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Methods for the detection of Paenibacillus larvae and Melissococcus plutonius using real-time PCR
Hrabák, Jaroslav ; Vlková, Kateřina ; Erban, Tomáš
The methodology is intended for diagnostic laboratories focused on bee infections. Detection of the causative agents of American foulbrood (Paenibacillus larvae) and European foulbrood (Melissococcus plutonius) is essential to prevent the spread of those highly contagious infections of a honey bee (Apis mellifera) diseases. In case of the suspicion, an examination of bee colonies in protective zones of the disease is indicated. The second possibility of use is the confirmation of the infection based on the detected clinical symptoms in the bee colony. The main target user in the Czech Republic are laboratories investigating animal diseases for the State Veterinary Administration (SVS) (accredited laboratories) in case of suspected disease and investigations in protective zones. Other target users are laboratories investigating bee colonies for reasons of preventive surveillance based on the request of beekeepers. The PCR method enables fast and reliable detection of P. larvae and M. plutonius from different samples taken from the bee colony. Compared to classic microbiological techniques, it allows to provide a result within a few hours of receiving the sample in the laboratory. In this way, it is possible to ensure, above all during the period of intense flight activity of bees, early intervention in bee colonies suspected of the occurrence of the mentioned diseases and to prevent the spread of the disease to nearby habitats of bee colonies.
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Molecular epidemiological analysis of carbapenem resistant isolates of Enterobacteriaceae and Pseudomonas spp.
Šrámková, Anna ; Hrabák, Jaroslav (advisor) ; Kolář, Milan (referee) ; Gniadkowski, Marek (referee)
Currently, the development of bacterial resistance is one of the major healthcare problems. Especially, the significant continuous increase of carbapenem-resistant Gram-negative bacteria, which most often affected seriously ill hospitalized patients, is a cause for great concern. Worldwide dissemination of carbapenemase-encoding genes is largely associated with mobile genetic elements and the clonal spread of high-risk clones. The dissertation thesis is focused on a molecular-epidemiological mapping of carbapenemase-positive Gram-negative isolates detected in hospital settings throughout the Czech Republic. Since 2015, a significant increase of carbapenemase-producing Enterobacteriaceæ (CPE) was detected in our country, mainly attributed to the dissemination of OXA-48 and NDM enzymes. Studies focusing on the first big outbreaks as well as sporadic cases of OXA-48 and NDM carbapenemases were performed. Results showed that blaOXA-48-carrying plasmids, which are derivatives of the archetypal IncL plasmid pOXA-48 originally described in Turkey, play a major role in the dissemination of OXA-48 enzymes in Czech hospitals. This finding is in agreement with the data from previous studies reported worldwide. The study of NDM-positive isolates revealed that IncX3 plasmids are the main factor contributing to the...
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Development and Validation of Methods for Typing of Bacteria by MALDI-TOF Mass Spectrometry
Chudějová, Kateřina ; Hrabák, Jaroslav (advisor) ; Dolejská, Monika (referee) ; Nyč, Otakar (referee)
1 ABSTRACT Healthcare-associated infections represent a significant cause of morbidity and mortality in hospital settings. The risk of nosocomial infection differs significantly in different group of patients, depending on the character of their primary illness, the co-morbidities, the type of care provided, the length of hospitalization, or the medical procedures used. Artificial surfaces such as central venous catheters, shunts, urinary catheters, valve and joint replacements or controlled lung ventilation play a major role. The majority of nosocomial infections is caused by several representative of Enterobacteriaceæ family, Pseudomonas spp., Acinetobacter spp., or some Gram-positives, especially Staphylococcus and Enterococcus spp. This is largely due to their ability to retain and transfer different types of resistance to antibiotics. The identification and subtyping of these pathogenic microorganisms is an essential tool of modern public health infectious disease surveillance not only for appropriate and efficient treatment of infections, but also in case of an outbreak. Understanding clonal continuity among investigated strains is essential to determine the source and routes of infections, confirm or rule out outbreaks, trace cross-transmission of healthcare-associated pathogens, or recognize virulent...
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Proteolytic Enzymes of Vegetative Forms and Spores of the Bacterium Paenibacillus larvae
Hrabák, Jaroslav ; Martínek, Karel (advisor) ; Kotyza, Jaromír (referee) ; Titěra, Dalibor (referee)
Due to the high resistance of the spores, the bacterium Paenibacillus larvae is the most dangerous bacterial pathogen of the honey bee (Apis mellifera). Thanks to its biological properties and restricted pathogenicity, this bacterium can be used as a model organism to study gram positive sporulating aerobic rods. This work is focused on completing information about secreted proteases of this bacterium and in a study of proteases bound in a spore structure. MYPGP medium was used for the cultivation of P. larvae. In this medium, lysis of the culture was shown after 40 hours of cultivation. The pH of the medium decreased below 6.4 by lysis. The induction of temperate bacteriophage BLA was detected as a causative agent of this lysis. A new sporulation medium called HCBB agar was proposed for the sporulation of P. larvae. In comparison with HCBB agar with MYPGP agar by 31 strains of P. larvae stored in our collection, HCBB agar was evaluated as an appropriate sporulation medium with a median of sporulatin 4.2 ' 106 spores per cm2 in aerobic conditions and 5.65 ' 106 spores per cm2 in aerobic conditions with 10 % CO2. For purification of the secreted proteases, a one-day culture incubated at room temperature was used. Optimal purification of 87/74 kDa and 42/40 kDa proteases was observed after application of this...
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Development and Validation of Methods for Rapid Detection of β-lactamases Using MALDI-TOF Mass Spectrometry for the Early Detection of Infectious Disease Agents and Their Antibiotic Resistance
Pašková, Veronika ; Hrabák, Jaroslav (advisor) ; Kolář, Milan (referee) ; Dolejská, Monika (referee)
The discovery of antibiotics and their subsequent clinical use has had a tremendous and beneficial impact on human health. β-lactam antibiotics are considered the most widely used therapeutic class of antibacterials prescribed in human and veterinary clinical practices due to their excellent safety profile and broad antimicrobial spectrum. β-lactams have undergone continuous development since their introduction in order to improve properties such as potency, spectrum of activity, pharmacokinetic and safety profiles and to counter the emergence of resistance. Resistance can occur by multiple mechanisms, including, notably, the production of β-lactamases and modification of β-lactam receptors - penicillin-binding proteins. The resistance of bacterial pathogens to common antimicrobial therapies and the emergence of multidrug- resistant bacteria are increasing at an alarming rate in Czechia. Understanding the mechanisms of resistance in clinical isolates is critical to the design of novel therapeutics and the improvement of detection techniques. Insight into the genetic basis of resistance can also reveal drug design strategies for curtailing the spread of resistance and combatting multidrug-resistant organisms. This dissertation thesis concerns different thematic investigations, beginning with the...
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The stool bacteriome during therapy for paediatric non-infectious diseases
Vodolánová, Lucie ; Cinek, Ondřej (advisor) ; Hrabák, Jaroslav (referee)
The intestinal microbiota is composed of up to 100 trillion microorganisms of which bacteria are overwhelming majority. The microbiota affects the development of the immune system, defence against pathogens, host nutrition, vitamin synthesis or fat storage and its composition is changing throughout life. Some studies point to an association between microbiota composition and the development of inflammatory bowel disease. One of the treatment options is anti-TNFα antibodies therapy, which uptake or antagonize the TNFα cytokine that otherwise mediates inflammation in the intestinal mucosa. The aim of the thesis was to examine how this treatment affects the composition of the intestinal bacteriome in paediatric patients with Crohn's disease, and to find specific bacterial taxa, whose abundance changes during the treatment. By inclusion of patients with juvenile idiopathic arthritis, also treated with anti-TNFα, the study aims to discern specific effects of therapeutically induced intestinal restitution (observable in patients with Crohn's disease) from general effects of anti-TNFα therapy. Stool samples from healthy children were used to determine "healthy" bacteriome. The composition of the bacteriome was studied by profiling the variable region of the V4 gene of 16S rDNA from patients stool samples...
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Genomic analysis of Paenibacillus larvae and its relation to virulence
Vlková, Kateřina ; Hrabák, Jaroslav (advisor) ; Fišer, Radovan (referee)
Paenibacillus larvae is a Gram-positive sporulating bacterium that causes American foulbrood (AFB). It is one of the most dangerous bacterial pathogens of the honeybee (Apis mellifera). P. larvae spores are highly infectious to bee larvae and resist physicochemical influences. P. larvae is subtyped using repPCR with ERIC primers (Enterobacterial Repetitive Integrance Consensus) into five genotypes (ERIC I-V), which possess different colony morphology, metabolism and especially virulence. There is a significant genetic variability among isolates of P. larvae, which may contribute to differences in virulence. P. larvae isolates used in this work were obtained from clinical cases of American foulbrood as well as from a debris collected from bee hives with no American foulbrood symptoms from all over the Czech Republic in cooperation with the Beekeeping Research Institute, s.r.o., Dol. The isolates were obtained from larvae and hive debris. Both virulet and avirulet strains were sequenced using the SMRT (single molecule real time) method on the Sequel platform (PacBio). This method is suitable for Whole Genome Sequencing (WGS), because it allows sequencing of long reads with high accuracy, eliminating the effect of a large number of repetitive sequences during the genome assembly. Furthermore,...
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Influence of pesticides on bees with focus on their endocrine disruptive effect
Bártová, Kateřina ; Erban, Tomáš (advisor) ; Hrabák, Jaroslav (referee)
The aim of this bachelor thesis is to assess the effect of imidacloprid in interaction with the mite Varroa destructor and deformed wing virus (DWV), transmitted by this mite, on the honeybee. Imidacloprid is a systematic pesticide belonging to the group of neonicotinoids acting as an agonists of acetylcholine receptor. The use of neonicotinoids in EU has been banned in 2013 due to its impact on honeybee health. However, residues of these substances still occur in the environment. Since 1950s, the mite V. destructor has spread from the original host Asian honeybee (Apis cerana) to the European honeybee (Apis mellifera). Varroa is serving as the vector for viruses, like DWV causing the crippled wings. Residues of pesticides, varroosis, viral and bacterial diseases together with other factors can cause honeybee colony collapse. These factors can interact via hidden mechanisms of interactions and affect honeybee health. The model experiment was performed using emerging worker bees. A total of four trials were performed. Two variants were treated with imidacloprid or the bees were exposed to Varroa collected from colonies with DWV clinical signs. Bees were fed with sugar solution or sugar solution with 2.5 μg/l of imidacloprid. Experiment lasted 72 hours and samples were analysed using a...
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Molecular-epidemiological analysis of Mycobacterium tuberculosis strains isolated in the West-Bohemian Region of the Czech Republic including detailed characterisation of anti-tuberculosis drugs-resistant strains
Amlerová, Jana ; Hrabák, Jaroslav (advisor) ; Čermák, Pavel (referee) ; Růžička, Filip (referee)
Molecular-epidemiological analysis of Mycobacterium tuberculosis strains isolated in the West-Bohemian Region of the Czech Republic including detailed characterisation of anti-tuberculosis drugs - resistant strains MUDr. Jana Amlerová Abstract Tuberculosis is contagious infectious disease that embodies significant epidemiological and clinical problem worldwide. Tuberculosis incidence differs considerably in various regions of the world but even the countries with low incidence engage strongly in epidemiology of tuberculosis. Tuberculosis belongs to one of the priorities of WHO, cooperation in this matter takes place on a global scale. Tuberculosis is a social illness; accordingly, the countries with high occurrence of tuberculosis are classified as developing countries. Mainly in Africa, there is the situation being complicated by coexistence of HIV. Generally, Europe represents a region with low incidence of tuberculosis. The Czech Republic is a country with the lowest incidence in the world with less than five new cases per 100 000 inhabitants every year. This situation is among others result of high-level tuberculosis surveillance and effective application of epidemiological arrangements based in legislation. This dissertation thesis examines several fields of tuberculosis, mainly focused on modern...
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Molecular epidemiological analysis of carbapenem resistant isolates of Enterobacteriaceae and Pseudomonas spp.
Šrámková, Anna ; Hrabák, Jaroslav (advisor) ; Kolář, Milan (referee) ; Gniadkowski, Marek (referee)
Currently, the development of bacterial resistance is one of the major healthcare problems. Especially, the significant continuous increase of carbapenem-resistant Gram-negative bacteria, which most often affected seriously ill hospitalized patients, is a cause for great concern. Worldwide dissemination of carbapenemase-encoding genes is largely associated with mobile genetic elements and the clonal spread of high-risk clones. The dissertation thesis is focused on a molecular-epidemiological mapping of carbapenemase-positive Gram-negative isolates detected in hospital settings throughout the Czech Republic. Since 2015, a significant increase of carbapenemase-producing Enterobacteriaceæ (CPE) was detected in our country, mainly attributed to the dissemination of OXA-48 and NDM enzymes. Studies focusing on the first big outbreaks as well as sporadic cases of OXA-48 and NDM carbapenemases were performed. Results showed that blaOXA-48-carrying plasmids, which are derivatives of the archetypal IncL plasmid pOXA-48 originally described in Turkey, play a major role in the dissemination of OXA-48 enzymes in Czech hospitals. This finding is in agreement with the data from previous studies reported worldwide. The study of NDM-positive isolates revealed that IncX3 plasmids are the main factor contributing to the...
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