Chemical identification of Legionella species by means of cellular fatty acid characterization.

Abstract

The identification method for Legionella species which was difficult to be identified with biochemical test was investigated by Comparing cellular fatty acid composition. First, a simple and rapid analytical method for the determination of cellular fatty acids of Legionella species by gas chromatography (GC) was developed. Cell cluster (about 50 mg) in 1 ml of 2 N sodium hydroxide-methanol solution was heated at 70°C with ultrasonication for 20 minutes. Then methyl esterification was carried out with 1 ml of 2 N hydrochloric acid-methanol solution. After extracting with 2 ml of hexane, the hexane layer was washed with water. The fatty acid composition was determined by GC and GC-MS on a Unisole-3000 column. This methyl esterification method was very useful in its simplicity, rapidity and reproducibility. Second, standard strains of Legionella species were investigated. All of 9 type strains of L. pneumophila (serogroup 1-9) contained plenty of i-C 16 : 0 and 5 type strains of L. bozemanii (serogroup 1, 2), L. dumoffii, L. gormanii and L. micdadei contained much of a-C 15 : 0. It was recognized that the composition ratio of fatty acid was not influenced by incubation period and medium. Next, radar charts of 4 axial coordinate for Legionella species were drawn with the peak area ratios of 4 branched fatty acids (i-C 14 : 0, i-C 16 : 0 as iso- and a-C 15 : 0, a-C 17 : 0 as antiiso-). Consequently this radar chart of L. pneumophila differed distinctly from others. These findings suggested that the proposed method in comparison with the composition patterns of 4 branched fatty acids was useful for the rapid and chemical identification of Legionella species.