Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment.

• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   MeSH
• denaturační gradientová gelová elektroforéza metody   MeSH
• diagnostické techniky molekulární metody   MeSH
• diagnostické techniky urologické MeSH
• DNA bakterií MeSH
• lidé MeSH
• moč chemie   mikrobiologie   MeSH
• močové katétry mikrobiologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• software * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• elektroforéza v agarovém gelu metody   využití   MeSH
• mutace genetika   MeSH
• Publikační typ
• kongresy MeSH

• MeSH
• DNA primery MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• genetické nemoci vrozené diagnóza   genetika   MeSH
• lidé MeSH
• mutace MeSH
• mutační analýza DNA metody   MeSH
• polymerázová řetězová reakce MeSH
• Check Tag
• lidé MeSH

More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations.

• MeSH
• alely MeSH
• Asijci * genetika   MeSH
• cystická fibróza etnologie   genetika   MeSH
• DNA primery MeSH
• elektroforéza v polyakrylamidovém gelu * metody   MeSH
• exony MeSH
• lidé MeSH
• modely genetické MeSH
• mutace * genetika   MeSH
• mutační analýza DNA * metody   MeSH
• polymerázová řetězová reakce MeSH
• protein CFTR * genetika   MeSH
• retrospektivní studie MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH

• MeSH
• klinické laboratorní techniky MeSH
• molekulární biologie MeSH
• polymerázová řetězová reakce MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Biologické vědy
• NLK Obory
• biologie

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

We isolated and amplified by PCR 16S rDNA from bacteria attached to the bovine rumen wall and analyzed it by denaturing gradient gel electrophoresis (DGGE) with subsequent sequence analysis. The attached bacterial community differed from the bacteria of rumen content; however, no differences were observed among the five epithelial sampling sites taken from each animal. The DGGE profile of the bacterial population attached to the rumen wall represented a high inter-animal variation.

• MeSH
• bachor mikrobiologie   MeSH
• Bacteria genetika   klasifikace   MeSH
• biodiverzita MeSH
• denaturace nukleových kyselin MeSH
• DNA bakterií genetika   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• financování organizované MeSH
• polymerázová řetězová reakce MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH

A feeding study was performed to monitor the effect of chitosan intake on the fecal microbiota of ten healthy human subjects. Diversity of microflora was monitored during 8 weeks including 4 weeks of chitosan supplementations. Using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons and quantitative PCR method we revealed possible changes originating in the overall bacterial composition and also in the subpopulation of Bifidobacterium group. DGGE profiles displayed high complexity and individuality for each subject. Considerable variations in the composition of band patterns were observed among different persons. A raised level of fecal Bacteroides in response to chitosan intake was found in all samples. Bifidobacterium levels following chitosan intake increased or remain unchanged. Non-significant increase was, surprisingly, found in the numbers of butyrate-producing bacteria.

• MeSH
• Bacteroides genetika   izolace a purifikace   MeSH
• Bifidobacterium genetika   izolace a purifikace   MeSH
• biodiverzita MeSH
• chitosan metabolismus   MeSH
• denaturace nukleových kyselin MeSH
• DNA bakterií genetika   MeSH
• dospělí MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• experimenty na lidech MeSH
• feces mikrobiologie   MeSH
• financování organizované MeSH
• lidé středního věku MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH

Social honey bees, Apis mellifera, host a set of distinct microbiota, which is similar across the continents and various honey bee species. Some of these bacteria, such as lactobacilli, have been linked to immunity and defence against pathogens. Pathogen defence is crucial, particularly in larval stages, as many pathogens affect the brood. However, information on larval microbiota is conflicting. Seven developmental stages and drones were sampled from 3 colonies at each of the 4 geographic locations of A. mellifera carnica, and the samples were maintained separately for analysis. We analysed the variation and abundance of important bacterial groups and taxa in the collected bees. Major bacterial groups were evaluated over the entire life of honey bee individuals, where digestive tracts of same aged bees were sampled in the course of time. The results showed that the microbial tract of 6-day-old 5th instar larvae were nearly equally rich in total microbial counts per total digestive tract weight as foraging bees, showing a high percentage of various lactobacilli (Firmicutes) and Gilliamella apicola (Gammaproteobacteria 1). However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence. Between emergence and day 6, imago reached the highest counts of Firmicutes and Gammaproteobacteria, which then gradually declined with bee age. Redundancy analysis conducted using denaturing gradient gel electrophoresis identified bacterial species that were characteristic of each developmental stage. The results suggest that 3-day 4th instar larvae contain low microbial counts that increase 2-fold by day 6 and then decrease during pupation. Microbial succession of the imago begins soon after emergence. We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level. Sampling and pooling adult bees or 6th day larvae may lead to high errors and variability, as both of these stages may be undergoing dynamic succession.

• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   MeSH
• denaturační gradientová gelová elektroforéza MeSH
• DNA bakterií genetika   MeSH
• ekosystém MeSH
• gastrointestinální trakt mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• Lactobacillaceae genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• střevní mikroflóra * MeSH
• včely embryologie   růst a vývoj   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucleotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis.

• MeSH
• bachor mikrobiologie   MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   MeSH
• denaturační gradientová gelová elektroforéza metody   MeSH
• DNA bakterií genetika   MeSH
• fylogeneze MeSH
• techniky typizace bakterií metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH