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Enviromental DNA-inexhaustible source of unique bacterial genes
Culka, Martin ; Černá, Věra (advisor) ; Vaněk, Ondřej (referee)
Search for new enzymes or variants of known ones is now a hot issue in enzymological research. The classical culture-based approach, however, often fails when applied on environmental samples, because they contain uncultured microorganisms at most. For this reason, a new approach has been developed - the metagenomics. This approach is based on direct isolation of total DNA (RNA) from specific environment and its subsequent sequence-based (genotype) or function-based (phenotype) analysis. In this work, the metagenomic approach has been used to find new forms of penicillin G acylase, the enzyme that catalyze cleavage or formation of acyl - β-lactam nucleus bond and is used in industry for synthesis of semi-synthetic β-lactam antibiotics, in eleven samples from 4.5 m soil horizon. Sequence analysis of PCR amplicons on metagenomic templates revealed nucleotide sequences of major part of potential structural penicillin acylase genes. After translation it has been found that the sequences are most homologous to penicillin amidase from Conexibacter woesei. Further perspective of this metagenomic study is amplification of at least one complete structural gene of environmental penicillin acylase. However, the cloned regions of the gene can also be used to create hybrid penicillin acylases using gene shuffling...
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Monitoring and detection of Escherichia coli and Klebsiella pneumoniae strains with Extended Spectrum Beta-Lactamase (ESBL) production in Hospital Prachatice, a. s. in 2014-2018.
TVRDKOVÁ, Pavla
The ever increasing resistance of bacteria is a grave issue world-wide. Some bacteria are resistant naturally but for other resistance is acquired. As a result, we can encounter bacterium that would formerly be quite easily killed by administering antibiotics but, in the course of time, as a result of, for example, communication between various bacteria species and strains in the form of various mutations and, frequently, after the administration of incorrect antibiotic and the effects of selective antibiotic pressure, the same bacterium becomes resistant to antibiotics. One of the types of such acquired resistance is the production of broad-spectrum beta-lactamases ESBL. The objective of this thesis is to establish the representation and development of ESBL production in Escherichia coli strains and Klebsiella pneumoniae strains as these make the most frequent producers of these broad-spectrum beta-lactamases, in certain hospital departments and in various types of clinical materials over the period of 5 years. In addition, the thesis focuses on the development of antibiotic resistance, accordingly, over the 5 year period. Data gathering and utilization of methods took place at the Medical Microbiology Department of the Prachatice Hospital (Nemocnice Prachatice, a.s.). To identify microbes to a more precise level, the commercial set ENTEROtest 24 N and INDOL test were used. The disk diffusion method and method for determining the minimum inhibitory concentration were used to determine sensitivity to antimicrobial agents. The commercial set MASTDISC AmpC and ESBL (D68C) were used to detect ESBL. The outcomes indicated that the number of Escherichia coli strains producing ESBL and Klebsiella pneumoniae strains producing ESBL grew almost each year (with the exception of 2016). The outcomes also indicated that the greatest number of ESBL producers were found with Klebsiella pneumoniae strains. Furthermore, the ESBL producers were grouped depending on hospital departments in which most frequent occurrences were found; the most frequent ESBL occurrence of both producers was found in the department of internal medicine. Additionally, the ESBL producers were grouped based on capture in various types of clinical materials; the greatest quantities of both microbes were found in urine. Based on these findings, the development of antibiotic resistance for both microbes over the period of 5 years was analyzed on samples of urine. When monitoring antibiotic resistance development, no considerable growths in bacterial strains resistant to individual anti-microbial agents was found over the 5 year period. Only for 3rd generation cephalosporins, there was a certain growth in resistance detected almost every year for both microbes. Namely, Escherichia coli isolates showed a growth from 6 % to 9 % and Klebsiella pneumoniae isolates showed a growth from 29 % to 33 % in the percentual quantity of strains resistant to these antibiotics.
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Occurrence of ESBL strains in Hospital Pisek, Inc.
KOTHÁNKOVÁ, Michaela
Resistance to ATB is becoming a big problem all round the world. Bacterial resistance occurs naturally or is obtained by gene mutations where genetic information is transmitted between genera or species. These strains are then resistant to antibiotics that are otherwise effective. The biggest problem is that in these cases the treatment is prolonged, the mortality and morbidity of the patients increases and the costs of the treatment as well. The initial theoretical part is devoted to the characteristics and types of effects of antimicrobial agents, beta-lactam antibiotics, antibiotic resistence, where resistance transfer process, multiresistance and resistance mechanisms are described. Further, this part is dedicated to beta-lactamases and their classification, broad-spectrum beta-lactamases (ESBL) and their producers, especially the genus Klebsiella, the genus Escherichia, the genus Enterobacter and the genus Proteus. The last chapter of this part is focused on the impact of resistance to therapy and treatment options. A methodical part of this work was performed at the Department of Clinical Microbiology of Písek Hospital, Inc., where the detection of ESBL positive strains of the genus Klebsiella, Escherichia, Enterobacter and Proteus was observed during one year (January-December 2016). This part focuses on microbes of the mentioned genera and their antibiotic susceptibility. For susceptibility testing, this is a disk diffusion test based on the inhibition zone and a bouillon microdilution method, which is based on minimum inhibitory concentration. Further, this part also deals with an ESBL production test where a screening test using Brilliance ESBL agar chromogenic soil and method of detection ESBL and AmpC production (known as the MAST test) are described. The last chapter of this part is dedicated to quality control. In the practical part of the thesis, we can compare the incidence of bacteria genus Klebsiella, genus Escherichia, genus Enterobacter, genus Proteus and their production of ESBL. Further, this part is focused on the distribution of isolates with ESBL production according to the type of hospital department, the highest incidence was found in the department of aftercare and department of social beds. This section also describes the distribution of ESBL-producing isolates according to the type of clinical material, where there was a huge catch in the urine. It was also compared the number of ESBL producers in the hospital and the community where it was found 85% incidence in the hospital, which was expected.
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Enviromental DNA-inexhaustible source of unique bacterial genes
Culka, Martin ; Černá, Věra (advisor) ; Vaněk, Ondřej (referee)
Search for new enzymes or variants of known ones is now a hot issue in enzymological research. The classical culture-based approach, however, often fails when applied on environmental samples, because they contain uncultured microorganisms at most. For this reason, a new approach has been developed - the metagenomics. This approach is based on direct isolation of total DNA (RNA) from specific environment and its subsequent sequence-based (genotype) or function-based (phenotype) analysis. In this work, the metagenomic approach has been used to find new forms of penicillin G acylase, the enzyme that catalyze cleavage or formation of acyl - β-lactam nucleus bond and is used in industry for synthesis of semi-synthetic β-lactam antibiotics, in eleven samples from 4.5 m soil horizon. Sequence analysis of PCR amplicons on metagenomic templates revealed nucleotide sequences of major part of potential structural penicillin acylase genes. After translation it has been found that the sequences are most homologous to penicillin amidase from Conexibacter woesei. Further perspective of this metagenomic study is amplification of at least one complete structural gene of environmental penicillin acylase. However, the cloned regions of the gene can also be used to create hybrid penicillin acylases using gene shuffling...
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