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Stress resistance of PHA accumulating bacteria against conditions associated with gastrointestinal tract
Kovářová, Radka ; Nováčková, Ivana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on stress resistance of PHA accumulating bacteria against unfavourable conditions associated with gastrointestinal tract. Two bacterial strains were chosen as model microorganisms for this purpose. Cupriavidus necator H16 producing PHA and its mutant strain Cupriavidus necator PHB-4 without the ability of PHA accumulation. Firstly, three gastrointestinal juices of different concentrations were chosen for the experimental part, namely gastric, bile and pancreatic juices. The stressed bacterial strain was then determined using a spread plate method and a flow cytometry. Other methods used include antimicrobial tests. The agar diffusion method was first tested to determine the size of the inhibition zones. The last method that has been performed is the broth dilution method, which serves to compare the absorbance of pure bacterial suspensions and stressed suspensions with the given juice of a certain concentration. The results show that the production of PHA represents a disadvantage in context of conditions associated with gastrointestinal tract of accumulating bacterial strain.
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Study of low temperature plasma direct application on yeasts Candida Glabrata and bacteries
Petrová, Veronika ; Kodedová,, Marie (referee) ; Krčma, František (advisor)
The subject of this thesis is to study the interactions of the low-temperature microwave plasma with selected microorganisms Candida glabrata, Staphylococcus epidermidis, and Escherichia coli and their combinations. The inhibitory effect of the plasma on these microorganisms is monitored on selected solid nutrient media and in suspension (nutrient medium and PBS). The inhibitory effect on the mixed cultures of these microorganisms is also studied on the solid nutrient media. The surface wave microwave discharge is used, with argon as a working gas. For the experiment on the solid nutrient media, a constant gas flow rate of 5,0 Slm and a power fluctuating between 8-10 W are maintained throughout the experiment. For the suspension experiment, a constant gas flow rate of 2,0 Slm and 13 W power are maintained throughout the experiment. The inhibitory effects of plasma on the solid nutrient media were observed on the individual microorganisms as well as on the mixed cultures. It was found that the microorganisms treated by plasma on the solid nutrient media were inhibited and uncultivable, while their cytoplasmic membrane remained intact. When the microorganisms were treated in suspension, individual cells were killed, but the efficiency was low. This was caused by the experimental setup, specifically due to the plasma only being formed in argon (no intermixing with air), as the capillary was submerged in the treated suspension.
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Stress resistance of PHA accumulating bacteria against conditions associated with gastrointestinal tract
Kovářová, Radka ; Nováčková, Ivana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on stress resistance of PHA accumulating bacteria against unfavourable conditions associated with gastrointestinal tract. Two bacterial strains were chosen as model microorganisms for this purpose. Cupriavidus necator H16 producing PHA and its mutant strain Cupriavidus necator PHB-4 without the ability of PHA accumulation. Firstly, three gastrointestinal juices of different concentrations were chosen for the experimental part, namely gastric, bile and pancreatic juices. The stressed bacterial strain was then determined using a spread plate method and a flow cytometry. Other methods used include antimicrobial tests. The agar diffusion method was first tested to determine the size of the inhibition zones. The last method that has been performed is the broth dilution method, which serves to compare the absorbance of pure bacterial suspensions and stressed suspensions with the given juice of a certain concentration. The results show that the production of PHA represents a disadvantage in context of conditions associated with gastrointestinal tract of accumulating bacterial strain.
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Laboratory diagnosis of disorders of phagocytosis
POLÍVKOVÁ, Ivona
Phagocytosis is one of the oldest processes of absorbing particles like amoeba, which is one of the basic mechanisms of the immune system to defend the body against infections. Failure of these processes is clinically manifested like immune deficiency and it can cause a very serious complication which could leads to the death of the patient. Phagocytosis is performed by specialized cells (macrophages, dendritic cells, neutrophils and other phagocytic myeloid cells) which are able to absorb the target particles, especially microorganisms, dead cells and foreign objects. These processes are essential for stability of homeostasis. Absorption of micro-organisms leads to activation of adaptive immunity response, elimination of apoptotic and destroyed cells and starts the reparation processes of damaged tissue. Phagocytosis as a complex process can be divided into several phases: active movement of phagocytes to the inflammation zone, adherence, ingestion and intracellular degradation which leads to the killing of pathogens. There are two mechanisms of killing pathogens. First is independent on oxygen where antimicrobial substances are stored in azurophilic granules which could be released into phagolysosom. Second mechanism is oxygen-dependent called oxidation (respiratory) flare which leads to the formation of biologically active mediators where oxygen molecules have considerable oxidation potential. Defects in phagocytic system are mainly caused by low number or malfunction of neutrophils which leads to severe infections mainly caused by staphylococci, Enterobacteriaceae or fungi. Among malfunctioning of phagocytosis processes belongs LAD I and LAD II syndrome. The other possible disorder of phagocytic malfunction is defect in enzymes. The NADPH oxidase is necessary for bacterial lyses mechanism of phagocytes and lack of these enzyme caused serious inherit disorder called chronic granulomatous disease. Lack or completely missing of enzyme myeloperoxidase stored in the azurophilic granules of neutrophils and monocytes is more and more common autosomal recessive disorder of phagocytosis. In this diploma work I focused on detection of these rare and serious disorders defects using flow cytometry to detect respiratory burst activity of neutrophils in blood samples. This laboratory procedure is called a burst test. In this test we quantitatively evaluate the respiratory burst activity of granulocytes in heparinized blood samples using the flow cytometery. Principle of this method is an oxidation-reduction reaction of dihydrorhodamine 123 to green fluorescent rhodamine 123 using peroxide, hydroxyl radicals and superoxide anions which are activated by respiratory burst. This process is one of the significant characteristic of phagocytic cells which are characterized by multi-stage activation of NADPH oxidase. This oxidase catalyzes electron reduction of molecular oxygen to superoxide. This step plays a very important role in our immune system and allows to kill and degrade particles and bacteria in phagocyte cells. Since 2009 - 2012 I analyzed the results of 611 patients who were tested for respiratory burst of neutrophils on a request of their physician. Among all of these results we obtain only two positive results of reduction of stimulation index (SI) and significant decrease percentage of activated granulocytes. Both of these parameters: reduced percentage of (activated granulocytes stimulated by PMA and E. coli) and low stimulation index pointing to a potentially serious disorder of phagocytosis mechanism. In specific tests of these cases proved the enzyme defect in phagocytosis mechanism which is the most common disorder in this type of immunodeficiency. These results indicate that the major primary defects of phagocytosis are very rare and their detection is usually in childhood for suspicion of primary immunodeficiency. This test is very helpful for discovering a prime immunodeficiency.
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