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Applied and Environmental Microbiology, November 2003, p. 6386-6392, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6386-6392.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Rapid Identification of Listeria Species by Using Restriction Fragment Length Polymorphism of PCR-Amplified 23S rRNA Gene Fragments

Delphine Paillard,1 Véronique Dubois,1* Robert Duran,2 Fany Nathier,1 Catherine Guittet,1 Pierre Caumette,2 and Claudine Quentin1

Laboratoire de Microbiologie, Faculté de Pharmacie, Université de Bordeaux 2, Bordeaux,1 Laboratoire d'Ecologie Moléculaire, Université de Pau et des Pays de l'Adour, Pau, France2

Received 28 March 2003/ Accepted 27 August 2003

A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.

* Corresponding author. Mailing address: Laboratoire de Microbiologie, U.F.R. des Sciences Pharmaceutiques, Université de Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. Phone: (33) 5 57 57 10 75. Fax: (33) 5 56 90 90 72. E-mail: veronique.dubois{at}bacterio.u-bordeaux2.fr.

Applied and Environmental Microbiology, November 2003, p. 6386-6392, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6386-6392.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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