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Species identification of Legionella via intergenic 16S-23S ribosomal spacer PCR analysis

¹Centre National de Référence des Legionella, UPRES EA1655, Faculté de Médecine R. T. H. Laënnec, rue Guillaume Paradin, 69372 Lyon Cedex 08, France
²Laboratoire d'Ecologie Microbienne du Sol, UMR CNRS 5557, Université Claude Bernard Lyon I, 43 Boulevard du 11 Novembre 1918,69622 Villeurbanne cedex, France

Author for correspondence: Serge Riffard. Tel: +33 478 77 86 57. Fax: +33 478 77 86 58. e-mail: derba{at}cimac-res.univ-lyonl.fr

ABSTRACT

Species identification of Legionella in routine laboratory testing is hampered by the lack of highly discriminatory phenotypic tests. Amplification polymorphism of the intergenic 16S-23S spacer regions (ISR) has been previously developed for identification of species within the Legionellaceae [Hookey. J. V., Birtles, R. J. & Saunders, N. A. (1995). J Clin Microbiol 33, 2377–2381], but it did not provide enough resolution to distinguish all members of the bluish-white autofluorescent species and the red autofluorescent group of the Legionellaceae. By choosing new primers that target regions 4 (positions 1521–1541 of Escherichia coli 16S rRNA gene) and 6 (positions 114–132 of E. coli 23S rRNA gene) within the rDNA operon close to the 16S-23S intergenic spacer, 34 profiles were determined among the 79 type and reference strains representing 42 species that were tested. Analysis of the RFLP generated after HintI restriction digestion of the PCR products further improved the method, allowing complete discrimination among the species and subspecies of Legionella tested. Twenty-three well-identified strains from unrelated origins belonging to seven species gave amplification patterns identical to that of their type strain. The technique was also tested on 80 field isolates that could not be unequivocally assigned to groups by phenotypic methods. Seventy-two per cent (58/80) of these isolates had a profile identical to that of a type strain, while 27% (22/80) may correspond to new taxa since their ISR-PCR profiles did not match any of the known profiles.

Key Words: Legionellaceae • 16S-23S intergenic spacer • ISR-PCR

The GenBank accession numbers for the sequences or partial sequences of Philadelphia-1, Los Angeles-1 and Micu-B3 strains of L. pneumophila are AF000654, AF000655 and AF000656, respectively.

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