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Detection of pathogens in joint infections, including total endoprostheses using the BioFire method (Biomérieux)
ŠTĚTINOVÁ, Lucie
This bachelor thesis deals with the detection of pathogens in joint infections including total joint replacements by the BioFire method (Biomérieux, France). Compared to conventional methods, the Biofire Joint Infection Panel (BioMérieux, France) offers simultaneous detection of up to 39 pathogens and antimicrobial resistance markers in about one hour of time.This method is a multiplex PCR system for rapid and comprehensive testing. It is an innovative method that, thanks to its speed and comprehensiveness, will allow clinical microbiologists and physicians to make timely decisions on appropriate antibiotic therapy. Early de-escalation of this antibiotic therapy will also be enabled through the timely and targeted deployment of the appropriate ATB. The theoretical part of the thesis discusses the major infections of joints and joint replacements, introduces the reader to the most important causative agents of these infections, and a section is also devoted to microbiological diagnosis. The practical part describes the classical multiplex PCR, the FilmArray multiplex PCR system and with it the BioFire Joint Infection Panel, which has been used in the analysis of synovial fluid in patients with suspected septic arthritis or periprosthetic infection. Data from the analysis are also evaluated in the practical part. The data are from March 2021 to June 2022 and are presented in the practical part in the form of tables and graphs. The conclusion of the paper summarizes the relevance of the method for routine diagnosis and answers the stated research questions.
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Comparison of detection of biofilm formation in clinical strains of Staphylococcus aureus from wounds using culture and genetic methods.
VÁLKOVÁ, Tereza
The main topic of this bachelor thesis is the bacterium Staphylococcus aureus, its ability to form a biofilm and the diagnostics of this formed biofilm. The theoretical part of the thesis first focuses on the bacterium itself, its morphology, antigenic structure and virulence factors. Furthermore, its pathogenesis and pathogenicity will be described, including the issue of the methicillin-resistant Staphylococcus aureus. The next part focuses on the biofilm. The composition, structure and formation of biofilm will be described, as well as its role in pathogenicity, treatment options and various methods of detection. The practical part describes methods of biofilm detection. The first method is crystal violet staining according to Christensen. This method came out positive for all 94 tested strains of Staphylococcus aureus. Subsequently, these strains were divided into categories according to the strength of biofilm production. The second method was Real-time PCR and the detection of selected genes responsible for biofilm formation: clfA, fnbA and fnbB. 2-3 genes were found in 93 samples, and only one sample contained none of the selected genes. Thus, it was shown that all strains are capable of biofilm formation to varying degrees, and no correlation was found between the presence of the examined genes and the level of biofilm production.
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Evaluation of the effects of biodegradable microplastics on biota of freshwater ecosystem via ecotoxicity tests
Procházková, Petra ; Maršálek,, Blahoslav (referee) ; Beklová, Miroslava (referee) ; Zlámalová Gargošová, Helena (advisor)
Microplastics, which are ubiquitous contaminants of freshwater ecosystems, have raised concerns regarding their potential impact on the environment. For this reason, there is an effort to replace conventional plastics with biodegradable alternatives that can easily degrade in the environment. However, the process of biodegradation of these materials requires specific conditions, such as temperature, humidity, pH, and the presence of microorganisms, which are not always met adequately. Consequently, they may fragment and form biodegradable microplastics that can affect the environment similarly to conventional microplastics. Therefore, the aim of this study was to assess the impact of microplastics of biodegradable poly(3-hydroxybutyrate) microplastics on freshwater organisms, specifically the alga Desmodesmus subspicatus, the plant Lemna minor and the crustacean Daphnia magna. Additionally, we aimed to develop a suitable analytical method for the direct detection of microplastics in the body of D. magna. Although the observed effects were not always significant, they highlighted certain secondary problems that may arise due to the presence of biodegradable microplastics in the environment These issues could be related to biodegradability and the formation of biofilm on the surface of biodegradable microplastics, leading to subsequent nutrient depletion in the surrounding environment. Therefore, the use of biodegradable plastics as a substitute for conventional materials must be approached critically.
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Role of extracellular DNA in microbial biofilms
Gromov, Anton ; Chytilová, Aneta (referee) ; Obruča, Stanislav (advisor)
Abstract This bachelor’s thesis deals with defining the role of extracellular DNA in microbial biofilms. The thesis includes a part about the basic characteristics and functions of biofilms, as well as the characteristics and functions of extracellular DNA. The theoretic part additionally deals with the process of formation of biofilms, and the influence of different types of stresses on it. Also, the paper discusses the role of such stresses. The role of biofilm in bacteria and their reproduction methods are also discussed. An interesting section of the thesis outlines the impact of antibiotics on biofilms, or the ways to gain DNA or use DNase. The eDNA functions and their effects on bacterial defence will be discussed thoroughly. In the experimental part, the biofilm content was examined and compared with cells growing in a liquid and solid medium. Measurement of DNA content in media was carried out and the effects of salt and temperature stresses on biofilm were also measured. Stress conditions affected the production of PHB, which is used as a reserve form of energy and carbon. Total sugars were measured using an anthracite reagent to study the effects of stresses on the content of macromolecules. The presence of eDNA in biofilms and planktonic cells was firstly checked using gel electrophoresis and then accurate concentrations in planktonic and biofilm cells were determined using different spectrophotometric methods.
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Biofilm formation in probiotic cultures and its application in pharmacy
Ryšávka, Petr ; Obruča, Stanislav (referee) ; Vorlová, Lenka (referee) ; Márová, Ivana (advisor)
The work was comprehensively focused on the development of adhesive forms of probiotics in the form of a biofilm on combined carriers with a prebiotic component. The second part dealed with the influence of food on the multiplication and survival of selected types of probiotic bacteria. Subsequently, the effect of individualized probiotic supplements on changes in the human intestinal microbiome was monitored. Suitable adherent probiotic strains for biofilm formation were selected and tested. Methods have been introduced and different variants of carriers for culturing and binding bacteria have been tested. In vitro experiments verified the stability of biofilm stucture and its resistance to low pH, bile and antibiotics in comparison with the planktonic cell form. The antimicrobial effect of probiotic strains in the form of a biofilm was studied. The cultivation of the multispecies biofilm on the combined carrier was optimized and the stability of the biofilm and the final viability of probiotic bacteria were confirmed. Furthermore, the influence of various foods and beverages on the viability of probiotic bacteria was evaluated with emphasis on the simulation of passage through the gastrointestinal tract. Both models, solutions with standardised concentrations of alcohol, sugar, salts, proteins or different pH and different types of real foods and beverages were tested. The effect of food and beverages was tested on monocultures of Lactobacillus acidophilus, Bifidobacterium breve and on probiotic capsules containing a mixed culture of probiotic microorganisms. The survival of probiotics in various food matrices in the simulated gastrointestinal tract was quantitatively different. We managed to define foods suitable for supporting the multiplication of probiotic bacteria. A separate part of the work was focused on the targeted modulation of the intestinal microbiome by individualized probiotics that were prepared on the basis of molecular biological analyzes of the intestinal microbiome aimed at detecting the percentage of lactobacilli, bifidobacteria and phylum Firmicutes and Bacteroidetes. Personalized probiotic supplementation confirmed the positive effect of this approach on microbiome changes, especially on the increase of the content of lactobacilli, bifidobacteria and the overall diversity of the microbiome.
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Scanning Electron Microscopy and its Applications for Sensitive Samples
Hrubanová, Kamila ; Nováček, Jiří (referee) ; Schröfel,, Adam (referee) ; Krzyžánek, Vladislav (advisor)
Předložená dizertační práce s názvem “Rastrovací elektronová mikroskopie a její aplikace pro senzitivní vzorky” pojednává o problematice rastrovací elektronové mikroskopie v kontextu instrumentálního a metodologického vývoje vedoucího k inovativnímu řešení, které je dobře aplikovatelné zejména v mikrobiologickém výzkumu. Součástí práce je rozprava o historii a současném stavu elektronové mikroskopie (EM) jakožto vědecké zobrazovací a analytické techniky, tato část se nachází v úvodních kapitolách. Nepopiratelný přínos EM v biologických a lékařských oborech je dokazován mnoha citovanými vědeckými publikacemi. Předložená dizertační práce přináší novinky z oblasti přípravy preparátů a kryogenní rastrovací elektronové mikroskopie (cryo-SEM) vyvinuté na pracovišti Ústavu přístrojové techniky AV ČR, v.v.i. v Brně. Jedná se především o návrhy a výrobu speciálních držáků vzorků a vývoj nových metodik v oblasti přípravy mikrobiologických preparátů vedoucích k nalezení optimálních parametrů jednotlivých procesů. V experimentální části se nachází ověření metodologických postupů při studiu hydratovaných a na elektronový svazek senzitivních preparátů. Následné srovnání různých přístupů na definovaném biologickém systému z oblasti mikrobiologie přispívá k rozšíření interpretace doposud známých výsledků. Mezi zkoumanými mikrobiologickými kmeny byly biofilm-pozitivní bakterie Staphylococcus epidermidis a kvasinky jako Candida albicans a Candida parapsilosis, jež jsou považovány za klinicky významné, protože se podílejí na vzniku závažných infekcí zejména u imunokompromitovaných pacientů. Dále byl studován vliv růstu biofilmu bakterie Bacillus subtilis na biodeteriorizaci a biodegradaci poly--kaprolaktonových fólií. Vývoj v oblasti cryo-SEM byl aplikován ve výzkumu mikrobů s biotechnologickým potenciálem, jako jsou např. Cupriavidus necator a Sporobolomyces shibatanus.
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Formation of biofilm by probiotic bacteria and its processing to solid drug form.
Grossová, Marie ; Rittich, Bohuslav (referee) ; Buňka, František (referee) ; Márová, Ivana (advisor)
The aim of present work is cultivation of probiotic bacteria L. acidophilus, B. breve and B. longum in such a way that the culture forms cells clusters or comprehensive biofilm on the variety of free carriers. Biofilm formation of L. acidophilus on the silica from point of view bile and acid tolerance in gastrointestinal tract was studied. While the number of living cells in planktonic form (planktonic form) at pH 1 fell by 30 %, the viability of the biofilm cells was maintained to 90 % under the same environmental conditions. The biofilm culture showed also the protection against environment contained bile. Furthermore, the possibilities of drying procedures of biofilm cultures used as commercial technologies in pharmaceutical industry were studied. The comparison of freeze-drying and fluidization bed drying showed, that freeze-drying is more suitable method, which is able to achieve higher amount of viable cells after drying than fluidization bed drying. The effectivity of freeze-drying method is dependent on the selection of suitable cryprotective medium. In this case, about 90 % higher viability after freeze drying was achieved in comparison with fluidization bed drying. Finally, the industrial processing of probiotic strains into the solid dosage form was studied. Tablets should be produced at hardness between 70 and 90 N and water activity of tablet mixture can be maintained below 0.3. Consequently, the drying step of the tablets in a hermetically closed space with at least 10 % of silica gel must be ensured. Thereafter, the tablets contain (5.4 ± 0.7)109 viable cells after 6 months of drying process. Capsule production technology has no significant effect on the cell‘s viability during production. The triplex blistering foil for primary blistering of probiotic capsules was chosen. The triplex foil, which has low values of water vapour transition rate (0.07 g H2O / (m2 × day) and oxygen transition rate (0.01 cm3/m2 × day), was chosen. Other studied blistering foils commonly used in the pharmaceutical industry are not suitable for long storage of solid dosage forms contained probiotics.
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Comparison of the biocidal activity of disinfectants against biofilm
Šlitrová, Barbora ; Votava,, Miroslav (referee) ; Omelková, Jiřina (advisor)
A common vector of nosocomial infections is Pseudomonas aeruginosa. This opportunistic pathogen is resistant against different classes of antibiotics as well as against biocides. Some P. aeruginosa strains are able to form biofilm. The biofilm formation on medical devices represent a common occurrence that can lead to serious illness and death. Bacteria in biofilms are more resistant than their planktonic counterparts. Therefore it is important to find disinfectants that will be effective also against biofilm. Bactericidal efficacy of four disinfectants has been evaluated on biofilm and on planktonic forms of P. aeruginosa.
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Study of the development of microbial biofilm within an experimental model, Galleria mellonella
Doubová, Dominika ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Dominika Doubová Supervisor: RNDr. Klára Konečná, Ph.D. Title: Study of the development of microbial biofilm within an experimental model, Galleria mellonella Aim of the thesis: The aim of this thesis is to introduce the invertebrate organism, Galleria mellonella (G. mellonella), that can be used within the scientific research as an alternative model organism to vertebrates. This model seems to be more financially favorable and more ethically acceptable, which is an essential fact in the current era of increasing pressure to abandon testing on vertebrates in particular. The experimental part of this thesis is focused on optimizing the utilization of the G. mellonella larvae as a model organism within the research of fungal burn wounds fungal infections induced by the yeast Candida albicans. Within these experiments, larvae were used to preparation of tissue explants on which the formation of a single- species yeast biofilm was investigated. Methods: Before obtaining of tissue explants, the larvae were decapitated. To reduce contamination of tissue samples primarily by the gut microbiota of the larvae, the approach of removing the digestive tube was optimized. Burn wounds were induced...
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