The Experts below are selected from a list of 147 Experts worldwide ranked by ideXlab platform
Jaime Romero - One of the best experts on this subject based on the ideXlab platform.
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Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)
FEMS microbiology ecology, 2010Co-Authors: Paola Navarrete, Fabien Magne, Pamela Mardones, Macarena Riveros, Rafael Opazo, Antonia Suau, Philippe Pochart, Jaime RomeroAbstract:The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpoB gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpoB-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpoB-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus, and Shewanella marinus. In contrast to 16S rRNA gene-TTGE, rpoB-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpoB leads to a single band in TTGE, the rpoB gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.
M. A. Lewinski - One of the best experts on this subject based on the ideXlab platform.
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Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system
European Journal of Clinical Microbiology & Infectious Diseases, 2013Co-Authors: S. S. Richter, L. Sercia, J. A. Branda, C.-a. D. Burnham, M. Bythrow, M. J. Ferraro, O. B. Garner, C. C. Ginocchio, R. Jennemann, M. A. LewinskiAbstract:This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l’Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates ( n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer’s instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7 % of the 965 isolates tested, with 83.8 % correct to the species level and 12.8 % limited to a genus-level identification. There was no identification for 1.7 % of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus , Escherichia hermannii , Hafnia alvei , and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca , Citrobacter koseri , Obesumbacterium proteus , and Enterobacter aerogenes , respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae .
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Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system
European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology, 2013Co-Authors: S. S. Richter, L. Sercia, J. A. Branda, C.-a. D. Burnham, M. Bythrow, M. J. Ferraro, O. B. Garner, C. C. Ginocchio, R. Jennemann, M. A. LewinskiAbstract:This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMerieux, Marcy l’Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer’s instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMerieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7 % of the 965 isolates tested, with 83.8 % correct to the species level and 12.8 % limited to a genus-level identification. There was no identification for 1.7 % of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.
Paola Navarrete - One of the best experts on this subject based on the ideXlab platform.
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Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)
FEMS microbiology ecology, 2010Co-Authors: Paola Navarrete, Fabien Magne, Pamela Mardones, Macarena Riveros, Rafael Opazo, Antonia Suau, Philippe Pochart, Jaime RomeroAbstract:The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpoB gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpoB-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpoB-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus, and Shewanella marinus. In contrast to 16S rRNA gene-TTGE, rpoB-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpoB leads to a single band in TTGE, the rpoB gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.
Sergei P Sineoky - One of the best experts on this subject based on the ideXlab platform.
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Expression and Characterization of Phytase from Obesumbacterium proteus in Pichia pastoris
Applied Biochemistry and Microbiology, 2019Co-Authors: T. L. Gordeeva, Sergei P Sineoky, L. N. Borshchevskaya, A. N. Kalinina, S. P. Voronin, M. D. KashirskayaAbstract:In^1 order to increase the expression level of gene for recombinant phytase in Pichia pastoris , a synthetic sequence of the phyOp-mod gene, which encodes phytase from Obesumbacterium proteus , has been designed and optimized by its codon composition. The GC content was adjusted to 48.33%, and AT-rich segments were eliminated. The main characteristics for the purified enzyme are established. The optimal pH and temperature values for РhyOp phytase functioning prove to be 4.0 and 50°C, respectively. The specific activity, Michaelis constant ( Km ), and maximal rate of reaction ( V _max) with sodium phytate as a substrate are 1452 U/mg, 0.31 mM, and 823 U/mg, respectively. It is shown that optimization of the codon composition increased the expression of the target product gene. The recombinant phytase designated as PhyOp demonstrated great potential for application as a feed additive due to its relatively high specific activity and good kinetic characteristics.
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gene cloning expression and characterization of novel phytase from Obesumbacterium proteus
Fems Microbiology Letters, 2004Co-Authors: Nickolay V Zinin, A V Serkina, Mikhail S Gelfand, Aleksei B Shevelev, Sergei P SineokyAbstract:The gene phyA encoding phytase was isolated from Obesumbacterium proteus genomic library and sequenced. The cleavage site of the PhyA signal peptide was predicted and experimentally proved. The PhyA protein shows maximum identity of 53% and 47% to phosphoanhydride phosphorylase from Yersinia pestis and phytase AppA from Escherichia coli, respectively. Based on protein sequence similarity of PhyA and its homologs, the phytases form a novel subclass of the histidine acid phosphatase family. To characterize properties of the PhyA protein, we expressed the phyA gene in E. coli. The specific activity of the purified recombinant PhyA was 310 U mg−1 of protein. Recombinant PhyA showed activity at pH values from 1.5 through 6.5 with the optimum at 4.9. The temperature optimum was 40–45 °C at pH 4.9. The Km value for sodium phytate was 0.34 mM with a Vmax of 435 U mg−1.
S. S. Richter - One of the best experts on this subject based on the ideXlab platform.
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Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system
European Journal of Clinical Microbiology & Infectious Diseases, 2013Co-Authors: S. S. Richter, L. Sercia, J. A. Branda, C.-a. D. Burnham, M. Bythrow, M. J. Ferraro, O. B. Garner, C. C. Ginocchio, R. Jennemann, M. A. LewinskiAbstract:This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l’Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates ( n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer’s instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7 % of the 965 isolates tested, with 83.8 % correct to the species level and 12.8 % limited to a genus-level identification. There was no identification for 1.7 % of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus , Escherichia hermannii , Hafnia alvei , and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca , Citrobacter koseri , Obesumbacterium proteus , and Enterobacter aerogenes , respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae .
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Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system
European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology, 2013Co-Authors: S. S. Richter, L. Sercia, J. A. Branda, C.-a. D. Burnham, M. Bythrow, M. J. Ferraro, O. B. Garner, C. C. Ginocchio, R. Jennemann, M. A. LewinskiAbstract:This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMerieux, Marcy l’Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer’s instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMerieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7 % of the 965 isolates tested, with 83.8 % correct to the species level and 12.8 % limited to a genus-level identification. There was no identification for 1.7 % of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.
