Cellular Fatty Acid Composition of the 

 Legionnaires' Disease Bacterium 



C.Wayne Moss. R.E. Wearer. S.B. Dees, and W.B. Cherry 



Identification and classification of microorganisms has been a central theme in the develop- 

 ment of microbiology as a science. Since the early work of Pasteur, classification has been based 

 largely on morphological, physiological, serological, and biochemical data. In recent years, a new 

 dimension-the use of chemical data-has been added to bacterial classification, and the term 

 "chemotaxonomy" is now well-established in the literature. To the microbiologist, tiie term 

 usually denotes techniques such as cell wall analyses, DNA composition, and DNA homologies. In 

 a broader sense, the tenn includes studies of cell composition with respect to sugars, proteins, 

 amino acids, and lipids, and studies of metabolic products of microorganisms. 



For the last several years our laboratory has done chemotaxonomic studies using gas liquid 

 chromatography, mass spectrometry, and associated analytical techniques to develop new meth- 

 odology for rapid and sensitive detection, identification, and classification of organisms. We have 

 done extensive studies with cellular fatty acids and have found that a number of closely related 

 bacterial species can be distinguished on the basis of qualitative differences in their fatty acids 

 ((S"). Moreover, we have obsei^ed that the cellular fatty acid compositions of strains of a species 

 are essentially identical. Our initial interest in the Legionnaires" disease bacterium (LDB) was to 

 examine the cellular fatty acid composition of all isolates to assess their degree of chemical 

 relatedness. At the time the study was begun, few data were available from conventional cultural 

 and biochemical tests. Thus, the data could provide valuable information for establishing the 

 relationship of isolates obtained in the future and those from the 1*^)76 Legionnaires' disease 

 outbreak. 



Four isolates from Philadelphia and an isolate each from Flint and Pontiac, Michigan, were 

 included in the initial study. Each of the six strains was inoculated onto a plate of Mueller-Hinton 

 agar supplemented with 1% hemoglobin and 1% (v/v) IsoVitaleX (BBL) and incubated in a candle 

 jar at 35°C for 72 h. After about 3 ml of sterile distilled water was added, the heavy cell growth 

 on the plates was removed with glass rod spreaders. Smears were prepared for Gram staining to 

 check the purity of the cultures. The cells were saponified, and the fatty acids were methylated 

 by the procedure described previously {10) and outlined in detail elsewhere in this manual. 

 Pseiidomonas cepacia, an organism of known cellular fatty acid composition (lV), was used as a 

 control; it was grown and processed under the same conditions as the six test cultures. 



Methyl esters were analyzed on a Perkin-Elmer Model 900 gas chromatograph (Perkin- 

 Elmer, Norwalk, CT) equipped with a tlame ionization detector and a Disc integrator recorder. 

 The instrument contained a 0.16-in (4.06 mm, l.D.) x 1 2-ft (3.Ci6 m) coiled glass column packed 

 with 3'j^ OV-101 methyl silicone which was coated on 100-120 mesh Gas-Chrom Q (Applied 

 Science Lab., State College, PA). The carrier gas was helium at a flow rate of 60 ml/min. The 

 initial column temperature was 160°C; after the sample was injected, it was increased to 265°C at 

 a rate of 5°C/min. Fatty acid methyl ester peaks were tentatively identified by comparing their 

 retention times to those of methyl ester standards (Applied Science Lab.; Analabs, North Haven, 

 CT; Supelco. Bellefonte, PA). Final identification was established by hydrogenation (10) and 



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