2011-2013. Using spa typing and PFGE we detected high clonal heterogenity of this collection with the exception of MRSA strains (resistant to oxacillin; 5% prevalence) which were clonally related. The prevalence of S. aureus MLSB resistance in our collection was high (69 %), which is a serious problem due to common usage of these antimicrobials in clinical practice. A half of the MLSB resistant strains lacked any known determinant of this resistance (ermA, ermC, ermT, msrA). Sequencing of the ribosomal genes revealed a high number of S. aureus strains carrying target site mutations resulting in MLSB resistance (37 %). This is new important information about the staphylococcal strains associated with chronic infections in Czech Republic. Focusing on mutability of analysed strains, we also detected several strains with point mutations or deletions in their mutator genes mutS a mutL. Hypermutability could be responsible for the high rate of ribosomal mutations and for the presence of...
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Mechanisms of bacterial persistence and clinical significance
Smělíková, Eva ; Melter, Oto (advisor) ; Pinkas, Dominik (referee)
Persistent bacteria, shortly persisters, are cells that are characterized by their tolerance to antibiotics without containing resistance genes. These are not resistant bacteria, because resistant bacteria are determined by genetic code. Persisters are indistinguishable from other antibiotic sensitive bacteria, and they are rather transient phenotypic subpopulations. Probably all types of bacteria can create a persistent stage, the ability is not species-bound and persistence has been described in a number of bacterial species. The reason for the formation of persistence may be sudden stress, then it is induced formation, or the reason may be an insurance for the future, then we call it stochastic phenotypic variability. Then a variety of phenotypes of different subpopulations within a genetically uniform population can be distinguished. They differ in growth properties and tolerance to antibiotics. Bet-hedging strategy is a hypothesis that describes persistence as insurance against the risk of external fluctuations. During stress an alarmon guanosine tetraphosphate or guanosine pentaphosphate is accumulated to alert the cell to unfavorable conditions and it initiates transition to a more tolerant state. The existence of these tolerant subpopulations is important in recurrent diseases because small...
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Bacterial REP elements: origins, variability and application.
Nunvář, Jaroslav ; Lichá, Irena (advisor) ; Pačes, Jan (referee) ; Melter, Oto (referee)
4 ABSTRACT (English) This thesis is based on three published research papers studying bacterial REP (repetitive extragenic palindrome) elements. REP elements are one of the best-characterized groups of bacterial DNA repeats, distributed mostly in gammaproteobacteria, including enterobacteria. They are present in noncoding parts of host genomes, usually occurring in hundreds of copies. REPs are typically aggregated in higher order repeats. In the Gram-negative model Escherichia coli, interactions of several proteins important for cell's physiology with REPs were described, indicating significant role for these elements for host cells. The first work (Nunvar et al. 2010) presents the discovery of a protein class, related to IS200/IS605 transposases. These proteins, termed RAYTs (REP-associated tyrosine transposases), contain characteristic motifs in their amino acid sequences, which are absent in canonical IS200/IS605 transposases. Another attribute of RAYTs is the arrangement of their encoding genes. These are single copy genes, always flanked at both termini by at least two REPs in inverted orientation. Based on the similarity between the REP-rayt-REP unit and insertion sequences of the IS200/IS605 family, between RAYTs and tyrosine transposases and between REPs and subterminal sequences of the IS200/IS605...
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Introduction of microbiological diagnostic agents of invasive infections - Bartonella henselae and Bartonella quintana
Nováková, Marie ; Melter, Oto (advisor) ; Chrenková, Vanda (referee)
Bartonellae are agents of infections with varied clinical symptomalogy, including serious, such as endocarditis. Direct detection of Bartonella so far in the Czech Republic and therefore does not need to address this issue. Proper diagnosis of these infections is a prerequisite for targeted antibiotic therapy for patients. The genus Bartonella today are more than 20 kinds of bacteria. They are small Gram-negative rod-shaped bacteria that cause the new emerging infections such as cat scratch disease (CSD), showing the typical regional lymphadenitis, as well as bacillary angiomatosis (BA), whose typical symptoms are skin and subcutaneous nodules (nodules). They also cause a disease called trench fever or South American bartonelosis. In the experimental part of the work was performed genotypic and phenotypic methods for the diagnosis of these bacteria, specifically on two types of Bartonella, Bartonella henselae and Bartonella quintana . The results show that by suitable methods to diagnose Bartonella are amplification genus- specific genes ribC and ftsZ, further embodiment of a method pulsed electrophoresis (PFGE) as typing method and the determination of susceptibility to antibiotics, the most appropriate testing showed E-test. This work was carried out optimization of genotypic and phenotypic...
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The introduction of diagnosis of new bacterial pathogens Ralstonia a Achromobacter isolated from patients with cystic fibrosis and determining their sensitivity to antibiotics
Michálková, Alice ; Melter, Oto (advisor) ; Bébrová, Eliška (referee)
Cystic fibrosis (mucoviscidosis) is an incurable genetic disease caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator Gene). The most common causes of increased mortality and morbidity of patients include bacterial respiratory infections which may occur even due to less frequent pathogens. Bacterial species of the Ralstonia and Achromobacter genera are not considered pathogenic for healthy people, but they have been established as pathogens in the sputum of patients with CF. However, due to their phenotypic similarity to other bacterial pathogens encountered in patients with CF, microbiologists often do not pay attention to them. The aim of this thesis was to propose some methods of identification of the genus Ralstonia, to determine both quantitative and qualitative susceptibility towards antibiotics, and to make a bibliographical search focused on the issue of the Achromobacter genus. Strains of Ralstonia spp. were identified using phenotypic and genotypic methods and were tested for the susceptibility towards antibiotics. The thesis also proposes a new method of genotypic indentification of R. respiraculi.
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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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