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Molecular identification, typing and adaptation of Achromobacter spp. during the course of chronic infection
Učíková, Barbora ; Dřevínek, Pavel (advisor) ; Nemec, Alexandr (referee)
Achromobacter spp. is an emerging pathogen, especially in chronic respiratory infections in patients with cystic fibrosis. MALDI-TOF mass spectrometry provides reliable identification only at the genus level. The nrdA gene sequence is used for species identification of representatives. Clonality studies using multilocus sequence typing can determine whether a patient is still infected with the same clone or whether reinfection with a new strain occurs over time. Time-collected isolates of Achromobacter spp. from patients with cystic fibrosis were included in our study. Patients were divided into three groups according to the time interval between collections. In the first group, the external interval between collections was approximately 10 years, in the second group 7 to 12 months, and the remaining group consisted of single isolates. In the course of chronic infection, Achromobacter spp. adapt to the exposed antibiotics and to the host. Isolates sampled at an interval of 10 years showed a higher number of mutations than isolates with a sampling interval of up to one year. During chronic infection, loss of motility occurs, which we demonstrated phenotypically at the level of motility, reduction in flagella number and changes in flagellar genes. Increased resistance was observed in some isolates by...
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Molecular and epidemiological characteristics of Burkholderia cenocepacia strain dominant in cystic fibrosis patients in the Czech Republic
Vilimovská Dědečková, Klára ; Dřevínek, Pavel (advisor) ; Macek, Milan (referee) ; Šmajs, David (referee)
Summary: Bacteria from BurkJwlderia cepacia complex represent the most serious pathogens for patients witb cystic fibrosis. l nc:ir scverity IS caused by their antibiotic resistance and ability to Sllrcad among patients wilh cystic fibrosis. Many cyslic fibrosis cenlrcs around IlIe world, also tbe biggest Czech Cystic Fibrosis Centre in University Hospital in Motol, faeed outbreak. In Ihe 1990's tlle epidernie strain HurMwlderia cenocepacia S1'32 spread in a population of cystic fibrosis patienlS. One of the goals of the tunent thesis was to study epidemiological situation of Blďkholderia cepada oornplex in the popolation of eystie fibrosis pmients allending Cysfie Fibrosis Centre in University Hospilal MOlol almost seventeen years after first cases of infected palients wilh Bec inrection. Otber goals were: to evaloate 3 usefulness of molccular genelie diagnosties of Burkholderia cepacia complex tetl years afier ils implementation in routine diagnosties, to improve surveillal1ee syslem not only by implementalion or simple and specifie tesl for detection of epidemie stmin, bul 3150 by implementation of genolyping melllods thal are able to detennine strain in olher cases of infeetion. The [asI goal was to characterize Ibe epidemie slrain frolll virolence point ofview. Dased oRthe thesis we: confirmed...
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Carbapenemases and their molecular genetic detection in bacteria causing severe hospital acquired infections
Vavrová, Jolana ; Dřevínek, Pavel (advisor) ; Zikánová, Blanka (referee)
The aim of this study is to characterize β-lactamases that hydrolyze carbapenems. They belong to molecular classes A, B and D and their new variations are still being identified. The production of enzymes hydrolyzing β-lactam antibiotics is common among Gram-negative bacteria which include also serious nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii or members of Enterobacteriaceae family. The importance of the carbapenemases in clinical practice underlines the fact that carbapenems are broad-spectrum antibiotics, which are considered the reserve antibiotics. An increase in the number of cases of the carbapenem resistance is caused mainly by spread of genes encoding carbapenemases. Detection of carbapenemases and surveillance of their occurrence are both essential not only for optimal management of patients' care, but also for control of the spread of bacterial strains producing carbapenemases.
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New approaches to determination pathophysiological changes in patients with cystic fibrosis
Doušová, Tereza ; Dřevínek, Pavel (advisor) ; Kreslová, Marcela (referee) ; Gayillyová, Renata (referee)
New approaches to determination pathophysiological changes in patients with cystic fibrosis Cystic fibrosis (CF) is a life-limiting disease caused by mutation in the cystic fibrosis transmembrane regulator (CFTR) gene. To date, more than 2,000 mutations in the CFTR gene have been described, of which only 360 are directly related to CF. In a group of patients carrying mutations of unknown or variable clinical significance, it may be difficult not only to diagnose CF but also to facilitate clinical studies to determine the efficacy of new low - molecular weight compounds targeting disrupted CFTR protein. These so-called CFTR modulators have opened a new era in causal treatment of CF. To maximize the effect of these new therapies, not only the patient's genotype, but also the individual rate of response is crucial. In recent years, intestinal organoids have been used as an ex vivo model to determine the degree of CFTR function and at the same time to predict the therapeutic response to available therapeutic molecules. In our project, using the patient's native tissue and cultures of intestinal organoids derived from this tissue, we demonstrated varying degrees of CFTR residual function in a total of 14 patients with CF (0-39.7% of healthy control function). We characterized de novo mutation of the CFTR gene in...
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A Validatation of non-invasive method for diagnostic of cystic fibrosis based on ion ratios determined by CE-C4D
Ďurč, P. ; Lačná, J. ; Dosedělová, V. ; Foret, František ; Homoľa, L. ; Pokojová, E. ; Malá, M. ; Skřičková, J. ; Dastych, M. ; Vinohradská, H. ; Dřevínek, P. ; Skalická, V. ; Kubáň, Petr
A validation of novel approach for diagnosis of cystic fibrosis (CF) is presented. The method is very simple and fast, since for collection of samples, only wiping of the skin of forearm is needed. The collected sample is analyzed by capillary electrophoresis with contactless conductometric detection (CE-C4D). Ion ratios are determined as ratios of peak area of chloride anion and peak areas of selected cations. It is possible to distinguish between cystic fibrosis patients and healthy individuals when ion ratio is applied as diagnostic factor. In here, we present analytical parameters of developed method (limits of detection, limits of quantification, repeatability of migration times, repeatability of peak areas) and ROC curve, which reveals high sensitivity and specificity of the developed method.
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The study of the pathogenesis of infection caused by bacteria from Burkholderia cepacia complex in patients with cystic fibrosis
Kalferstová, Lucie ; Dřevínek, Pavel (advisor) ; Kolář, Milan (referee) ; Šebo, Peter (referee)
Summary: Bacteria from Burkholderia cepacia complex (Bcc) and bacteria Pseudomonas aeruginosa belong among the most serious pathogens causing lung infections in patients with cystic fibrosis (CF). These bacteria are highly resistant to almost all of the available antibiotics. Another serious problem is the ability of certain strains to spread among the patients, which can cause an epidemic infection. Some of the Bcc strains are capable of entering the bloodstream and causing serious septic condition called cepacia syndrome. One of these strains is the Czech epidemic strain B. cenocepacia ST32, which spread among Czech patients with CF in the 90s of the 20th century. The aim of this study was to compare transcriptome profiles of isolates gained from blood of patients with cepacia syndrome with transcriptome profiles of isolates gained from sputum of patients in the stable phase eventually exacerbation, and to choose the most appropriate genes with the different expression, which could be used as a possible marker for detection of arising cepacia syndrome. Another aim of this study was to do further study of function and influence on virulence of chosen marker (which is coding generally known virulence factor) in the time of cepacia syndrome. The last aim was to assess the epidemiologic situation of bacteria...
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Finalizing the full genome sequence of epidemic strain Burkholderia cenocepacia ST32 and identification of a prognostic marker for infections that are caused by the ST32 strain in patients with cystic fibrosis
Vavrová, Jolana ; Dřevínek, Pavel (advisor) ; Wiedermannová, Jana (referee)
Burkholderia cenocepcia is one of the serious infectious agents of respiratory tract among cystic fibrosis patients. There are problems mainly with strains which are capable of epidemic spread. The known epidemic in the Czech Republic was caused by ST32 strain in the past. In this work, there was completed whole genome sequence of referential isolate 1232 of B. cenocepacia ST32 in cooperation with bioinformatics by new generation sequencing techniques and by determining the problematic areas by a combination of Sanger sequencing bioinformatics approaches and manual assembling of sequence reads localized in these areas. The final version of the genome sequence was annotated by PGAAP and at the present time it is finalized. Second part of this work is dedicated to looking for a prognostic marker of infection caused by ST32 strain in patients with cystic fibrosis. We analysed the results of ST32 trancriptomic experiment and chose genes possibly connected with the cepacia syndrome - serious, mostly fatal state of infection. By quantitative PCR we compared their expression in isolates from 4 patients from time of cepacia syndrome and month before that. We checked the possibility of direct detection of the expression of these genes in clinical material. We identified genes for type III secretion system as...
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Molecular and epidemiological characteristics of Burkholderia cenocepacia strain dominant in cystic fibrosis patients in the Czech Republic
Vilimovská Dědečková, Klára ; Dřevínek, Pavel (advisor) ; Macek, Milan (referee) ; Šmajs, David (referee)
Summary: Bacteria from BurkJwlderia cepacia complex represent the most serious pathogens for patients witb cystic fibrosis. l nc:ir scverity IS caused by their antibiotic resistance and ability to Sllrcad among patients wilh cystic fibrosis. Many cyslic fibrosis cenlrcs around IlIe world, also tbe biggest Czech Cystic Fibrosis Centre in University Hospital in Motol, faeed outbreak. In Ihe 1990's tlle epidernie strain HurMwlderia cenocepacia S1'32 spread in a population of cystic fibrosis patienlS. One of the goals of the tunent thesis was to study epidemiological situation of Blďkholderia cepada oornplex in the popolation of eystie fibrosis pmients allending Cysfie Fibrosis Centre in University Hospilal MOlol almost seventeen years after first cases of infected palients wilh Bec inrection. Otber goals were: to evaloate 3 usefulness of molccular genelie diagnosties of Burkholderia cepacia complex tetl years afier ils implementation in routine diagnosties, to improve surveillal1ee syslem not only by implementalion or simple and specifie tesl for detection of epidemie stmin, bul 3150 by implementation of genolyping melllods thal are able to detennine strain in olher cases of infeetion. The [asI goal was to characterize Ibe epidemie slrain frolll virolence point ofview. Dased oRthe thesis we: confirmed...
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Carbapenemases and their molecular genetic detection in bacteria causing severe hospital acquired infections
Vavrová, Jolana ; Dřevínek, Pavel (advisor) ; Zikánová, Blanka (referee)
The aim of this study is to characterize β-lactamases that hydrolyze carbapenems. They belong to molecular classes A, B and D and their new variations are still being identified. The production of enzymes hydrolyzing β-lactam antibiotics is common among Gram-negative bacteria which include also serious nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii or members of Enterobacteriaceae family. The importance of the carbapenemases in clinical practice underlines the fact that carbapenems are broad-spectrum antibiotics, which are considered the reserve antibiotics. An increase in the number of cases of the carbapenem resistance is caused mainly by spread of genes encoding carbapenemases. Detection of carbapenemases and surveillance of their occurrence are both essential not only for optimal management of patients' care, but also for control of the spread of bacterial strains producing carbapenemases.
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