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Introduction of an invertebrate animal model, Galleria Galleria mellonellamellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics
Kubíček, Jiří ; Konečná, Klára (advisor) ; Němečková, Ivana (referee)
Charles university, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Pharmacy Author: Jiří Kubíček Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Introduction of an invertebrate animal model, Galleria mellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics. Background: The aim of this diploma thesis has been to present an invertebrate organism, Galleria mellonella (G. mellonella), Pyralidae, as an alternative, more economically advantegous and morally more acceptable model for in vivo studies of an antituberculotics (anti-TB) efficacy. In the experimental part of this work, attention was paid to the optimization of methodological steps, that should lead to a higher reproducibility and validity of the obtained data. The effect of the disaggregating supplement, tyloxapol, on the growth of Mycobacterium aurum (M. aurum) was evaluated. The growth of this microbial strain in the culture medium, Middlebrook 7H9, was also mapped to determine the exponential growth phase. In the second part of the experiments, G. mellonella larvae were infected with a M. aurum bacterial strain, followed by an administration of the selected, commercially available anti-TB. Methods: To map the exponential phase of M....
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Trends in incidence of Tuberculosis and Nontuberculous Mycobacterial Infections in years 2001-2020
Doležalová, Karolína ; Göpfertová, Dana (advisor) ; Skřičková, Jana (referee) ; Svatoň, Martin (referee)
Trends in incidence of tuberculosis and non-tuberculous mycobacteriosis in children and adolescents in Czech republic in years 2001-2020 BCG vaccination programs were introduced into Czechoslovakia more than 60 years ago under an entirely different epidemiological situation than that of today. Compulsory mass BCG vaccination was abolished in November 2010 and changed to a selective vaccination program for infants at high risk of contracting tuberculosis. This work wants to ascertain the risk of tuberculosis and non-tuberculous mycobacterial infection in 10 years following the change of the vaccination program and a comparison with the same period of mass BCG vaccination. The design of this work is a descriptive study, comparison, and statistical analysis of data for the period 2001-2020 about cases of tuberculosis and mycobacteriosis as reported in the Tuberculosis Register. The conclusions are that the trend of TB incidence in children in both compared periods is identical and statistically significantly decreasing (p <0,001). The trend of incidence of NTM cervical lymphadenitis in children is in the first period degressive and non-significant (p=0,561). However, in the second period, the trend is significantly increasing (p <0,001), in every, compared two subsequent years is an increase of 8 %....
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Introduction of an invertebrate animal model, Galleria Galleria mellonellamellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics
Kubíček, Jiří ; Konečná, Klára (advisor) ; Němečková, Ivana (referee)
Charles university, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Pharmacy Author: Jiří Kubíček Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Introduction of an invertebrate animal model, Galleria mellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics. Background: The aim of this diploma thesis has been to present an invertebrate organism, Galleria mellonella (G. mellonella), Pyralidae, as an alternative, more economically advantegous and morally more acceptable model for in vivo studies of an antituberculotics (anti-TB) efficacy. In the experimental part of this work, attention was paid to the optimization of methodological steps, that should lead to a higher reproducibility and validity of the obtained data. The effect of the disaggregating supplement, tyloxapol, on the growth of Mycobacterium aurum (M. aurum) was evaluated. The growth of this microbial strain in the culture medium, Middlebrook 7H9, was also mapped to determine the exponential growth phase. In the second part of the experiments, G. mellonella larvae were infected with a M. aurum bacterial strain, followed by an administration of the selected, commercially available anti-TB. Methods: To map the exponential phase of M....
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Růst Mycobacterium smegmatis na agarovém médiu a agarovém médiu pokrytém celofánovou folií - morfologická a proteomová studie
Ramaniuk, Volha ; Weiser, Jaroslav (advisor) ; Beranová, Jana (referee)
Biofilm formation is one of the most common bacterial survival strategies. Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature, although they can cause serious problems when they appear in medical equipment and artificial replacements of the human body. Non-pathogenic Mycobacterium smegmatis mc2 155 was used as a model organism in our experiments. We investigated morphology of the three- and six-day-old colonies (in fact biofilms) on agar and agar covered with cellophane using Stereo microscope and Scanning Electron Microscope. We found that a type of surface as well as a carbon source has a great influence on the morphology of the M. smegmatis colonies. We isolated proteomes from the agar and cellophane cultures and from planktonic culture. Two-dimensional electrophoresis was used as the main proteomic method. Proteomic data were analyzed using PDQuest software. Then the sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 7 unique proteins that might be specific for recognition and adhesion of bacteria to the cellophane, no unique protein in agar proteome and 46 unique...
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The diagnosis of pulmonary tuberculosis
CHALOUPKOVÁ, Lenka
Even in 21st century tuberculosis (TBC) pose a significant medical and social problem. Open borders intensified migrations, expansin of AIDS pandemic and related increase of patients with reduced immunity are all reasons why tubercuosis still remains one of the main causes of death all over the world. The disease originator is Mycobacterium tuberculosis, an acidoresistent bacteria that is able to survive in a human organism for a lot of years. Symptoms of the disease are not characteristic, that is why the correct diagnosis is essential to reveal the new cases of the disease. One of the standards to stay is bacteriological examination based on microscopic examination and cultivation of the material. Also rapid molecular genetic techniques and methods based on the immune response are availabe. This work uses data gaied from Plicní léčebna Nemocnice České Budějovice, a.s. (Pulmonary Department of Hospital in České Budějovice) for the period of 2008-2016, as well as data gained from Pracoviště imunologie (Immunolgy Department) in Nemocnice České Budějovice, a.s. The goal of the work was to create a statistical overview of patients with confirmed pulmonary tuberculosis according to age and gender for the period of 2008-2016 . Next it aims at comparison of tuberculin skin test and quantiferon test in patients with pulmonary tuberculosis for the period of 2008 - 2016. Implementation and evaluation of the quantiferon test in patients in 2016 are the end of my work. The methodology was mainly based on gathering the necessary data and their evaluation. Results show that pulmonary tuberculosis was confirmed in 81 patients in relation to data obtained from the Pulmonary Department. Of that 75% were male and only 25% female. For both sexes, the occurrence of tuberculosis was on record in all age categories. In men most often at the age of 31-50 and 51-70. Tuberculin skin test (PPD) and quantiferon (QF) test were also accomplished in 62% of patients. Only in 7% patients none of the tests was done. When the two tests were compared, both PPD and QF tests were positive in 64%. Also the results show that the quantiferon test is more sensitive than the tuberculin skin test. The quantiferon test was evaluated separately for the positive and negative results, positive result was compared with bacteriological examination. Next the QF test was evaluated from the point of view of gender, age, diagnosis and engaged department. A relatively signifcant finding was that even though the QF test is more sensitive, for more than a third of patients with the positive QF test result, mycobacterial identification was not required at all. Compared to bacteriology examination TB was cofirmed in 14% of patients. Despite the fact that the Czech Republic ranks among the countries with a low incidence of tuberculosis, this fact can not be underestimated. The public's feeling that there is no need to worry about this illness has also been supported by the approval of the cancellation of flat vaccinations, effective from 1. 11. 2010. Also, a number of physicians are no longer taking this disease into account while making diagnosis. However, education of lay public is essential above all in this issue.
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Růst Mycobacterium smegmatis na agarovém médiu a agarovém médiu pokrytém celofánovou folií - morfologická a proteomová studie
Ramaniuk, Volha ; Weiser, Jaroslav (advisor) ; Beranová, Jana (referee)
Biofilm formation is one of the most common bacterial survival strategies. Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature, although they can cause serious problems when they appear in medical equipment and artificial replacements of the human body. Non-pathogenic Mycobacterium smegmatis mc2 155 was used as a model organism in our experiments. We investigated morphology of the three- and six-day-old colonies (in fact biofilms) on agar and agar covered with cellophane using Stereo microscope and Scanning Electron Microscope. We found that a type of surface as well as a carbon source has a great influence on the morphology of the M. smegmatis colonies. We isolated proteomes from the agar and cellophane cultures and from planktonic culture. Two-dimensional electrophoresis was used as the main proteomic method. Proteomic data were analyzed using PDQuest software. Then the sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 7 unique proteins that might be specific for recognition and adhesion of bacteria to the cellophane, no unique protein in agar proteome and 46 unique...
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Vaccination - yes or no? Public perception of the benefits of vaccines
Chudomelová, Iva ; Drda Morávková, Alena (advisor) ; Jelínek, Michael (referee)
Thanks to the vaccination were saved millions of lifes around the world, but this topic raises some controversy. Many European countries abolished the obligatory tuberculosis vaccination. The main argument was steadily decreasing incidence of infectious diseases and burdening BCG vaccination for the newbors and more adverse reactions detected in recent years. The cause of their formation is probably connected with other antigens. The downtrend in the incidence of TB is recorded only in some states, mainly in Western Europe. With respect to the current demographic situation and legal or illegal migration, it is continue necessary to take this disease seriously and to investigate the new vaccination strategies.
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Antituberculotic susceptibility testing of Mycobacterium tuberculosis isolates
SUCHÁ, Denisa
Mycobacterium tuberculosis was first described by a German physician and microbiologist Robert Koch in the year 1882. It is an obligate pathogen, which causes a very serious infectious disease - tuberculosis. This disease is an important epidemiological problem all over the world. It is necessary to diagnose all the clinical cases of the disease, isolate the patiens and apply an adequate treatment to reduce the incidence of the disease and to prevent its spread. Essential for the diagnosis of tuberculosis is a confirmation by cultivation, i.e. isolation of Mycobacterium tuberculosis from clinical specimens, exact identification of the isolates and antitubercular drug susceptibility testing. The theme of this thesis was to determine the susceptibility to the basic anti-tubercular agents (streptomycin, isoniazid, rifampicin, ethambutol and pyrazinamide). The theoretical part provides an overview of susceptibility testing methods. A group of 275 isolates of Mycobacterium tuberculosis isolated from January 2008 to June 2013 from clinical specimens, in the Department of Microbiology of the Faculty Hospital in Pilsen, the Laboratory for Diagnosis of Mycobacteria was examined within the practical part. The susceptibility to antitubercular drugs was tested in all the isolates by two methods the proportion method (Canetti, 1963) and the method in semi-closed system BACTEC? MGITÔ 960. The results showed 100% concordance between both methods. The resistance was proved to at least one of the basic antitubercular drugs in 24 of tested isolates. The file was also evaluated from the demographical and epidemiological point of view. There was a higher incidence of tuberculosis among men (75% men and 25% women). In terms of age, the highest incidence has been shown between 40 and 60 years of age. The percentage of foreigners in the investigated group was 30 %. The Czech Republic is a country with a low incidence of tuberculosis,however, this situation can not be underestimated, if only due to the fact that there is a constant migration of people into the Czech Republic from the countries with a high incidence of tuberculosis and its drug-resistant strains.
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