JOURNAL OF CLINICAL MICROBIOLOGY, June 1993, p. 1413-14190095-1137/93/061413-07$02.00/0Copyright X) 1993, American Society for Microbiology

Hydroxy-Fatty Acid Profiles of Legionella Species: DiagnosticUsefulness Assessed by Principal Component Analysis

ERIK JANTZEN,* ANDERS SONESSON, TORILL TANGEN, AND JAN ENGStatens Institutt for Folkehelse, Geitmyrsveien 75, N-0462 Oslo, Norway

Received 22 July 1992/Accepted 2 March 1993

Twenty-nine species (76 strains) of members of the genus Legionella were analyzed for their cellularhydroxylated fatty acids (OH-FAs). The individual patterns were unusually complex and included bothmonohydroxylated and dihydroxylated chains of unbranched or branched (iso and anteiso) types. Comparisonof the strain profiles by SIMCA (Soft Independent Modelling of Class Analogy) principal component analysisrevealed four main groups. Group 1 included Legionella pneumophila plus L. israelensis strains, and group 2included L. micdadei and L. maceacherneii strains. These two closely related groups were characterized by theoccurrence of di-OH-FAs and differed mainly in the amounts of 3-OH-a21:0, 3-OH-n21:0, 3-OH-n22:0, and3-OH-a23:0. Group 3 (13 species) was distinguished by i14:0 at less than 3%, 3-OH-3-OH-n14:0 at greater than5%, 3-OH-n15:0 at greater than 2%, and minute amounts of OH-FAs with chains longer than 21:0. Group 4(12 species) was heterogeneous. Its main characteristics were the presence of 3-OH-n12:0 and 3-OH-n13:0,3-OH-i14:0 at greater than 5%, as well as significant amounts of 3-OH-a21:0 and 3-OH-n21:0. The groupingsobtained by OH-FA profiles were found to reflect DNA-DNA homology groupings reasonably well, and theprofiles appear to be useful for differentiation of LegioneUla species.

Members of the genus Legionella include fastidious, wa-

ter-borne gram-negative bacteria that occasionally causerespiratory disease in humans, e.g., Legionnaires' disease.The first recognized species of the genus, Legionella pneu-mophila, was described in 1977 (5). Subsequently, more than32 additional species have been allocated to the genus,mainly on the basis of DNA homology, and the list of speciesis continuously growing (1, 2). A splitting of the genus intotwo additional genera, Tatlockia and Fluonbacter, has beenproposed (4, 7). On the basis of 16S rRNA sequence deter-minations, it was more recently demonstrated that the familyLegionellaceae forms a relatively homogeneous taxonomicentity and the continued use of a single genus was recom-mended (6).

L. pneumophila is the dominating Legionella agent thatcauses human respiratory infections; however, the numberof clinical isolates of other Legionella species is steadilyincreasing (3). A diagnostic report specifying both speciesname and serotype may therefore be of interest both forclinical and epidemiological purposes.

Differentiation of the numerous Legionella species bysimple laboratory tests is currently not possible. However,more elaborate fatty acid and ubiquinone analyses haveproved to be useful alternatives to genetic techniques (10,13, 16). The profiles of these cellular constituents permitallocation of a strain to the Legionella genus as well as intoat least five distinct subgroups.The 3-hydroxy-fatty acids (3-OH-FAs) that we focused on

in the present study probably originated from the lipid part ofouter membrane lipopolysaccharides (14; unpublished data).Chromatographic profiles of such lipopolysaccharide-de-rived constituents have generally proved valuable for clas-sification studies and identification of gram-negative bacteria(8, 12). Mayberry (11) described members of the species ofLegionella as being characterized by very complex patternsof 3-OH-FAs, all apparently bonded in stable amide link-

* Corresponding author.

ages. This implies that fairly strong acidic conditions are

required for cleavage, and only trace amounts are detectedafter the traditionally used alkaline conditions.We developed a technique suitable for routine analysis of

amide-linked OH-FAs and tested this on a panel of 76 strainscomprising most of the recognized Legionella species. Forthe comparison of the OH-FA patterns we used a computermethod, a special version of principal component analysisnamed SIMCA (Soft Independent Modelling of Class Anal-ogy [17]). On the basis of fatty acid data, this method haspreviously proved to be capable of disclosing small devia-tions from regular patterns, thus recognizing distinct groupsand subgroups within collections of strains (9). The groupingwe obtained on the basis of OH-FA profiles is discussed inrelation to the classification obtained by DNA-DNA hybrid-ization (1).

MATERLALS AND METHODS

Bacterial strains. The 76 strains examined in the presentstudy are listed in Table 1. Bacterial cells were grown for 2days at 37°C on buffered charcoal yeast extract agar (Oxoid,Basingstoke, United Kingdom).

Chemical procedures. Bacterial cells from one cultureplate were transferred with a platinum loop directly to a

screw-cap vial containing 2 M HCl in methanol (1 ml). Theclosed vial was then heated at 85°C for 18 h to liberate fattyacid methyl esters (FAMEs). The FAMEs were then ex-tracted twice with hexane (2 ml), after the addition of waterthat was half saturated with NaCl (1 ml). This extract wasconcentrated to about 0.2 ml by nitrogen bubbling and wasthen applied to a silica extraction column (AnalytichemInternational, Harbor City, Calif.) prewashed with 2 ml eachof hexane, CH2Cl2, and hexane-CH2Cl2 (1:1). The nonhy-droxylated FAMEs were first removed with 3 ml of hexane-CH2Cl2 (1:1), whereupon 3 ml of dry diethyl ether eluted theOH-FAMEs. The solvent was removed by nitrogen bubblingbefore derivatization of the residue by adding 200 ,ul of a 50%solution of trifluoroacetic anhydride (TFAA) in acetonitrile

1413

Vol. 31, No. 6

on June 5, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

1414 JANTZEN ET AL.

TABLE 1. Strains analyzed

Species Sourcea Strain no. OH-FA' groupb FA group' Ubiquinone group' DNA groupd

L. pneumophila Berdal, FML, Oslo Weaver-Phil-1 1 I A 5Falsen, CCUG, G0teborgKallings, SBL, StockholmKallings, SBL, StockholmKallings, SBL, StockholmKallings, SBL, StockholmKallings, SBL, StockholmATCCATCCATCCATCCATCCATCCATCCATCCATCCATCCATCCATCCATCC

CCUG 13400LD 9/86LD 80/87LD 84/83LD 120/85LD 155/85ATCC 33154ATCC 33155ATCC 33156ATCC 33215ATCC 33216ATCC 33823ATCC 35096ATCC 35289ATCC 43130ATCC 43283ATCC 43290ATCC 43736ATCC 43703

1111111111111111111

IIIIIII

IIIIIIIII

AAAAAA-AAAAAAAAAAAAA

5555SSSSS5SSSSSSSS5

L. micdadei

L. maceachernii

L. israelensis

L. anisa

Fallon, GlasgowFox, ColumbiaFox, ColumbiaFox, ColumbiaFox, ColumbiaFox, ColumbiaFox, ColumbiaFox, ColumbiaFox, ColumbiaFox, ColumbiaATCC

Fox, ColumbiaFox, ColumbiaFox, ColumbiaFox, ColumbiaATCC

ATCC

Fallon, GlasgowATCC

L. bozemanii

L. chemii

L. cincinnatiensis

L. dumoffi

L. gormanii

Fox, ColumbiaFox, ColumbiaFox, ColumbiaATCC

ATCC

ATCC

ATCCFox, Columbia

L. gratiana

L. longbeachae

L. parisiensis

L. sainthelensi

Bornstein, Lyon

Bornstein, LyonATCCATCC

ATCC

ATCC

Continued on following page

DDDDDDDDDDD

66666666666

88/1058QuigleyLR7WSICU 5RubinsEKHEBAMcCoyPGH-12ATCC 33218

D437-A2-4Sc-73-C2Sc-73-C3Pxl-G2-E2ATCC 35300

ATCC 43119

88/22485ATCC 35292

ATCC 33217ATCC 35545

ORZORBSc-65-C3ATCC 35252

ATCC 43753

ATCC 33279

ATCC 3329786A5796

22222222222

22222

1

33

33

3333

3

3

33

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIII

III

IIII

22

IIIIIIII

I

II

IIII

DDDDD

A

BB

BB

BBBB

B

B

BB

99999

11

22

22

2222

2

2

22

38420412

Serogroup 1ATCC 33484ATCC 33462

ATCC 35299

ATCC 35248

333

3

3

III

II

I

BBB

B

B

222

2

2

J. CLIN. MICROBIOL.

on June 5, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

HYDROXY-FATI'Y ACIDS OF LEGIONELLA SPECIES 1415

TABLE 1-Continued

Species Sourcea Strain no. OH-FA1 groupb FA groupc Ubiquinone groupc DNA groupd

L. steigerwaltii ATCC ATCC 35302 3 II B 2

L. tucsonensis ATCC ATCC 49180 3 ? ? ?

L. wadsworthii Bornstein, Lyon 81.716A 3 III C ?ATCC ATCC 16415 3 III C ?

L. birminghamensis ATCC ATCC 43702 4 IV A ?

L. brunensis ATCC ATCC 43878 4 ? ? ?

L. erythra ATCC ATCC 35303 4 IV A 1

L. feeleii ATCC ATCC 35072 4 IV E 3ATCC ATCC 35849 4 IV E 3Falsen, CCUG, G0teborg CCUG 16417 4 IV E 3

L. hackeliae ATCC ATCC 35999 4 III D 10ATCC ATCC 35250 4 III D 10

L. jamestowniensis ATCC ATCC 35298 4 III D 4

L. jordanis ATCC ATCC 33623 4 III D 7Falsen, CCUG, G0teborg CCUG 16413 4 III D 7

L. moravica ATCC ATCC 43877 4 ? ? ?

L. oakridgensis Bornstein, Lyon OR-10 4 V C 8Falsen, G0teborg CCUG 16414 4 V C 8

L. quinlivanii ATCC ATCC 43830 4 IV A ?

L. rubrilucens ATCC ATCC 35304 4 IV A 1

L. spintensis Fallon, Glasgow ML-76 4 I D 1a Strain numbers were those designated by the source. See Acknowledgments section for the complete sources. ATCC, American Type Culture Collection,

Rockville, Md.; CCUG, Culture Collection of the University of G0teborg, G0teborg, Sweden; FML, Forsvarets Mikrobiologiske Laboratorium (NorwegianDefence Microbial Laboratory), Oslo, Norway; SBL, Statens Bakteriologiske Laboratorium (National Bacteriological Laboratory), Stockholm, Sweden.

b Results of this study, see Fig. 1.C Results of Wilkinson et al. (16).d Results of Brenner (1).

(AN) and heating (3 min) with a hair dryer. Prior to injectiononto the gas chromatographic column, the solvent wasexchanged with 10% TFAA in AN.Gas chromatography. Gas chromatography was performed

by using a DANI 6500-HR instrument (Monza, Italy)equipped with a flame ionization detector and a 25-m fused-silica column cross-linked with an SE-30 liquid phase (SGE;Ringwood, Victoria, Australia), a carrier gas (He) flow rateof 1.2 ml/min, an injector temperature of 250°C, a detectortemperature of 300°C, and an oven temperature program of120 to 280°C at 4°C/min. The identities of the individualFAMEs were established by use of retention time data(underivatized and trifluoroacetylated) and mass spectrom-etry.

Principal component analysis. Principal component analy-sis of the quantitative FAME data was done as describedpreviously (9) by using the SIRIUS program (Pattern Rec-ognition Systems Ltd., Bergen, Norway), which includes aSIMCA principal component analysis part. The principles ofSIMCA have been described previously (17). Briefly, eachstrain represented by its 29 variable values (OH-FAMEconcentrations) can be viewed (and mathematically treated)as a single point in the 29-dimensional space. Collections of

strains can then be described by the lines (principal compo-nents, factors, eigenvectors) that pass through the center ofgravity of the point cloud. These new, orthogonal variablesare the linear combinations of the original data, conservingmost of the variations among the strains. Projections ontoprincipal components (score plots, eigenvector projections)show the relative positions of the strains, i.e., the degree ofsimilarity among them (see Fig. 1). Interpretation of suchscore plots are possible through the connection between theoriginal variables and the principal components. A loadingplot (see Fig. 2) displays this information by showing thecontribution of each variable (OH-FAME) to the groupingthat was obtained.The SIMCA procedure is performed in the following

manner. (i) The raw gas chromatographic data (OH-FAMEprofile of each strain) are loaded into the computer program.(ii) These raw data are transformed into the correspondinglogarithmic values after addition of the value 1.0 to eachdatum. (iii) The computer program calculates the number ofstatistically significant principal components and their rela-tive contribution to the variation within the strain materialand then creates the plots.The fuzzy clustering routine of the SIRIUS program

VOL. 31, 1993

on June 5, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

1416 JANTZEN ET AL.

estimates the "degree of membership" of each strain to a

preconceived number of clusters or groups. We used thescores from the principal component analysis as the basis forthe calculations and a weighting exponent of 2.0.

RESULTS AND DISCUSSION

The unusually complex FA patterns of Legionella speciesare known to include a wide range of methyl-branched (isoand anteiso), unsaturated, and cyclopropane-substitutedFAs as well as 15 to 29 monohydroxylated and dihydroxy-lated FAs (11, 15). However, in our view OH-FAs have notbeen taken into full account for diagnostic purposes. Thereason for this may be that only trace amounts of theamide-linked OH-FAs are liberated at standard alkalineconditions for FA release (8). Acidic conditions, on the otherhand, provide cleavage of amide linkages but degrade cyclo-propane-substituted FAs. In the case of the legionellae, theuse of acidic conditions results in very complex gas chro-matographic profiles, often with several (partly) overlappingpeaks. However, separate FAME and OH-FAME profilescan readily be obtained by using a bonded-silica column forfractionation (see Materials and Methods).We found that, altogether, 29 OH-FAs were distributed

among the 76 strains of Legionella (representing 29 species)examined (Table 2). These were identified by comparingtheir chromatographic and mass spectrometric propertieswith those of standards, and the data obtained were gener-

ally in agreement with those obtained by Mayberry (11, lla).The plots shown in Fig. 1 were the result of the SIMCA

principal component analysis of the OH-FA data. In thisprocess, each of the 76 OH-FA strain profiles was reduced tosingle points (numbers). Hence, the numbers indicate theDNA group affiliation of the strains, according to Brenner(1), and the distance between points reflects OH-FA relat-edness between individual strains.

Also, as shown in Fig. 1, the separation along principalcomponents 1, 2, and 3 accounted for 56.2, 14.2, and 8.1%,respectively, of the original FA variation among the strains.With the aid of these plots and the results of the "fuzzy"clustering process (Table 3), four OH-FA groups weredefined.Group 1, defined by the 20 strains of L. pneumophila

(DNA group 5) plus the L. israelensis strain (DNA group 11),was found to be relatively distinct, although affiliation ingroup 1 was seen for strains of both group 4 (Fig. 1A) andgroup 2 (Fig. 1B). The L. israelensis strain showed a

distinctly weaker association to the group than did the L.pneumophila strains (see also Table 3). Compared with theother 2,3-di-OH-FA-containing group, group 2, the maindifferences were lower amounts of 2,3-di-OH-al5:0, 3-OH-a21:0, 3-OH-n21:0, 3-OH-n22:0, and 3-OH-a23:0 in group 1strains (Table 2). The main distinguishing OH-FA character-istics of group 1 versus groups 3 and 4 were the presence ofthe three 2,3-di-OH-FAs in group 1.Group 2, the other cluster with 2,3-di-OH-FA-containing

strains, comprised the strains L. micdadei (DNA group 6)and L. maceacherneii (DNA group 9), i.e. the two speciesthat have been proposed to be transferred to a separategenus, Tatlockia (4). In addition to the presence of 2,3-di-OH-FAs, the distinguishing features of group 2 strains werethe presence of relatively large amounts of the long-chainconstituents 3-OH-a21:0, 3-OH-n21:0, 3-OH-n22:0, and3-OH-a23:0.Group 3 appeared as a homogeneous cluster well sepa-

rated from the others. All 23 strains except the 1 strain of L.

TABLE 2. OH-FA characteristics of groups 1 to 4a

PercenthFatty acidc Group 1 Group 2 Group 3 Group 4

(21)d (16) (22) (17)

3-OH-n12:0 - - 0-0.5 0-43-OH-n13:0 0-1 0-1 0-2 0.5-42,3-di-OH-i14:0 5-14 2-11 - -

3-OH-i14:0 14-34 14-25 tr-2 5-412,3-di-OH-n14:0 0.5-2 0-3 -

3-OH-n14:0 0.5-3 2-16 6-20 2-122,3-di-OH-al5:0 tr-3 3-9 -

3-OH-i15:0 - - - 0-13-OH-a15:0 1-5 1-13 1-14 2-383-OH-n15:0 - 0-2 4-15 0-23-OH-i16:0 tr-3 0-2 4-30 0-23-OH-n16:0 tr-2 tr 4-15 0-43-OH-i17:0 tr-6 0-1 0-11 0-23-OH-a17:0 0-3 0-1 1-13 0-43-OH-n17:0 tr-7 - 0-6 0-23-OH-i18:0 tr-3 0-3 0-4 0-63-OH-n18:0 1-8 tr-2 6-15 1-133-OH-i19:0 tr-2 0-2 0-1 0-23-OH-a19:0 0.5-6 0-2 0-10 0-103-OH-n19:0 1-4 0-4 1-8 0.5-73-OH-i20:0 0.5-5 tr-2 0-1 0-63-OH-n20:0 6-20 7-12 1-15 7-283-OH-i21:0 tr-1 0-1 0-1 0-23-OH-a21:0 1-4 3-10 - 1-103-OH-n21:0 2-6 4-16 0-5 1-113-OH-i22:0 tr-6 1-4 0-0.5 0-43-OH-n22:0 1-4 5-12 0-0.5 0-113-OH-a23:0 tr-2 4-11 - 0-33-OH-n23:0 - 0-1 - 0-0.5

a See Table 1 and text for definition of the groups.b The amounts are given as ranges among strains (percent [wt/wt] of the

total); tr, less than 0.5%; -, not detected.I 3-OH, a hydroxyl group in the 3 position; 2,3-di-OH, hydroxyl groups in

the 2 and 3 positions; n, normal chain (unbranched); i, isomethyl branchedchain; a, anteisomethyl branched chain; number before colon, number ofcarbon atoms in chain; number after colon, number of double bonds.

d Values in parentheses are the number of strains investigated.

dumoffii gave an intragroup affinity value of greater than 80%(Table 3). The group comprised all strains of the 11 speciesbelonging to DNA group 2 that we examined, together withstrains of the two novel species L. gratiana and L. tucson-ensis, which have an unknown DNA group affiliation. Thethree species L. bozemanii, L. dumoffli, and L. gormanii,which have been proposed to be transferred to the genusFluoribacter (11), were found to be in this group. Its maindistinguishing features were a lack of 2,3-di-OH-FAs, 3-OH-i14:0 at less than 3%, 3-OH-n14:0 at greater than 5%,3-OH-n15:0 at greater than 2%, and minute amounts ofOH-FAs with chain lengths of more than 20 carbon atoms.Group 4 encompassed the strains of the remaining species,

representing 10 DNA groups plus the four species L. bir-minghamensis, L. brunensis, L. moravia, and L. quinlivaniiof unknown DNA association. As seen in Table 3, this groupwas more heterogeneous than the three others and wouldprobably split into several subgroups if more strains of eachspecies were added to the data matrix and reanalyzedseparately. Group 4 lacked 2,3-di-OH-FAs, contained 3-OH-i14:0 at concentrations of greater than 5%, and, in mostcases, significant amounts of 3-OH-n12:0, 3-OH-n13:0,3-OH-a21:0, and 3-OH-n21:0.

Figure 2 provides the relative contribution of the OH-FAsto the separations along the three principal components

J. CLIN. MICROBIOL.

on June 5, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

HYDROXY-FAITY ACIDS OF LEGIONELLA SPECIES 1417

A

8 ° °-1. o0

0.60

80.0

-0.6so0

-A. W0

BA1.0

A

8 0.00

j-0. so

-1.60I

01.00.

^ 0.so

r

u

-1.00

1

l n ° pA

3

Iaa&\x-

-1.W0 -0.*0 0.00 0.60 1.W0Co.qponnt 1. Scores (5. 2%)

3bt

22

2.Sr2

2p 24~~~1Ic46 62 2 2rj b

-1.O -.60 0.0 0.022o~nn .Soe 5.%

-0. 0.00 0. 50

-it 2. Score 14.2%)

1.00

FIG. 1. Principal component plots based on the OH-FA compo-sitions of 76 strains representing 29 Legionella species. (A) Compo-nent 1 versus component 2. (B) Component 1 versus component 3.(C) Component 2 versus component 3. Four clusters (OH-FAgroups 1 to 4) are marked. Each number represents one strain and its

10

10

lr

TABLE 3. OH-FA group affiliation of Legionella speciesobtained by fuzzy clustering

% Group affinity for the followingSpecies DNA OH-FA groupb:group'

1 2 3 4

L. pneumophila (20)C 5 70-100 0-12 0-4 0-14L. micdadei (10) 6 0-10 79-99 0-2 0-8L. maceachernii (5) 9 2-7 83-95 1-3 2-7L. israelensis (1) 11 53 19 9 20L. anisa (3) 2 0-1 0-1 96-98 0-2L. bozemanii (2) 2 2-3 2-2 89-93 4-6L. cherrii (4) 2 0-3 0-2 91-99 1-4L. cincinnatiensis (1) 2 1 1 96 2L. dumoffii (1) 2 7 5 77 12L. gormanii (1) 2 4 3 87 6L. gratiana (1) 4 2 89 5L. longbeachae (4) 2 0-5 0-3 86-99 1-6L. parisiensis (1) 2 0 0 100 0L. sainthelensi (1) 2 3 2 90 4L. steigerwaltii (1) 2 2 1 95 2L. tucsonensis (1) 3 2 91 4L. wadsworthii (2) 2 5-5 3-4 83-85 7-8L. birminghamensis (1) 22 21 6 51L. brunensis (1) 12 15 10 64L. erythra (1) 1 8 3 4 84L. feeleii (3) 3 7-13 6-8 4-5 74-83L. hackeliae (2) 10 10-14 10-10 8-12 64-72L. jamestowniensis (1) 4 12 12 11 64L. jordanis (2) 7 27-34 10-11 10-12 46-50L. moravica (1) 25 9 15 51L. oakridgensis (2) 8 18-19 6-6 7-7 68-69L. quinlivanii (1) 3 2 2 93L. rubnlucens (1) 1 12 5 4 79L. spintensis (1) 1 25 12 24 40

a DNA homology group (25%) according to Brenner (1).b The four groups (classes) defined on the basis of principal component

analysis (Table 1 and Fig. 1) were specified for the fuzzy clustering routine ofthe SIRIUS program (see Materials and Methods). Values in boldface typeindicate best fit. Group affinity numbers (percent), recorded as range amongstrains were calculated by the fuzzy clustering program on the basis of thescores from the principal component analysis.

c Values in parentheses are number of strains examined.

given in Fig. 1. Thus, Fig. 2A shows the contribution toseparation along principal component 1, and the large barsshow that 2,3-diOH-i14:0, 3-OH-i14:0, 3-OH-n22:0 (positive)and 3-OH-n15:0, 3-OH-i16:0, and 3-OH-n16:0 (negative)were the most important contributors to the separation alongthis component (x axes in Fig. 1A and B). Values withpositive and negative signs suggest that the two groups ofFAs were negatively correlated. Evidently, the three uniquedi-OH-FAs contributed substantially to the separation ofboth principal components 1 and 3, whereas 3-OH-i14:0 and3-OH-a23:0 were the two major contributors to the separa-tion along principal component 2.

In a recent taxonomic study based on 16S rRNA sequence

DNA group affiliation: 1, L. erythra; lb, L. rubrilucens lc, L.spiritensis; 2, L. anisa, L. cincinnatiensis, L. dumoffii, L. gormanii,L. longbeacheae, L. parisiensis, L. sainthelensi, L. steigerwaltii; 3,L. feeleii; 4, L. jamestowniensis; 5, L. pneumophila; 6, L. micdadei;7, L. jordanis; 8, L. oakridgensis; 9, L. maceachemeii; 10, L.hackeliae; 11, L. israelensis. Abbreviations for species of unknownDNA group affiliation are as follows: bi, L. birminghamensis; br, L.brunensis; gr, L. gratiana; mo, L. moravica; qu, L. quinlivanii; tu,L. tucsonensis.

VOL. 31, 1993

on June 5, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

A0.30-

S 0.20

0 O.10

0 -O. 1.0

-I

WI

-0. 20

-0. 30

0.20

..I 0.10.

IJ, 0.00

Cl44'

I -0.10

-0. 20

-0. 30

h VI * * 0V4

If V4VI VI 0V4 V4

0 0C C C 4 S4 C - C " 5 C C 4S C < C 4S C u C a C

1 ~ ~ A LLkb a -ZAkXX X.1 -22

C c . CC a5. C - C -. M e C -. C eC C a CM C I C

c 0.60 -

0.50

p 0.40C

20.3.14

-0. 10-

-0.20-

FIG. 2. Contribution of each OH-FA to the principal component plots of Fig. 1. See Table 2 for more complete assignments of OH-FAs(corresponding order). Large bars such as il4 (3-OH-il4:O, positive) and i16 (3-OH-il6:O, negative) in panel A represent the most importantcontributions. The sign implies only that the two groups of OH-FAs are negatively correlated.

1418

7 FLUr

I

F--.

on June 5, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

HYDROXY-FATTY ACIDS OF LEGIONELLA SPECIES 1419

determinations, it was demonstrated that members of thefamily Legionellaceae form a relatively homogeneous taxonlocated separately and distantly to its closest neighbors,Coxiella burnetii and Wolbachia persica (6). The results ofthe present study reinforce this taxonomic concept, since allLegionella strains that we examined, as well as C. burnettii(although to a lesser extent, with just nine 3-OH-FAs [18]),shared a very complex OH-FA pattern not seen in othergroups of gram-negative bacteria.The high degree of similarity of the OH-FA patterns of the

species associated in DNA group 2 (group 3, Fig. 1) is alsonoteworthy. Both phylogenetic evidence (6) and the pheno-typic data presented here indicate that the number of specieswithin this taxon may be unnecessarily high. Additionally,the arguments for creating a genus (Fluonbacter) comprisingL. bozemanii, L. dumofii, and L. gormanii was not sup-ported by the results of the present study.The two species L. micdadei and L. maceachemeii, which

have been proposed to be transferred to a new genus,Tatlockia (4), formed OH-FA group 2 and, accordingly, werealso closely related in terms of their OH-FA compositions.However, the high degree of resemblance of the OH-FAs ofthe two species to those of the other legionellae did notsupport a new genus subdivision of the family Legionel-laceae.The reproducibility of the OH-FA profile method is indi-

rectly demonstrated in Fig. 1 and Table 3. Thus, all 20 strainsof L. pneumophila and 10 strains of L. micdadei that weexamined clustered in two distinct and fairly close groups.The tight clustering of all DNA group 2-associated species(OH-FA group 3) also supports the reliability of the method.Thus, OH-FA profiling provides a useful diagnostic tool forthe allocation of a strain to the genus Legionella as well as toa distinct Legionella species or group of species.

ACKNOWLEDGMENTS

We thank B.-P. Berdal, Norwegian Defence Microbial Labora-tory, Oslo, Norway; N. Bornstein, National Public Health Labora-tory, Lyon, France; R. J. Fallon, Ruchill Hospital, Glasgow, UnitedKingdom; E. Falsen, Department of Clinical Bacteriology, Uni-viversity of G0teborg, G0teborg, Sweden; K. Fox, Department ofMicrobiology and Immunology, University of South Carolina, Co-lumbia; and I. Kallings, National Bacteriological Laboratory, Stock-holm, Sweden, for providing strains.The work was supported by the Royal Norwegian Concil for

Science and Industrial Research.

REFERENCES1. Brenner, D. J. 1987. Classification of the Legionellae. Semin.

Respir. Infect. 2:190-205.2. Fallon, R. J. 1990. Legionella, p. 275-287. In M. T. Parker and

L. H. Collier (ed.), Topley & Wilson's Principles of Bacteriol-ogy, Virology, and Immunology, vol. 2 Edward Arnold, Lon-don.

3. Fang, G.-D., V. L. Yu, and R. M. Vickers. 1989. Disease due tothe Legionellaceae (other than Legionella pneumophila). His-

torical, microbiological, clinical, and epidemiological review.Medicine 68:116-132.

4. Fox, K. F., A. Brown, A. Fox, and G. Schnitzer. 1991. Tatlockia,a genetically and chemically distinct group of bacteria. Proposalto transfer Legionella maceachemii (Brenner et al.) to the genusTatlockia, as Tatlockia maceachernii comb. nov. Syst. Appl.Microbiol. 14:52-56.

5. Fraser, D. W., T. R. Tsai, W. Orenstein, W. E. Parkin, H. J.Beecham, R. G. Sharrar, J. Harris, G. F. Mallison, S. M.Martin, J. E. McDade, C. C. Shepard, P. S. Brachman, and FieldInvestigation Team. 1977. Legionnaires' disease: description ofan epidemic of pneumonia. N. Engl. J. Med. 297:1189-1197.

6. Fry, N. K., S. Warwick, N. A. Saunders, and T. M. Embly. 1991.The use of 16S ribosomal RNA analyses to investigate thephylogeny of the family Legionellaceae. J. Gen. Microbiol.137:1215-1222.

7. Garity, G. M., A. Brown, and R. M. Vickers. 1980. Tatlockiaand Fluoribacter: two new genera of organisms resemblingLegionella pneumophila. Int. J. Syst. Bacteriol. 30:609-614.

8. Jantzen, E., and K. Bryn. 1985. Whole-cell and lipopolysaccha-ride fatty acids and sugars of gram-negative bacteria, p. 145-171. In M. Goodfellow and D. Minnikin (ed.), Chemical meth-ods in bacterial systematics. Academic Press, London.

9. Jantzen, E., 0. M. Kvalheim, T. A. Hauge, N. Hagen, and K.B0vre. 1987. Grouping of bacteria by SIMCA pattern recogni-tion on gas chromatographic lipid data: patterns among Mo-raxella and rod-shaped Neisseria. Syst. Appl. Microbiol. 9:142-150.

10. Lambert, M. A., and C. W. Moss. 1989. Cellular fatty acidcompositions and isoprenoid quinone contents of 23 Legionellaspecies. J. Clin. Microbiol. 27:465-473.

11. Mayberry, W. R. 1984. Monohydroxy and dihydroxy fatty acidcomposition of Legionella species. Int. J. Syst. Bacteriol.34:321-326.

11a.Mayberry, W. R. Personal communication.12. Mayer, H., J. H. Krauss, A. Yokota, and J. Weckesser. 1990.

Natural variants of lipid A. Adv. Exp. Med. Biol. 256:45-70.13. Mitchell, K., and R. J. Fallon. 1990. The determination of

ubiquinone profiles by reversed-phase high-performance thin-layer chromatography as an aid to the speciation of Legionel-laceae. J. Gen. Microbiol. 136:2035-2041.

14. Sonesson, A., E. Jantzen, K. Bryn, L. Larsson, and J. Eng. 1989.Chemical composition of a lipopolysaccharide from Legionellapneumophila. Arch. Microbiol. 153:72-78.

15. Thacker, W. L., R. F. Benson, L. Hawes, H. Gidding, B. Dwyer,W. R. Mayberry, and D. J. Brenner. 1991. Legionella fairfield-ensis sp. nov. isolated from cooling tower waters in Australia. J.Clin. Microbiol. 29:475-478.

16. Wilkinson, I. J., N. Sangster, R. M. Ratcliff, P. A. Mugg, D. E.Davos, and J. A. Lanser. 1990. Problems associated with iden-tification ofLegionella from the environment and isolation of sixpossible new species. Appl. Environ. Microbiol. 56:796-802.

17. Wold, S., C. Albano, W. J. Dunn m, U. Edlund, K. Esbensen, P.Geladi, S. Hellberg, E. Johanson, W. Linberg, and M. Sj0str0m.1984. Multivariate data analysis in chemistry, p. 17-95. In B. R.Kowalski (ed.), Chemometrics, mathematics and statistics inchemistry. Reidel, Dordrecht, The Netherlands.

18. Wollenweber, H.-W., S. Schramek, H. Moll, and E. T. Rietschel.1985. Nature and linkage type of fatty acids present in lipopoly-saccharides of phase I and II Coxiella bumetti. Arch. Microbiol.142:6-11.

VOL. 31, 1993

on June 5, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from