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Antimicrobial resistance in clinical isolates of Clostridioides difficile derived from hospitalised patients in the Czech Republic and Slovakia.
Zíková, Jaroslava ; Krůtová, Marcela (advisor) ; Najmanová, Lucie (referee)
Clostridioides difficile is one of the most common pathogens of healthcare-associated colitis (CDI). However, in recent years there has been an increasing number of reported cases in the community. Causative strain characterization allows us to track the occurrence and spread of certain C. difficile types. To update the current CDI epidemiological situation, between October and November 2021, 342 C. difficile isolates or stool samples from CDI patients for culture were sent from 23 Czech (n=200) and 17 Slovak (n=142) hospitals. All C. difficile isolates were characterized by ribotyping and toxin genes detection. From 342 isolates, 52 different ribotyping profiles were identified. The frequent ribotypes detected were 001, 176, 014, 018 and 020. Genes for toxins A and B were found in all isolates. Genes for binary toxins were detected in 43 (21,5 %) Czech isolates and in 117 (82,4 %) Slovak isolates. Further, selected isolates (n=140, 40,9 %) were subjected to whole-genome sequencing (WGS) and antimicrobial susceptibility testing to 17 antibiotics (E-test). Multilocus sequencing typing determined 30 different sequence types from the 39 included ribotypes. All C. difficile isolates were susceptible to vancomycin, tetracycline, amoxicillin, and meropenem. The most frequently detected antimicrobial...
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The directed search of genes for secondary metabolites in streptomycetes.
Bakal, Tomáš ; Najmanová, Lucie (advisor) ; Janderová, Blanka (referee)
Discoveries of new natural antibiotics are now relatively rare, therefore the construction of strains producing hybrid substances seems to be a very promising opportunity to gain new interesting biologically active compounds. This work is part of a larger project focused on the preparation of new biologically active substances derived from the antibiotic lincomycin. Lincomycin is composed of saccharide (MTL) and amino acid (propylhygric acid) moieties condensed by amide bond. Various modifications of amino acid moiety, especially of the side alkyl chain, are known to improve the antibiotic properties of final molecule. The bottleneck of biosynthesis of such modified compounds is the condensing enzyme NDL-synthetase, and especially its A-domain, which, similarly to nonribosomal peptide synthetases (NRPS), specifically recognizes and activates the amino acid precursor. In this work a set of degenerate primers for PCR searching of NRPS A-domains was proposed and the conditions of PCR reaction were optimized. In the first step a collection approximately 800 isolates of soil actinomycetes will serve as a source of genetic information for search of interesting NRPS A-domains, applicable for the construction of hybrid biosynthetic clusters. The isolates of this collection have been also characterized taxonomically...
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Analysis of the gene lmbX included propylproline biosynthesis
Smutná, Yvona ; Najmanová, Lucie (referee)
Analysis of the gene lmbX included propylproline biosynthesis Abstract Lincomycin and its derivates are potent antibiotics used in human and veterinary medicine, they are antibiotics exibiting biological activity against Gram-positive bacteria. Lincomycin molecule is composed of two amide-bonded subunits, methylthiolincosamide and propylproline. The aim of the study was to assign the gene lmbX to the propylproline biosynthesis based on gene inactivation experiments. The gene lmbX was inactivated by help of REDIRECT technology: PCR targeting system in Streptomyces coelicolor. The respective S. lincolnensis inactivants were checked for lincomycin production by means of biological test and UPLC analysis of their fermentation broth and then exploited in feeding experiments. It was found, that inactivation of the lmbX gene caused suppression of the lincomycin production which was restored upon PPL feeding therefore the respective gene was assigned to reaction of PPL branch of LM biosynthesis.
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The use of parallel sequencing methods in microbiology.
Pavlíková, Magdaléna ; Najmanová, Lucie (advisor) ; Vopálenský, Václav (referee)
The thesis describes the history of development of sequencing methods with special focus on the modern effective parallel sequencing methods and their application in microbiology. The development and improvements of sequencing systems lead to the acceleration of the process and considerable decrease of price, which consequently allow wider spectrum of applications. Each of the sequencing systems has its characteristic features including drawbacks stemming from the principle of the respective method. Not every method suitable for all the applications. In the thesis the sequencing methods are compared and examined with respect to their appropriateness for certain application fields in microbiology. The currently available sequencing methods are usually categorized into three "generations", distinguished by sets of typical features. First generation methods include the systems of Sanger and Maxam-Gilbert; "next generation" is represented by methods 454, Illumina, SOLiD and Helicos; and finally SMRT, Ion Torrent and the commercially not yet available nanopore sequencing are usually called "next-next generation". Now the sequencing becomes a standard technology of molecular biology, not only in the basic microbiological research, but it is also widely applied in medicine (quick identification of patogenes,...
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Complex characterization of subgingival plaque - use of modern sequencing methods in diagnostics and monitoring of treatment of periodontal diseases
Těšínská, Barbora ; Najmanová, Lucie (advisor) ; Konopásek, Ivo (referee)
Periodontitis is a multifactorial inflammatory disease which can result in a complete loss of teeth. Its main cause is the accumulation of bacteria from the dental plaque followed by massive reaction of the host immune system. It was proved that the composition of oral microbiome (OM) differs in periodontally healthy individuals and patients with periodontitis. This work aims to solve specific parts of the long-term project concerning the taxonomic composition of the OM of periodontally healthy individuals and patients with chronic and aggressive form of the disease. The OM was characterized based on 16s rDNA sequencing. It is evident from the results that the shift in the OM composition occurs prior the development of clincal signs of the disease and that the precise dental care can significantly postpone or even avoid the onset of the disease. When comparing the OM composition in individuals with chronic and aggressive periodontitis, no remarkable differences were detected to explain the faster progress of the aggressive form of the disease. This work also aimed to compare the results obtained by 454 pyrosequencing and Illumina. Both sequencing methods were found to provide statistically comparable results. Illumina MiSeq thus can be employed to build on the former results of the long-term study...
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The identification of biosynthetic clusters in the soil isolates of actinomycetes.
Pavlíková, Magdaléna ; Najmanová, Lucie (advisor) ; Kopecký, Jan (referee)
Pyrrolobenzodiazepines (PBDs), predominantly produced by microorganisms from the genus Streptomyces, belong to a group of secondary metabolites with significant antitumor activity. A key role in the biosynthesis of PBDs have nonribosomal peptide synthetases, which catalyze the condensation of anthranilate subunit with prolin or proline derivative resulting in tricyclic PBD molecule, which can be subsequently further substituted. Five biosynthetic gene clusters have been published yet. The aim of this diploma thesis was to identify and characterize the biosynthetic gene cluster (or clusters) for the biosynthesis of PBDs limazepines. The producer of limazepines is the only yet identified strain, which produces both the PBDs hydroxylated at C9 position and compounds unsubstituted in this position simultaneously. According to already published data, in the biosynthesis of PBDs hydroxylated at position C9 the kynurenine pathway is involved, while the biosynthesis of the C9 unsubstituted PBDs employes the chorismate biosynthetic pathway. Moreover, according to the previously published data, the PBDs produced by this model strain incorporate three different proline derivatives, two of them being described as a structural part of a PBD molecule for the first time. Based on the results of this diploma...
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The use of parallel sequencing methods in microbiology.
Pavlíková, Magdaléna ; Najmanová, Lucie (advisor) ; Vopálenský, Václav (referee)
The thesis describes the history of development of sequencing methods with special focus on the modern effective parallel sequencing methods and their application in microbiology. The development and improvements of sequencing systems lead to the acceleration of the process and considerable decrease of price, which consequently allow wider spectrum of applications. Each of the sequencing systems has its characteristic features including drawbacks stemming from the principle of the respective method. Not every method suitable for all the applications. In the thesis the sequencing methods are compared and examined with respect to their appropriateness for certain application fields in microbiology. The currently available sequencing methods are usually categorized into three "generations", distinguished by sets of typical features. First generation methods include the systems of Sanger and Maxam-Gilbert; "next generation" is represented by methods 454, Illumina, SOLiD and Helicos; and finally SMRT, Ion Torrent and the commercially not yet available nanopore sequencing are usually called "next-next generation". Now the sequencing becomes a standard technology of molecular biology, not only in the basic microbiological research, but it is also widely applied in medicine (quick identification of patogenes,...
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Molecular analysis of resistance gene vga(A)LC identification of key aminoacid residues.
Kroová, Michaela ; Najmanová, Lucie (advisor) ; Vopálenský, Václav (referee)
Protein Vga(A) gives staphylococci resistance to streptogramins A. The recently discovered protein Vga(A)LC differs from Vga(A) only by 7 amino acid residues, but this difference is sufficient for shift of its substrate specificity towards lincosamides. The group of four amino acids in the central part of protein (LGAG in Vga(A) and SVTS in Vga(A)LC) was detected to be crucial for the substrate specificity. In this diploma thesis 5 alternativesets of vga(A)LC gene point mutations were prepared in order to determine the impact of individual amino acids of the aforementioned group on the resistance phenotype. Mutations were prepared in vector pGEM® -T and cloned into shuttle vector pRB374. The prepared constructs were transformed by electroporation into the sensitive strain of Staphylococcus aureus RN4220 and values of minimum inhibitory concentration (MIC) were measured for lincomycin, clindamycin and pristinamycin IIA by the agar dilution method. The transformation was not successful in one of the mutations. Results of setting MIC for the remaining four mutations do not make it possible to specify uniquely the ratio of individual amino acids for determining substrate specificity. Two of the amino acids were found to be important. We anticipate preparation of more mutations.
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The directed search of genes for secondary metabolites in streptomycetes.
Bakal, Tomáš ; Najmanová, Lucie (advisor) ; Janderová, Blanka (referee)
Discoveries of new natural antibiotics are now relatively rare, therefore the construction of strains producing hybrid substances seems to be a very promising opportunity to gain new interesting biologically active compounds. This work is part of a larger project focused on the preparation of new biologically active substances derived from the antibiotic lincomycin. Lincomycin is composed of saccharide (MTL) and amino acid (propylhygric acid) moieties condensed by amide bond. Various modifications of amino acid moiety, especially of the side alkyl chain, are known to improve the antibiotic properties of final molecule. The bottleneck of biosynthesis of such modified compounds is the condensing enzyme NDL-synthetase, and especially its A-domain, which, similarly to nonribosomal peptide synthetases (NRPS), specifically recognizes and activates the amino acid precursor. In this work a set of degenerate primers for PCR searching of NRPS A-domains was proposed and the conditions of PCR reaction were optimized. In the first step a collection approximately 800 isolates of soil actinomycetes will serve as a source of genetic information for search of interesting NRPS A-domains, applicable for the construction of hybrid biosynthetic clusters. The isolates of this collection have been also characterized taxonomically...
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Analysis of the gene lmbX included propylproline biosynthesis
Smutná, Yvona ; Najmanová, Lucie (referee)
Analysis of the gene lmbX included propylproline biosynthesis Abstract Lincomycin and its derivates are potent antibiotics used in human and veterinary medicine, they are antibiotics exibiting biological activity against Gram-positive bacteria. Lincomycin molecule is composed of two amide-bonded subunits, methylthiolincosamide and propylproline. The aim of the study was to assign the gene lmbX to the propylproline biosynthesis based on gene inactivation experiments. The gene lmbX was inactivated by help of REDIRECT technology: PCR targeting system in Streptomyces coelicolor. The respective S. lincolnensis inactivants were checked for lincomycin production by means of biological test and UPLC analysis of their fermentation broth and then exploited in feeding experiments. It was found, that inactivation of the lmbX gene caused suppression of the lincomycin production which was restored upon PPL feeding therefore the respective gene was assigned to reaction of PPL branch of LM biosynthesis.
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