Fluorescence spectroscopy is an emerging tool for the analysis of biomolecules from complex matrices. We explored the potentialities of the method for the pseudomonad taxonomic purpose at the genus and species level. Emission spectra of three intrinsic fluorophores (namely, NADH, tryptophan, and the complex of aromatic amino acids and nucleic acid) were collected from whole bacterial cells. Their comparisons were performed through principal component analysis and factorial discriminant analysis. Reference strains from the Xanthomonas, Stenotrophomonas, Burkholderia, and Pseudomonas genera were well separated, with sensitivity and selectivity higher than 90%. At the species level, P. lundensis, P. taetrolens, P. fragi, P. chlororaphis, and P. stutzeri were also well separated, in a distant group, from P. putida, P. pseudoalcaligenes, and P. fluorescens. These results are in agreement with the generally admitted rRNA and DNA bacterial homology grouping but they also provide 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 20NE identification. Rapid, easy to perform, and low cost, fluorescence spectroscopy provides substantial information on cell components. Statistical analysis of collected data allows in-depth comparison of strains. Our results strongly support the view that fluorescence spectroscopy fingerprinting can be used as a powerful tool in a polyphasic approach to pseudomonad taxonomy.

The development of experimental conditions for rapid bacterial discrimination using fluorescence spectroscopy fingerprinting is presented. Colonies of Pseudomonas and related reference strains on agar plates were analyzed directly using an optic fiber coupled to a laboratory spectrofluorimeter. Spectra were collected using either classic fluorescence spectroscopy after excitation at 250 nm and 340 nm for aromatic amino acids and nucleic acids (AAA + NA) and nicotinamide adenine dinucleotide (NADH) respectively, or synchronous scanning in the excitation wavelength range 250-500 nm. Factorial discriminant analysis (FDA) showed 100% correct classification at the genus and species level from NADH spectra and 100% correct classification at the genus and species level for 6l ¼ 30, 70, 90 and 110 nm (cross-validation). Analysis of variance (ANOVA) confirmed that culture time (48 or 72 h) colony and optic fiber positioning had non-significant impacts on differences between species. The use of optical fiber-fluorescence spectroscopy for bacterial discrimination directly on colonies is fast, simple and reliable. The results are independent of culture growth phase and neither need reagent addition nor prior manual preparation of cells, thus eliminating all risk of human error or contamination during sample work-up.a UPRES 2008.03.0101 Typicit e des Produits alimentaires, VetAgro