Cellular Fatty Acid Composition of the Legionnaires' Disease Bacterium 



mass spectrometry iJO. 1(\ IS). Combined gas chromatography-mass spectrometry of metliyl 

 esters was done with a DuPont instrument type 2 1^9 IB equipped witli a combination electron 

 impact(EI)-chemical ionization (CI) source. Isobutane was used as the reagent gas for CI. Details 

 of the gas chromatographic procedure and identification of fatty acids are presented elsewhere in 

 the manual. 



The cellular fatty acid profile (as methyl esters) of the Flint 1 strain of the LDB is shown in 

 the chromatogram in Fig. 1. The single most abundant acid in the chromatogram is a saturated 

 branched-chain 16 carbon acid (i-!6:0) with the methyl branch at the iso-( penultimate )carbon 

 atom. The next most aboundant are a mono-unsaturated 16 carbon straight chain acid ( 16: 1 ), a 

 saturated 15 carbon branched-chain acid (a-15:0) with the methyl branch at the anteiso-(anti- 

 penultimate)carbon atom, a saturated 14 carbon branched-chain acid (i-14:0), a saturated 17 

 carbon branched-chain acid (a- 17:0), and a mono-unsaturated 16 carbon iso-branched-chain acid 

 (i-l6:l). With the exception of 16:1, the nonnal straight-chain saturated (15:0, 16:0, etc.) and 

 unsaturated (14:1) acids were present in only small to trace amounts. The identities of the 

 labeled fatty acid methyl esters in the chromatognun were confimied by both EI and CI mass 

 spectrometry. The EI spectra of anteiso-branched methyl esters were clearly distinguished from 

 iso-branched and nomial straight-chain esters by comparing the ratio of the m/e = M-29 and m/e 

 = M-3 1 peaks in the spectra. With anteiso esters the M-29 is equal to or greater than the M-31, 

 whereas the M-31 peak is approximately twice the size of the M-29 peak in iso-branched and 

 normal straight-chain esters (76, IS). Unsaturation was confinned by hydrogenating the methyl 

 ester sample (10), which resulted in the disappearance of the i-16: 1 and 16:1 peaks with concom- 

 itant increases in the size of the i-16:0 and 16:0 peaks, respectively. The absence of hydroxy 

 acids was confirmed by the fact that there was no change in retention time of any peak in the 

 chromatogram when the methyl ester sample was treated with trinuoroacetic anhydride (10). 

 The profiles of the other five strains were strikingly similar, as shown in Fig. 2. The fatty acid 

 profile of the control culture, P. cepacia was essentially the same as that reported previously {10). 



The cellular fatty acid composition of each of the six strains is shown in Table 1 . Peak areas 

 from gas-liquid chromatography (GLC) were determined with the Disc integrator, and the per- 

 centage content of each acid was calculated from the ratio of the area of its peak to the total area 

 of all peaks. Relative response factors detemiined for each acid were used in the calculation. The 

 data clearly show the similarity of the fatty acid compositions of the six strains. In each strain 

 the iso-l6:0 acid was the major component; concentrations ranged from 32% to 43% of the total 

 acid content. With the exception of 16:1, the other four major acids in each strain were also 

 branched-chain acids. These acids (i-14:0, a-15:0, i-16:l, a-17:0) were present in each strain but 

 in different proportions. The total proportion of branched-chain acids in each of the six strains 

 ranged from 81% to 90%. The only qualitative difference in the cellular fatty acid profiles of the 

 six strains was the presence of relatively small amounts (2%-4%') of a 1 7-carbon cyclopropane 

 acid (17A) in the Pontiac and the Philadelphia 2, 3, and 4 strains, which was not detected in the 

 other two strains. Results of retesting the cellular fatty acid composition of each strain through 

 the entire procedure (growth, saponification, extraction, GLC) were virtuahy identical (see Table 

 1). 



Since the initial study, 62 additional strains of the LDB have been isolated from clinical 

 materials and from environmental sources at diverse geographical locations. We examined each of 

 these for cellular fatty acids after growing them on enriched Mueller-Hinton (M-H) agar or on 

 other recently developed growth media [Feeley-Gomian (F-G) agar (7), charcoal yeast extract 

 (CYE) agar (7), semisynthetic medium (14)]. The cellular fatty acid composition of each strain 

 was similar to that observed in the initial study (12, Figs. 1 and 2, and Table 1). No major 

 differences in cellular fatty acids were observed among strains of the four recognized serogroups 

 ( 7). 



49 



