Legionella oakridgensis: Unusual New Species Isolated from

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1983, p /83/ $02.00/0 Vol. 45, No. 2 Legionella oakridgensis: Unusual New Species Isolated from Cooling Tower Watert LETA H. ORRISON,l* WILLIAM B. CHERRY,1 RICHARD L. TYNDALL,2 CARL B. FLIERMANS,3 STEPHEN B. GOUGH,2 MARY A. LAMBERT,4 L. KIRVEN McDOUGAL,1 WILLIAM F. BIBB,4 AND DON J. BRENNER,4 Hospital Infections Program' and Division of Bacterial Diseases,4 Centers for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333; Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee ; and Savannah River Laboratory, E.l. du Pont de Nemours & Co., Inc., Aiken, South Carolina Received 1 June 1982/Accepted 18 November 1982 We describe a new species of Legionella represented by 10 strains isolated from industrial cooling towers. Legionella oakridgensis differed genetically from the other seven species of Legionella in DNA hybridization studies and differed serologically in direct fluorescent-antibody tests. The new species, unlike all other species except L. jordanis, did not require added L-cysteine for growth in serial transfer on charcoal-yeast extract agar. L. oakridgensis, as well as three other species tested, required L-cysteine for primary isolation from animal tissues. L. oakridgensis was the only species of Legionella that failed to produce alkaline phosphatase at ph 8.5. In all other respects, it resembled other species of Legionella, including having a high content of branched-chain cellular fatty acids and being pathogenic for guinea pigs. These bacteria have not yet been associated with human disease, but they are potential causes of legionellosis. Colonization of air conditioning equipment by legionellae is recognized in the United States and abroad as a potential major public health problem because strong circumstantial evidence indicates that cooling towers and evaporative condensers have frequently been the source of legionellae involved in outbreaks of both mild and severe respiratory disease (2, 11, 12, 16). The maintainence of equipment free of these organisms is a desirable objective but one that has not yet been achieved by practical means. In this study we describe 10 strains isolated from thermally altered water of large industrial cooling towers in two different geographic locations (R. Tyndall, S. B. Gough, C. B. Fliermans, E. Dominque, and C. Duncan, submitted for publication). These cultures were characterized by morphological, cultural, biochemical, genetic, serological, and pathogenicity tests. They represent a new species, for which the name Legionella oakridgensis sp. nov. is proposed. The type strain of L. oakridgensis is Oak Ridge 10 (OR-10; ATCC 33761). MATERIALS AND METHODS Cultures. Tyndall and co-workers in Oak Ridge, Tenn., isolated the strains of L. oakridgensis designatt Publication 2103, Environmental Sciences Division, Oak Ridge National Laboratory. ed OR-10, OR-12, OR-15, OR-16, OR-18, and OR-19 from water obtained in Pennsylvania; those labeled OR-6, OR-23, OR-24, and OR-30 were from water obtained in Minnesota. Cultures of all other described species and serogroups of legionellae were included in the study. These were L. pneumophila serogroups 1 through 6 (13, 25, 26), L. bozemanii (4), L. dumoffii (4), L. micdadei (20), L. gormanii (27), L. longbeachae serogroups 1 and 2 (3, 24), and L. jordanis (8). Because the OR series of strains were received at different times and because preliminary tests indicated their similarity or identity, not all cultures were subjected to every test procedure. Some cultures originating from each of the two different geographic areas were, however, tested in all procedures. Staining procedures and morphological studies. The cultures were stained by the Gram method (Hucker's modification), by a fat stain (Sudan black B) for the presence of storage material, and by the Wirtz-Conklin method for demonstrating spores. Acid-fast stains were performed by the Ziehl-Neelsen procedure. Flagella stains were done by a simplified Leifson procedure (10). Cultures on charcoal-yeast extract (CYE) agar slants (14) that were incubated for 24 to 48 h at both 25 and 35 C were examined for motility by darkfield microscopy. Measurements of cell size were made on Gram-stained smears, using a calibrated ocular micrometer. Cultural tests. Cultures were plated on CYE agar and on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with and without the addition of 5% sheep blood. They also were cultured on CYE agar slants, and the growth was examined for 536

2 VOL. 45, 1983 autofluorescence at 366-nm wavelength by using a Woods lamp. The basic CYE agar medium was that described by Feeley et al. (14). Cultures were tested for their ability to grow in serial transfers on agar slants of the basic medium from which charcoal (Norite SG), L-cysteine, or ferric pyrophosphate (Fe3) or both of the latter two additives were omitted. Except for serial transfers, inocula for the tests were taken from 1- to 2-day-old cultures on complete CYE. After the last serial transfer, all cultures were examined by Gram stain and by direct fluorescent-antibody (DFA) tests and were plated 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 of some legionellae when cultured directly from fresh tissues or from frozen tissue homogenates. Cultures on CYE agar slants were tested for ability to grow at 25 and 35 C in the air incubator and at 42.5 to 43.0 C in the water bath. The incubator temperature for all other tests was 35 C. Plated cultures were incubated in candle extinction jars until succeeding transfers were able to grow well in air. Isolates also were observed for growth in both stagnant and shaken (100 rpm) yeast extract broth prepared according to Ristroph et al. (32). DFA studies. DFA tests were performed as described previously (9), using fluorescein-labeled antibodies for the species and serogroups of legionellae listed above. Preparation of antisera and fluorescent-antibody conjugates for DFA tests. Heavy cell suspensions (about 4 x 109 cells per ml) of OR-10, the type strain of L. oakridgensis, were harvested from CYE agar slants and killed with 1% Formalin in 0.85% NaCl. These antigens were used to immunize rabbits according to a published protocol, except that Freund adjuvant was not used (9). Immunoglobulin G was separated from the serum on a column of Staphylococcus protein A- Sepharose (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) as described by Goding (17). The antibody was labeled with fluorescein isothiocyanate, and the protein was adjusted to 5 mg/ml by methods previously described (19). Testing the OR-10 conjugate with homologous and heterologous cultures of legionellae. Titrations of the OR-10 conjugate with the 10 isolates were performed with formalinized (1%) cell suspensions (McFarland no. 2 standard) in 0.85% NaCl. Twofold dilutions of the conjugate were made in saline (0.85%) containing 0.2 M sodium borate and adjusted to ph 7.6. When a working dilution of the conjugate was obtained, this dilution was tested on smears of cultures of all species and serogroups of the legionellae. Physiological tests. Cultures were tested for oxidase and catalase by the methods of Weaver and Feeley (34). Urea hydrolysis was determined by heavy inoculation of Christensen urea agar slants with 48-h cultures grown on CYE agar. The cultures were tested for the production of a brown, water-soluble pigment after growth on yeast extract agar medium that had been absorbed with charcoal by the method of Ristroph et al. (32) and supplemented with 1% L-tyrosine (1). The chromogenic cephalosporin test for the detection of 1- lactamase production was performed as described by Thomsberry and Kirven (33). The isolates were grown on CYE agar slants containing 0.2% KNO3, to test for LEGIONELLA OAKRIDGENSIS SP. NOV. 537 nitrate reduction. The ability to hydrolyze gelatin was tested in CYE medium in which the agar was replaced by 3% gelatin. The hydrolysis of sodium hippurate was tested by the method of Hebert (18). The API ZYM system (Analytab Products, Plainview, N.Y.) was used to test the legionellae cultures for 19 different enzyme activities. The recommendations of the manufacturers were followed for preparing the cell suspensions and for conducting, incubating, and reading the tests. Cell suspensions were made from cultures that were grown on CYE agar slants for 24 to 48 h. Alkaline phosphatase activity was determined by two methods. In the first test, 0.01 M disodium p- nitrophenyl phosphate (Sigma Chemical Co., St. Louis, Mo.) was dissolved in 0.04 M glycine-naoh buffer at ph 10.5 (21). The tests were repeated on the same substrate under the same conditions except that the buffer was adjusted to ph 8.5. The tests were incubated for 3 h at 35 C and examined for the yellow color of p-nitrophenol, indicating alkaline phosphatase activity. The second substrate was 100 nm 2-napthyl phosphate (API ZYM test). This test was performed at ph 8.5, and a positive result was a violet color given by the detector reagents. Appropriate positive and negative 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 growth on CYE agar slants. Growth from one slant was removed with about 1 ml of sterile distilled water and transferred to a test tube (20 by 150 mm) containing 4 ml of 5% NaOH in 50%o aqueous methanol. The tubes were sealed with Teflon-lined caps, and the cellular lipids were saponified for 30 min at 100 C. Fatty acids were extracted and methylated as described previously (28). The fatty acid methyl esters were analyzed by gas-liquid chromatography (GLC) on both packed and capillary columns, using a flame ionization detector (28). The peaks were tentatively identified by retention time data, and quantitation was done with a model 3390A reporting integrator (Hewlett-Packard, Avondale, Pa.). The identity of peaks was confirmed by the results obtained from hydrogenation, acetylation, and GLC-mass spectrometry (30). Antimicrobial susceptibility testing. Eight L. oakridgensis strains and seven other legionellae were tested for in vitro susceptibility to the following seven antimicrobial agents by the agar dilution method, using buffered CYE (31): erythromycin, rifampin, chloramphenicol, trimethoprim, cefoxitin, doxycycline, and penicillin. DNA studies. The guanine-plus-cystosine content of L. oakridgensis DNA was determined spectrophotometrically by thermal denaturation (22). Strains used in DNA relatedness studies were grown on 12 to 20 standard plastic petri dishes containing CYE agar. Plates were incubated at 35 C either in candle jars or in an atmosphere containing 2.5% CO2 until growth was confluent (4 to 5 days). Cells from each of the several plates were harvested, pooled, and resuspended in 10 ml of buffer containing 0.1 M NaCI-0.05 M Tris-0.05 M EDTA-50 plg of pronase per ml. The extraction and purification of DNA, the in vitro labeling method, and the determination of DNA relatedness by the hydroxyapatite procedure have been described (6). Animal pathogenicity studies. All 10 strains of L. oakridgensis were isolated from guinea pigs injected

3 538 ORRISON ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Phenotypic characteristics of three Legionella species Characteristic L. oakridgensis L. jordanis L. pneumophila Growth on agar CYE TSA' + blood TSA Growth in YEBb Blue autofluorescencec Staining Gram Acid fast Spore Leifson or DFAd Motility Brown pigment' Requires added L-Cysteine Nof Yesfg Yesf Fe3 Nof Nof Nof Biochemical reaction Oxidase + vh Catalase Urease Gelatin liquefaction NO3-NO2 1-Lactamase +(w)' + + Hippurate hydrolysis + API ZYM test Alkaline phosphatase, ph Acid phosphatase Phosphoamidase a TSA, Trypticase soy agar. b YEB, Yeast extract broth. I Long-wavelength (366-nm) UV d light. 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 pathogenicity, we selected for further testing strain OR-23 that had been transferred serially several times on CYE agar. A portion of cells grown to log phase in yeast extract broth on a shaker at 30 C was formalinized (1%), and a quantitative microscopic count was obtained on smears stained with the OR-10 conjugate. When the cell count was known, appropriate dilutions of the broth culture were made in sterile distilled water, and each of two mature male guinea pigs (-800 g) was injected intraperitoneally with 1.0 ml of the respective dilutions. When they showed evidence of illness or a pronounced rise in temperature, at least one of each pair was killed, autopsied, and cultured, and smears were prepared for DFA examination. Cultures were made on CYE agar plates from either peritoneal swabs or the spleen. Ability of legionellae to grow on CYE lacking L- cysteine or Fe3 when cultured directly from animal tissue. Fresh unfrozen guinea pig spleens, spleen homogenates, and spleens frozen at -60 C were cultured on CYE agar slants from which either L-cysteine or Fe3 was omitted. An homogenate from the spleen of one of the guinea pigs infected with the OR-23 strain was made in distilled water (1:16, wt/vol). The homogenate was diluted and plated to obtain an estimate of viable Legionella cells. Slants of both complete and deficient CYE medium were inoculated with 0.03 ml of the homogenate. The frozen and thawed spleens of three other guinea pigs infected with various sizes of inocula of the OR-10 culture were swabbed, and the swab was used to inoculate multiple tubes of the complete and deficient media. Spleens of guinea pigs infected with L. micdadei (strain TATLOCK), L. dumoffli (strain Tex-KL), or human lung tissue infected with L. pneumophila (serogroup 1) were also cultured on these deficient media as described above. Cultures were incubated in candle jars for up to 22 days. Complete CYE agar media served as a control in all tests. RESULTS Morphology and staining characteristics. All cultures of L. oakridgensis were gram-negative rods consistent in size with members of the genus, i.e., 0.3 to 0.9,m wide by 2 to 20,um in

4 VOL. 45, 1983 LEGIONELLA OAKRIDGENSIS SP. NOV. 539 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 _b L. jordanis + + L. pneumophila serogroups 1, 2, 3, 4, 5, 6 L. dumoffii L. micdadei +- L. bozemanii L. gormanii L. longbeachae serogroups 1, 2 a 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 ± b Occasional streaks of growth where the inoculum was very heavy; growth never confluent. length (7). They took the safranin counterstain readily (20 s). Their cells appeared thicker than those of most legionellae in DFA-stained smears, apparently because of a large amount of extracellular envelope material. When they were stained with Sudan black B, very few cells contained stained storage material. All cultures were nonmotile by microscopic examination, and flagella were not observed. All cultures were nonsporeforming, non-encapsulated, and non-acid fast (Table 1). Cultural tests. All strains of L. oakridgensis grew slowly on CYE agar when first isolated and were stimulated by incubation in candle jars. They failed to grow on Trypticase soy agar with and without the addition of blood. Cultures on CYE agar did not give blue autofluorescence. Colonies appeared on CYE agar plates after 3 to 5 days of incubation and were typical of the legionellae (34). The basic CYE agar medium was tested for its ability to support the growth of legionellae under four conditions: (i) when added L-cysteine-HCl was omitted, (ii) when Fe3 was omitted, (iii) when both were omitted, and (iv) when L-cysteine, Fe3, and Norite SG all were omitted. Only L. oakridgensis and L. jordanis among the species and serogroups of legionellae, including five environmental isolates of L. pneumophila, grew on CYE without L-cysteine. L. oakridgensis adapted to the deficient medium on the first transfer from complete CYE medium and appeared to grow as well as it did on the complete CYE agar. L. jordanis (strains ABB-9 and BL- 540) grew slowly at first and never as vigorously as did L. oakridgensis (Table 2). Three of the L. oakridgensis strains (OR-10, -23, and -24) were transferred serially 10 times on CYE agar slants without added L-cysteine; the other seven OR strains were transferred serially 5 times. All strains gave excellent growth (Table 2). The eight strains of L. oakridgensis that were tested were transferred serially five times on CYE agar slants without added Fe3. The omission of Fe3 did not appear to diminish growth (Table 2). All seven species, including the six serogroups of L. pneumophila and the two serogroups of L. longbeachae, also grew well through two serial transfers on CYE agar slants from which Fe3 was omitted (Table 2). They were not examined further. Eight of the OR strains were transferred serially five times on CYE agar slants from which both L-cysteine and Fe3 were omitted. Growth appeared to be equal to that on the complete CYE agar medium (Table 2). When Norite SG, L-cysteine, and Fe3 were omitted from CYE agar medium, L. oakridgensis cultures either did not grow at all or grew poorly in streaks where the inoculum was very heavy (Table 2). A total of 5% sheep blood could not replace the Norite SG in the L-cysteine- and Fe3-deficient media. Eight OR strains, strains of three different serogroups of L. pneumophila, and a strain of L. jordanis grew poorly in yeast extract broth incubated stagnantly. Cultures of all of these strains gave good to heavy growth when shaken during 48 h of incubation. All strains of L. oakridgensis grew slowly on CYE agar at 25 C, and in 4 to 5 days the growth equaled that obtained at 35 C in 2 days. They failed to grow at 42.5 to 43 C within 7 days or after removal to 35 C for an additional 3 days. DFA tests of isolates with conjugates for recognized serogroups and species of legionellae. No fluorescence was observed when cultures of the 10 strains of L. oakridgensis were tested with

5 540 ORRISON ET AL. APPL. ENVIRON. MICROBIOL. LUJ z 0 C- LU a-i i-14: MINUTES 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 designated as 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 monovalent conjugates for the seven species and eight serogroups of legionellae listed in Materials and Methods. The OR-10 conjugate was tested against OR-10 and seven other OR isolates. The homologous 4+ staining titer was 1:64: heterologous 4+ titers were either 1:32 or 1:64. The isolates were identical or very similar by these tests. Smears of the cultures of the seven species (including eight serogroups) of legionellae were stained with the working dilution (1:32) and with a fourfold-higher concentration (1:8 dilution) of the OR-10 conjugate. All tests were negative. Thus, by these reciprocal DFA tests L. oakridgensis is unrelated to any other described legionellae. Physiological tests. In Table 1, we list the important phenotypic characteristics of L. oakridgensis 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. oakridgensis and the other described species of Legionella was its lack of a requirement for added L- cysteine for serial transfer on laboratory media. Lack of a requirement for added Fe3 was shared by all other legionellae species and serogroups under the same conditions. The OR strains also differed from all other legionellae in alkaline phosphatase activity by being the only described species in which this enzyme was not active at ph 8.5. At ph 10.5 the alkaline phosphatase of L. jordanis was very weak; at ph 8.5 it was strong. L. micdadei strains (TATLOCK, HEBA, and PPA) have alkaline phosphatase activity at ph 8.5 but not at ph Like all other species and serogroups of legionellae except L. pneumophila, L. oakridgensis failed to hydrolyze sodium hippurate. In the 19 API ZYM tests, the L. oakridgensis strains resembled other legionellae in having strong acid phosphatase and phosphoamidase activity, moderately strong leucine aminopeptidase activity, weak valine aminopeptidase and C4 and C8 esterase activity, and lack of both proteolytic (trypsin and chymotrypsin) and carbohydrate-degrading enzyme activity. GLC studies of cellular fatty acids. A representative chromatogram of the cellular fatty acids of L. oakridgensis is shown in Fig. 1. The major fatty acid was identified as a saturated branched-chain C16 fatty acid with the methyl branch in the iso position of the carbon chain (i- 16:0). The other acids present in large to moderate 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

6 VOL. 45, 1983 acids of L. oakridgensis and seven other species of Legionella are shown in Table 3. The values for L. oakridgensis are averages calculated after each of six strains (OR-10, OR-12, OR-15, OR- 16, OR-18, OR-19) was grown four different times and analyzed for cellular fatty acids. Small amounts (1% or less) of hydroxy acids were found in some of the OR strains, but they were not included because their identities have not been confirmed. The data in Table 3 show that L. oakridgensis can be readily distinguished from L. micdadei, L. bozemanii, L. dumoffii, and L. jordanis by the 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. oakridgensis, whereas in the other four species they were the two most abundant acids and together accounted for 48 to 64% of the total fatty acids. L. pneumophila, L. longbeachae, and L. gormanii contained a combined total of 20 to 38% of these two fatty acids. All of the strains of L. oakridgensis contained lower concentrations (2 to 3%) of a-15:0 acid and higher concentrations (9%) of a 17CYC acid than have been reported in other species (Table 3). The fatty acid composition of L. oakridgensis was qualitatively similar to L. pneumophila and L. longbeachae, as shown by the presence of large to moderate amounts of i-16:0, 16:1, and 16:0 acids. It could be differentiated from these two as well as the other five species, however, by the presence of moderately large amounts (13%) of the 18:0 acid that never exceeded 3% in any of the other seven Legionella species. The fatty acid composition of the OR-23 and OR-24 isolates was essentially the same as that shown in Table 3 for the other six strains of L. oakridgensis. When isolates OR-6 and OR-30 were tested, however, quantitative differences appeared in the concentrations of the i-16:1 and 16:1 acids that seemed to be related to the age of the culture. In cultures incubated for 60 h or less, the major fatty acid was 16:1, and its relative concentration was 26%; in cultures incubated for a longer period of time (at least 72 h), i- 16:0 was the major fatty acid, with a relative concentration of 21%. Regardless of the age of the OR-6 and OR-30 strains, the relative percentages of all other fatty acids found were essentially the same as those reported for L. oakridgensis (Table 3). Although a variation in the relative amounts of i-16:0 and 16:1 acids has also been reported in isolates of L. longbeachae, these changes were not consistent and did not appear to be related to the age of the culture (29). Experiments to define these variations in the ratios of the C16 acids are currently under study. F,_ LEGIONELLA OAKRIDGENSIS SP. NOV. 541 ce 'R _2. i., _. _ I. 0- t t 0w0 0' - IA r.. -I - -I - -& --I o (_ 0r _. ~- ON 0 Po f Pf0. 2. _.. 0 "_. 00 _.. 4& -4 %10 t4j W 0-0. p a, " 1. 3 o *_ _ w. 00 0% 0) _ % %o ti 0o _ o. P, 0' 0- I. la < 0-0 _. -4 0' 3 0 C) w 0' 0- o0 " w V c - 00 U, O- 0. t- _-, ".'3 - w ' O' C, 0' C, --i -3 -q -- I1) - I- pp I.. oo 00 0 % Xo o)..o

7 542 ORRISON ET AL. APPL. ENVIRON. MICROBIOL. TABLE 4. DNA relatedness of L. oakridgensis (strain OR-10) to other L. oakridgensis strains and to other Legionella species Source of unlabeled DNA Source of labeled DNA (L. oakridgensis OR-10) RBR RBR (600C)a Divergence RR(5C ~~~~(%[6OoC])b RBR (750C) L. oakridgensis OR OR OR-1S OR OR OR L. pneumophila Philadelphia 1 8 L. bozemanii WIGA 8 L. dumoffli NY-23 7 L. jordanis ABB-9 7 L. micdadei TATLOCK 6 L. gormanii LS-13 6 L. longbeachae LB-4 5 a RBR, Relative binding ratio = (percent heterologous DNA bound to hydroxyapatite/percent homologous DNA bound to hydroxyapatite) x100. Reassociation was 75 ± 5% in homologous OR-10 DNA reactions. In control reactions in which labeled OR-10 DNA was incubated in the absence of any unlabeled DNA, an average of <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 of a heterologous DNA duplex compared with that of the homologous DNA duplex is caused by 1% of the bases within the duplex that are unpaired. Antimicrobial susceptibility. The in vitro antimicrobial susceptibility data for OR strains varied little from the patterns of susceptibility of some of the other legionellae (31). The OR strains were more susceptible to trimethoprim, being similar to L. bozemanii, L. dumoffii, L. gormanii, and L. longbeachae. The OR strains and L. jordanis were the least susceptible to erythromycin, with minimal inhibitory concentrations of 4,.g/ml, compared with L. pneumophila, the most erythromycin-susceptible species with geometric mean minimum inhibitory concentrations of 0.75 jig/ml. DNA studies. The guanine-plus-cytosine content was determined in triplicate on DNAs from five L. oakridgensis strains. The guanine-pluscytosine content range was 42.3 to 43.9%, with an average value of 43.2%. The DNA from the OR-10 strain of L. oakridgensis was labeled with 32p in vitro and tested for relatedness to DNAs from five other Oak Ridge isolates and to strains of all named Legionella species (Table 4). The OR isolates were 90 to 100% related to the OR-10 strain in tests done at both optimal and stringent incubation temperatures. Divergence within related DNA sequences was 0.5% or less. In contrast, DNA from OR-10 was only 5 to 8% related to DNAs from the seven other Legionella species. Animal pathogenicity. Two guinea pigs each received intraperitoneal injections of the following numbers of cells of the OR-23 strain: 1.2 x 104, 1.2 x 105, 1.2 x 106, and 1.2 x 107. All animals showed symptoms typical of Legionella infection; i.e., within 1 to 2 days, they had temperatures above 40 C, ruffled hair, lethargy, and lacrimation. Tissues from one animal of each pair were examined by DFA staining tests and cultured. L. oakridgensis cells were present in large numbers in the peritoneal exudate of all four animals by DFA tests, and L. oakridgensis was cultured from all animals. Two of the remaining four guinea pigs died of the infections, and the other two were killed while moribund. Inhibition of growth of legionellae on CYE lacking L-cysteine or Fe3 when inoculated directly from animal tissue. In Table 5 the growth of legionellae from spleen homogenates of guinea pigs and from a human lung homogenate when inoculated on complete CYE agar is compared with that obtained on deficient CYE agar. L. oakridgensis strains required added L-cysteine for isolation from tissue, although it was not needed for growth when the culture was transferred from the complete CYE isolation medium to deficient CYE medium. Unless the inoculum was very heavy, the OR-23 isolate also required added Fe3 for normal good growth from tissue (Table 5). L. micdadei, L. dumoffli, and L. pneumophila required the addition of both Fe3 and L-cysteine to the CYE agar for growth from tissue, and they required added L-cysteine, but

8 VOL. 45, 1983 LEGIONELLA OAKRIDGENSIS SP. NOV. 543 TABLE 5. Inhibition of growth of legionellae on CYE agar slants lacking added L-cysteine or Fe3 when inoculated directly from animal tissue Growth on CYE agar mediaa Legionella sp./specimen Inoculum Complete No Fe3 No L-cysteine L. oakridgensis OR-23 Guinea pig 1 Spleen homogenate b Guinea pig 2 Spleen swab 1+ - Guinea pig 3 Spleen swab 3+ - Guinea pig 4 Spleen swab b L. micdadei (TATLOCK) Guinea pig spleen homogenate 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 of growth and its identity were determined by streaking on blood agar and by DFA tests. 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 further transfers on CYE agar. DISCUSSION Although the 10 strains of L. oakridgensis were isolated from the water of industrial cooling towers in two widely separated locations, they were identical in the characteristics examined in this study. They differed from all described Legionella species except L. jordanis in not requiring added L-cysteine for growth on CYE agar except upon initial isolation from animal tissues. Added L-cysteine was stimulatory but not required by L. jordanis for routine culture on CYE agar. L. oakridgensis also required added Fe3 for primary isolation from animal tissues, as do the other three legionellae species tested. The rapid loss of a requirement for L-cysteine and Fe3 for the growth of L. oakridgensis after isolation from animal tissue suggests a complex host-parasite relationship not yet defined. Also, it is possible that L- cysteine and Fe3 are required for growth of the bacteria directly from animal tissue because, in some way, they neutralize tissue factors that inhibit in vitro growth during the first transfer on CYE agar. Because fairly large inocula were used in the serial transfer experiments, quantitative differences may be revealed when the amount of growth obtained on L-cysteine- and Fe3-deficient CYE media is compared with that on the complete medium. Clearly, however, L. oakridgensis and, to a lesser extent, L. jordanis differed from all other Legionella species in their requirements for L-cysteine. No described species of legionellae required added Fe3 for good growth in routine serial culture on CYE agar slants. Although the qualitative fatty acid composition of L. oakridgensis was similar to that of other species of legionellae, several quantitative differences existed that could separate this species from other members of the genus. Strains of L. oakridgensis contained relatively large amounts (12% or greater) of 18:0 acid that had not been previously reported as a major cellular fatty acid in species of Legionella (8, 29; Table 3). Although the relative concentrations of the i- 16:0 and 16:1 acids in some young cultures of L. oakridgensis did vary somewhat from that shown in Table 3 and Fig. 1, the other characteristic fatty acids did not change. Thus, L. oakridgensis can be distinguished from all other known species of Legionella by quantitative differences in its cellular fatty acid composition. The presence of hydroxy fatty acids has not been reported previously in whole-cell hydrolysates of legionellae, but very low concentrations of these acids have been found in various cell fractions of L. pneumophila (23). We were able to detect hydroxy acids in the present study because a high-resolution capillary column was used for the GLC analysis. These acids are present at low concentrations, however, and may not be useful for differentiating members of this genus. The level of DNA relatedness between strains of L. oakridgensis is very high, leaving no doubt that these strains represent a single species. Relatedness between L. oakridgensis and other legionellae, although low, is not substantially different from that between several other Legionella species. The guanine-plus-cytosine con-

9 544 ORRISON ET AL. tent of L. oakridgensis DNA (43%) is within the range (39 to 44%) reported for other Legionella species (5, 15). L. oakridgensis did not differ importantly from other legionellae in morphology, staining characteristics, pathogenicity for guinea pigs, or antimicrobial susceptibility. Neither motility nor the presence of flagella could be demonstrated, but strains of other species of legionellae have been reported to be nonmotile (7). L. oakridgensis has not been incriminated in human infection, but its pathogenicity for guinea pigs suggests that it may be an unrecognized human pathogen. On the basis of phenotypic similarity to other legionellae and in keeping with previously established practice (4, 8, 24, 27), we propose that this new species be placed with the other legionellae in the genus Legionella. The name Legionella oakridgensis sp. nov. is proposed for the species represented by the 10 strains isolated from industrial cooling tower water: oak * ridg * en' sis sp. nov., M.L. fem. adj. oakridgensis coming from Oak Ridge, Tenn., the city where the organism was first isolated. The type strain of L. oakridgensis is OR-10 (ATCC 33761). ACKNOWLEDGMENTS We gratefully acknowledge and deeply appreciate the technical assistance of A. G. Steigerwalt, Elizabeth Dominque, and Carol Duncan. We thank C. W. Moss for the identification of cellular fatty acids by mass spectrometry. We thank Thomas Ozro MacAdoo of the Department of Foreign Languages and Literature, Virginia Polytechnic Institute and State University, Blacksburg, for help in arriving at the name of L. oakridgensis and for advice on naming this organism in accordance with the rules of Latin grammar and the requirements of the Bacteriological Code. This research was partially sponsored by the Electric Power Research Institute (contract RP 1909) and the U.S. Nuclear Regulatory Commission (under interagency agreement with the Department of Energy) through the Oak Ridge National Laboratory, which is operated by Union Carbide Corp. for the Department of Energy (contract W-7405-eng-26). LITERATURE CITED 1. Baine, W. B., and J. K. Rasheed Aromatic substrate specificity of browning by cultures of the Legionnaires' disease bacterium. Ann. Intern. Med. 90: Band, J. D., M. LaVenture, J. P. Dais, G. F. Mallison, P. Skally, P. S. Hayes, W. L. Schell, H. Weiss, D. J. Greenberg, and D. W. Fraser Epidemic Legionnaires' Disease: airborne transmission down a chimney. J. Am. Med. Assoc. 245: Bibb, W. F., R. J. Sorg, B. M. Thomsason, M. D. Hicklin, A. G. Stelgerwalt, D. J. Brenner, and M. R. Wulf Recognition of a second serogroup of Legionella longbeachae. J. Clin. Microbiol. 14: Brenner, D. J., A. G. Steigerwalt, G. W. Gorman, R. E. Weaver, J. C. Feeley, L. G. Cordes, H. W. WilkInson, C. Patton, B. M. Thomason, and K. R. Lewallen-SassevDlle Legionella bozemanii sp. nov. and Legionella dumoffii sp. nov.: classification of two additional species of Legionella associated with human pneumonia. Curr. Microbiol. 4: Brenner, D. J., A. G. Stelgerwalt, and J. E. McDade Classification of the Legionnaires' disease bacteri- APPL. ENVIRON. MICROBIOL. um: Legionella pneumophila, genus novum species nova of the family Legionellaceae, familia nova. Ann. Intern. Med. 90: Brenner, D. J., A. G. Steigerwalt, R. E. Weaver, J. C. Feeley, and M. Mandel Classification of the Legionnaires' disease bacterium: an interim report. Curr. Microbiol. 1: Chandler, F. W., J. A. Blackmon, M. D. Hicklin, R. M. Cole, and C. S. Calloway Ultrastructure of the agent of Legionnaires' disease in the human lung. Am. J. Clin. Pathol. 71: Cherry, W. B., G. W. Gorman, L. H. Orrison, C. W. Moss, A. G. Steigerwalt, H. W. Wilkinson, S. E. Johnson, R. M. McKinney, and D. J. Brenner Legionella jordanis: a new species of Legionella isolated from water and sewage. J. Clin. Microbiol. 15: Cherry, W. B., and R. M. McKlnney Detection of Legionnaires' disease bacteria in clinical specimens by direct immunofluorescence, p In G. L. Jones and G. A. Hebert (ed.), "Legionnaires' ": the disease, the bacterium and methodology. Center for Disease Control, Atlanta. 10. Clark, W. A A simplified Leifson flagella stain. J. Clin. Microbiol. 3: Cordes, L. G., D. W. Fraser, P. Skaliy, C. A. Perlino, W. R. Elsea, G. F. Mallison, and P. S. Hayes Legionnaires' disease outbreak at an Atlanta, Georgia country club: evidence for spread from an evaporative condenser. Am. J. Epidemiol. 111: Dondero, T. J., R. C. Rendtorff, G. W. MalHlson, R. M. Weeks, J. S. Levy, E. W. Wong, and W. Schaffner An outbreak of Legionnaires' Disease associated with a contaminated air-conditioning cooling tower. N. EngI. J. Med. 302: England, A. C., HI, R. M. McKinney, P. Skaliy, and G. W. Gorman A fifth serogroup of Legionella pneumophila. Ann. Intern. Med. 93: Feeley, J. C., R. J. Gibson, G. W. Gorman, N. C. Langford, J. K. Rasheed, D. C. Mackel, and W. B. Baine CYE agar: a primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10: Garrity, G. M., A. Brown, and R. M. Vickers Tatlockia and Fluoribacter: two new genera of organisms resembling Legionella pneumophila. Int. J. Syst. Bacteriol. 30: Glick, T. H., M. B. Gregg, B. Berman, G. Mallison, W. W. Rhodes, Jr., and I. Kassanoff Pontiac fever. An epidemic of unknown etiology in a health department: clinical and epidemiological aspects. Am. J. Epidemiol. 107: Goding, J. W Conjugation of antibodies with fluorochromes: modification to the standard methods. J. Immunol. Methods 13: Hebert, G. A Hippurate hydrolysis by Legionella pneumophila. J. Clin. Microbiol. 13: H6bert, G. A., B. Pittman, R. M. McKinney, and W. B. Cherry The preparation and physiochemical characterization of fluorescent antibody reagents. Center for Disease Control, Atlanta. 20. H6bert, G. A., A. G. Steigerwalt, and D. J. Brenner Legionella micdadei species nova: classification of a third species of Legionella associated with human pneumonia. Curr. Microbiol. 3: Kersterks, K., and J. DeLey Enzymatic tests with resting cells and cell free extracts, p. 44. In J. R. Norris and D. W. Robbins (ed.), Methods in microbiology, vol. 6A. Academic Press, Inc., New York. 22. Marmur, J., and P. Doty Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol. 5: Mayberry, W. R Dihydroxy and monohydroxy fatty acids in Legionella pneumophila. J. Bacteriol. 147: McKinney, R. M., R. Porchen, P. H. Edelstein, M. J. Blssett, P. P. Harris, S. P. Bondell, A. G. Steigerwalt,

10 VOL. 45, 1983 R. E. Weaver, M. Ein, D. S. Lindquist, and D. J. Brenner Legionella longbeachae species novum, another etiologic agent of human pneumonia. Ann. Intern. Med. 94: McKinney, R. M., L. Thacker, P. P. Harris, K. R. Lewallen, G. A. Hebert, P. H. Edelstein, and B. M. Thomason Four serogroups of Legionnaires' disease bacteria defined by direct immunofluorescence. Ann. Intern. Med. 90: McKinney, R. M., H. W. Wilkinson, H. M. Sommers, B. J. Fikes, K. R. Sassevilie, M. M. Yungbluth, and J. S. Wolf Legionella pneumophila serogroup 6: isolation from cases of legionell6sis, identification by immunofluorescence staining, and immunological response to infection. J. Clin. Microbiol. 12: Morris, G. K., A. Stelgerwalt, J. C. Feeley, E. S. Wong, W. T. Mardn, C. M. Patton, and D. J. Brenner Legionella gormanii sp. nov. J. Clin. Microbiol. 12: Moss, C. W Gas-liquid chromatography as an analytical tool in microbiology. J. Chromatogr. 203: LEGIONELLA OAKRIDGENSIS SP. NOV Moss, C. W., D. E. Karr, and S. B. Dees Cellular fatty acid composition of Legionella longbeachae sp. nov. J. Clin. Microbiol. 14: Moss, C. W., S. B. Samuels, J. Liddle, and R. M. McKinney Occurrence of branched-chain hydroxy fatty acids in Pseudomonas maltophila. J. Bacteriol. 114: Orrison, L. H., W. B. Cherry, C. B. Fliermans, S. B. Dees, L. K. McDougal, and D. J. Dodd Characteristics of environmental isolates of Legionella pneumophila. Appl. Environ. Microbiol. 42: Rlstroph, J. D., K. W. Hedlund, and R. G. Allen Liquid medium for growth of Legionella pneumophila. J. Clin. Microbiol. 11: Tborasberry, C., and L. A. Kirven ,-Lactamase of the Legionnaires' bacterium. Curr. Microbiol. 1: Weaver, R. E., and J. C. Feeley Cultural and biochemical characterization of the Legionnaires' disease bacterium, p In G. L. Jones and G. A. Hdbert (ed.), "Legionnaires' ": the disease, the bacterium and methodology. Center for Disease Control, Atlanta. Downloaded from on October 19, 2018 by guest