Catheterization of patient is common during many interventions. This is one of reasons, why over 40% of nosocomial infections are infections of urinary tract, esp. of catheterized patients. Poly-microbial wound infection are known to be only partially cultivable, part of microbes can be proved only by molecular techniques. The microbial diversity of biofilm communities of urethral stents and catheters was not satisfactory examined. Suitable method for poly-microbial diagnostic is Denaturing Gradient Gel Electrophoresis (DGGE). In most studies the PCR-DGGE proved also non-cultivable bacteria and even bacteria unknown with those infections. The aim of this project is to study of species composition of biofilm communities by cultivation and molecular methods and assessment of the differences in poly-microbial communities in relation to therapy failure, length of insertion of catheter, its type and other factors including predominating microbial species.

Katétrizace patří k běžným postupům při řadě zákroků, s čímž souvisí fakt že přes 40 % nozokomiálních infekcí jsou právě inf. močového traktu, zejména inf. katétrizovaných pacientů. U polymikrobiálních infekcí ran je známo, že kultivačně lze zachytit pouze část mikrobů a část lze prokázat pouze metodami molekulárními. Mikrobiální diverzita biofilmových společenstev močových stentů a katétrů nebyla dosud uspokojivě prozkoumána. Vhodnou metodou molekulární analýzy se zdá denaturační gradientová gelová elektroforéza (DGGE). Většinou, když byla PCR-DGGE použita, byly zachyceny i bakterie nekultivovatelné, navíc byly prokázány bakterie u daných infekcí dosud neznámé. Cílem projektu je studium druhového složení biofilmových společenstev kultivačně a molekulárními technikami a zhodnocení rozdílů v polymikrobiálních společenstvech ve vztahu k selhání terapie, délce zavedení katétru, jeho druhu a dalším faktorům včetně predominujících mikrobiálních druhů.

• MeSH
• antibiotická profylaxe MeSH
• biofilmy MeSH
• denaturační gradientová gelová elektroforéza MeSH
• katétrové infekce MeSH
• močové katétry mikrobiologie   MeSH
• stenty mikrobiologie   MeSH

• Konspekt
• Mikrobiologie
• NLK Obory
• mikrobiologie, lékařská mikrobiologie
• infekční lékařství
• urologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Early diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically important Candida species, utilizing the variability in the length of the ITS2 rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 various Candida reference strains from which 26 Candida species were clearly identified, while Candida guilliermondii, C. fermentati, and C. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically important Candida strains, including species of the "psilosis" complex.

• MeSH
• Candida izolace a purifikace   MeSH
• časové faktory MeSH
• DNA fungální chemie   genetika   MeSH
• elektroforéza kapilární metody   MeSH
• kandidóza diagnóza   MeSH
• lidé MeSH
• mezerníky ribozomální DNA chemie   genetika   MeSH
• mikrobiologické techniky metody   MeSH
• polymerázová řetězová reakce metody   MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.

• MeSH
• algoritmy MeSH
• analýza hlavních komponent MeSH
• Bacteria metabolismus   MeSH
• biofilmy * MeSH
• buněčná adheze MeSH
• fagocytóza MeSH
• fyziologie bakterií MeSH
• optická pinzeta * MeSH
• polysacharidy chemie   MeSH
• Ramanova spektroskopie * MeSH
• Staphylococcus epidermidis metabolismus   MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Colonies of Candida parapsilosis on culture plates were probed directly in situ using Raman spectroscopy for rapid identification of specific strains separated by a given time intervals (up to months apart). To classify the Raman spectra, data analysis was performed using the approach of principal component analysis (PCA). The analysis of the data sets generated during the scans of individual colonies reveals that despite the inhomogeneity of the biological samples unambiguous associations to individual strains (two biofilm-positive and two biofilm-negative) could be made.

• MeSH
• biofilmy * MeSH
• Candida klasifikace   cytologie   ultrastruktura   MeSH
• Ramanova spektroskopie * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• elektroforéza kapilární metody   MeSH
• oxid křemičitý chemie   MeSH
• rezistence na methicilin * MeSH
• Staphylococcus aureus účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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

The nosocomial pathogen Pseudomonas aeruginosa is equipped with a large arsenal of cell-associated and secreted virulence factors which enhance its invasive potential. The complex relationships among virulence determinants have hitherto not been fully elucidated. In the present study, 175 catheter-related isolates were observed for the presence of selected virulence factors, namely extracellular enzymes and siderophore production, biofilm formation, resistance to antibiotics, and motility. A high percentage of the strains produced most of the tested virulence factors. A positive correlation was identified between the production of several exoproducts, and also between the formation of both types of biofilm. An opposite trend was observed between the two types of biofilm and the production of siderophores. Whereas the relationship between the submerged biofilm production (i.e. the biofilm formed on the solid surface below the water level) and the siderophore secretion was negative, the production of air-liquid interface (A-L) biofilm (i.e. the biofilm floating on the surface of the cultivation medium) and the siderophore secretion were positively correlated. All correlations were statistically significant at the level P = 0.05 with the correlation coefficient γ ≥ 0.50. Our results suggest that: (1) the co-production of the lytic enzymes and siderophores can play an important role in the pathogenesis of the catheter-related infections and should be taken into account when the virulence potential is assessed; (2) biofilm-positive strains are capable of forming both submerged and non-attached A-L biofilms; and (3) the different micro-environment in the submerged biofilm and A-L biofilm layers have opposite consequences for the production of other virulence factors.

• MeSH
• bakteriální léková rezistence MeSH
• biofilmy růst a vývoj   MeSH
• dítě MeSH
• dospělí MeSH
• enzymy sekrece   MeSH
• faktory virulence analýza   genetika   MeSH
• katétrové infekce mikrobiologie   MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• lokomoce MeSH
• mladiství MeSH
• mladý dospělý MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• pseudomonádové infekce mikrobiologie   MeSH
• Pseudomonas aeruginosa genetika   izolace a purifikace   patogenita   fyziologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• siderofory metabolismus   MeSH
• virulence MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cíl práce: Močové katétry kolonizuje celá řada mikroorganizmů, včetně kvasinek. Na kolonizaci se obvykle podílí více mikrobiálních druhů, které zde vytváří společenstvo - mnohodruhový biofilm. Tato kolonizace katétru většinou neovlivňuje významným způsobem klinický stav pacienta. Na druhou stranu biofilmové ložisko se může stát zdrojem endogenní infekce či může ovlivňovat funkčnost katétru a tvorbu močových kamenů. Materiál a metody: Celkem bylo zpracováno 721 močových katétrů. Mikroorganizmy byly z katétrů uvolněny sonikací a následně kultivovány. Identifikace byla provedena pomocí běžných fenotypových testů, dále pomocí hmotnostní spektrometrie MALDI-TOF a nejednoznačně určené kvasinky byly sekvenovány. Tvorba biofilmu byla prokazována kultivací v mikrotitrační destičce. Výsledky a závěr: Kolonizaci kvasinkami se podařilo prokázat u 244 (33,8 %) vyšetřených močových katétrů. Celkem však bylo izolováno 274 kmenů kvasinek, které se ve většině případů vyskytovaly na katétrech spolu s dalšími kvasinkami či jinými mikroorganizmy a tvořily zde vícedruhový biofilm. Nejčastěii byla izolována Candida albicans (n = 144), následovala Candida glabrata (n = 41), Candida tropicalis (n = 41) a Candida parapsilosis sensu stricto (n = 14). Dále byly zachyceny Candida kefyr (n = 10), Candida krusei (n = 9), Candida fabianii (n = 6), Candida lusitaniae (n = 5), Candida dubliniensis (n = 3) a v jednom případě Saccharomyces cerevisiae. Většina kvasinek byla schopna poměrně snadno vytvořit pevně lpící vrstvu biofilmu na umělých površích.

Objective: Urinary catheters are colonized by a wide range of microorganisms, including numerous yeasts. The catheters are usually colonized by more microbial species forming a community - multispecies biofilm. Catheter colonization usually does not affect the patient's clinical status in any significant way. On the other hand, the biofilm can become a source of endogenous infection and its presence can affect functionality of the catheter and formation of urinary stones. Material a Methods: A total of 721 urinary catheters were studied. Microorganisms were released from catheters by sonication and subsequently cultured. Their identification was performed with the use of common phenotypic tests, as well as using MALDI TOF. Yeasts whose identification was ambiguous were recognized by sequencing. Biofilm formation was assessed by growth in a microtiter plate. Results and Conclusion: Yeast colonization was proved in 244 urinary catheters. However, a total of 274 yeast strains were isolated. Most of them occurred together with other yeast species and/or bacteria on the catheters, producing multispecies biofilm there. The most frequent species was Candida albicans (a total of 144 isolated strains), followed by Candida glabrata (41), Candida tropicalis (41) and Candida parapsilosis sensu stricto (14). Other isolated species were as follows: Candida kefyr (10), Candida krusei (9), Candida fabianii (6), Candida lusitaniae (5), Candida dubliniensis (3) and Saccharomyces cerevisiae (one case). Most of the yeasts rather readily formed a firmly adhering biofilm layer on artificial surfaces.

• Klíčová slova
• močové cesty, sonikace,
• MeSH
• biofilmy růst a vývoj   MeSH
• Candida klasifikace   růst a vývoj   MeSH
• infekce močového ústrojí etiologie   mikrobiologie   MeSH
• infekce MeSH
• katétrové infekce mikrobiologie   MeSH
• katétry mikrobiologie   MeSH
• lidé MeSH
• Saccharomyces cerevisiae růst a vývoj   MeSH
• Check Tag
• lidé MeSH