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Applied and Environmental Microbiology, January 2005, p. 29-38, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.29-38.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

Anne-Gaëlle Le Bourhis,1 Katiana Saunier,2 Joël Doré,2 Jean-Philippe Carlier,3 Jean-François Chamba,1* Michel-Robert Popoff,3 and Jean-Luc Tholozan4

Institut Technique Français des Fromages, La Roche-sur-Foron,1 Unité d'Ecologie et de Physiologie du Système Digestif, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy-en-Josas,2 Centre National de Référence des Bactéries Anaérobies, Institut Pasteur, Paris,3 Laboratoire de Microbiologie, Institut de Recherche pour le Développement, Université de Provence, Marseille, France4

Received 2 June 2004/ Accepted 23 August 2004

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.

* Corresponding author. Mailing address: 419 Route des Champs Laitiers, BP 30, 74801 La Roche-sur-Foron Cedex, France. Phone: 33 (0)4 50 03 33 03. Fax: 33 (0)4 50 25 82 26. E-mail: itff.jfchamba{at}wanadoo.fr.

Applied and Environmental Microbiology, January 2005, p. 29-38, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.29-38.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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