Vol. 29, No. 1JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1991, p. 183-1890095-1137/91/010183-07$02.00/0Copyright C 1991, American Society for Microbiology

Multivariate Analyses of Cellular Fatty Acids in Bacteroides,Prevotella, Porphyromonas, Wolinella, and Campylobacter spp.

ILIA BRONDZlt* AND INGAR OLSEN2

Research Department, National Institute of Occupational Health, Umea, Sweden,' and Department of Microbiology,Dental Faculty, University of Oslo, Oslo, Norway2

Received 5 March 1990/Accepted 2 October 1990

The genera Bacteroides, Wolinella, and Campylobacter contain several similar species that require taxonomicrevision. Fatty acid profiles of whole bacterial cells have proven useful for taxonomy. In this study, cellularfatty acids from Bacteroides, Prevotella, Porphyromonas, Wolinella, and Campylobacter spp. were identified andquantitated by gas chromatography and gas chromatography-mass spectrometry, and the data were subjectedto principal component analyses. Bacteroides fragilis, the type species of the genus Bacteroides, was distinctfrom the other organisms. While Bacteroides gracilis, Wolinella succinogenes, WolineUa curva, Wolinella recta,and Campylobacterfetus subsp. venerealis were close to each other, Prevotella (Bacteroides) buccae, Prevotellaoralis, Prevotella oris, Prevotella disiens, Prevotella veroralis, Prevotella heparinolyticus, Porphyromonas(Bacteroides) endodontalis, and Bacteroides ureolyticus could be distinguished. B. fragilis was characterized bythe presence of C30H-i-17, C. 15, and C,-15 and the absence of C12:0 and unsaturated fatty acids. For comparison,B. gracilis, B. ureolyticus, W. succinogenes, W. curva, W. recta, and Campylobacter fetus subsp. venerealis

contained C12:0, C16:1, C18:1, and C30H-14 acids but lacked branched hydroxy and branched nonhydroxy acids.B. gracilis and B. ureolyticus are not "true" bacteroides.

Species of the genera Bacteroides, Wolinella, and Cam-pylobacter are often detected by culture of specimens frompatients with periodontal diseases (14, 25, 37, 41). Wolinellarecta has attracted much attention as an agent recoveredfrom active periodontal lesions. Also, Bacteroides gracilisand Bacteroides ureolyticus have been isolated from dis-eased periodontal sites (14, 37). W. recta, Wolinella curva,B. gracilis, and B. ureolyticus have also been implicated asetiologic agents in extraoral infections, such as superficialulcers, soft tissue infections, and urethritis, and have beenisolated from chest wall masses (13, 15, 16, 19, 20, 31, 38).Campylobacters are recognized as important agents of re-productive diseases in sheep and cattle, but some specieshave been associated with gastroenteritis, diarrhea, bloodinfections, proctitis, proctocolitis, enteritis, and bacteremiain humans (for a review, see reference 46).

Recently, it was proposed that only members of theso-called "Bacteroides fragilis group," which includes B.fragilis and closely related species, be retained in the genusBacteroides because these species share several basic bio-chemical and physiological characteristics that are distinctfrom those of other species that were formerly included inthe genus (12, 34). For pigmented, asaccharolytic species,the genus Porphyromonas was recently created (33), and formoderately saccharolytic species like Bacteroides melanino-genicus, Bacteroides oralis, and related species, the genusPrevotella was proposed (35). Reclassification of other Bac-teroides species has also been proposed (for a review, seereference 35). Furthermore, rRNA sequencing studies havesuggested that B. gracilis and B. ureolyticus are not "true"Bacteroides species (26).By partial 16S rRNA sequencing, Wolinella succinogenes

was found to be closely related to Campylobacter pylori (23,

* Corresponding author.t Present address: Norwegian Plant Protection Institute, Box 70,

1432 As-NLH, Norway.

26, 46), while W. recta, W. curva, B. gracilis, and B.ureolyticus belonged to the same cluster of organisms as thatof the true campylobacters (26). Similar studies showed thatspecies of the genus Campylobacter consisted of threerRNA homology groups which could not easily be differen-tiated by phenotypic characteristics (46).

Phenotypic characterization of asaccharolytic gram-nega-tive rods is often not differential. Usually, classification ofsuch organisms is based on colony and cell morphologies,growth characteristics, antibiotic susceptibilities, biochemi-cal tests, and G+C contents (1, 18, 36, 39, 40, 42, 43).Because these organisms do not catabolize carbohydratesand are inert in most traditional identification tests, differ-entiation between them has been based on only a few traits.For example, W. recta differs from W. succinogenes in itscellular and ultrastructural morphology, serological reac-tions, and susceptibility to dyes and antibiotics (40). W.recta and B. gracilis are metabolically identical and differonly in motility and cell morphology (42). W. recta is motileby means of a single polar flagellum (22), whereas B. gracilishas no flagella but moves by twitching cell motility. Whereascells of B. gracilis often have tapered ends, cells of W. rectahave rounded ends. Problems with species identification inWolinella, Bacteroides, and Campylobacter spp. have beensolved by using serological techniques (1), protein profiles(39), and DNA-DNA hybridization (30, 39, 41, 44), whileassignment of species to the genus level has been checked byrRNA sequencing (23, 26, 46). In the present study, we usedgas chromatography and gas chromatography-mass spec-trometry of whole-cell fatty acids to assess the taxonomicrelationships between W. succinogenes, W. recta, W. curva,B. gracilis, B. ureolyticus, and Campylobacter fetus subsp.venerealis. Another purpose of this study was to increase thenumber of reliable characteristics for classification and iden-tification of organisms within the genera Wolinella, Cam-pylobacter, Bacteroides, Prevotella, and Porphyromonas.Fatty acids are well-established as being useful criteria formaking taxonomic distinctions between microorganisms

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184 BRONDZ AND OLSEN

TABLE 1. Bacteria investigated in this study

Species Straina Isolation site

Prevotella (Bacteroides) buccae ES9-1 Dental root canalES12-B Dental root canalES17-1 Dental root canalATCC 33574T Gingival sulcus

Prevotella (Bacteroides) oris ES14B-3A Dental root canalES9-3 Dental root canalATCC 33573T Gingival sulcusRPG Dental root canal

Prevotella (Bacteroides) oralis ES4-B Dental root canalES14B-3A Dental root canalES15-2 Dental root canal

Prevotella (Bacteroides) disiens DSM 20516T Bartholin abscessPrevotella (Bacteroides) veroralis ATCC 33779T Oral cavityPrevotella (Bacteroides) heparinolyticus ATCC 35895T Periodontal pocketBacteroides fragilis ATCC 25285T Appendix abscessPorphyromonas (Bacteroides) endodontalis HG 412 Dental root canal

HG 370T (ATCC 35406T) Dental root canalHG 182 (BN 11a-f) Dental root canalHG 181 (H 11a-e) Dental root canal

Bacteroides gracilis CCUG 13143T (ATCC 33236T) Periodontal pocketCampylobacterfetus subsp. venerealis CCUG 538T (ATCC 19438T) Vaginal mucosa of heiferBacteroides ureolyticus CCUG 7319T (ATCC 33387T) Amniotic fluidWolinella succinogenes CCUG 12550T (ATCC 29543T) Bovine rumenWolinella curva CCUG 13146T (ATCC 35224T) Human alveolar (or jaw) abscessWolinella recta FDC 371T (ATCC 33238T) Periodontal pocket

a Abbreviations: ES, phenotypically characterized by M. Haapasalo; ATCC, American Type Culture Collection, Rockville, Md.; DSM, German Collection ofMicroorganisms and Tissue Cultures, Braunschweig, Federal Republic of Germany; HG, phenotypically characterized by A. J. van Winkelhoffand G. Sundqvist;CCUG, Culture Collection, University of Gothenburg, Gothenburg, Sweden; FDC, Forsyth Dental Center, Boston, Mass.

(24), and they circumvent the problem of biochemical inert-ness of cells. Fatty acid data were subjected to multivariatestatistical analyses. Previously, we have used multivariateanalyses to define genera of bacteria and yeasts such asPorphyromonas (2); Bacteroides (7a); Actinobacillus, Hae-mophilus, and Pasteurella (9); Treponema (2a); and Can-dida, Torulopsis, and Saccharomyces (7, 8).

MATERIALS AND METHODS

Organisms. The strains examined in this study are listed inTable 1. Except for W. recta, which was received fromA. C. R. Tanner, Forsyth Dental Center, Boston, Mass., allorganisms were obtained directly from the American TypeCulture Collection or the Culture Collection, University ofGothenburg, Gothenburg, Sweden. The fatty acid profiles ofthese organisms were compared with those of the Bacteroi-des, Prevotella, and Porphyromonas spp. that we examinedpreviously (7a). All organisms were cultured anaerobicallyand in duplicate on different days at 37°C for 2 to 5 days incommercially available Mycoplasma Base broth (BBL Mi-crobiology Systems, Cockeysville, Md.) supplemented withcysteine (0.5 g/liter). For culturing B. fragilis, the broth wasalso supplemented with hemin (5 mg/liter) and menadione(0.5 mg/liter), and for the remaining organisms, the brothwas supplemented with hemin, ammonium formate (2 g/li-ter), and disodium fumarate (3 g/liter). Bacterial cells wereharvested by centrifugation, washed in deionized, distilledwater, lyophilized, and stored at -20°C under N2.

Methanolysis and derivatization. Freeze-dried whole cells(1 mg) were methanolyzed with 1 ml of 2 M hydrochloricacid in anhydrous methanol for 24 h at 95°C (4). The resultingmethanolysate was dried with a stream of N2 in an ice bathand then extracted with 1 ml of n-hexane. Hydrochloric acid,methanol, and n-hexane, all pro analysis, were obtained

from E. Merck AG, Darmstadt, Federal Republic of Ger-many. The above procedure was used for routine examina-tion of fatty acid contents. For assessment of hydroxy fattyacids, the hydroxy groups of these acids were derivatizedwith trifluoroacetic acid anhydride after evaporation ofmethanol. Derivatization occurred in 1 ml of a solutioncontaining 1 part of trifluoroacetic acid anhydride (FlukaChemie AG, Buchs, Switzerland) and 3 parts of acetonitrile(Rathburn Chemicals, Walkerburn, United Kingdom) at90°C for 3 min. After derivatization, this solution was dilutedbefore gas chromatography with 1.5 ml of acetonitrile so thatit contained 10% trifluoroacetic acid anhydride (3).Gas chromatography and gas chromatography-mass spec-

trometry. A model 8700 gas chromatograph (Perkin-ElmerCorp., Norwalk, Conn.) was used for fatty acid analyses.The HP Ultra Performance Column Ultra 1 used was 25 m by0.20 mm (inner diameter). Helium served as the carrier gas ata flow rate of 2.0 ml/min. The temperature of the injector was200°C, and the temperature of the flame ionization detectorwas 275°C. The program was as follows. The temperaturewas held for 1 min at 90°C and was then increased from 90 to275°C at a rate of 6°C/min. The attenuator was set at 16. Thepaper speed was 5 mm/min. The sample (0.2 RI) was deliv-ered as a splitless injection. The identities of the methano-lyzed and derivatized hydroxy fatty acids were establishedby cochromatography of authentic standards and gas chro-matography-mass spectrometry (2, 3, 5). Three independentderivatizations were prepared from each duplicate sample.Each derivative was injected three times. The quantity of thesubstances, expressed as relative percent, was calculatedfrom the area under each peak and corrected with the molarresponse factor (3). The sum of the identified substances wasconsidered to be 100%. For identification of fatty acids,methyl ester standards of decanoic, dodecanoic, tetrade-canoic, pentadecanoic, hexadecanoic, and octadecanoic ac-

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MULTIVARIATE ANALYSES OF CELLULAR FATTY ACIDS 185

ids were obtained from Sigma Chemical Co., St. Louis, Mo.Bacterial acid methyl esters mixture CP (catalog no. 4-7080),gas-liquid chromatography standard mixture GLC 70 (cata-log no. 4-7044), American Oil Chemists Society oil referencemixture, RM-1 rapeseed (catalog no. 4-7019), National Insti-tutes of Health reference mixtures (catalog no. A-NHI-C4-7010, A-NHI-D 4-7011, and A-NHI-F 4-7013), and FA-FAME Kit 14 (catalog no. 1039) were obtained from SupelcoInc., Bellefonte, Pa. Furthermore, 13-methyltetradecanoicacid methyl ester, methyl-12-methyltetradecanoic acid ester,methyl-14-methylhexadecanoic acid ester, methyl-14-meth-ylpentadecanoic acid ester, methyl-15-methylhexadecanoicacid ester, and methyl-11-methyltridecanoic acid ester werereceived from Larodan Fine Chemicals, Malmo, Sweden.Phenol-water-extracted lipopolysaccharides from Porphy-romonas (Bacteroides) gingivalis ATCC 33277' (2) andActinobacillus actinomycetemcomitans ATCC 33384T (3)were also used as standards for identification of fatty acids.

Statistical analyses. In this study, principal componentanalysis (21, 49) was used by which the original space forvariable measurements was projected down onto two low-dimensional subspaces. One of these was sample related; theother was variable related. By plotting the sample-orientedparameters t, and t2 against each other, information on thedominating behavior of the samples was gained, and simi-larly, by plotting the variable loadings Pi versus P2' infor-mation was obtained on which variables contributed to thecorresponding sample-oriented projection. The complexitiesof the models were determined by cross-validation (48, 49).

RESULTS

Gas chromatography and gas chromatography-mass spec-trometry. The distributions of fatty acids in whole-cell meth-anolysates from species of Bacteroides, Prevotella, Porphy-romonas, Wolinella, and Campylobacter are shown in Table2. Eighteen different fatty acids, including normal fattyacids, saturated and unsaturated fatty acids, iso- and an-teiso-branched fatty acids, and hydroxy straight-chain andbranched-chain fatty acids, were detected. The distributionsof cellular fatty acids in Prevotella (Bacteroides) buccae,Prevotella oris, Prevotella oralis, Prevotella disiens, Prevo-tella veroralis, Prevotella heparinolyticus, B. fragilis, andPorphyromonas (Bacteroides) endodontalis have been de-scribed elsewhere (7a). B. gracilis, B. ureolyticus, W. suc-cinogenes, W. curva, W. recta, and C. fetus subsp. venere-alis differed from these organisms by containing dodecanoicacid (C12:0), 3-hydroxytetradecanoic acid (C30H-14), hexade-cenoic acid (C16:1), and octadecenoic acid (C18:1). Contraryto the other Bacteroides, Prevotella, or Porphyromonas spp.examined, B. gracilis and B. ureolyticus, as well as W.succinogenes, W. curva, W. recta, and C. fetus subsp.venerealis, lacked 13-methyltetradecanoic acid (Ci-15), 12-methyltetradecanoic acid (Cai15), and 3-hydroxy-15-methyl-hexadecanoic acid (C30H i 17). It was also noteworthy that inB. ureolyticus octadecenoic acid constituted 52.8% of thecellular fatty acid content. Hexadecanoic acid was the majorfatty acid in W. recta (35.6%), W. curva (31.9%), and C.fetus subsp. venerealis (36.5%). The principal acid in B.gracilis was tetradecanoic acid (28.0%), and the principalacid in W. succinogenes was hexadecenoic acid (29.6%).

Statistical analyses. In the principal component analysis,the two principal score vectors t1 and t2 were plotted againsteach other (Fig. 1). This sample-oriented (i.e., bacterialstrain-oriented) projection described the two largest variantsof the data matrix (Table 2). The two principal components

t1 and t2 constituted 32.0 and 14.8%, respectively, of thetotal variance in the matrix. In this projection, C. fetussubsp. venerealis, W. succinogenes, W. curva, and W. rectaformed a homogeneous cluster that was fairly close to B.gracilis, while B. ureolyticus was more distant. B. fragilisand the remaining species tested, P. buccae, P. oris, P.oralis, P. disiens, P. veroralis, P. heparinolyticus, andPorphyromonas endodontalis, appeared to be remote fromC. fetus subsp. venerealis, W. succinogenes, W. curva, W.recta, B. gracilis, and B. ureolyticus. In this projection,previous Bacteroides spp. that were proposed to belong tothe genera Prevotella (35) and Porphyromonas (33) weredistant from B. fragilis in an intermediate position.

In Fig. 2 the first principal component t, and the thirdprincipal component t3 were plotted against each other,representing 32.0 and 10.8%, respectively, of the variance inthe fatty acid data. This sample-oriented projection de-scribed the largest and third largest variants in the datamatrix. In this projection, B. ureolyticus appeared to bedistant from all the other species. Campylobacter fetussubsp. venerealis, W. succinogenes, W. curva, and W. rectaformed a homogeneous cluster that was distinguished fromall the other organisms, except B. gracilis, which was close.B. ureolyticus, B. gracilis, C. fetus subsp. venerealis, W.succinogenes, W. curva, and W. recta appeared to be remotefrom B. fragilis and the Prevotella and Porphyromonas spp.examined (P. buccae, P. oris, P. oralis, P. disiens, P.veroralis, P. heparinolyticus, and Porphyromonas endodon-talis). B. fragilis was clearly distinct from Bacteroides spp.proposed previously to belong to the genera Prevotella (35)and Porphyromonas (33).

In Fig. 3 the second principal component t2 was plottedagainst the third principal component t3. This sample-relatedprojection described the second and third largest variants ofthe fatty acid data, representing 14.8 and 10.8%, respec-tively, of the data variance. In this projection B. ureolyticuswas remote from all the other species examined. B. gracilis,C. fetus subsp. venerealis, W. succinogenes, W. curva, andW. recta formed a homogeneous cluster fairly close to P.oris. P. buccae and P. oralis appeared to be more remote.Most distant were B. fragilis and Porphyromonas endodon-talis, P. heparinolyticus, P. veroralis, and P. disiens. Also inthe projection in Fig. 3, B. fragilis was distant from previousBacteroides spp. proposed to belong to the genera Prevotella(35) and Porphyromonas (33).

DISCUSSION

The present study, which used multivariate statisticalanalyses of cellular fatty acids to study the inter- andintrageneric relationships of the genera Bacteroides, Prevo-tella, Porphyromonas, Wolinella, and Campylobacter, dem-onstrated that B. fragilis, the type species of the genusBacteroides, is distinct from all the Prevotella and Porphy-romonas spp. examined. There is biochemical, chemical,and molecular biochemical evidence that the genus Bacteroi-des should be restricted to the type species and closelyrelated organisms such as B. caccae, B. distasonis, B.eggerthii, B. merdae, B. ovatus, B. stercoris, B. thetaio-taomicron, B. uniformis, and B. vulgatus (12, 34). Results ofthe present study supported such a restriction of the genusBacteroides.A characteristic feature of B. fragilis was its tendency to

form branched-chain hydroxy (C30H i 17) and branched-chain nonhydroxy (Ca 15 and Ci 15) fatty acids. WhileC30H-17 was the predominant fatty acid, Ca 15 was more

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186 BRONDZ AND OLSEN

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MULTIVARIATE ANALYSES OF CELLULAR FATTY ACIDS 187

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FIG. 1. Sample-oriented (i.e., bacterial strain-oriented) principalcomponent projection (t1 versus t.). These two principal compo-nents describe 32.0 and 14.2%, respectively, of the variance in thefatty acid data given in Table 2. Samples include P. (Bacteroides)buccae (samples 1 to 4), P. oris (samples 5 to 8), P. oralis (samples9 to 11), P. disiens (sample 12), P. ileroralis (sample 13), P.heparinolyticus (sample 14), B. fragilis (sample 15), Porphyrornonas(Bacteroides) endodontalis (samples 16 to 19), B. gracilis (sample20), C. fetlus subsp. v'enerealis (sample 21), B. ureolyticus (sample22), W. succinogenes (sample 23), W. curva (sample 24), and W.recta (sample 25). Samples representing the same or closely relatedspecies are surrounded by circles. For further description of sam-ples, see Table 1.

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prevalent than Ci-15. This may be important characters oftrue Bacteroides species, since none of the other speciesexamined fulfilled all these criteria. Only a small amount ofbranched-chain fatty acids has been found in species previ-ously classified as Bacteroides such as Ruminobacter amy-lophilus and Fibrobacter succinogenes, while no branched-chain acids have been detected in Anaerorhabdius fiurcosus,Megamonas hypermegas, or Mitsuokella miultiacidus (47).On the principal component projections, B. fragilis was

distant from P. biuccae, P. oris, P. oralis, P. disiens, P.veroralis, and P. heparinolyticius. These moderately saccha-rolytic, predominantly oral organisms form a group of phe-notypically and phylogenetically coherent species whichdiffer so significantly from the emended description of thegenus Bacteroides sensu stricto (34) that a new genus,Prevotella, was recently proposed for them (35). The multi-variate fatty acid analyses presented here supported such adistinction, as well as that of pigmented asaccharolyticorganisms grouped in the genus Porphyromonas (33).

B. iureolyticius was remote from B. fragilis. The predomi-nant fatty acid of B. iureolyticius was Cl8.l which constituted52.8% of the fatty acid content. B. ureolyticlus has beenreported to be atypical in that C18.1 accounted for about 70%of the total fatty acids (27) and because it had a DNA basecomposition (28 to 30 mol% G+C) much lower than those ofother Bacteroides spp. (typically 40 to 55 mol%) (19). Also,DNA-DNA homologies, biochemical tests, protein profiles,and rRNA studies (26, 32, 45) have suggested that B.ureolyticius should be removed from the genus Bacteroides.Oral isolates of B. uireolyticus may represent a distinct groupof species that have not yet been defined (13).The fact that B. gracilis differed markedly from B. fragilis

suggested that B. gracilis is not a true Bacteroides spp.either. This agreed with rRNA sequencing studies (26).While B. gracilis differed from B. ureolyticus, it was close

to C. fetus subsp. i'enerealis, W. siuccinogenes, W. curva,and W. recta. Optimally, the fatty acid contents of at least

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188 BRO&DZ AND OLSEN

five strains from each species should be subjected to multi-variate analyses in order to determine their exact positions.This objection was met to some extent by the use of typestrains and subdivisions of the type strains. In order toclarify further the relationships between B. gracilis and thewolinellas and campylobacters, we are extending our chem-ical analyses to lipopolysaccharides, which seem to beexcellent preparations for determination of the chemotaxon-omies of closely related organisms (6).

In the present study, W. recta and W. currva were veryclose to W. succinogenes, the type species of the genusWolinella, and to C. fetus subsp. i'enerealis, which is asubdivision of C. fetus, the type species of the genusCampylobacter. The similarities in the lipid contents of theseorganisms were also reflected through the discovery that W.succinogenes contains both menaquinone-6 and a novelmethyl 1 derivative (ring substituted at position 5 or 8 of thenaphthoquinone nucleus) of menaquinone-6 as in Campylo-bacter spp. (and in B. gracilis) (11). The multivariate analy-ses of fatty acid data supported the rRNA sequencing studiesby Lau et al. (23), who found that W. succinogenes is relatedto the campylobacters. Other rRNA sequencing studies haveconcluded that W. recta and W. curva belong to the samecluster of organisms as true campylobacters, whereas W.succinogenes is closely related to C. pylori, which is not atrue Campylobacter spp. (10, 17, 23, 26, 29, 46). The presentfatty acid data from type species and subdivisions of typespecies did not support the maintenance of Wolinella andCampylobacter as distinct genera. More species and testsshould be included, however, before this issue can besettled.Another striking feature of B. ureolyticus, B. gracilis, W.

succinogenes, W. cuirva, W. recta, and C. fetus subsp.venerealis was their unsaturated cellular fatty acids whichcould not be detected in the other species examined, al-though straight-chain fatty acids with one or more doublebonds have been detected in most microorganisms (28).From our findings in B. fragilis, it seems that true Bacteroi-des spp. lack unsaturated fatty acids. This view is supportedby studies from other representatives of the B. frtagilisgroup, such as B. distasonis, B. ot'atus, B. thetaiotaomi-cron, and B. vulgatus, which all lack unsaturated fatty acids(47). On the other hand, organisms previously classified asBacteroides spp., such as Ruminobacter amylophilus, Fibro-bacter succinogenes, Anaerorhabdus furcosuts, Megamonashypermegas, and Mitsuokella multiacidus, contain variousunsaturated fatty acids (47).

C30H-14 was found in B. gracilis and B. ureolyticus, andalso in W. succinogenes, W. recta, W. curma, and C. fetussubsp. venerealis, but not in the other species. Previously,C3OHl14 has been detected as a major fatty acid in Rumino-bacter amylophilus and Mitsuokella multiacidus (47).A major fatty acid (22.2%) in B. gracilis was dodecanoic

acid (C12:0). Also, B. iureolyticuts, W. succinogenes, W.recta, W. curva, and C. fetlus subsp. venerealis (0.6%)contained this fatty acid, but none of the other species did.Interestingly, Campylobacter sputoruim subsp. mucosalisdiffers from the other campylobacters by its significantamount (about 10% of the total acid) of C12:0 (47).

Although care should be taken when drawing firm taxo-nomic conclusions from fatty acid content alone, because itdepends to some extent on medium composition and growthconditions, it seems that multivariate analyses of data fromcellular fatty acid contents may contribute significantly toour understanding of the taxonomy of biochemically inertgram-negative rods.

ACKNOWLEDGMENTS

We thank Norsk Dental Depot's Fond for Odontologisk Forskn-ing, Tannlegeundervisningens Fond, and Nordisk Ministerr'ad: Nor-diska Stipendkurs no. 35/90, for financial support.

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