SPECIES IDENTIFICATION OF LEGIONELLA VIA INTERGENIC 16S-23S RIBOSOMALSPACER PCR ANALYSIS

Species identification of Legionella in routine laboratory testing is hampered by the lack of highly discriminatory phenotypic tests, Amplif ication polymorphism of the intergenic 16S-23S spacer regions (ISR) ha s been previously developed for identification of species within the L egionellaceae [Hookey, J. V., Birtles, R. J. & Saunders, N. A. (1995). J. Clin Microbiol 33, 2377-2381], but it did not provide enough resol ution to distinguish all members of the bluish-white autofluorescent s pecies and the red autofluorescent group of the Legionellaceae. By cho osing new primers that target regions 4 (positions 1521-1541 of Escher ichia 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 re presenting 42 species that were tested. Analysis of the RFLP generated after HinfI restriction digestion of the PCR products further improve d the method, allowing complete discrimination among the species and s ubspecies of Legionella tested. Twenty-three well-identified strains f rom unrelated origins belonging to seven species gave amplification pa tterns 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 no t match any of the known profiles.