Development of the BIOLOG substrate utilization system for identification of Legionella spp.

The genus Legionella consists of 51 serogroups comprising 34 species. Biochemical reactions and cell wall fatty acid and quinone analyses may confirm that an isolate is a Legionella sp. and indicate to which species it belongs, but DNA hybridization studies have been necessary for a definitive identification. Recently, the commercially available BIOLOG identification system has offered a standardized, easily reproducible system of substrate metabolism by bacteria resuspended in multiwell plates. A tetrazolium dye acts as an electron acceptor during the oxidation of the wide range of substrates and forms an irreversible, highly colored formazan when reduced. The 95 substrate wells are read rapidly with a conventional plate reader, and the results are downloaded for comparison with a computer data base, allowing quick identification. The BIOLOG system's ability to test more diverse classes of substrates, including amino acids, peptides, carboxylic acids, and carbohydrates, was used in this study to establish a new data base and identify the asaccharolytic Legionella spp. In particular, Legionella pneumophila behaved as a microaerophile, and the fastest, most diverse metabolic activities occurred after the development of a low-oxygen incubation environment. Alternatively, bacteria could be successfully incubated in air when their concentration was double that recommended by the manufacturer. Similar results were obtained by using either Page's amoebal saline or distilled water as the resuspending and incubation medium. Type strains did not cross-identify with any of the strains already in the manufacturer's data base. The results indicate that this modified system has value in being able to identify Legionella isolates to the species level.