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Ampicillin-Resistant β-lactamase-negative Haemophilus Influenzae Strains Isolated in the Czech Republic in 2010-2018
Jakubů, Vladislav ; Žemličková, Helena (advisor) ; Růžička, Filip (referee) ; Nyč, Otakar (referee)
Ampicillin-resistant β-lactamase- negative Haemophilus influenzae strains isolated in the Czech Republic in 2010-2018 According to the results of regular surveillance of resistance of bacterial pathogens of respiratory tract infections, the number of Haemophilus influenzae strains with non-enzymatic resistance to β-lactam antibiotics is increasing in the Czech Republic. Non-enzymatic resistance is caused by mutations in the ftsI gene encoding penicillin-binding protein (PBP3), which result in a reduced ability to bind β-lactam antibiotics. Demonstration of this type of resistance is more difficult than detection of β-lactamase, which is still the most common cause of aminopenicillin resistance in H. influenzae. Analysis of a set of 228 H. influenzae strains revealed that the highest possible capture (99.5%) of isolates with non-enzymatic resistance due to mutations in the ftsI gene can be achieved in routine practice by simultaneous examination of penicillin, ampicillin, amoxicillin and cefuroxime disc diffusion method according to the EUCAST methodology. The currently recommended EUCAST method using only penicillin had a lower mutation detection rate of 95.7%. Sequence analysis showed considerable variability in ftsI gene mutations. In the set of 228 strains, a total of 37 different combinations of amino...
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Dependence of porin and beta-lactamases gene expression on the innoculum size of Klebsiella pneumoniae during the beta-lactam antibiotic treatment
Hepnar, David ; Žemličková, Helena (advisor) ; Fišer, Radovan (referee)
Gene expression of porin and beta-lactamases genes during the beta-lactam antibiotic treatment and effect of inoculum size on Klebsiella pneumoniae clinical isolates ABSTRACT In recent years, Klebsiella pneumoniae has been increasingly reported to be one of the most important nosocomial pathogens, and it is usually resistant to many antibiotics. In this work, we focused on the expression of the AmpC group β- lactamase DHA-1 and its negative regulator AmpR, as well as the porins OmpK35 and OmpK36 and on effect of inoculum. We used well-characterized Klebsiella pneumoniae strains in this study. Plasmids obtained from these strains were also transformed into different wild-type Klebsiella pneumoniae strains, which were typed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Gene expression analysis was performed by RT-PCR using specific primers and TaqMan probes. In most strains, expression was dependent on the presence of an inducer. The highly resistant strain showed a different expression pattern, but the expression of blaDHA-1 remained inducible by cefoxitin. Different regulation was also observed in the transformants. Based on our data, we suggest that the previously described regulatory pathway for AmpC is not generally suitable, and we propose that there are more...
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Antimicrobial susceptibility testing of newly synthesized compounds
Benková, Markéta ; Marek, Jan (advisor) ; Žemličková, Helena (referee) ; Hanovcová, Irena (referee)
In the dissertation thesis we dealt with the antimicrobial susceptibility testing (against gram-positive and gram-negative bacteria, yeasts and filamentous fungi) of newly synthesized compounds and their structure-antimicrobial activity relationship monitoring. These compounds based on quaternary ammonium salts with various structural modifications were prepared in cooperation with the Department of Toxicology and Military Pharmacy of the Faculty of Military Health Sciences of the University of Defence and Biomedical Research Centre at the University Hospital Hradec Králové on a joint project of the Czech Health Research Council. Selection of a methodology for antimicrobial susceptibility testing was the first part of the thesis. The microdilution broth method, which was successfully implemented at the department, was chosen for this purpose. The in vitro testing of selected synthesized compounds against several bacterial and fungal strains was the next phase of the work. Several substances have also been evaluated for the green microalgae growth inhibition (in terms of environmental friendliness). In addition, cytotoxic effects were tested on mammalian cell line to assess whether the compounds were more suitable as antiseptics or surface disinfectants. Several substances active against individual...
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Using of MALDI-TOF Mass Spectrometry and Other Methods for Identification of Microorganisms
Šafránková, Renáta ; Žemličková, Helena (advisor) ; Sedláček, Ivo (referee) ; Scharfen, Josef (referee)
Using of MALDI-TOF mass spectrometry and other methods for identification of microorganisms. Background MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry) method for identification of microorganisms appeared not long ago. It is based on analysis of cell proteome. The method has undergone rapid development recently and it gradually complemented or even substituted classical routine biochemical methods. It became highly important part of microbiological laboratories. An increase in its use contributed considerably to starting of effective therapies timely and resulted in shortening of time needed for medical care. Numerous laboratory techniques, that are available nowadays, can be applied for more detailed characterization of isolated microbes, when it is necessary. The molecular-biological methods are of the most importance, namely polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), 16S rRNA gene sequence analysis or whole gene sequencing (WGS) analysis. Aims Four particular groups of infectious agents (Campylobacter spp., Plesiomonas shigelloides, Citrobacter spp., Staphylococcus intermedius/Staphylococcus pseudintermedius) were described and/or reidentified by MALDI-TOF MS and other methods (PCR, RFLP, extended biochemical...
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Antimicrobial susceptibility testing of newly synthesized compounds
Benková, Markéta ; Marek, Jan (advisor) ; Žemličková, Helena (referee) ; Hanovcová, Irena (referee)
In the dissertation thesis we dealt with the antimicrobial susceptibility testing (against gram-positive and gram-negative bacteria, yeasts and filamentous fungi) of newly synthesized compounds and their structure-antimicrobial activity relationship monitoring. These compounds based on quaternary ammonium salts with various structural modifications were prepared in cooperation with the Department of Toxicology and Military Pharmacy of the Faculty of Military Health Sciences of the University of Defence and Biomedical Research Centre at the University Hospital Hradec Králové on a joint project of the Czech Health Research Council. Selection of a methodology for antimicrobial susceptibility testing was the first part of the thesis. The microdilution broth method, which was successfully implemented at the department, was chosen for this purpose. The in vitro testing of selected synthesized compounds against several bacterial and fungal strains was the next phase of the work. Several substances have also been evaluated for the green microalgae growth inhibition (in terms of environmental friendliness). In addition, cytotoxic effects were tested on mammalian cell line to assess whether the compounds were more suitable as antiseptics or surface disinfectants. Several substances active against individual...
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Occurrence of LA-MRSA and CA-MRSA in a Population at Higher Carriage Risk
Neradová, Kateřina ; Žemličková, Helena (advisor) ; Karpíšková, Renáta (referee) ; Melter, Oto (referee)
Occurrence of LA-MRSA and CA-MRSA in a population at higher carriage risk Bacterial resistance is one of the most serious phenomenon of modern medicine. Staphylococcus aureus is one of the bacteria in which the incidence of antibiotic resistance is a serious complication in the effective treatment of infectious diseases. Typically, methicillin- resistant Staphylococcus aureus (MRSA) occurs in a hospital settings where infectious complications appear in hospitalized patients. Community strains can be distinguished from typical nosocomial strains, which affect young people without anamnestic link to health care facilities and have different genetic characteristics. In livestock, a third distinct group of MRSA has been identified and now represents an epidemiological risk to humans. Cases of colonization or infection caused by MRSA are frequently reported in people who work with animals, including veterinary personnel. The aim of this study was to determine the prevalence of MRSA colonization among veterinary professionals. A total of 134 nasal swabs from healthy attendees of a veterinary conference held in Hradec Kralove in the Czech Republic were tested for presence of MRSA. The strains were further genotypically and phenotypically characterized. Nine isolated MRSA strains were characterized with sequence...
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Dependence of porin and beta-lactamases gene expression on the innoculum size of Klebsiella pneumoniae during the beta-lactam antibiotic treatment
Hepnar, David ; Žemličková, Helena (advisor) ; Fišer, Radovan (referee)
Gene expression of porin and beta-lactamases genes during the beta-lactam antibiotic treatment and effect of inoculum size on Klebsiella pneumoniae clinical isolates ABSTRACT In recent years, Klebsiella pneumoniae has been increasingly reported to be one of the most important nosocomial pathogens, and it is usually resistant to many antibiotics. In this work, we focused on the expression of the AmpC group β- lactamase DHA-1 and its negative regulator AmpR, as well as the porins OmpK35 and OmpK36 and on effect of inoculum. We used well-characterized Klebsiella pneumoniae strains in this study. Plasmids obtained from these strains were also transformed into different wild-type Klebsiella pneumoniae strains, which were typed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Gene expression analysis was performed by RT-PCR using specific primers and TaqMan probes. In most strains, expression was dependent on the presence of an inducer. The highly resistant strain showed a different expression pattern, but the expression of blaDHA-1 remained inducible by cefoxitin. Different regulation was also observed in the transformants. Based on our data, we suggest that the previously described regulatory pathway for AmpC is not generally suitable, and we propose that there are more...
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