|
|
Subcellular localization of resistant proteins Vga(A)LC and Msr(A) using fluorescence microscopy
Nguyen Thi Ngoc, Bich ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
Vga(A)LC and Msr(A) are clinically significant resistant proteins in staphylococci that confer resistance to translational inhibitors. They belong to ARE ABC-F protein subfamily, which is part of ABC transporters. Unlike typical ABC transporters, ABC-F proteins do not have transmembrane domains that are responsible for the transport of substances through the membrane. Therefore, they do not have characteristic transport function but regulatory or resistance function. Their mechanism of action on the ribosome has been described only recently, where these proteins displace the antibiotic from the ribosome. However, some aspects of their function are still unclear. For example, what is the function of the Vga(A) location on a membrane that has been detected in the membrane fraction but not in the ribosomal. In this work, using fluorescence microscopy, I observed subcellular localization of the Vga(A)LC-mEos2, Vga(A)LC-GFP and Msr(A)-eqFP650 resistant fusion proteins in live cells of S. aureus under different culture conditions . It has been shown that Vga(A)LC-GFP and Msr(A)-eqFP650 occur in a foci near the membrane. Depending on ATPase activity or the presence of an antibiotic, the localization of Msr(A)-eqFP650 in the cell changes from focal to diffuse, presumably on ribosomes, suggesting a...
|
|
|
Antibiotic resistance conferred by members of ARE subfamily of ABC proteins
Veselá, Ludmila ; Balíková Novotná, Gabriela (advisor) ; Borčin, Kateřina (referee)
The main topic of this thesis is the ARE subfamily of ABC transporters. The importance of the proteins of this subfamily lies in the fact that they confer resistance to several classes of clinically important antibiotics: macrolides, lincosamides, streptogramines and pleuromutilines and they do it in significant pathogens, as for example Staphylococcus aureus. Compared to canonical ABC transporters, the structure of ABC proteins lacks the transmembrane domain (TMD) and so far, there where not even found an integrating transmembrane protein. Due to these facts, the mechanism of resistance conferred by these proteins remains unclear. In the thesis, both suggested hypotheses of the mechanism of how these proteins work are discussed. The first hypothesis presumes the active efflux of antibiotics out of the bacteria. The second hypothesis suggests release of antibiotic from its binding site initiated by ARE proteins, followed by its passive diffusion out of the cell. Keywords: ABC proteins, ARE proteins, resistance, MLS, Vga
|
|
|
Mechanism of inducible gene expression of resistance protein Vga(A)LC from Staphylococcus haemolyticus.
Novotná, Michaela ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
The staphylococcal protein VgaA belongs to ARE ABCF family, which confers resistance to ribosome binding antibiotics by the target protection mechanism. VgaA confers resistance to lincosamides, streptogramins A and pleuromutilins and thus provides the so-called LSAP resistance phenotype. The expression of resistance genes often reduces fitness in the absence of an antibiotic, therefore the expression of resistance genes is often tightly controlled and triggered only in response to the presence of an antibiotic to which the protein confers resistance. The inducible expression has also been observed for the vgaA gene, nevertheless, its mechanism has not been elucidated. In the diploma thesis, it was shown that the vgaALC gene from Staphylococcus haemolyticus is regulated by ribosome-mediated attenuation. The mechanism is based on the detection of translation inhibitors via a ribosome translating a special regulatory open reading frame (uORF), which is part of an attenuator located in the 5' untranslated region of the mRNA. The vgaALC gene is regulated at the transcriptional level in response to LSAP antibiotics. Antibiotic specificity of induction is affected not only by the nature of the peptide encoded by uORF but also by the antibiotic specificity of the resistance protein. Fluorescence microscopy...
|
|
|
Enterococcus spp. as a reservoir of resistance genes.
Zajíčková, Adéla ; Lichá, Irena (advisor) ; Balíková Novotná, Gabriela (referee)
Bacteria of the genus Enterococcus are categorised among common nosocomial pathogens. They are a significant reservoir of resistance genes to a majority of antibiotics and exhibit an intrinsic resistance to low levels of beta-lactams, glycopeptides, aminoglycosides, streptogramines and lincosamides. The aim of this paper is to review the main resistance genes and other mechanisms involved in the resistance of bacteria of this genus to antibiotics. The paper is mainly focused on the resistance to beta-lactam antibiotics, which is provided by the expression and mutations of low-affinity PBPs, the individual van resistance types mediating resistance to vancomycin, and the expression of enzymes capable of modifying the functional groups of aminoglycoside antibiotics. The paper also describes the resistance to newer antibiotics that are used to treat vancomycin-resistant isolates. The resistance to individual antibiotics can arise from the coding of their own chromosomal genes or entire signaling pathways leading to a reduction in the effect of antibiotics, the acquisition of genetic mutations, and especially the spread of new resistance genes by horizontal transfer.
|
|
|
Membrane vesicles in bacteria and their role in antibiotic resistance
Benešová, Anna ; Konopásek, Ivo (advisor) ; Pospíšil, Jiří (referee)
Membrane vesicles are produced by both Gram-positive and Gram-negative bacteria. Mechanism of their formation differs between these two groups of bacteria. It is caused by the different structure of their cell envelope. Gram-negative bacteria contain outer membrane and membrane vesicles can originate from this membrane. Membrane vesicles of Gram-positive bacteria are derived from the cytoplasmic membrane. They have to cross the barrier of the thick layer of peptidoglycan. Membrane vesicles contain cellular components whose properties enable vesicles to fulfil various functions. Antibiotic resistance can be counted as one of these functions. This thesis discusses three ways used by the membrane vesicles to protect the cells from antibiotics: transport of enzymes that degrade antibiotics, removal of antibiotics from cell's surroundings and role of membrane vesicles in horizontal gene transfer.
|
|
|
Removal of antibiotics from wastewater by advanced oxidation technologies
Macsek, Tomáš ; Rusník,, Igor (referee) ; Bodík,, Igor (referee) ; Hlavínek, Petr (advisor)
Antibiotics are substances that inhibit the growth of microorganisms and are widely used in modern medicine. High consumption of antibiotics correlates with their elevated occurence in sewage systems, from where they are further released into the environment. The threat of their occurrence in the environment is in triggering the formation and spread of antibiotic resistance. This thesis focuses on the removal of selected antibiotics and partly on the antibiotic resistance from wastewater by advanced oxidation processes (AOPs). AOPs are based on the creation of highly reactive hydroxyl radicals, which are able to oxidize even highly persistent substances. The thesis focuses on the purification of effluents from municipal wastewater treatment plants (WWTP), which are identified as the main source of pharmaceutical pollution in the environment, by AOPs mainly based on ozonation. These processes were tested under laboratory conditions on model water as well as under real conditions as the tertiary stage of the treatment at Brno-Modřice WWTP under various operating states. As the results of the performed experiments show, it could be concluded that the studied AOPs are capable of effective degradation of studied antibiotics from the treated medium. Under real conditions, the antibiotics sulfamethoxazole, trimethoprim, azithromycin and clarithromycin were monitored. Output concentrations in each operational state were achieved to be below the limit of detection for all four antibiotics. The application of AOPs based on ozonation as the tertiary step of the treatment also had a positive effect on the reduction of microbial contamination and antibiotic resistance. Within the pilot plant experiments, a reduction of up to 4 orders of magnitude of E.coli, coliform microorganisms and a reduction of their resistant strains in the range of 1.4 - 4.0 logs were observed, compared to the effluent from the WWTP.
|
|
|
Functional membrane microdomains in the bacterial cytoplasmic membrane
Švecová, Magdaléna ; Mikušová, Gabriela (advisor) ; Koukalová, Alena (referee)
Functional membrane microdomains are structural heterogeneities in bacterial cytoplasmic membrane with up to few tens of nanometers in size. Same as in the case of eukaryotic lipid rafts the lipid and protein composition and fluidity of bacterial membrane microdomains differ from the rest of the membrane. Membrane microdomains contain the structural analogues of eukaryotic flotilin as well as hopanoids and carotenoids as functional analogues of cholesterol in eukaryotic lipid rafts. In functional membrane microdomains there are located proteins associated with membrane trafficking, signaling, secretion, biofilm formation, and sporulation. Functional membrane microdomains are specific sites for the entry of certain antibiotics into cells. What is more, disassembly of functional membrane microdomains might be regarded as a possible novel mechanism of bacterial infections suppression that is caused by antibiotic resistant bacterial pathogens. In the absence of membrane domains proteins which require for their functions the membrane domain localization lose their activity. This may result in inhibition of bacterial cell growth. Key words: Functional membrane microdomains, bacterial cytoplasmic membrane, cardiolipin, hopanoids, flotilins, antibiotic resistance
|
|
|
The importance of prompt diagnostic of MRSA patients and in the nosocomial environment of the Strakonice Hospital JSC
KUBIČKOVÁ, Andrea
Methicillin-resistant Staphylococcus aureus strains (MRSA) belong among the dangerous agents of nosocomial infections. Bacteremia-related mortality is reported 20-40%. Treatment of nosocomial infections is complicated and financially demanding. To reduce the risk for the patients, the introduction of anti-epidemic measures contributes for the effective searching of MRSA carriers and preventing the spread of resistant clones within the hospital, as well as rapid and reliable diagnosis of MRSA infections. The aim of this bachelor thesis is to describe the occurrence of MRSA strains in the Hospital Strakonice a.s. in 2012-2018 and to verify the usefulness of the Alere PBP2a SA Culture Colony Test for rapid MRSA diagnosis. The overall incidence of MRSA strains in the period of 2012-2018 was 3,27/1000 patients. The highest incidence of MRSA strains in reporting period was reported in 2018 (4,2/1000 patients). The higher incidence was detected in patients over 60 years of age and mainly in male patients. The wards where the highest number of new MRSA cases were recorded were surgical and internal ones, respectively. With the help of targeted screening from 29 up to 51 % new MRSA cases were revealed. The most common specimens were wound specimens (deep as well as superficial). In 2018, 74 isolated Staphylococcus aureus strains from clinically relevant materials such as blood and other body fluid specimens and tracheal aspirates were tested with the help of Alere PBP2a SA Culture Colony Test. 8 isolates were detetected positive for PBP2a protein and 66 were negative. Obtained results were in all cases confimed by the disc diffusion technique with the same results. The results of analysis were known within 6 minutes after the identification of Staphylococcus aureus strain and could have been actively reported to the physician. This lead to the significant speed-up of early initiation of appropriate targered antibiotic therapy. Alere PBP2a SA Culture Colony Test for rapid distinction between MRSA and MSSA Staphylococcus aureus strains reported 100% sensitivity as well as specificity. This test should be included in routine microbiological examination at ÚKMAS.
|
|
|
Extensively resistant Acinetobacter baumannii in the Czech Republic: population genetic structure and mechanisms of resistance to carbapenems and aminoglycosides
Švandová, Ladislava ; Nemec, Alexandr (advisor) ; Melter, Oto (referee)
This study focuses on the question of the epidemiology of resistance to antibiotics in Acinetobacter baumannii, which is nowadays one of the most problematic bacterial patho- gens associated with failing antimicrobial therapy. Its aim was to define population-genetic properties, epidemiology and the nature of multidrug resistance for a sample of the current population of A. baumannii from Czechia. A total of 55 isolates were collected in eight medi- cal facilities in central Bohemia from October 2016 to May 2018. The isolates were assessed for their identity at the species, clonal and strain levels as well as resistance phenotype and genotype; they were classified into five clonal groups, each of which encompassed isolates that were likely to be epidemiologically related. The 55 isolates studied belonged, nearly exclusively, to global clone ECII, with 53 % of them forming a genetically relatively homoge- neous group characterized by extensive resistance to antibiotics (susceptible only to col- istin), the presence of genes encoding ArmA a OXA-23 (resistance to all aminoglycosides and carbapenems) and spread in all locations. The in-depth epidemiological analysis of isolates from the city of Příbram and its vicinity indicated the regional spread of two strains, one of which belonged to the...
|
|
|
Subcellular localization of resistant proteins Vga(A)LC and Msr(A) using fluorescence microscopy
Nguyen Thi Ngoc, Bich ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
Vga(A)LC and Msr(A) are clinically significant resistant proteins in staphylococci that confer resistance to translational inhibitors. They belong to ARE ABC-F protein subfamily, which is part of ABC transporters. Unlike typical ABC transporters, ABC-F proteins do not have transmembrane domains that are responsible for the transport of substances through the membrane. Therefore, they do not have characteristic transport function but regulatory or resistance function. Their mechanism of action on the ribosome has been described only recently, where these proteins displace the antibiotic from the ribosome. However, some aspects of their function are still unclear. For example, what is the function of the Vga(A) location on a membrane that has been detected in the membrane fraction but not in the ribosomal. In this work, using fluorescence microscopy, I observed subcellular localization of the Vga(A)LC-mEos2, Vga(A)LC-GFP and Msr(A)-eqFP650 resistant fusion proteins in live cells of S. aureus under different culture conditions . It has been shown that Vga(A)LC-GFP and Msr(A)-eqFP650 occur in a foci near the membrane. Depending on ATPase activity or the presence of an antibiotic, the localization of Msr(A)-eqFP650 in the cell changes from focal to diffuse, presumably on ribosomes, suggesting a...
|
guest ::
