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Mise au point de la spectroscopie de fluorescence pour la taxonomie des Pseudomonads. Application à la caractérisation d'isolats d'ateliers carnés

Abstract : In the methodological field relating to microorganisms characterization, spectroscopic methods such as the fluorescence spectroscopy appear to be particularly appreciable tools. Their power is strengthened by the use of chemometrics which permit to treat all the spectral information. This work presents the elaboration stages of the method for Pseudomonads characterization, which form one of the most adaptative flora and present the biggest phenotypic and genotypic diversity so far depicted. Through the use of intrinsic fluorophores (NADH, aromatic amino acids + nucleic acids and tryptophan), the analysis of bacterial suspensions directly prepared from colonies permits to obtain more than 90% of good classification for reference strains even when divided in very related taxa. Synchronous fluorescence spectroscopy was successfully developed coupled with acquisition of spectra directly from colonies on agar plates using an optic fiber. The variance analysis has shown an excellent repeatability of the results, but also no significant effects of "the optical fibre position in the colony" nor of the incubation time before reading (between 48 and 72 hours). Sensitivity and selectivity reached 100% for bacterial discrimination at the genus, species or subspecies level. In these conditions, the method appears as a very reliable tool for a taxonomic purpose since our results are in agreement with the generally admitted rRNA and DNA bacterial homology grouping but they also bring out additional information about strain relatedness. In the case of environmental isolates, the method allows good discrimination, even for strains for which ambiguity still remained after PCR and API 20 NE identification. It can also be a powerfull tool to study bacterial traceability.
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Belal Tourkya. Mise au point de la spectroscopie de fluorescence pour la taxonomie des Pseudomonads. Application à la caractérisation d'isolats d'ateliers carnés. Ingénierie des aliments. Université Blaise Pascal - Clermont-Ferrand II; Université d'Auvergne - Clermont-Ferrand I, 2009. Français. ⟨NNT : 2009CLF21942⟩. ⟨tel-00725447⟩

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