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Evaluation of quantitative PCR measurement of bacterial colonization of epithelial cells

100%

Schmidt M. T. , Olejnik-Schmidt A. K. , Myszka K. , Borkowska M. , Grajek W.

Polish Journal of Microbiology

2010

tom 59

nr 2

89-93

Microbial colonization is an important step in establishing pathogenic or probiotic relations to host cells and in biofilm formation on industrial or medical devices. The aim of this work was to verify the applicability of quantitative PCR (Real-Time PCR) to measure bacterial colonization of epithelial cells. Salmonella enterica and Caco-2 intestinal epithelial cell line was used as a model. To verify sensitivity of the assay a competition of the pathogen cells to probiotic microorganism was tested. The qPCR method was compared to plate count and radiolabel approach, which are well established techniques in this area of research. The three methods returned similar results. The best quantification accuracy had radiolabel method, followed by qPCR. The plate count results showed coefficient of variation two-times higher than this of qPCR. The quantitative PCR proved to be a reliable method for enumeration of microbes in colonization assay. It has several advantages that make it very useful in case of analyzing mixed populations, where several different species or even strains can be monitored at the same time.

Impact of DNA microarray data transformation on gene expression analysis - comparison of two normalization methods

86%

Schmidt M. T. , Handschuh L. , Zyprych J. , Szabelska A. , Olejnik-Schmidt A. K. , Siatkowski I. , Figlerowicz M.

tom 58

nr 4

Two-color DNA microarrays are commonly used for the analysis of global gene expression. They provide information on relative abundance of thousands of mRNAs. However, the generated data need to be normalized to minimize systematic variations so that biologically significant differences can be more easily identified. A large number of normalization procedures have been proposed and many softwares for microarray data analysis are; available. Here, we have applied two normalization methods (median and loess) from two packages of microarray data analysis softwares. They were examined using a sample data set. We found that the number of genes identified as differentially expressed varied significantly depending on the method applied. The obtained results, i.e. lists of differentially expressed genes, were consistent only when we used median normalization methods. Loess normalization implemented in the two software packages provided less coherent and for some probes even contradictory results.In general, our results provide an additional piece of evidence that the normalization method can profoundly influence final results of DNA microarray-based analysis. The impact of the normalization method depends greatly on the algorithm employed. Consequently, the normalization procedure must be carefully considered and optimized for each individual data set.

Isolation and identification of Clostridium spp. from natural samples that performs effective conversion of glycerol to 1,3-propanediol

86%

Leja K. , Myszka K. , Kubiak P. , Wojciechowska J. , Olejnik-Schmidt A. K. , Czaczyk K. , Grajek W.

Acta Scientiarum Polonorum. Biotechnologia

2011

tom 10

nr 2

Indigenous bacteria in the natural environment can product a wide range of metabolites more efficiently. The aim of this work was to isolate from the natural environment non-pathogenic Clostridium strains that are able to convert glycerol to 1,3-propanediol and other metabolites of potential uses in industry. The effective methods of selection and maintenance of anaerobic cultures in the laboratory conditions were also investigated. Samples were pre-cultured on modified PY medium consisted 50 g/l of glycerol. Isolated colonies growth on TSC medium were screened on the basis of morphological characters typical for Clostridium sp. Isolated bacterial strains were allowed to growth in selective media such as RCM and modified PY. The metabolites of bacteria were investigated by the HPLC technique. The bacteria strains were identified by 16S rRNA technique. The highest percentage of isolates of the genus Clostridium were obtained from excrements of animals, compost, and silages. Nearly 60% were able to convert glycerol to 1,3-propanediol. The highest capacity for utilization of glycerol to 1,3-propanediol was observed in case of the species of Clostridium bifermentans and Clostridium sordelli. The most of examined microflora were also able to short-chain organic acids and ethanol synthesis.

Szereg procesów metabolicznych jest efektywniej przeprowadzanych przez mikroflorę kolonizującą środowisko naturalne. Celem pracy była selekcja niepatogennych kultur bakterii z rodzaju Clostridium ze środowiska naturalnego zdolnych do konwersji glicerolu do 1,3-propanodiolu i innych metabolitów o znaczeniu przemysłowym. Badania dotyczyły także opracowania efektywnych procedur izolacji oraz hodowli mikroorganizmów beztlenowych w warunkach laboratoryjnych. Wstępną adaptację mikroflory obecnej w próbach środowiskowych przeprowadzono na podłożu PY zawierającym glicerol w stężeniu 50 g/l. Hodowle prowadzono w warunkach beztlenowych w anaerostatach. Wyraźnie oddzielone kolonie o morfologii typowej dla Clostridium sp. na podłożu TSC posiewano na podłoża wybiórczo-namnażające RCM oraz PY. Zawartość metabolitów w płynie pohodowlanym oceniano za pomocą techniki HPLC. Identyfikacji gatunkowej dokonano metodą amplifikacji sekwencji kodującej 16S rRNA. Najwyższy odsetek kultur bakteryjnych Clostridium sp. pozyskano z odchodów zwierzęcych, kompostów i obornika. Blisko 60% uzyskanych izolatów wykazywało zdolność syntezy 1,3-propanodiolu z glicerolu. Najwyższą zdolnością do utylizacji glicerolu do 1,3-propanopdiolu charakteryzowały się szczepy Clostridium bifermentans oraz Clostridium sordelli. Większość przebadanych drobnoustrojów wykazywała także zdolność do syntezy krótkołańcuchowych kwasów organicznych i etanolu.