Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
The increasing resistance in agents of nosocomial infections is one of the main problems of current medicine. The efficient treatment is also complicated by their ability to form biofilm. One of perspective alternatives to the treatment of these infections is the use of bacteriophages. The project is focused on the isolation of new phages and their subtypes effective against multiresistant strains of staphylococci and pseudomonades. Phages will be biologically and genetically characterized, including complete genome sequence. Capillary electrophoresis methods will be employed for the detection of phages in cultures. The lytic effect of the virulent phages as well as the synergistic effect of bacteriophages with antimicrobials will be assessed on nosocomial isolates. Within the analysis of tested phages and their ability to induce infections in bacteria persisting intracellulary in macrophages and bacteria growing in biofilm, the optimal therapeutic strategy for the treatment of chronic infections caused by intracellular-living and/or biofilm-forming bacteria will be proposed.
Rostoucí výskyt rezistencí u původců nozokomiálních infekcí je jedním z hlavních problémů současné medicíny. Úspěšnou léčbu dále komplikuje schopnost těchto bakterií růst ve formě biofilmu. Mezi perspektivní alternativy léčby takových infekcí patří využití bakteriofágů. V rámci projektu budou izolovány nové fágy či jejich subtypy účinné vůči nozokomiálním kmenům stafylokoků a pseudomonád. Fágy budou podrobně charakterizovány z hlediska jejich biologických a genetických vlastností, včetně kompletní sekvence genomu. Pomocí metod kapilární elektroforézy bude ověřena možnost jejich kvantifikace ve vzorku pro účely kontroly kvality fágové kultury. Účinky těchto fágů či jejich synergické kombinace s antimikrobiálními látkami budou ověřovány na nozokomiálních izolátech, zejm. na multirezistentních kmenech. Na základě analýzy schopnosti testovaných fágů infikovat bakterie perzistující uvnitř makrofágů a bakterie rostoucí ve formě biofilmu bude navržena optimální terapeutická strategie pro léčbu chronických infekcí spojených s intracelulárním přežíváním bakterií či s tvorbou biofilmu.
- MeSH
- antiinfekční látky terapeutické užití MeSH
- biofilmy účinky léků MeSH
- elektroforéza MeSH
- fágová terapie metody MeSH
- infekce spojené se zdravotní péčí terapie MeSH
- lidé MeSH
- mnohočetná bakteriální léková rezistence účinky léků MeSH
- Pseudomonas aeruginosa izolace a purifikace MeSH
- Staphylococcus aureus izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- infekční lékařství
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Amphotericin B (AmB) is still, despite its severe nephrotoxicity, the first-line agent in the management of serious systemic fungal infections. A sensitive and reliable method is therefore required to control AmB concentration in body fluids of a patient. This study demonstrates the potential of the off-line combination of preparative isoelectric focusing (IEF) with capillary isoelectric focusing (CIEF) or capillary zone electrophoresis (CZE) in the determination of AmB in human blood serum. The required value of the isoelectric point of AmB was determined to be 6.1 using the CIEF technique. Preparative IEF served as a pre-separation and concentration technique. The pH gradient was traced by colored low molecular pI markers. The collected fraction with AmB was easily processed and then analyzed by CIEF and CZE. Tens of picograms of AmB in human blood serum sample can be determined by a combination of preparative IEF with CZE. The method was linear in the AmB concentration range of 0.3-600ngmL
Pre-separation and pre-concentration of bacteria is an important step especially when they are uncultured and bacterial concentration in the matrix is low. This study describes a preparative method based on isoelectric focusing of colored microorganisms in a cellulose-based separation medium from a high conductivity matrix. The isoelectric points found for the examined cells were 1.8 for Micrococcus luteus, 3.5 for Dietzia sp., and 4.7 for Rhodotorula mucilaginosa using capillary isoelectric focusing. The final positions of the zones of colored microbial cells in the cellulose-bed are indicated by colored pI markers. Segments of the separation medium with cells were harvested by a spatula, simply purified using centrifugation and analyzed by capillary isoelectric focusing and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The determined recovery ranged from 78% to 93%. The viability of the harvested cells was verified by their cultivation.
Identification and prevention of Staphylococcus aureus-caused infections may benefit from a fast and dependable method to distinguish between the methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains. The current methods involving polymerase chain reaction and/or other molecular tests are usually laborious and time-consuming. We describe here a fast and low-cost method employing capillary zone electrophoresis (CZE) to distinguish between MRSA and MSSA. The method makes use of a supercritical water-treated fused silica capillary, the inner surface of which has subsequently been modified with (3-glycidyloxypropyl)trimethoxysilane. With optimized proportions of suitable additives to the background electrolyte, a CZE separation of MRSA from MSSA may be completed within 12 min. The cells were baseline-resolved, and resolution was determined to be 3.61. The isoelectric points of MSSA and MRSA were found to be the same for both groups of these strains, pI = 3.4.
The effect of antibiotics on the microbial cells and concentration of antibiotics in the human body is essential for the effective use of antimicrobial therapy. The capillary isoelectric focusing is a suitable technique for the separation and the detection of bacteria, and amphoteric substances from nature. However, the determination of isoelectric points of ampholytic antibiotics by conventional techniques is time consuming. For this reason, capillary isoelectric focusing seems to be appropriate as a simple and reliable way for establishing them. The separation conditions for the capillary isoelectric focusing of selected ampholytic antibiotics with known isoelectric points and pK as, ampicillin (pI 4.9), ciprofloxacin (pI 7.4), ofloxacin (pI 7.1), tetracycline (pI 5.4), tigecycline (pI 9.7), and vancomycin (pI 8.1), were found and optimized in the suitable pH ranges pH 2.0-5.3, 2.0-9.6, and 9.0-10.4. The established values of isoelectric points correspond with those found in the literature except tigecycline. Its pI was not found in the literature. As an example of a possible procedure for direct detection of both ampholytic antibiotics and bacteria, Staphylococcus epidermidis, in the presence of culture media or whole human blood, was found. The changes of the bacterial cells after their treatment with tetracycline were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Capillary isoelectric focusing allows the fast and simple determination of isoelectric points of relevant antibiotics, their quantification from the environment, as well as studying their effectiveness on microorganisms in biological samples.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- isoelektrická fokusace přístrojové vybavení metody MeSH
- koncentrace vodíkových iontů MeSH
- krev mikrobiologie MeSH
- kultivační média chemie MeSH
- lidé MeSH
- Staphylococcus epidermidis chemie účinky léků izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
Přeruš. str. : il., tab. ; 30 cm
Project deals with problems of biofilm infections of urinary tract. On the basis of our previous and preliminary studies we focused on the study of mixed-species biofilm, which is often present in these infections. By means of capillary isoelectric focusing we can differenciate microbes on the basis of their surface properties. This method opens new possibilities in the disgnosis of urinary and other infections. The capillary isoelectric focusing can be also used in differenciation of biofilm-positive and bifilm-negative microbes. There are strains isolated from infections of urinary catheters in this study. It focuses on the study and understanding of mixed-species communities and their characteristics. For the choice of optimal therapy we have to know susceptibility of particular microorganisms, growing in biofilms, to antimicrobial agents - not only MIC, but also MBIC. This is one of the conditions for ffective action against biofilms as the sources of urinary tract infections.
Projekt se zaměřuje na problematiku biofilmových infekcí močového traktu. Na základě našich předchozích prací a předběžných studií jsme se zaměřili na studium vlastností mnohodruhového biofilmu, který se u těchto infekcí vyskytuje. Kapilární izoelektrickou fokusací lze odlišit mikroby na základě jejich povrchových vlastností. Ověření této metody pro močové infekce otevírá další v diagnostice další možnosti. Stejně tak lze kapilární izoelektrickou fokusaci využít při rozlišení biofilmpozitivních a biofilmnegativních mikrobů. Studie zahrnuje kmeny izolované z infekcí močových katétrů a zaměřuje se na studium smíšených společenstev těchto mikrobů a jejich vlastností. Pro volbu vhodné terapie je nutno zjistit citlivost jednotlivých mikroorganizmů rostoucích v biofilmu k antimikrobiálním látkám, tedy ne pouze MIC, ale i MBIC. To je podmínkou účinného zásahu proti biofilmům jako zdroji močových infekcí.
- MeSH
- bakteriální léková rezistence MeSH
- biofilmy MeSH
- gramnegativní bakteriální infekce diagnóza MeSH
- grampozitivní bakteriální infekce diagnóza MeSH
- infekce močového ústrojí MeSH
- isoelektrická fokusace metody využití MeSH
- katétrové infekce MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- infekční lékařství
- urologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
- Klíčová slova
- kapilární elektroforéza, izoelektrická fokusace,
- MeSH
- elektroforéza kapilární metody využití MeSH
- houby * izolace a purifikace patogenita MeSH
- isoelektrická fokusace * metody využití MeSH
- kandidóza * diagnóza MeSH
- kvasinky * izolace a purifikace patogenita MeSH
- molekulární sondy - techniky * využití MeSH
- Ramanova spektroskopie * metody MeSH
An original method based on capillary zone electrophoresis with fluorimetric detection has been developed for the determination of terpenic compounds. The method is based on the separation of a terpenes dynamically labeled by the non-ionogenic tenside poly(ethylene glycol) pyrenebutanoate, which was used previously for the labeling of biopolymers. The background electrolytes were composed of taurine-Tris buffer (pH 8.4). In addition to the non-ionogenic tenside aceton and poly(ethylene glycol) were used as the additives. The capillary zone electrophoresis with fluorometric detection at the excitation wavelength 335 nm and the emission wavelength 463 nm was successfully applied to the analysis of tonalid, cholesterol, vitamin A, ergosterol, estrone and farnesol at level of 10(-17) mol L(-1). Farnesol, is produced by Candida albicans as an extracellular quorum-sensing molecule that influences expression of a number of virulence factors, especially morphogenesis and biofilm formation. It enables this yeast to cause serious nosocomial infections. The sensitivity of this method was demonstrated on the separation of farnesol directly from the cultivation medium.
- MeSH
- biofilmy MeSH
- butyráty chemie MeSH
- Candida albicans chemie metabolismus MeSH
- cholesterol chemie MeSH
- elektroforéza kapilární metody MeSH
- ergosterol chemie MeSH
- estron chemie MeSH
- farnesol chemie izolace a purifikace metabolismus MeSH
- fluorometrie metody MeSH
- polymery chemie MeSH
- quorum sensing MeSH
- senzitivita a specificita MeSH
- terpeny chemie izolace a purifikace MeSH
- tetrahydronaftaleny chemie MeSH
- vitamin A chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In virological practice, the pre-concentration, purification and subsequent determination of the purity and concentration of the viruses from the cultural medium and/or from the real sample are required. The conventional techniques used today are equipment demanding, time-consuming and laborious. In this study, the CIEF of influenza viruses with UV detection has been developed and subsequently used to test the purification of the virus from the biological samples. The equine and swine influenza viruses present in infected allantoic fluid of specific pathogen free embryonated chicken eggs were precipitated by using PEG 6000 and sodium chloride. The precipitated viruses were centrifuged at 14 000 x g, and the impurities of different densities were removed by using the sucrose gradients. The efficiency of the virus purification technique was examined by the CIEF and compared to the results of real-time PCR. The pIs of both influenza viruses were determined. Simultaneously, the CIEF was found to be a suitable method for the rapid testing of the efficiency of the virus purification.
- MeSH
- isoelektrická fokusace metody MeSH
- izoelektrický bod MeSH
- koncentrace vodíkových iontů MeSH
- koně MeSH
- kultivace virů metody MeSH
- Orthomyxoviridae chemie růst a vývoj izolace a purifikace MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- prasata MeSH
- sacharosa MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The isoelectric points of 39 Candida parapsilosis strains were determined by means of capillary isoelectric focusing. The value of the isoelectric point corresponded well with cell surface hydrophobicity, as well as with the ability to form biofilm in these yeasts.
- MeSH
- biofilmy MeSH
- Candida chemie fyziologie MeSH
- časové faktory MeSH
- druhová specificita MeSH
- hydrofobní a hydrofilní interakce MeSH
- isoelektrická fokusace MeSH
- izoelektrický bod MeSH
- kandidóza mikrobiologie MeSH
- lidé MeSH
- povrchové vlastnosti MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
