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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1999 Jul;37(7):2248–2254. doi: 10.1128/jcm.37.7.2248-2254.1999

Usefulness of Fatty Acid Composition for Differentiation of Legionella Species

Alexandra Diogo 1, António Veríssimo 2,*, M Fernanda Nobre 2, Milton S da Costa 1
PMCID: PMC85129  PMID: 10364593

Abstract

Numerical analysis of fatty acid methyl ester (FAME) profiles of 199 isolates and 76 reference strains, belonging to all validly described species of the genus Legionella that can be cultured in laboratory media, was used to differentiate between the species of this genus. With the exception of the strains that autofluoresced red, it was possible to differentiate all the other Legionella species. The strains of the species L. bozemanii, L. dumoffii, L. feeleii, L. gormanii, L. maceachernii, L. micdadei, and L. quinlivanii did not form single clusters, showing some degree of variability in the fatty acid compositions. The strains of the blue-white autofluorescent species had very similar fatty acid compositions and were difficult to distinguish from each other. Nine isolates had fatty acid profiles unlike those of any of the validly described species and may represent different FAME groups of known species or undescribed Legionella species. The method used in this study was useful for screening and discriminating large number of isolates of Legionella species. Moreover, the results obtained can be included in a database of fatty acid profiles, leading to a more accurate automatic identification of Legionella isolates.

Forty additional species and one genomospecies of the genus Legionella have been described since the initial description of Legionella pneumophila (2, 5, 10). Many of these species originate from environmental sources and have not been implicated in human disease. Genetic techniques, including whole-genome DNA-DNA hybridization, as well as computer-assisted whole-cell protein profiling, appear to be the only methodologies to identify strains of the species of this genus, since most species cannot be identified by using conventional biochemical and physiological characteristics (4, 8, 11, 23, 24, 27).

Fatty acid analysis has been shown to be a useful alternative or adjunct to phenotypic and genetic methodologies for the identification of many bacteria and has been used extensively for the identification of clinically important bacteria, having become established in many laboratories involved in taxonomy and diagnostic microbiology (13, 18, 22, 26). Particular attention must be paid to the influence of culture conditions on bacterial fatty acid composition, but standardization of media and growth conditions leads to highly reproducible fatty acid profiles that may be used as chemotaxonomic markers to distinguish strains of different species (21, 25). Fatty acid analysis has been used to discriminate the species of the genus Legionella (12, 15, 16, 17, 28), but there have been no recent studies that include all the known species of this genus or that have used a standardized procedure that can be used by many laboratories for the tentative identification of isolates.

In this study, we used one commercial system to analyze the fatty acid profiles of all validly described Legionella species and genomospecies, with the exception of L. lytica (10), to evaluate the ability of fatty acid composition to differentiate Legionella spp. and to produce a database to facilitate the automatic identification of isolates.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Seventy-six type and reference strains, representing Legionella species and one genomospecies, and 199 isolates of the genus Legionella were used in this study (Table 1). The isolates obtained from France, the United Kingdom, and the United States were identified by the laboratory of origin or by other laboratories (1, 9, 23, 24), while the remaining isolates had been previously identified by indirect immunofluorescence and/or numerical analysis of whole-cell protein profiles in our laboratory (27). Cultures were maintained at −80°C in 5.0% (wt/vol) yeast extract containing 15.0% (vol/vol) glycerol. Legionella strains were spread, as recommended by a Microbial ID, Inc. (MIDI; Newark, Del.) protocol (Microbial Identification System) on buffered charcoal-yeast extract agar (7). All plates were incubated in sealed plastic bags, to prevent evaporation, at 36 ± 1°C for 72 ± 2 h, in a humidified incubator.

TABLE 1.

Legionella strains examined in this study, organized on the basis of clusters obtained from numerical analysis of FAME profiles

Cluster Species as determined by FAME analysis No. of strains Reference strains and isolatesa
1 Legionella group I 2 Isolates LC3644 and LC4381
2 L. oakridgensis 18 L. oakridgensis ATCC 33761T; isolates Ch-18, Cu-5, Felg-30, Felg-32, Felg-44, Felg-92, Felg-99, Felg-100, Felg-110, Felg-111, Felg-119, Iz-221, Iz-323, Iz-324, Iz-330, LC3780, and NantesIV no. 2
3 L. jordanis 4 L. jordanis ATCC 33623T; isolates Ch-13, Ch-14, and LC3940
4 L. brunensis 5 L. brunensis ATCC 43878T; isolates Ch-9, Ch-10, Ch-11, and Ch-12
5 L. micdadei 13 L. micdadei ATCC 33218T; isolates Aço-69, Aix73G9, Iz-225, Iz-277, Iz-327, Iz-328, Nant-931001936, SG-224, SG-238, SG-245, SG-250, and Toul29-9
6 L. micdadei 3 L. micdadei ATCC 33204; isolates LC0858 (=Bari 2/158) and LugII-124
7 L. maceachernii 2 L. maceachernii ATCC 35300T; isolate LC4349
8 L. hackeliae 2 L. hackeliae ATCC 35250T and ATCC 35999
9 L. nautarum 2 L. nautarum ATCC 49506T; isolate Ch-5
10 L. adelaidensis 2 L. adelaidensis ATCC 49625T; isolate F552
11 L. moravica 2 L. moravica ATCC 43877T; isolate Ch-19
12 L. worsleiensis 2 L. worsleiensis ATCC 49508T; isolate Ch-27
13 L. birminghamensis 5 L. birminghamensis ATCC 43702T; isolates Ch-25, Ch-26, LC2720, and 88202373
14 L. quinlivanii 2 L. quinlivanii ATCC 43830T; isolate Edt-8
15 L. feeleii 4 L. feeleii ATCC 35072T and ATCC 35849; isolates CAMR 1717 and Ly166.96
16 L. spiritensis 2 L. spiritensis ATCC 35249T and ML0076 (=NCTC 12082)
17 L. erythra and L. rubrilucens 20 L. erythra ATCC 35303T; isolates Ch-1, Ch-2, Ch-3, Ch-4, Ch-15, LC0709, LC1317, LC3719, and 930201758; L. rubrilucens ATCC 35304T; isolates Ch-16, Ch-17, Dijon48 no. 2, LC0704, LC0805H, LC1092, LC4042, TurinI no. 1, and LC4557
18 L. londiniensis 13 L. londiniensis ATCC 49505T; isolates Alf-41, Alf-50, Ch-20, Cu-2, Iz-9, Iz-195, Iz-233, Iz-310, Mulhouse B26, SG-142, 920201357, and 93203797
19 L. dumoffii 3 Isolates Léchère 2F10, Toulouse23 no. 6, and 930201759
20 L. cherrii 2 L. cherrii ATCC 35252T; isolate SGT-14
21 L. dumoffii 7 L. dumoffii ATCC 33279T; isolates Iz-111, Iz-188, Iz-322, LC0455, SG-155, and Strasbourg-92101226
22 Legionella group II 5 Isolates Ch-32, Ch-33, Ch-36, Ch-37, and Ch-39
23 L. gormanii 5 Isolates Ch-21, Greoux9C3, LC0777C, Toulouse29 no. 16, and 89202694
24 L. anisa 9 L. anisa ATCC 35292T; isolates CAMR 1363, CAMR 1728, Ch-35, D4550a, D4559a, F1496, 87101758, and 920201241
25 L. parisiensis 2 L. parisiensis ATCC 35299T and Lyon 96010011 (=ATCC 700174)
26 L. bozemanii 10 Isolates Ch-34, Ch-40, Iz-43, Iz-313, Iz-315, Iz-331, Iz-332, Iz-333, LC2763, and SGT-13
27 L. bozemanii 9 L. bozemanii ATCC 33217T, ATCC 35545, and NCTC 11975; isolates Ch-38, Iz-36, Iz-40, LC4348, Ly86.88, and 88101908
28 L. shakespearei 3 L. shakespearei ATCC 49655T; isolates CAMR 1862 and LC3043
29 L. pneumophila 58 L. pneumophila ATCC 33152T, ATCC 33153, ATCC 33154, ATCC 33155, ATCC 33156, ATCC 33215, ATCC 33216, ATCC 33735, ATCC 33736, ATCC 33737, ATCC 33823, ATCC 35096, ATCC 35251, ATCC 35289, ATCC 43106, ATCC 43107, ATCC 43108, ATCC 43109, ATCC 43110, ATCC 43112, ATCC 43113, ATCC 43130, ATCC 43283, ATCC 43290, ATCC 43703, ATCC 43736, and NCTC 11404; isolates Aço-24, Aço-44, Agn-10, Alf-73, Ed-8, Ed-12, Ed-21, Ed-30, Edt-2, Edt-3, Edt-5, Edt-6, Felg-75, Felg-76, Felg-84, Felg-105, Felg-115, HRD-1, Ice-50, IMC-1, Iz-6, Iz-12, Iz-28, Iz-35, Iz-51, Iz-69, LC1329, NMex-19, SG-13, SG-100, and SG-222
30 L. feeleii 4 Isolates Ch-7, LC4210, Ly126.92b, and UNH-1
31 L. longbeachae 6 L. longbeachae ATCC 33462T and ATCC 33484; isolates Ch-29, Cu-3, D4154, and 88010337
32 L. cincinnatiensis 12 L. cincinnatiensis ATCC 43753T; isolates E1549, Felg-2, Felg-16, Felg-27, Felg-28, La-7, La-10, La-15, La-18, La-22, and LC3936
33 L. sainthelensi 19 L. sainthelensi ATCC 35248T; isolates Aço-66, Ed-4a, Felg-1, Felg-20, Felg-73, Felg-74, Felg-80, Felg-95, Felg-106, Felg-122, Felg-145, Felg-152, Iz-21, Iz-86, La-2, La-5, La-20, and LC4261
Unclustered L. fairfieldensis ATCC 49588T, L. geestiana ATCC 49504T, L. genomospecies 1 ATCC 51913, L. gormanii ATCC 33297T, L. gratiana ATCC 49413T, L. israelensis ATCC 43119T, L. jamestowniensis ATCC 35298T, L. lansingensis ATCC 49751T, L. quateirensis ATCC 49507T, L. quinlivanii LC0870 (=NCTC 12434), L. santicrucis ATCC 35301T, L. steigerwaltii ATCC 35302T, L. tucsonensis ATCC 49180T, L. wadsworthii ATCC 33877T, and L. waltersii ATCC 51914T; isolates Montbeliard A1, Greoux 11D13, and Toulouse20 no. 5
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a

Abbreviations for strain origins: ATCC, American Type Culture Collection, Rockville, Md.; CAMR, Center for Applied Microbiology and Research, Salisbury, United Kingdom; NCTC, National Collection of Type Cultures, London, United Kingdom; Aço, hydrothermal area, Azores, Portugal; Agn, hydrothermal area, southern Italy; Alf, hydrothermal area, central Portugal; Ch, man-made environments, kindly donated by V. Drasar, Vyskov, Czech Republic; Cu, hydrothermal area, central Portugal; Ed and Edt, hydrothermal area, central Portugal; Felg, hydrothermal area, central Portugal; HRD, lung aspirate, Coimbra, Portugal; Ice, hydrothermal area, Iceland; IMC, neonatology unit, hospital, Coimbra, Portugal; Iz, hydrothermal area, northern Portugal; La, hydrothermal area, northern Portugal; NMex, hydrothermal area, New Mexico; SG and SGT, hydrothermal area, central Portugal; UNH, hydrothermal area, central Portugal. Strains with prefix LC, strain ML0076, and strain CAMR1862 were kindly donated by T. G. Harrison (London, United Kingdom). Other previously identified strains were kindly donated by N. Bornstein and F. Lo Presti (Lyon, France) and R. F. Benson (Atlanta, Ga.). 

FAME analysis.

Cells were harvested, and the fatty acid methyl esters (FAMEs) were obtained by saponification, methylation, and extraction, as described previously (14) and as recommended by the MIDI standard protocol. The separation of FAMEs was achieved by using a Hewlett-Packard model 5890 gas chromatograph with a flame ionization detector fitted with a 5% phenylmethyl silicone capillary column (0.2 mm by 25 m; Hewlett-Packard), controlled by the Sherlock Single Tower Library Generation software (version 1.06; MIDI). The carrier gas was high-purity H2, the column head pressure was 60 kPa, the septum purge rate was 5 ml/min, the split ratio was 55:1, and the injection port temperature was 300°C. The temperature of the oven was programmed to increase from 170 to 270°C in 5°C/min increments. Peaks were integrated, and FAMEs were quantified and identified by using the peak-naming table component of the MIDI software package (Aerobe Method versions 3.8 and 3.9) Quantities were expressed as percentages of the total named FAME peak area.

Numerical analysis and mean profiles.

A dendrogram, based on the fatty acid patterns of the strains, was generated by clustering the Euclidean distances of the fatty acids, using mapped features to increase differentiation, with the unweighted pair group method with arithmetic average algorithm that is provided by the MIDI software package. Clusters were delineated in the dendrogram to differentiate each species. The mean profile of each cluster in the dendrogram was calculated on the basis of the individual profiles of all strains in the cluster. For the unclustered strains, a mean profile was obtained from two or more replicate analyses performed on different occasions.

Reproducibility.

Reproducibility was tested by repeated analyses of a standard quantitative FAME mixture (MIDI). The reproducibility of the fatty acid profiles was determined by analyzing 129 strains, on two or more occasions under standardized growth conditions, and by the inclusion of a control strain (L. pneumophila OLDA; ATCC 43109) in each batch of strains analyzed. The coefficient of variation, measured as (standard deviation/mean) × 100, was calculated for each fatty acid representing at least 20% of the total fatty acid content in the mean profile (21).

RESULTS

The coefficient of variation [(standard deviation/mean) × 100)] for each of the most abundant fatty acids was less than 12% among replicate analyses. Furthermore, the control strain, L. pneumophila ATCC 43109, had a maximum coefficient of variation of 4%.

At least 68 different fatty acids were detected in the 275 strains tested, but 15 of them appeared, as minor components, in fewer than 1% of the strains (Table 2). Seven fatty acids were detected in all strains, while nine other fatty acids appeared in at least 72% of the strains. These fatty acids were the most common fatty acids detected in the strains examined and may, therefore, represent the qualitative fatty acid pattern of the genus Legionella.

TABLE 2.

Frequency of fatty acids and alcohols in strains of the genus Legionellaa

Fatty acids Frequency in strains (%)
Saturated
 14:0 86.2
 15:0 99.3
 16:0 100.0
 17:0 92.7
 18:0 78.9
 19:0 53.8
 20:0 74.2
Saturated hydroxy
 10:0 3OH 2.2
 11:0 3OH 1.4
 12:0 3OH 1.4
 15:0 2OH 16.4
 15:0 3OH 8.4
 16:0 3OH 5.8
Unsaturated
 a15:1 Ab 3.6
 15:1ω6c 100.0
 i16:1 Hb 56.7
 16:1ω11c 2.9
 16:1ω7c 100.0
 16:1ω5c 51.3
 i17:1ω9c 15.6
 a17:1 Ab 1.1
 a17:1ω9c 41.8
 17:1ω8c 4.4
Unsaturated hydroxy
 16:1 2OH 5.4
Cyclopropane
 cyc17:0 77.4
Methyl branched
 a11:0 4.0
 i12:0 1.8
 i13:0 3.6
 a13:0 9.8
 i14:0 100.0
 i15:0 84.0
 a15:0 100.0
 i16:0 100.0
 16:0 10 methyl 1.1
 i17:0 73.1
 a17:0 100.0
 i18:0 52.7
 i19:0 13.4
 a19:0 24.4
 i20:0 8.4
Branched-chain hydroxy
 i14:0 3OH 57.8
 i15:0 3OH 1.1
 i16:0 3OH 24.0
Alcohols
 16:1ω7t 1.4
 16:1ω7c 2.9
 16:0 N 4.0
Others
 Summed feature 1c 72.0
 Summed feature 2d 8.0
 Summed feature 3e 25.8
 Summed feature 5f 1.8
 Summed feature 7g 8.0
 Summed feature 9h 3.3
 Unknown 13.566i 3.6
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a

The fatty acids 12:0, 13:0, 11:0 2OH, 13:0 2OH, 17:0 2OH, i16:1 G, i17:1ω10c, i18:1 G, 18:1ω9c, 18:1ω5c, 19:1ω12t, cyc19:0ω8c, unknown 11.541, unknown 14.966, and unknown 16.580 occurred in less than 1% of the strains tested and were not listed in the table. 

b

The double-bond position indicated by the capital letters is unknown. 

c

Group of fatty acids (14:1ω5c and/or 14:1ω5t) that could not be separated by this method. 

d

Group of fatty acids (i15:1 H and/or 13:0 3OH and/or i15:1 I) that could not be separated by this method. 

e

Group of fatty acids (i16:1 I and/or 14:0 3OH) that could not be separated by this method. 

f

Group of fatty acids (i17:1 I and/or a17:1 B) that could not be separated by this method. 

g

Group of fatty acids (18:1ω7c and/or 18:1ω9t and/or 18:1ω12t and/or 18:1ω9c) that could not be separated by this method. 

h

Group of fatty acids (unknown 18.846 and/or unknown 18.858 and/or cyc19:0ω10c) that could not be separated by this method. 

i

Fatty acid with unknown identity (the number indicates its equivalent chain length). 

The numerical analysis of the FAME profiles of 275 strains resulted in the formation of 33 clusters, with 18 strains remaining unclustered (Fig. 1). Nineteen of the clusters corresponded directly to Legionella species, while 13 of the unclustered strains belonged to species represented by type strains alone.

FIG. 1.

FIG. 1

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Dendrogram based on unweighted pair group average linkage of FAME profiles, using mapped features, of 275 Legionella strains. With the exceptions of Toulouse 20 no. 5, LC0870, Montbeliard A1, and Greoux 11D13, all of the unclustered strains are type strains.

L. geestiana ATCC 49504T had a fatty acid composition that was different from those of all other Legionella strains (Fig. 1). This strain had 16:1ω7c as the major fatty acid and higher relative proportions of i15:0 and i17:0 than of the corresponding a15:0 and a17:0 isomers, in addition to an unusually large amount of i15:0 (Table 3). Cluster 2 was composed of the 18 L. oakridgensis strains. The relative proportions of 16:1ω7c and 18:0, associated with the lowest relative amount of a15:0, clearly differentiated L. oakridgensis from all other species (Table 3). The L. jordanis strains were grouped in cluster 3 (Fig. 1). The major fatty acid detected was a15:0, followed by i16:0. These two fatty acids also constituted the major acyl chains of L. birminghamensis (cluster 13), L. israelensis, and several species that autofluoresced blue-white. However, L. jordanis could be distinguished from the other species by the low amounts of cyc17:0 and its biosynthetic precursor, 16:1ω7c (Table 3). The five strains identified as L. brunensis formed cluster 4 (Fig. 1), the major fatty acid detected being a15:0, followed by a17:0. These were also the major fatty acids in L. jamestowniensis. However, these two species could be distinguished by the relative amounts of i17:0, a17:1ω9c, and the sum of cyc17:0 and 16:1ω7c, which is higher in the type strain of L. jamestowniensis than in L. brunensis (Table 3).

TABLE 3.

Fatty acid composition of Legionella species based on numerical analysis of FAME profilesa

Species and cluster Fatty acid composition (% of total)b
i14:0 14:0 i15:0 a15:0 15:1ω6c 15:0 Alcohol 16:1ω7c i16:1 Hc i16:0 16:1ω11c 16:1ω7c 16:1ω5c 16:0 i17:1ω9c a17:1ω9c i17:0 a17:0 cyc17:0 17:0 18:0 19:0
L. adelaidensis, cluster 10 3.7 ± 0.7  trd tr 15.8 ± 2.0 4.9 ± 0.6 3.9 ± 0.1  —e — 17.2 ± 2.9 — 27.0 ± 1.7f — 13.0 ± 2.9 — — tr 3.4 ± 0.2 — tr tr tr
L. anisa, cluster 24 6.7 ± 1.3 tr tr 24.2 ± 2.2 tr tr — — 24.0 ± 2.0 — 8.0 ± 2.4 tr 6.9 ± 1.6 — — tr 6.4 ± 1.1 10.0 ± 2.9 tr tr tr
L. birminghamensis, cluster 13 3.2 ± 0.5 — tr 32.2 ± 4.6 6.8 ± 3.6 2.3 ± 0.9 — tr 21.5 ± 1.7 — 14.0 ± 1.9 — 3.3 ± 0.9 — — tr 10.6 ± 2.0 — tr — tr
L. bozemanii
 Cluster 26 2.4 ± 0.5 tr tr 26.3 ± 1.5 tr 2.3 ± 0.4 — — 12.0 ± 1.8 — 6.3 ± 0.5 tr 11.8 ± 1.6 — — tr 10.6 ± 1.5 14.1 ± 1.8 4.8 ± 0.9 tr tr
 Cluster 27 3.4 ± 1.0 tr tr 32.8 ± 2.0 tr tr — — 16.3 ± 2.1 — 6.4 ± 2.2 tr 7.0 ± 1.5 tr — tr 11.8 ± 3.3 8.7 ± 1.3 2.3 ± 1.1 tr tr
L. brunensis, cluster 4 tr tr 4.2 ± 1.9 36.7 ± 2.5 tr tr — tr 11.8 ± 1.1 — 5.6 ± 0.7 — 2.8 ± 0.4 tr tr 4.4 ± 2.0 23.1 ± 1.9 tr tr tr —
L. cherrii, cluster 20 7.3 ± 0.5 tr tr 30.1 ± 1.7 tr tr — tr 24.4 ± 4.2 — 10.9 ± 2.4 tr 3.7 ± 1.4 — — tr 9.2 ± 1.3 4.7 ± 0.4 tr tr —
L. cincinnatiensis, cluster 32 9.8 ± 1.7 tr tr 15.8 ± 2.1 2.5 ± 0.2 tr — — 23.9 ± 1.8 — 18.6 ± 2.1 tr 7.4 ± 1.3 — — tr 3.6 ± 0.5 11.0 ± 2.5 tr tr —
L. dumoffii
 Cluster 19 2.9 ± 0.6 tr tr 37.2 ± 2.5 tr tr — — 13.6 ± 1.1 — 13.1 ± 1.3 tr 3.7 ± 0.2 — — — 13.2 ± 0.7 4.8 ± 1.4 2.5 ± 0.9 tr —
 Cluster 21 3.2 ± 0.9 tr tr 32.4 ± 6.1 tr tr — tr 14.6 ± 2.8 — 7.2 ± 2.0 tr 7.6 ± 3.6 — tr — 11.0 ± 0.8 11.6 ± 1.4 2.4 ± 0.8 tr tr
L. erythra and L. rubri-lucens, cluster 17 3.2 ± 0.6 tr tr 14.1 ± 2.5 tr tr — tr 29.9 ± 3.2 — 22.8 ± 3.7 — 7.8 ± 3.2 tr tr tr 10.2 ± 2.8 — tr tr tr
L. fairfieldensisT 5.5 ± 0.3 tr tr 10.8 ± 2.7 tr tr — tr 37.7 ± 2.7 — 18.4 ± 2.1 tr 5.0 ± 0.5 — — — 3.3 ± 0.7 10.7 ± 2.0 tr tr —
L. feeleii
 Cluster 15 5.6 ± 1.0 tr tr 24.7 ± 2.3 3.1 ± 1.0 tr 2.8 ± 0.4 — 22.6 ± 2.6 tr 18.3 ± 1.9 — 7.5 ± 0.6 — — tr 6.0 ± 2.7 tr tr tr tr
 Cluster 30 4.6 ± 1.1 tr tr 20.6 ± 2.2 2.3 ± 0.6 tr 3.4 ± 1.4 — 17.3 ± 2.1 2.4 ± 0.4 23.8 ± 2.2 — 10.2 ± 2.0 — — tr 6.8 ± 0.5 3.4 ± 1.0 tr tr —
L. geestianaT tr tr 20.8 ± 0.5 9.2 ± 0.3 tr tr — — tr — 33.0 ± 0.2 — 13.5 ± 1.6 tr — 5.6 ± 0.6 2.8 ± 0.1 — tr tr —
L. genomospecies 1 2.8 ± 0.2 tr tr 20.7 ± 1.3 4.5 ± 0.2 tr — — 21.9 ± 1.0 — 27.1 ± 0.8 — 8.0 ± 1.4 — — tr 7.8 ± 0.4 — tr tr —
L. gormanii
 Cluster 23 5.0 ± 0.6 tr tr 24.5 ± 2.1 tr 3.0 ± 0.7 — — 17.5 ± 2.2 — 10.7 ± 2.2 tr 8.2 ± 2.1 — — tr 8.9 ± 1.0 11.0 ± 1.9 2.6 ± 0.2 tr tr
 ATCC 33297T 7.9 ± 1.2 tr tr 26.9 ± 3.1 4.1 ± 1.0 3.7 ± 0.7 — tr 19.5 ± 2.9 — 8.1 ± 2.0 tr 4.8 ± 0.9 — — tr 6.6 ± 1.7 8.0 ± 1.0 2.3 ± 0.5 tr tr
L. gratianaT 18.0 ± 0.8 tr tr 14.8 ± 0.5 4.1 ± 0.2 tr — tr 26.1 ± 1.5 — 21.0 ± 2.8 tr 3.0 ± 0.4 — — tr tr 4.0 ± 1.2 tr tr —
L. hackeliae, cluster 8 3.3 ± 0.3 4.1 ± 1.9 3.5 ± 0.1 35.7 ± 2.8 tr tr — tr 13.1 ± 1.5 — 12.2 ± 1.5 — 5.3 ± 2.2 tr — tr 8.4 ± 0.2 3.7 ± 0.0 — tr —
L. israelensisT 3.1 ± 0.4 tr tr 37.9 ± 1.4 tr tr — 3.2 ± 0.2 19.4 ± 0.6 — 7.2 ± 0.1 — 4.8 ± 0.2 tr tr tr 11.2 ± 0.7 5.8 ± 0.2 tr tr —
L. jamestowniensisT tr tr tr 39.0 ± 1.9 tr tr — 2.4 ± 0.1 11.8 ± 1.3 — 4.7 ± 0.4 tr 3.5 ± 0.2 tr 3.8 ± 0.2 tr 16.6 ± 1.3 5.8 ± 0.5 tr tr —
L. jordanis, cluster 3 2.9 ± 0.1 — 2.3 ± 0.2 54.4 ± 2.1 tr tr — tr 17.3 ± 0.9 — 2.6 ± 0.4 tr tr tr tr tr 11.1 ± 0.3 2.3 ± 0.8 tr tr —
L. lansingensisT tr — tr 27.0 ± 0.4 tr tr — tr 13.6 ± 1.6 — 3.1 ± 0.1 tr 3.5 ± 0.2 tr 4.5 ± 0.1 3.4 ± 0.2 33.7 ± 0.8 tr tr tr tr
L. londiniensis, cluster 18 tr tr tr 10.7 ± 1.9 3.7 ± 1.1 tr — tr 18.1 ± 3.3 — 20.2 ± 2.1 — 5.1 ± 2.1 3.1 ± 1.6 — 2.7 ± 1.4 21.6 ± 3.1 — 3.6 ± 0.8 2.5 ± 0.8 tr
L. longbeachae, cluster 31 7.3 ± 0.9 tr tr 14.8 ± 2.7 tr tr — tr 23.1 ± 1.6 — 22.3 ± 2.6 3.6 ± 1.1 5.7 ± 1.7 — — tr 4.8 ± 0.7 9.6 ± 3.1 tr tr —
L. maceachernii
 Cluster 7 tr — — 32.2 ± 0.6 3.1 ± 1.9 tr — 2.4 ± 0.1 9.0 ± 0.4 — 17.4 ± 0.1 — 5.6 ± 0.4 — 5.5 ± 0.4 — 19.3 ± 1.5 tr tr — —
 Toulouse20 n°5 tr tr tr 38.3 ± 1.7 4.4 ± 0.2 5.1 ± 0.7 — — 6.3 ± 0.4 — 18.4 ± 0.8 — 9.2 ± 1.7 — — — 14.2 ± 0.7 — tr tr —
L. micdadei
 Cluster 5 tr tr tr 39.2 ± 1.8 2.4 ± 1.2 2.5 ± 0.9 — tr 11.9 ± 1.5 — 8.2 ± 1.6 tr 4.9 ± 0.8 — 4.8 ± 0.7 tr 16.5 ± 2.6 2.8 ± 0.8 tr — —
 Cluster 6 tr tr tr 34.9 ± 1.8 5.8 ± 1.0 6.6 ± 0.6 — tr 6.5 ± 1.1 — 14.6 ± 2.5 — 7.4 ± 0.4 — 5.4 ± 0.4 — 9.7 ± 1.5 tr tr — —
L. moravica, cluster 11 7.1 ± 0.9 tr — 14.8 ± 5.3 2.7 ± 0.3 tr — — 21.9 ± 1.6 — 31.8 ± 5.4 — 7.0 ± 1.8 — — — 4.2 ± 1.8 — tr 3.7 ± 0.1 tr
L. nautarum, cluster 9 4.0 ± 1.7 tr tr 38.1 ± 1.8 5.1 ± 1.6 tr — — 15.0 ± 3.9 — 16.5 ± 4.6 tr 5.3 ± 3.6 — — — 6.3 ± 0.4 2.4 ± 0.4 — — —
L. oakridgensis, cluster 2 tr tr — 2.8 ± 0.8 tr tr — tr 24.6 ± 3.6 — 18.8 ± 5.3 tr 10.4 ± 3.5 tr tr tr 5.9 ± 1.2 7.8 ± 4.8 2.8 ± 0.5 12.0 ± 1.4 2.5 ± 0.6
L. parisiensis, cluster 25 3.2 ± 0.1 tr tr 27.6 ± 2.7 tr tr — — 15.5 ± 0.2 — 7.2 ± 1.0 tr 12.8 ± 0.5 — — tr 9.2 ± 0.2 11.6 ± 2.0 2.4 ± 0.3 tr tr
L. pneumophila, cluster 29 8.4 ± 1.0 tr tr 15.3 ± 2.6 3.1 ± 0.6 tr — 3.6 ± 1.0 32.8 ± 3.7 — 13.9 ± 3.9 tr 4.2 ± 1.5 — tr tr 5.1 ± 1.0 5.5 ± 2.5 tr tr tr
L. quateirensisT 7.9 ± 1.8 tr tr 16.9 ± 2.4 4.4 ± 0.8 tr — — 17.6 ± 2.6 — 32.0 ± 1.6 — 5.1 ± 1.6 — — tr 4.4 ± 0.5 — tr 3.2 ± 0.5 tr
L. quinlivanii
 Cluster 14 2.6 ± 0.9 tr tr 26.2 ± 0.8 6.0 ± 0.4 2.4 ± 0.8 — — 20.2 ± 2.8 — 19.0 ± 0.8 — 6.2 ± 2.4 — — tr 11.3 ± 1.0 — tr — —
 LC0870 2.3 ± 0.5 tr tr 16.4 ± 0.7 4.2 ± 0.1 tr — — 20.4 ± 0.5 — 29.0 ± 0.8 — 9.0 ± 2.6 — — tr 9.8 ± 0.2 — 2.4 ± 0.2 tr —
L. sainthelensi, cluster 33 6.5 ± 0.9 tr tr 12.0 ± 2.4 3.1 ± 0.7 2.3 ± 0.5 — tr 20.6 ± 2.2 — 28.7 ± 2.6 tr 10.4 ± 2.0 — — tr 3.1 ± 0.6 8.4 ± 2.9 tr tr —
L. santicrucisT 14.8 ± 1.7 tr tr 13.8 ± 0.9 2.9 ± 0.2 tr — tr 26.9 ± 0.8 — 24.4 ± 0.2 — 7.6 ± 1.5 — — tr 2.4 ± 0.3 tr tr tr —
L. shakespearei, cluster 28 14.8 ± 2.0 tr tr 11.1 ± 0.9 6.1 ± 0.5 2.6 ± 0.8 — — 36.5 ± 1.3 — 13.6 ± 2.4 — 4.1 ± 0.2 — — tr 2.4 ± 0.4 — 2.6 ± 1.1 tr tr
L. spiritensis, cluster 16 2.7 ± 0.6 tr tr 20.6 ± 1.8 tr tr — 3.7 ± 2.1 30.0 ± 2.7 — 14.1 ± 0.9 — 5.2 ± 1.5 tr tr tr 14.6 ± 2.1 tr tr tr tr
L. steigerwaltiiT 3.7 ± 0.2 tr tr 26.6 ± 1.9 tr 3.4 ± 0.5 — — 9.8 ± 1.7 — 7.4 ± 1.1 tr 20.7 ± 0.4 — — tr 4.8 ± 0.9 12.1 ± 0.6 tr tr tr
L. tucsonensisT 3.6 ± 0.2 — — 34.6 ± 3.9 tr tr — tr 16.6 ± 0.6 — 17.3 ± 0.7 tr 4.5 ± 0.2 — tr tr 11.6 ± 0.6 4.2 ± 1.6 tr tr —
L. wadsworthiiT tr — tr 48.0 ± 0.9 tr tr — — 13.9 ± 0.8 — 5.0 ± 0.2 tr tr — — tr 16.7 ± 1.0 5.8 ± 0.4 tr tr —
L. waltersiiT 22.6 ± 1.1 tr tr 9.7 ± 0.7 9.1 ± 0.8 5.2 ± 1.1 — — 21.2 ± 1.0 — 15.0 ± 0.5 — 7.1 ± 1.0 — — tr tr — tr tr tr
L. worsleiensis, cluster 12 7.4 ± 0.4 tr tr 16.4 ± 1.2 tr tr — — 18.8 ± 2.2 — 33.4 ± 0.6 — 9.6 ± 1.6 — — — 4.8 ± 1.6 — tr tr —
Legionella group I, cluster 1 tr tr 36.7 ± 1.5 17.7 ± 1.4 9.1 ± 0.8 3.8 ± 0.2 — — 3.8 ± 0.3 — 4.9 ± 0.6 — tr tr — 6.5 ± 0.6 4.9 ± 0.1 — tr — —
Legionella spp.
 Montbeliard A1 tr tr tr 18.2 ± 1.6 tr tr — tr 14.0 ± 0.9 — 34.5 ± 3.3 — 4.2 ± 0.2 — — tr 17.5 ± 1.2 — tr tr —
 Greoux 11D13 tr tr tr 9.6 ± 0.3 3.6 ± 0.8 tr — tr 14.5 ± 1.5 — 44.4 ± 2.4 — 5.9 ± 1.6 tr — tr 7.1 ± 0.6 — tr tr tr
Legionella group II, cluster 22 2.6 ± 0.5 tr tr 31.4 ± 4.5 tr tr — tr 15.2 ± 2.8 — 8.1 ± 2.8 tr 7.3 ± 0.4 — tr tr 12.0 ± 1.4 9.8 ± 3.7 tr tr tr
Open in a new tab
a

The strains included in each cluster are shown in Table 1. 

b

The fatty acids shown are those present in at least 2.0% of the total fatty acids. Values are means ± standard deviations. 

c

The double-bond position indicated by the capital letter is unknown. 

d

tr, fatty acid with less than 2.0% of the total. 

e

—, fatty acid not detected. 

f

Bold type indicates the major fatty acid of the unclustered strain or cluster of strains. 

Two reference strains and 14 isolates previously identified as L. micdadei were split into clusters 5 and 6 (Fig. 1), despite the fact that a15:0 was the major fatty acid in all strains of this species. Cluster 5, containing the type strain of L. micdadei, had higher amounts of i16:0 and a17:0 and lesser quantities of 16:1ω7c than cluster 6 did (Table 3). One important characteristic of the L. micdadei strains was the presence of moderate amounts of a17:1ω9c, previously identified as a17:1ω7c (19, 20). This fatty acid was also detected in the type strains of L. jamestowniensis and L. lansingensis and two of the three strains assigned to L. maceachernii (cluster 7); nevertheless, a17:1ω9c was not detected in strain Toulouse20 no. 5 (Table 3).

L. hackeliae and L. nautarum strains constituted clusters 8 and 9, respectively (Fig. 1). The mean fatty acid profiles of these species were similar (Table 3), but they could be distinguished clearly from each other by numerical analysis. L. adelaidensis and L. moravica strains formed clusters 10 and 11, respectively (Fig. 1). In both species, the major fatty acid was 16:1ω7c, followed by i16:0 and a15:0 in similar amounts (Table 3). The L. worsleiensis strains available formed cluster 12 and had a fatty acid composition that was similar to that of L. quateirensis ATCC 49508T, resulting in a high similarity value between these two species (Fig. 1).

Legionella genomospecies 1 (ATCC 51913) was responsible for the separation of the two L. quinlivanii strains used (Fig. 1). While strains ATCC 51913 and LC0870 (L. quinlivanii serogroup 2) had similar mean fatty acid profiles, the type strain of L. quinlivanii had a slightly different profile (Table 3), being included in cluster 14 with a Portuguese isolate (Edt-8); this strain produced strong cross-reactions between L. quinlivanii and L. feeleii when analyzed by indirect immunofluorescence.

The eight strains assigned to L. feeleii were split into clusters 15 and 30 (Fig. 1). Cluster 30 included four strains identified as L. feeleii by several methods, while cluster 15 contained both the type and a reference strain of L. feeleii. The mean profile of each cluster had different major fatty acids, but all of the strains contained two fatty acids (16:1ω11c and alcohol 16:1ω7c) that were not found in any other of the strains examined (Table 3).

Twenty strains belonging to the red autofluorescent species L. rubrilucens and L. erythra (cluster 17) had very similar fatty acid compositions, leading to the formation of a single cluster (Fig. 1). The type strain of L. londiniensis and 12 strains assigned to this species formed cluster 18 (Fig. 1). The major fatty acid detected in most strains was a17:0, although in some strains i16:0 and 16:1ω7c could reach similar levels or could be even slightly higher than the level of a17:0 (Table 3). This fatty acid was also the major fatty acid in L. lansingensis, but this species could be easily distinguished from L. londiniensis by differences in the relative amounts of a15:0, i16:0, and 16:1ω7c and by the presence of a17:1ω9c.

The blue-white autofluorescent species formed a well-defined group, at a Euclidean distance of approximately 23 units (Fig. 1). The type strain of L. bozemanii, two reference strains, and 16 isolates previously assigned to this species were split into clusters 26 and 27. Strains assigned to L. dumoffii also formed two clusters (clusters 19 and 21). Cluster 19 did not contain the type strain of this species, but two of the three isolates had been identified as L. dumoffii (24). Five strains previously identified as L. gormanii were included in cluster 23. However, the type strain of L. gormanii remained unclustered, despite the similar fatty acid compositions of the strains of this species (Table 3).

The fatty acid composition of the type strain of L. waltersii was unique because i14:0 was the major fatty acid. Moreover, this organism and the type strain of L. gratiana, unlike all the other strains examined, possessed only trace amounts of a17:0 (Table 3).

All 58 L. pneumophila strains examined formed a single cluster (cluster 29), at a Euclidean distance of approximately 17 units (Fig. 1). Cluster 32 included the type strain of L. cincinnatiensis, two strains previously assigned to this species (E1549 and LC3936), four unidentified strains recently isolated from a Portuguese spa (designated Felg), and five other isolates (designated La) shown to be similar to the type strain of L. sainthelensi (27). Clusters 31 and 33 contained only strains previously identified as L. longbeachae and L. sainthelensi, respectively (Fig. 1). For L. cincinnatiensis, L. sainthelensi, and L. longbeachae, the major fatty acids were the same, with only small quantitative differences in the minor acyl components (Table 3).

The fatty acid profiles of eight previously unidentified isolates and one isolate initially assigned to L. geestiana (LC3644) did not conform to any of the profiles of recognized species. Legionella group I (cluster 1) comprised two of these isolates for which the presence of i15:0 and a15:0 as the major fatty acids represents a unique profile among Legionella spp. (Table 3). Two other strains (Montbeliard A1 and Greoux 11D13) had distinct fatty acid compositions, while Legionella group II (cluster 22) included five unidentified isolates with colonial blue-white autofluorescence.

DISCUSSION

The strains of the genus Legionella are notoriously difficult to identify by traditional methods. Fatty acid analysis of Legionella strains is useful for characterizing these organisms at the genus level but has been difficult to use in identifying individual species (15, 16). This study shows that the application of numerical analysis, instead of simple quantitative comparison of fatty acid compositions, coupled with the utilization of a standardized identification system greatly enhances the utilization of fatty acid profiles for the identification of individual Legionella species.

The identification of some legionellae may not be straightforward, since the strains of some Legionella species, namely, L. micdadei, L. maceachernii, L. quinlivanii, L. feeleii, L. dumoffii, L. gormanii, and L. bozemanii, form more than one cluster. The variability of the fatty acid composition of L. micdadei strains was noticed previously by Moss and Lambert-Fair (19), who showed that strain Bari 2/158 had a different fatty acid composition than the type strain of L. micdadei. Our results extend this characteristic to two other strains of L. micdadei and clearly show that a similar degree of variation can occur in other species. However, automatic identification of the isolates of these species is possible, as long as these fatty acid subgroups are taken into account in the construction of databases.

Variations in the major acyl components may also occur within one cluster, as with the L. londiniensis strains examined. Previous results for the fatty acid composition of this species were based on the type strain alone, and variations in the levels of the fatty acids could not be observed (6). This example shows the importance of examining a large number of strains, when available, for a reliable analysis of chemotaxonomic markers of a species.

Despite these variations, some species like L. pneumophila had a very stable fatty acid profile. The formation of a single cluster by a large number of L. pneumophila strains provides a suitable example of the resolution and ability of this method to distinguish this species from all other species of the genus Legionella. Some Legionella species, on the other hand, had very similar fatty acid compositions and may be difficult to differentiate by this method. The numerical analysis of FAME profiles showed a high similarity between some species, such as L. moravica, L. quateirensis, and L. worsleiensis; L. erythra and L. rubrilucens; and L. sainthelensi, L. cincinnatiensis, and L. longbeachae. Moreover, these similarities were also found by so-dium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiling (27), mip gene sequencing studies (23), and intergenic 16S-23S ribosomal spacer PCR analysis (24), corroborating recent phylogenetic studies (3, 10). With the exception of the red autofluorescent strains, it was possible to differentiate all the Legionella species and unambiguously identify, by numerical analysis of FAME profiles, the vast majority of the strains used. Indeed, the identification of the vast majority of the strains that were also used in other recent studies could be confirmed by fatty acid analysis (23, 24, 27).

Nine isolates had fatty acid profiles unlike any of those of the validly described species and may represent distinct FAME groups of known species or undescribed Legionella species, as has been suggested recently by others, based on genetic typing methods (23, 24) or protein profiling (27).

The lack of strains other than the type strain of some species was probably the major difficulty in assessing the resolution of this identification method for species of the genus Legionella. The inclusion of more strains of some species, preferably from widely separated geographical areas and diverse ecological habitats, is clearly necessary for an enhanced database for this method. However, on the basis of our results, we conclude that the numerical analysis of fatty acid profiles, by a standardized system, is helpful in identifying Legionella species, especially when a large number of Legionella isolates are being screened, as is the case in many clinical or reference laboratories.

ACKNOWLEDGMENTS

This work was supported, in part, by the Sociedade das Águas de Luso, SA, and Companhia das Águas Medicinais da Felgueira, SA.

We thank Nicole Bornstein and François Lo Presti (Centre National de Référence des Legionella, Lyon, France), Tim Harrison (Central Public Health Laboratory, London, United Kingdom), Robert Benson (Centers for Disease Control, Atlanta, Ga.), and Vladimir Drasar (National Legionella Reference Laboratory, Vyskov, Czech Republic) for the kind donation of strains.

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