Summary auto-generated
This study describes a PCR-based method for rapidly identifying Legionella species using intergenic 16S-23S ribosomal DNA spacer (ISR-PCR) analysis. The authors designed new primers targeting conserved regions of the 16S and 23S rRNA genes that could amplify the spacer region between them. When tested on 79 reference strains representing 42 Legionella species, the method generated 34 distinct profiles with high reproducibility. HinfI restriction digestion of PCR products further improved discrimination between closely related species or subspecies. Among 80 field isolates that were difficult to identify by conventional phenotypic methods, 72% had ISR-PCR profiles matching known species, while 27% had novel profiles potentially representing new taxa. The technique proved faster and more discriminatory than existing phenotypic methods, particularly for distinguishing bluish-white and red autofluorescent Legionella species that were previously difficult to differentiate. Results from 23 well-characterized strains from multiple origins confirmed the stability of species-specific patterns.
Key findings
- ISR-PCR analysis identified 34 distinctive profiles among 42 Legionella species using new primers closer to the intergenic spacer region than previous methods
- HinfI restriction digestion of PCR products further improved discrimination, allowing complete differentiation among all tested Legionella species and subspecies
- The method successfully identified 72% (58/80) of phenotypically atypical field isolates with profiles matching known type strains, while 27% (22/80) may represent novel species or subspecies
- Intraspecies stability was confirmed with multiple epidemiologically unrelated isolates of the same species showing identical patterns to their type strains
- The technique provides rapid identification (less than 2 days) with greater discriminatory power than conventional phenotypic methods, particularly for bluish-white autofluorescent species
This summary was generated automatically from the article PDF and is not part of the original publication. Refer to the PDF for the authoritative text.
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.