Vol. 45, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1983, p. 536-5450099-2240/83/020536-10$02.00/0

Legionella oakridgensis: Unusual New Species Isolated fromCooling Tower Watert

LETA H. ORRISON,l* WILLIAM B. CHERRY,1 RICHARD L. TYNDALL,2 CARL B. FLIERMANS,3STEPHEN B. GOUGH,2 MARY A. LAMBERT,4 L. KIRVEN McDOUGAL,1 WILLIAM F. BIBB,4 AND

DON J. BRENNER,4Hospital Infections Program' and Division of Bacterial Diseases,4 Centers for Infectious Diseases, Centers

for Disease Control, Atlanta, Georgia 30333; Environmental Sciences Division, Oak Ridge NationalLaboratory, Oak Ridge, Tennessee 378302; and Savannah River Laboratory, E.l. du Pont de Nemours & Co.,

Inc., Aiken, South Carolina 298083

Received 1 June 1982/Accepted 18 November 1982

We describe a new species of Legionella represented by 10 strains isolated fromindustrial cooling towers. Legionella oakridgensis differed genetically from theother seven species of Legionella in DNA hybridization studies and differedserologically in direct fluorescent-antibody tests. The new species, unlike all otherspecies except L. jordanis, did not require added L-cysteine for growth in serialtransfer on charcoal-yeast extract agar. L. oakridgensis, as well as three otherspecies tested, required L-cysteine for primary isolation from animal tissues. L.oakridgensis was the only species of Legionella that failed to produce alkalinephosphatase at pH 8.5. In all other respects, it resembled other species ofLegionella, including having a high content of branched-chain cellular fatty acidsand being pathogenic for guinea pigs. These bacteria have not yet been associatedwith human disease, but they are potential causes of legionellosis.

Colonization of air conditioning equipment bylegionellae is recognized in the United Statesand abroad as a potential major public healthproblem because strong circumstantial evidenceindicates that cooling towers and evaporativecondensers have frequently been the source oflegionellae involved in outbreaks of both mildand severe respiratory disease (2, 11, 12, 16).The maintainence of equipment free of theseorganisms is a desirable objective but one thathas not yet been achieved by practical means.

In this study we describe 10 strains isolatedfrom thermally altered water of large industrialcooling towers in two different geographic loca-tions (R. Tyndall, S. B. Gough, C. B. Flier-mans, E. Dominque, and C. Duncan, submittedfor publication). These cultures were character-ized by morphological, cultural, biochemical,genetic, serological, and pathogenicity tests.They represent a new species, for which thename Legionella oakridgensis sp. nov. is pro-posed. The type strain of L. oakridgensis is OakRidge 10 (OR-10; ATCC 33761).

MATERIALS AND METHODSCultures. Tyndall and co-workers in Oak Ridge,

Tenn., isolated the strains of L. oakridgensis designat-

t Publication 2103, Environmental Sciences Division, OakRidge National Laboratory.

ed OR-10, OR-12, OR-15, OR-16, OR-18, and OR-19from water obtained in Pennsylvania; those labeledOR-6, OR-23, OR-24, and OR-30 were from waterobtained in Minnesota. Cultures of all other describedspecies and serogroups of legionellae were included inthe study. These were L. pneumophila serogroups 1through 6 (13, 25, 26), L. bozemanii (4), L. dumoffii(4), L. micdadei (20), L. gormanii (27), L. longbeachaeserogroups 1 and 2 (3, 24), and L. jordanis (8). Becausethe OR series of strains were received at differenttimes and because preliminary tests indicated theirsimilarity or identity, not all cultures were subjected toevery test procedure. Some cultures originating fromeach of the two different geographic areas were,however, tested in all procedures.

Staining procedures and morphological studies. Thecultures were stained by the Gram method (Hucker'smodification), by a fat stain (Sudan black B) for thepresence of storage material, and by the Wirtz-Conklinmethod for demonstrating spores. Acid-fast stainswere performed by the Ziehl-Neelsen procedure. Fla-gella stains were done by a simplified Leifson proce-dure (10). Cultures on charcoal-yeast extract (CYE)agar slants (14) that were incubated for 24 to 48 h atboth 25 and 35°C were examined for motility by dark-field microscopy. Measurements of cell size weremade on Gram-stained smears, using a calibratedocular micrometer.

Cultural tests. Cultures were plated on CYE agarand on Trypticase soy agar (BBL Microbiology Sys-tems, Cockeysville, Md.) with and without the addi-tion of 5% sheep blood. They also were cultured onCYE agar slants, and the growth was examined for

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autofluorescence at 366-nm wavelength by using aWoods lamp.The basic CYE agar medium was that described by

Feeley et al. (14). Cultures were tested for their abilityto grow in serial transfers on agar slants of the basicmedium from which charcoal (Norite SG), L-cysteine,or ferric pyrophosphate (Fe3) or both of the latter twoadditives were omitted. Except for serial transfers,inocula for the tests were taken from 1- to 2-day-oldcultures on complete CYE. After the last serial trans-fer, all cultures were examined by Gram stain and bydirect fluorescent-antibody (DFA) tests and were plat-ed on Trypticase soy-blood agar to determine purity.Media without either Fe3 or L-cysteine added also

were tested for their ability to support the growth ofsome legionellae when cultured directly from freshtissues or from frozen tissue homogenates.

Cultures on CYE agar slants were tested for abilityto grow at 25 and 35°C in the air incubator and at 42.5to 43.0°C in the water bath. The incubator temperaturefor all other tests was 35°C. Plated cultures wereincubated in candle extinction jars until succeedingtransfers were able to grow well in air. Isolates alsowere observed for growth in both stagnant and shaken(100 rpm) yeast extract broth prepared according toRistroph et al. (32).DFA studies. DFA tests were performed as de-

scribed previously (9), using fluorescein-labeled anti-bodies for the species and serogroups of legionellaelisted above.

Preparation of antisera and fluorescent-antibody con-jugates for DFA tests. Heavy cell suspensions (about 4x 109 cells per ml) of OR-10, the type strain of L.oakridgensis, were harvested from CYE agar slantsand killed with 1% Formalin in 0.85% NaCl. Theseantigens were used to immunize rabbits according to apublished protocol, except that Freund adjuvant wasnot used (9). Immunoglobulin G was separated fromthe serum on a column of Staphylococcus protein A-Sepharose (Pharmacia Fine Chemicals, Inc., Pis-cataway, N.J.) as described by Goding (17). Theantibody was labeled with fluorescein isothiocyanate,and the protein was adjusted to 5 mg/ml by methodspreviously described (19).

Testing the OR-10 conjugate with homologous andheterologous cultures of legionellae. Titrations of theOR-10 conjugate with the 10 isolates were performedwith formalinized (1%) cell suspensions (McFarlandno. 2 standard) in 0.85% NaCl. Twofold dilutions ofthe conjugate were made in saline (0.85%) containing0.2 M sodium borate and adjusted to pH 7.6. When aworking dilution of the conjugate was obtained, thisdilution was tested on smears of cultures of all speciesand serogroups of the legionellae.

Physiological tests. Cultures were tested for oxidaseand catalase by the methods of Weaver and Feeley(34). Urea hydrolysis was determined by heavy inocu-lation of Christensen urea agar slants with 48-h cul-tures grown on CYE agar. The cultures were tested forthe production of a brown, water-soluble pigment aftergrowth on yeast extract agar medium that had beenabsorbed with charcoal by the method of Ristroph etal. (32) and supplemented with 1% L-tyrosine (1). Thechromogenic cephalosporin test for the detection of 1-lactamase production was performed as described byThomsberry and Kirven (33). The isolates were grownon CYE agar slants containing 0.2% KNO3, to test for

nitrate reduction. The ability to hydrolyze gelatin wastested in CYE medium in which the agar was replacedby 3% gelatin. The hydrolysis of sodium hippurate wastested by the method of Hebert (18).The API ZYM system (Analytab Products, Plain-

view, N.Y.) was used to test the legionellae culturesfor 19 different enzyme activities. The recommenda-tions of the manufacturers were followed for preparingthe cell suspensions and for conducting, incubating,and reading the tests. Cell suspensions were madefrom cultures that were grown on CYE agar slants for24 to 48 h.

Alkaline phosphatase activity was determined bytwo methods. In the first test, 0.01 M disodium p-nitrophenyl phosphate (Sigma Chemical Co., St. Lou-is, Mo.) was dissolved in 0.04 M glycine-NaOH bufferat pH 10.5 (21). The tests were repeated on the samesubstrate under the same conditions except that thebuffer was adjusted to pH 8.5. The tests were incubat-ed for 3 h at 35°C and examined for the yellow color ofp-nitrophenol, indicating alkaline phosphatase activi-ty. The second substrate was 100 nM 2-napthyl phos-phate (API ZYM test). This test was performed at pH8.5, and a positive result was a violet color given bythe detector reagents. Appropriate positive and nega-tive controls were used in all tests.GLC studies of ceDlular fatty acids. Cells for fatty

acid analyses were obtained after 1 to 3 days of growthon CYE agar slants. Growth from one slant wasremoved with about 1 ml of sterile distilled water andtransferred to a test tube (20 by 150 mm) containing 4ml of 5% NaOH in 50%o aqueous methanol. The tubeswere sealed with Teflon-lined caps, and the cellularlipids were saponified for 30 min at 100°C. Fatty acidswere extracted and methylated as described previous-ly (28). The fatty acid methyl esters were analyzed bygas-liquid chromatography (GLC) on both packed andcapillary columns, using a flame ionization detector(28). The peaks were tentatively identified by retentiontime data, and quantitation was done with a model3390A reporting integrator (Hewlett-Packard, Avon-dale, Pa.). The identity of peaks was confirmed by theresults obtained from hydrogenation, acetylation, andGLC-mass spectrometry (30).

Antimicrobial susceptibility testing. Eight L. oakrid-gensis strains and seven other legionellae were testedfor in vitro susceptibility to the following seven antimi-crobial agents by the agar dilution method, usingbuffered CYE (31): erythromycin, rifampin, chloram-phenicol, trimethoprim, cefoxitin, doxycycline, andpenicillin.DNA studies. The guanine-plus-cystosine content of

L. oakridgensis DNA was determined spectrophoto-metrically by thermal denaturation (22). Strains usedin DNA relatedness studies were grown on 12 to 20standard plastic petri dishes containing CYE agar.Plates were incubated at 35°C either in candle jars or inan atmosphere containing 2.5% CO2 until growth wasconfluent (4 to 5 days). Cells from each of the severalplates were harvested, pooled, and resuspended in 10ml of buffer containing 0.1 M NaCI-0.05 M Tris-0.05M EDTA-50 pLg of pronase per ml. The extraction andpurification of DNA, the in vitro labeling method, andthe determination ofDNA relatedness by the hydroxy-apatite procedure have been described (6).Animal pathogenicity studies. All 10 strains of L.

oakridgensis were isolated from guinea pigs injected

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TABLE 1. Phenotypic characteristics of three Legionella speciesCharacteristic L. oakridgensis L. jordanis L. pneumophila

Growth on agarCYE + + +TSA' + bloodTSA

Growth in YEBb + + +Blue autofluorescencec - - -

StainingGramAcid fastSporeLeifson or DFAd - + +

Motility - + +Brown pigment' + + +Requires added

L-Cysteine Nof Yesfg YesfFe3 Nof Nof Nof

Biochemical reactionOxidase + vhCatalase + + +UreaseGelatin liquefaction + + +NO3-NO21-Lactamase +(w)' + +Hippurate hydrolysis +

API ZYM testAlkaline phosphatase, pH 8.5 - + +Acid phosphatase + + +Phosphoamidase + + +

a TSA, Trypticase soy agar.b YEB, Yeast extract broth.I Long-wavelength (366-nm) UV light.d Flagella stains.e Medium was charcoal-treated yeast extract agar.f Stock cultures only.g Poor growth if omitted.h v, Variable.i w, Weak.

with the bacteria concentrated from water samples(see above). As an additional measure of pathogenici-ty, we selected for further testing strain OR-23 thathad been transferred serially several times on CYEagar. A portion of cells grown to log phase in yeastextract broth on a shaker at 30°C was formalinized(1%), and a quantitative microscopic count was ob-tained on smears stained with the OR-10 conjugate.When the cell count was known, appropriate dilutionsof the broth culture were made in sterile distilledwater, and each of two mature male guinea pigs (-800g) was injected intraperitoneally with 1.0 ml of therespective dilutions. When they showed evidence ofillness or a pronounced rise in temperature, at leastone of each pair was killed, autopsied, and cultured,and smears were prepared for DFA examination.Cultures were made on CYE agar plates from eitherperitoneal swabs or the spleen.

Ability of legionellae to grow on CYE lacking L-cysteine or Fe3 when cultured directly from animaltissue. Fresh unfrozen guinea pig spleens, spleen ho-mogenates, and spleens frozen at -60°C were culturedon CYE agar slants from which either L-cysteine orFe3 was omitted. An homogenate from the spleen of

one of the guinea pigs infected with the OR-23 strainwas made in distilled water (1:16, wt/vol). The homog-enate was diluted and plated to obtain an estimate ofviable Legionella cells. Slants of both complete anddeficient CYE medium were inoculated with 0.03 ml ofthe homogenate. The frozen and thawed spleens ofthree other guinea pigs infected with various sizes ofinocula of the OR-10 culture were swabbed, and theswab was used to inoculate multiple tubes of thecomplete and deficient media. Spleens of guinea pigsinfected with L. micdadei (strain TATLOCK), L.dumoffli (strain Tex-KL), or human lung tissue infect-ed with L. pneumophila (serogroup 1) were also cul-tured on these deficient media as described above.Cultures were incubated in candle jars for up to 22days. Complete CYE agar media served as a control inall tests.

RESULTSMorphology and staining characteristics. All

cultures of L. oakridgensis were gram-negativerods consistent in size with members of thegenus, i.e., 0.3 to 0.9 ,m wide by 2 to 20 ,um in

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TABLE 2. Growth of legionellae during routine culture on complete and deficient media

L-Cysteine ~~~L-Cysteine L-Cysteine, Fe3,Legionella sp. Completea omitted Fe3omitted and Fe3 and Norite SG

omitted omitted

L. oakridgensis + + + + _bL. jordanis + ++L. pneumophila

serogroups 1, 2, 3, 4, 5, 6L. dumoffiiL. micdadei +-L. bozemaniiL. gormaniiL. longbeachae

serogroups 1, 2a Complete CYE agar = yeast extract, 10 g/liter (GIBCO Diagnostics, Madison, Wis.); agar, 10 g/liter (Oxoid

Ltd., London, U.K.); Norite SG charcoal, 2 g/liter; L-cysteine-HCI, 0.4 g/liter; and ferric pyrophosphate, 0.25 g/liter, pH 6.9 ± 0.05.

b Occasional streaks of growth where the inoculum was very heavy; growth never confluent.

length (7). They took the safranin counterstainreadily (20 s). Their cells appeared thicker thanthose of most legionellae in DFA-stainedsmears, apparently because of a large amount ofextracellular envelope material. When they werestained with Sudan black B, very few cellscontained stained storage material. All cultureswere nonmotile by microscopic examination,and flagella were not observed. All cultureswere nonsporeforming, non-encapsulated, andnon-acid fast (Table 1).

Cultural tests. All strains of L. oakridgensisgrew slowly on CYE agar when first isolated andwere stimulated by incubation in candle jars.They failed to grow on Trypticase soy agar withand without the addition of blood. Cultures onCYE agar did not give blue autofluorescence.Colonies appeared on CYE agar plates after 3 to5 days of incubation and were typical of thelegionellae (34).The basic CYE agar medium was tested for its

ability to support the growth of legionellae underfour conditions: (i) when added L-cysteine-HClwas omitted, (ii) when Fe3 was omitted, (iii)when both were omitted, and (iv) when L-cyste-ine, Fe3, and Norite SG all were omitted. OnlyL. oakridgensis and L. jordanis among the spe-cies and serogroups of legionellae, including fiveenvironmental isolates of L. pneumophila, grewon CYE without L-cysteine. L. oakridgensisadapted to the deficient medium on the firsttransfer from complete CYE medium and ap-peared to grow as well as it did on the completeCYE agar. L. jordanis (strains ABB-9 and BL-540) grew slowly at first and never as vigorouslyas did L. oakridgensis (Table 2). Three of the L.oakridgensis strains (OR-10, -23, and -24) weretransferred serially 10 times on CYE agar slantswithout added L-cysteine; the other seven OR

strains were transferred serially 5 times. Allstrains gave excellent growth (Table 2).The eight strains of L. oakridgensis that were

tested were transferred serially five times onCYE agar slants without added Fe3. The omis-sion of Fe3 did not appear to diminish growth(Table 2).

All seven species, including the six sero-groups of L. pneumophila and the two sero-groups of L. longbeachae, also grew wellthrough two serial transfers on CYE agar slantsfrom which Fe3 was omitted (Table 2). Theywere not examined further.

Eight of the OR strains were transferred seri-ally five times on CYE agar slants from whichboth L-cysteine and Fe3 were omitted. Growthappeared to be equal to that on the completeCYE agar medium (Table 2). When Norite SG,L-cysteine, and Fe3 were omitted from CYEagar medium, L. oakridgensis cultures either didnot grow at all or grew poorly in streaks wherethe inoculum was very heavy (Table 2). A totalof 5% sheep blood could not replace the NoriteSG in the L-cysteine- and Fe3-deficient media.

Eight OR strains, strains of three differentserogroups of L. pneumophila, and a strain of L.jordanis grew poorly in yeast extract broth incu-bated stagnantly. Cultures of all of these strainsgave good to heavy growth when shaken during48 h of incubation.

All strains of L. oakridgensis grew slowly onCYE agar at 25°C, and in 4 to 5 days the growthequaled that obtained at 35°C in 2 days. Theyfailed to grow at 42.5 to 43°C within 7 days orafter removal to 35°C for an additional 3 days.DFA tests of isolates with conjugates for recog-

nized serogroups and species of legionellae. Nofluorescence was observed when cultures of the10 strains of L. oakridgensis were tested with

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FIG. 1. Gas chromatogram of methylated fatty acids from saponified cells of L. oakridgensis strain OR-10.Analysis was on a fused silica-glass capillary column (50 m by 0.2 mm) coated with OV-101. Peaks are designatedas follows: number to the left of colon, number of carbon atoms; number to right, number of double bonds; i,methyl branch at the iso carbon atom; a, methyl branch at the anteiso carbon atom; CYC, cyclopropane acid.

the working dilutions of polyvalent and monova-lent conjugates for the seven species and eightserogroups of legionellae listed in Materials andMethods. The OR-10 conjugate was testedagainst OR-10 and seven other OR isolates. Thehomologous 4+ staining titer was 1:64: heterolo-gous 4+ titers were either 1:32 or 1:64. Theisolates were identical or very similar by thesetests. Smears of the cultures of the seven species(including eight serogroups) of legionellae werestained with the working dilution (1:32) and witha fourfold-higher concentration (1:8 dilution) ofthe OR-10 conjugate. All tests were negative.Thus, by these reciprocal DFA tests L. oakrid-gensis is unrelated to any other described legion-ellae.

Physiological tests. In Table 1, we list theimportant phenotypic characteristics of L. oak-ridgensis and compare them with those of L.jordanis and L. pneumophila. All 10 strains ofL.oakridgensis gave identical reactions in all tests.The major difference between L. oakridgensisand the other described species of Legionellawas its lack of a requirement for added L-cysteine for serial transfer on laboratory media.Lack of a requirement for added Fe3 was sharedby all other legionellae species and serogroupsunder the same conditions. The OR strains alsodiffered from all other legionellae in alkaline

phosphatase activity by being the only describedspecies in which this enzyme was not active atpH 8.5. At pH 10.5 the alkaline phosphatase ofL. jordanis was very weak; at pH 8.5 it wasstrong. L. micdadei strains (TATLOCK,HEBA, and PPA) have alkaline phosphataseactivity at pH 8.5 but not at pH 10.5. Like allother species and serogroups of legionellae ex-cept L. pneumophila, L. oakridgensis failed tohydrolyze sodium hippurate.

In the 19 API ZYM tests, the L. oakridgensisstrains resembled other legionellae in havingstrong acid phosphatase and phosphoamidaseactivity, moderately strong leucine aminopepti-dase activity, weak valine aminopeptidase andC4 and C8 esterase activity, and lack of bothproteolytic (trypsin and chymotrypsin) and car-bohydrate-degrading enzyme activity.GLC studies of cellular fatty acids. A repre-

sentative chromatogram of the cellular fattyacids of L. oakridgensis is shown in Fig. 1. Themajor fatty acid was identified as a saturatedbranched-chain C16 fatty acid with the methylbranch in the iso position of the carbon chain (i-16:0). The other acids present in large to moder-ate amounts were a monounsaturated C16 acid(16:1), saturated C16 (16:0) and C18 (18:0) acids,and a C17 cyclopropane acid (17CYC).The average percentages of the cellular fatty

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LEGIONELLA OAKRIDGENSIS SP. NOV. 541

acids of L. oakridgensis and seven other speciesof Legionella are shown in Table 3. The valuesfor L. oakridgensis are averages calculated aftereach of six strains (OR-10, OR-12, OR-15, OR-16, OR-18, OR-19) was grown four differenttimes and analyzed for cellular fatty acids. Smallamounts (1% or less) of hydroxy acids werefound in some of the OR strains, but they werenot included because their identities have notbeen confirmed.The data in Table 3 show that L. oakridgensis

can be readily distinguished from L. micdadei,L. bozemanii, L. dumoffii, and L. jordanis bythe low concentrations of the anteiso-C15 and-C17 branched-chain acids (a-15:0 and a-17:0).These two acids accounted for only 2 and 5%,respectively, of the total fatty acids in L. oakrid-gensis, whereas in the other four species theywere the two most abundant acids and togetheraccounted for 48 to 64% of the total fatty acids.L. pneumophila, L. longbeachae, and L. gor-manii contained a combined total of 20 to 38% ofthese two fatty acids. All of the strains of L.oakridgensis contained lower concentrations (2to 3%) of a-15:0 acid and higher concentrations(9%) of a 17CYC acid than have been reported inother species (Table 3). The fatty acid composi-tion of L. oakridgensis was qualitatively similarto L. pneumophila and L. longbeachae, asshown by the presence of large to moderateamounts of i-16:0, 16:1, and 16:0 acids. It couldbe differentiated from these two as well as theother five species, however, by the presence ofmoderately large amounts (13%) of the 18:0 acidthat never exceeded 3% in any of the otherseven Legionella species.The fatty acid composition of the OR-23 and

OR-24 isolates was essentially the same as thatshown in Table 3 for the other six strains of L.oakridgensis. When isolates OR-6 and OR-30were tested, however, quantitative differencesappeared in the concentrations of the i-16:1 and16:1 acids that seemed to be related to the age ofthe culture. In cultures incubated for 60 h orless, the major fatty acid was 16:1, and itsrelative concentration was 26%; in cultures incu-bated for a longer period of time (at least 72 h), i-16:0 was the major fatty acid, with a relativeconcentration of 21%. Regardless of the age ofthe OR-6 and OR-30 strains, the relative per-centages of all other fatty acids found wereessentially the same as those reported for L.oakridgensis (Table 3). Although a variation inthe relative amounts of i-16:0 and 16:1 acids hasalso been reported in isolates ofL. longbeachae,these changes were not consistent and did notappear to be related to the age of the culture(29). Experiments to define these variations inthe ratios of the C16 acids are currently understudy.

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TABLE 4. DNA relatedness of L. oakridgensis (strain OR-10) to other L. oakridgensis strains and to otherLegionella species

Source of labeled DNA (L. oakridgensis OR-10)Source of unlabeled DNA

RBR (600C)a Divergence RR(5CRBR ~~~~(%[6OoC])b RBR (750C)L. oakridgensisOR-10 100 0.0 100OR-24 100 0.5 94OR-1S 100 0.5 96OR-12 100 0.5 90OR-19 99 0.0 98OR-23 96 0.5 100

L. pneumophila Philadelphia 1 8L. bozemanii WIGA 8L. dumoffli NY-23 7L. jordanis ABB-9 7L. micdadei TATLOCK 6L. gormanii LS-13 6L. longbeachae LB-4 5

a RBR, Relative binding ratio = (percent heterologous DNA bound to hydroxyapatite/percent homologousDNA bound to hydroxyapatite) x100. Reassociation was 75 ± 5% in homologous OR-10 DNA reactions. Incontrol reactions in which labeled OR-10 DNA was incubated in the absence of any unlabeled DNA, an averageof <1% of the labeled DNA bound to hydroxyapatite.

b Divergence was calculated (to the nearest 0.5%) on the assumption that a 10C decrease in thermal stability ofa heterologous DNA duplex compared with that of the homologous DNA duplex is caused by 1% of the baseswithin the duplex that are unpaired.

Antimicrobial susceptibility. The in vitro anti-microbial susceptibility data for OR strains var-ied little from the patterns of susceptibility ofsome of the other legionellae (31). The ORstrains were more susceptible to trimethoprim,being similar to L. bozemanii, L. dumoffii, L.gormanii, and L. longbeachae. The OR strainsand L. jordanis were the least susceptible toerythromycin, with minimal inhibitory concen-trations of 4 ,.g/ml, compared with L. pneumo-phila, the most erythromycin-susceptible spe-cies with geometric mean minimum inhibitoryconcentrations of 0.75 jig/ml.DNA studies. The guanine-plus-cytosine con-

tent was determined in triplicate on DNAs fromfive L. oakridgensis strains. The guanine-plus-cytosine content range was 42.3 to 43.9%, withan average value of 43.2%.The DNA from the OR-10 strain of L. oakrid-

gensis was labeled with 32p in vitro and testedfor relatedness to DNAs from five other OakRidge isolates and to strains of all named Le-gionella species (Table 4). The OR isolates were90 to 100% related to the OR-10 strain in testsdone at both optimal and stringent incubationtemperatures. Divergence within related DNAsequences was 0.5% or less. In contrast, DNAfrom OR-10 was only 5 to 8% related to DNAsfrom the seven other Legionella species.Animal pathogenicity. Two guinea pigs each

received intraperitoneal injections of the follow-

ing numbers of cells of the OR-23 strain: 1.2 x104, 1.2 x 105, 1.2 x 106, and 1.2 x 107. Allanimals showed symptoms typical of Legionellainfection; i.e., within 1 to 2 days, they hadtemperatures above 40°C, ruffled hair, lethargy,and lacrimation. Tissues from one animal ofeach pair were examined by DFA staining testsand cultured. L. oakridgensis cells were presentin large numbers in the peritoneal exudate of allfour animals by DFA tests, and L. oakridgensiswas cultured from all animals. Two of the re-maining four guinea pigs died of the infections,and the other two were killed while moribund.

Inhibition of growth of legionellae on CYElacking L-cysteine or Fe3 when inoculated directlyfrom animal tissue. In Table 5 the growth oflegionellae from spleen homogenates of guineapigs and from a human lung homogenate wheninoculated on complete CYE agar is comparedwith that obtained on deficient CYE agar. L.oakridgensis strains required added L-cysteinefor isolation from tissue, although it was notneeded for growth when the culture was trans-ferred from the complete CYE isolation mediumto deficient CYE medium. Unless the inoculumwas very heavy, the OR-23 isolate also requiredadded Fe3 for normal good growth from tissue(Table 5). L. micdadei, L. dumoffli, and L.pneumophila required the addition of both Fe3and L-cysteine to the CYE agar for growth fromtissue, and they required added L-cysteine, but

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TABLE 5. Inhibition of growth of legionellae on CYE agar slants lacking added L-cysteine or Fe3 wheninoculated directly from animal tissue

Growth on CYE agar mediaaLegionella sp./specimen Inoculum

Complete No Fe3 No L-cysteine

L. oakridgensis OR-23Guinea pig 1 Spleen homogenate 4+ 3+ b

Guinea pig 2 Spleen swab 1+ -

Guinea pig 3 Spleen swab 3+ -

Guinea pig 4 Spleen swab 4+ 3+ 3+bL. micdadei (TATLOCK) Guinea pig spleen homogenate 2+ - -

L. dumoffli (Tex-KL) Guinea pig spleen homogenate 3-4+L. pneumophila Human lung homogenate 3-4+ d

(serogroup 1)a Average of two experiments with multiple tubes inoculated for each specimen. Growth on complete medium

occurred in 3 to 5 days. Purity ofgrowth and its identity were determined by streaking on blood agar and by DFAtests. Growth graded 1 + = 100 colonies per plate.bColonies barely visible with a hand lens. These spleens contained very large numbers of Legionella sp. by

DFA staining and by the amount of growth on complete media.c -, No growth; no colonies.d After 22 days of incubation, many (2 to 3+) tiny colonies appeared.

not added Fe3, for normal growth in furthertransfers on CYE agar.

DISCUSSIONAlthough the 10 strains of L. oakridgensis

were isolated from the water of industrial cool-ing towers in two widely separated locations,they were identical in the characteristics exam-

ined in this study. They differed from all de-scribed Legionella species except L. jordanis innot requiring added L-cysteine for growth onCYE agar except upon initial isolation fromanimal tissues. Added L-cysteine was stimula-tory but not required by L. jordanis for routineculture on CYE agar. L. oakridgensis also re-

quired added Fe3 for primary isolation fromanimal tissues, as do the other three legionellaespecies tested. The rapid loss of a requirementfor L-cysteine and Fe3 for the growth of L.oakridgensis after isolation from animal tissuesuggests a complex host-parasite relationshipnot yet defined. Also, it is possible that L-

cysteine and Fe3 are required for growth of thebacteria directly from animal tissue because, insome way, they neutralize tissue factors thatinhibit in vitro growth during the first transfer onCYE agar.Because fairly large inocula were used in the

serial transfer experiments, quantitative differ-ences may be revealed when the amount ofgrowth obtained on L-cysteine- and Fe3-defi-cient CYE media is compared with that on thecomplete medium. Clearly, however, L. oakrid-gensis and, to a lesser extent, L. jordanis dif-fered from all other Legionella species in theirrequirements for L-cysteine. No described spe-cies of legionellae required added Fe3 for good

growth in routine serial culture on CYE agarslants.Although the qualitative fatty acid composi-

tion of L. oakridgensis was similar to that ofother species of legionellae, several quantitativedifferences existed that could separate this spe-cies from other members of the genus. Strains ofL. oakridgensis contained relatively largeamounts (12% or greater) of 18:0 acid that hadnot been previously reported as a major cellularfatty acid in species of Legionella (8, 29; Table3). Although the relative concentrations of the i-16:0 and 16:1 acids in some young cultures of L.oakridgensis did vary somewhat from thatshown in Table 3 and Fig. 1, the other character-istic fatty acids did not change. Thus, L. oakrid-gensis can be distinguished from all other knownspecies of Legionella by quantitative differencesin its cellular fatty acid composition.The presence of hydroxy fatty acids has not

been reported previously in whole-cell hydroly-sates of legionellae, but very low concentrationsof these acids have been found in various cellfractions of L. pneumophila (23). We were ableto detect hydroxy acids in the present studybecause a high-resolution capillary column wasused for the GLC analysis. These acids arepresent at low concentrations, however, andmay not be useful for differentiating members ofthis genus.The level ofDNA relatedness between strains

of L. oakridgensis is very high, leaving no doubtthat these strains represent a single species.Relatedness between L. oakridgensis and otherlegionellae, although low, is not substantiallydifferent from that between several other Le-gionella species. The guanine-plus-cytosine con-

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APPL. ENVIRON. MICROBIOL.

tent of L. oakridgensis DNA (43%) is within therange (39 to 44%) reported for other Legionellaspecies (5, 15).

L. oakridgensis did not differ importantlyfrom other legionellae in morphology, stainingcharacteristics, pathogenicity for guinea pigs, orantimicrobial susceptibility. Neither motility northe presence of flagella could be demonstrated,but strains of other species of legionellae havebeen reported to be nonmotile (7). L. oakridgen-sis has not been incriminated in human infec-tion, but its pathogenicity for guinea pigs sug-gests that it may be an unrecognized humanpathogen. On the basis of phenotypic similarityto other legionellae and in keeping with previ-ously established practice (4, 8, 24, 27), wepropose that this new species be placed with theother legionellae in the genus Legionella. Thename Legionella oakridgensis sp. nov. is pro-posed for the species represented by the 10strains isolated from industrial cooling towerwater: oak * ridg * en' sis sp. nov., M.L. fem.adj. oakridgensis coming from Oak Ridge,Tenn., the city where the organism was firstisolated. The type strain of L. oakridgensis isOR-10 (ATCC 33761).

ACKNOWLEDGMENTSWe gratefully acknowledge and deeply appreciate the tech-

nical assistance of A. G. Steigerwalt, Elizabeth Dominque,and Carol Duncan. We thank C. W. Moss for the identifica-tion of cellular fatty acids by mass spectrometry. We thankThomas Ozro MacAdoo of the Department of Foreign Lan-guages and Literature, Virginia Polytechnic Institute and StateUniversity, Blacksburg, for help in arriving at the name of L.oakridgensis and for advice on naming this organism inaccordance with the rules of Latin grammar and the require-ments of the Bacteriological Code.

This research was partially sponsored by the Electric PowerResearch Institute (contract RP 1909) and the U.S. NuclearRegulatory Commission (under interagency agreement 40-550-75 with the Department of Energy) through the Oak RidgeNational Laboratory, which is operated by Union CarbideCorp. for the Department of Energy (contract W-7405-eng-26).

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