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Applied and Environmental Microbiology, September 2004, p. 5628-5643, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5628-5643.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Molecular Fingerprinting of Dairy Microbial Ecosystems by Use of Temporal Temperature and Denaturing Gradient Gel Electrophoresis

J.-C. Ogier,1* V. Lafarge,2 V. Girard,1,2 A. Rault,2 V. Maladen,2 A. Gruss,1 J.-Y. Leveau,3 and A. Delacroix-Buchet1

Unité de Recherches Laitières et Génétique Appliquée, Institut National de la Recherche Agronomique, Jouy-en-Josas,1 Unité Composition et Hygiène des Produits Laitiers, Agence Française de Sécurité Sanitaire des Aliments, Maisons-Alfort,2 Département de Microbiologie, Ecole Nationale Supérieure des Industries Alimentaires, Massy, France3

Received 25 November 2003/ Accepted 16 May 2004

Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of ~150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.

* Corresponding author. Mailing address: Unité de Recherches Laitières et Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas, France. Phone: (33) 1 34 65 20 97. Fax: (33) 1 34 65 20 65. E-mail: ogier{at}jouy.inra.fr.

Applied and Environmental Microbiology, September 2004, p. 5628-5643, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5628-5643.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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