Legionella anisa: a New Species of Legionella Isolated from Potable

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1985, p /85/ $02.00/0 Vol. 49, No. 2 Legionella anisa: a New Species of Legionella Isolated from Potable Waters and a Cooling Tower GEORGE W. GORMAN,' JAMES C. FEELEY,'* ARNOLD STEIGERWALT,' PAUL H. EDELSTEIN,2 C. WAYNE MOSS,' AND DON J. BRENNER' Division of Bacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia ; Medical and Research Services, Veterans Administration Medical Center, Wadsworth Division, Los Angeles, California ; and Department of Medicine, UCLA School of Medicine, Los Angeles, California Received 25 May 1984/Accepted 30 October 1984 Between March 1980 and June 1981, five strains of Legionella-like organisms were isolated from water. Four were recovered from potable water collected from hospitals in Chicago, Ill., and Los Angeles, Calif., during outbreaks of nosocomial legionellosis. The fifth strain was isolated from water collected from an industrial cooling tower in Jamestown, N.Y. The strains exhibited biochemical reactions typical of Legionella species and were gram-negative motile rods which grew on buffered charcoal-yeast extract agar but not on blood agar, required cysteine, and were catalase positive, urease negative, nitrate negative, hippurate negative, and nonfermentative. All strains were positive for oxidase and beta-lactamase and produced a brown, diffusible pigment. Of the five strains, four exhibited blue-white autofluorescence under long-wavelength UV light. The fatty-acid composition and ubiquinone content of these strains were consistent with those of other Legionella species. Direct fluorescent-antibody examination of the five strains with conjugates to previously described Legionella species demonstrated no cross-reactions except with the conjugates to L. longbeachae serogroup 2 and L. bozemanii serogroup 2. Four strains gave a 4+ reaction to the L. Iongbeachae serogroup 2 conjugate and the fifth strain gave a 1 + reaction. Each of the five strains gave a 4+ reaction with the conjugate to L. bozemanii serogroup 2. DNAs from the five strains were highly related (84 to 99%) and showed 5 to 57% relatedness to other Legionella species. These strains constitute a new species in the genus Legionella, and the name Legionella anisa sp. nov. is proposed. The type strain of L. anisa is WA-316-C3 (ATCC 35292). In 1979, Brenner et al. reported that the agent responsible for the outbreaks of Legionnaires disease in Philadelphia and several other locations was a new bacterium, Legionella pneumophila (5). Since then, several additional Legionella species have been reported (2, 4, 6, 7, 11, 13, 14, 16, 17, 23). In our laboratory, the use of direct plating techniques rather than animal inoculation has reduced the complexity of Legionella isolations. We describe a direct plating method that we use after the concentration and selective decontamination of water samples. This procedure has aided in the recovery of existing Legionella species as well as the detection of new species. We also now describe another Legionella species for which the name Legionella anisa sp. nov. is proposed. The type strain of L. anisa is WA-316-C3 (ATCC 35292). MATERIALS AND METHODS Isolation procedures for environmental specimens. Specimens of water were collected from three separate sites associated with outbreaks of legionellosis. Hot water (10 liters) was collected from a sink in a hospital in Los Angeles, Calif. Of the unconcentrated water sample, 1 ml was added to an equal volume of sterile KCI-HCI buffer (ph 2.0), mixed by inversion, and then incubated for 15 min at room temperature (3). The ph of the sample was then neutralized by adding 1 ml of sterile 0.01 N KOH solution, the sample was again mixed, and 0.1 ml was plated onto charcoal-yeast extract agar (CYE) and buffered charcoal-yeast extract agar (BCYE) media with and without antimicrobial agents (3, 24). * Corresponding author. 305 In addition, the bacteria in a 1-liter volume of this sample were concentrated by filtration on a polycarbonate membrane filter (pore size, 0.2,um) supported on a steam-sterilized, 47-mm polycarbonate filter-funnel assembly (Nuclepore Corp., Pleasanton, Calif.). After filtration, we removed the membrane with sterile forceps and placed it soiled side down into a screw cap jar containing 10 ml of sterile tap water. The particulates collected on the filter face were suspended by placing the sealed jar containing the filter in an ultrasonic cleaning bath for 10 min. Portions (0.1 ml each) of the resulting suspension were plated onto CYE and BCYE media with and without antimicrobial agents, directly and after exposure to acid treatment, and incubated at 35 C in a humidified incubator with an atmosphere of 2.5% CO2 in air. We rubbed sterile cotton swabs around the insides of shower heads in a Chicago hospital during a nosocomial outbreak of Legionnaires disease caused by L. pneumophila. We then placed each swab in 5 ml of water that was simultaneously collected from the shower head and transported these samples in a cool (<10 C) container to the Centers for Disease Control (CDC). After 2 months of storage in a refrigerator (5 C), the swabs were vortexed in the collecting water, and 0.1-ml samples of the resulting suspensions were cultured directly and after acid treatment onto CYE and BCYE media with and without antimicrobial agents, as described above but without filter concentration. Water (1 liter) was collected from a cooling tower of an industrial manufacturing plant in Jamestown, N.Y. The cooling tower of the plant was associated with an outbreak of legionellosis caused by L. pneumophila. The water was transported to CDC in a cool (<10 C) container, and 1-mI

2 306 GORMAN ET AL. TABLE 1. Intensity of fluorescence staining of L. anisa strains with DFA conjugates Intensity6 of fluorescence staining of strain: Conjugate' WA-316- CH-47- CH-47- E327F- JA-19- C3 C1 C3 DP JS-C12 L. bozemanii Serogroup Serogroup L.feeleii L. gormanii L. jordanis L. Iongbeachae Serogroup Serogroup L. micdadei L. anisa CH-47-C CH-47-C L. oakridgensis L. wadsvorthii CDCpoolA" CDCpoolBd _ CDC pool CDCpool D a Conjugates listed for individual Legionella species are monovalent. Those listed as CDC pools are polyvalent. b Symbols: 4+, brilliant staining; 3+, bright staining; 1+, barely visible staining; -, no detectable staining. c Contains conjugates to L. pneumophila serogroups 1 through 4. d Contains conjugates to L. pneiumophila serogroups 5 and 6, L. duimoffli, and L. longbeac hae serogroup 1. e Contains conjugates to L. bozemnanii, L. gormanii, L. micdadei, and L. longbeachae serogroup 2. f Contains conjugates to L. pneuimophila serogroups 7 through 9. samples (unconcentrated) were acid treated and plated on CYE agar (12) and incubated as described above. Minimally chlorinated tap water (0.1 mg/liter) was taken from a sink fixture at a hospital in Los Angeles and plated directly onto BCYE and BMPA (10) agar media. In addition, a 30-ml sample was centrifuged at 4,000 x g for 10 min to obtain an estimated 10-fold concentration of particulates. The sediment was suspended in 3 ml of the supernatant and inoculated intraperitoneally into two guinea pigs and onto BCYE and BMPA plates with and without an acid treatment that consisted of one part of the sample to nine parts acid buffer, ph 2.0, for 4 min. DFA tests. Direct fluorescent-antibody (DFA) tests were performed with formalized antigen preparations of the five isolates as previously described (8), by using routine test dilutions of currently available Legionella conjugates which included L. pneumophila serogroups 1 through 9, L. micdadei, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. Iongbeachae serogroups 1 and 2, L. jordanis, L. wadsworthii, L. oakridgensis, and L. feeleii. DFA conjugates for L. anisa were prepared with antisera against strains CH-47-C1 and CH-47-C3. APPL. ENVIRON. MICROBIOL. Phenotypic characterization. The five strains of L. anisa were examined for staining characteristics and physiological activity by previously published methods (7, 11, 13, 17, 23). DNA hybridization studies. DNAs from L. anisa strains WA-316-C3 and E327F-DP, L. oakridgensis OR-10 (23), and L. wadsworthii A (11) were isolated, purified, labeled with 32P04, and reacted as previously described (4, 5) with purified, unlabeled DNAs from L. anisa strains CH-47-C1, CH-47-C3, and JA-19-JS-C12 and from all described Legionella species. The method used to determine the guanineplus-cytosine content of DNA has been described (5). Gas-liquid chromatography studies. Cells for fatty acid analysis were obtained after 1 to 3 days of growth on CYE agar slants incubated at 37 C. Growth from one slant was removed with ca. 1 ml of sterile water and transferred to a test tube (20 by 150 mm) containing 4 ml of 15% NaOH in 50% aqueous methanol. The cellular lipid was saponified, and the resulting fatty acids were methylated as described previously (18). Methyl esters were analyzed by gas-liquid chromatography with a Hewlett-Packard series 5880A gas chromatograph equipped with a flame ionization detector, an automatic sample injector, and a fused-silica capillary column (50 m by 0.2 mm) with cross-linked OV-1 as stationary phase (Hewlett-Packard Co., Avondale Div., Avondale, Pa.). Fatty acids (as methyl esters) were identified by comparison of the gas-liquid chromatography retention time with known standards and were further confirmed by both electron impact and chemical ionization mass spectrometry (18, 22). Determination of isoprenoid quinones. For isoprenoid-quinone determinations, cultures were grown for 48 to 72 h on CYE plates. Cells were removed by scraping and were saponified by the modified procedure of Abe et al. (1), as described previously (15). Isoprenoid quinones were removed from the saponified cells by extracting four times, each time with 5 ml of a 1:1 mixture of diethylether-hexane. The combined diethylether-hexane layer was reduced to ca. 1.0 ml by evaporation, and the extracted isoprenoid quinones were analyzed by reverse-phase, thin-layer chromatography and by reverse-phase, high-pressure liquid chromatography as previously described (15, 20). Identification was established with both of these techniques by comparison with ubiquinone standards (Sigma Chemical Co., St. Louis, Mo.) and ubiquinones from other bacteria (9). Identification was confirmed by eluting the isolated ubiquinones from a reverse-phase, thin-layer chromatography plate followed by analysis by both electron impact and chemical ionization mass spectrometry (15). RESULTS Isolation of strains. We isolated five strains of L. anisa from four samples. The type culture strain, WA-316-C3, was isolated from the unconcentrated, acid-treated, 10-liter, potable-water sample collected in Los Angeles. The isolation was made on BCYE agar without antimicrobial agents. In addition, several Legionella-like colonies were also observed on CYE and BCYE media, both with and without antimicrobial agents, that had been inoculated with acidtreated filtrates. Strains CH-47-C1 and CH-47-C3 were recovered from the same swab sample from a shower head in Chicago. Growth occurred only on BCYE agar without antimicrobial agents, and L. anisa was the only Legionella species isolated from the sample. Strain JA-19-JS-C12 was isolated only after acid treatment of the Jamestown coolingtower water sample and plating on plain CYE agar (12). Strain E327F-DP was isolated from the sediment of the

3 VOL. 49, 1985 centrifuged sample of chlorinated tap water taken in Los Angeles. A single colony was recovered from this sample after acid treatment and plating onto BCYE medium. No legionellae were recovered from the guinea pigs inoculated with this sample. All of the isolated L. anisa strains had phenotypic characteristics typical of legionellae. With a Woods light (366 nm), we found that colonies of four of the strains autofluoresced blue-white, whereas the remaining strain, CH-47-C3, did not. All strains gave identical reactions in all other tests (see below). DFA test results for all five L. anisa isolates were negative with all conjugates except L. Iongbeachae serogroup 2 and L. bozemanii serogroup 2. Strong cross-reactions (3+ to 4+) were observed with four of the strains to both the monovalent and the polyvalent pooled conjugate which contained the L. Iongbeachae serogroup 2 component, and all strains exhibited strong cross-reactions with the monovalent conjugate to L. bozemanii serogroup 2 (Table 1). The JA-19-JS- C12 strain was notably less cross-reactive (1+). Conjugates prepared from hyperimmune rabbit sera to strains CH-47-C1 and CH-47-C3 produced similar strong reactions with each of the five strains of L. anisa. In reactions at 60 C, labeled DNAs from L. anisa strains WA-316-C3 and E327F-DP were 84 to 99% related to unlabeled DNA from other L. anisa strains (Table 2). Divergence in related DNA sequences from L. anisa strains was 0.5 to 1.0%. In reactions at 75 C, DNAs from L. anisa strains were 84 to 92% related. L. anisa DNA was 5 to 57% related to DNAs from other Legionella species. Divergence in related sequences between L. anisa and other Legionella species was 7.0 to 14.5%, and relatedness in reactions at 75 C fell to 3 to 27%. L. anisa showed the highest relatedness to L. bozemanii (57%) and to the other autofluorescing species, L. dumoffii (25%) and L. gormanii (39%). L. anisa was 22% related to the nonfluorescing species L. wadsworthii. L. wadsworthii was 22 to 30% related to the autofluorescing species other than L. bozemanii (11). The guanine-plus-cytosine content of L. anisa WA-316-C3 was 42 mol%. The cellular-fatty acid compositions of the five isolates of L. anisa were qualitatively similar to other Legionella species (Table 3). These compositions had characteristic features such as the presence of iso-branched-chain 14- and 16-carbon acids (i-14:0 and i-16:0, respectively) and anteisobranched-chain 15- and 17-carbon acids (a-15:0 and a-17:0, respectively) together with moderate amounts of mono-unsaturated straight-chain 16-carbon acid (C16:1) and 17-carbon cyclopropane acid (17-cyc). Like other legionellae, L. anisa contained relatively large amounts (63 to 71%) of the total acids as branched-chain acids but no individual hydroxy acids at concentrations greater than 1% (7, 11, 18, 21-23). Quantitatively, the fatty acid composition of L. anisa was most similar to that of L. bozemanii (7, 18), L. longbeachae (21), L. dumoffii (7, 23), L. jordanis (7, 13), L. gormanii (7, 23), and L. wadsworthii (11) but was distinct from that of L. pneumophila (18, 21), L. oakridgensis (23), and L. micdadei (7, 23). Each of the five isolates of L. anisa contained ubiquinones Q-9, Q-10, Q-11, and Q-12. Menaquinones were not detected in any strain (Table 4). This ubiquinone pattern is similar to that of L. bozemanii, L. dumoffii, and L. Iongbeachae but is distinct from that of L. pneumophila, L. micdadei, L. jordanis, L. wadsworthii, L. oakridgensis, and L. feeleii (15, 19). The patterns were reproducible, as essentially identical results were obtained with each of the isolates grown and processed three separate times. The presence of Q-10, Q-11, LEGIONELLA ANISA, A NEW LEGIONELLA SP. 307 TABLE 2. DNA relatedness of L. anisa strains 32PO4-labeled DNA from: L. (ianisa L. ainisa Source of unlabeled DNA WA-316-C3 E327F-DP RBR" D" RBR RBR (60 C) (75 C) (60 C) L. anisa WA-316-C3 100b i 94 L. anisa E327F-DP lbob L. anisa CH-47-C L. anisa JA-19-JS-C L. anisa CH-47-C L. bozemanii WIGA L. gormanii LS L. dumoffii NY L. oakridgensis OR-10 11' L. pneumophila-philadelphia L. longbeachae-long Beach L. micdadei TATLOCK 5 6 L. jordanis BL L. wadsworthii A 22' a RBR, Relative binding ratio; RBR = (DNA bound to hydroxyapatite in heterologous DNA reactions/dna bound to hydroxyapatite in homologous DNA reactions) x 100. D, Divergence, calculated to the nearest 0.5%. D is the amount of unpaired bases in a related DNA sequence, on the assumption that each 1% of unpaired bases causes ca. 10C decrease in the thermal stability of a DNA duplex. bbefore normalization some 55% of DNA bound to hydroxypatite in homologous reactions. In control reactions containing only labeled DNA, 0.2 to 1.5% of the DNA bound to hydroxypatite. These control reaction values were subtracted from all experimental values before RBR was calculated. All reactions were done at least twice. ' In these experiments labeled L. oakridgensis OR-10 and L. wadsworthii A were reacted with unlabeled DNA from strain WA-316-C3. and Q-12 as major components in L. anisa is consistent with previous data which show that all Legionella species contain ubiquinones with more than 10-isoprene units in the side chain (15, 19). DISCUSSION L. anisa strains were isolated from widely separated geographic areas of the United States and from both industrial and drinking water. Use of acid treatment, alone or with the particulates concentrated in water by either filtration through polycarbonate filters or centrifugation, proved effective in the isolation of these strains. Increased numbers of autofluorescent colonies were observed after filtration of a large-volume water sample, and the successful isolation of another strain occurred only after centrifugation. We have found that particulates in 1-liter volumes of tap water are easily collected on a single filter disk. Recovery of L. anisa isolates and other Legionella species by culturing the suspended bacteria is practical and more sensitive than recovery from unconcentrated samples. Biochemical reactions, cellular-fatty acid composition, and ubiquinone content did not reveal any unique characteristic of L. anisa to differentiate this species from other Legionella species. Since one strain of L. anisa did not autofluoresce blue-white under long-wave UV light, the use of this phenotypic characteristic for placing autofluorescent species of Legionella into a separate genus should be questioned. DFA techniques that include standard methods with routine test dilutions of available Legionella conjugates resulted in strong cross-reactions to pooled and monovalent conjugates with L. Iongbeachae serogroup 2 and to the monovalent conjugate with L. bozemanii serogroup 2. This finding should serve as a caution for possible misidentifica-

4 308 GORMAN ET AL. APPL. ENVIRON. MICROBIOL. TABLE 3. Cellular fatty acid composition of L. anisa Fatty acid (% of total acids)" Strain i-14:0 i-15:0 a-15:0 15:1 15:0 i-16:0 16:1 un 16:0 i-17:0 a-17:0 17-cyc 17:0 18:0 20:0 WA-316-C Tb E-327F-DP 5 T CH-47-C T CH-47-C T JA-19-JS-C T T a Mean of at least two determinations. Fatty acids are designated as follows: number to the left of the colon, number of carbon atoms; number to the right of the colon, number of double bonds; i-, methyl branch at the iso carbon atom; a-, methyl branch at the anteiso carbon atom; cyc, cyclopropane ring; un, unidentified. b T, Less than 1%. tion of species on the basis of current serological tests and suggests that DFA procedures probably are only presumptive for species identification. Since DNA relatedness studies showed that the five strains are highly related to each other and closely related to other Legionella species, a new species should be established for them in the genus Legionella. We propose the name Legionella anisa sp. nov. for this new species [(a'ni.sa) Gr. adj. anisos unequal; N.L. fem. adj. anisa unequal, referring to the fact that blue-white autofluorescence is seen in most but not all strains]. Description of L. anisa sp. nov. The phenotypic characteristics of L. anisa fit the definition of the family Legionellaceae and of the genus Legionella. L. anisa grew on CYE and BCYE agars but not on media lacking L-cysteine HCI. It is a gram-negative rod that is motile by means of a few polar, subpolar, or lateral flagella. It is positive in catalase, gelatinase, and reactions for beta-lactamase and produces a brown, diffusible pigment on tyrosine-yeast extract agar. It is negative in reactions for oxidase, urease, tyrosine decarboxylase, ornithine decarboxylase, reduction of nitrate to nitrite, hydrolysis of hippurate, and acid production from carbohydrates (D-glucose, D-xylose, D-mannitol, lactose, sucrose, and maltose). With a Woods light (366 nm), we found that colonies of four of the strains autofluoresced blue-white, whereas the remaining strain, CH-47-C3, did not. The guanine-plus-cytosine content of DNA from the type strain was 42 mol% (thermal denaturation). All L. anisa strains contain predominantly branched-chain fatty acids and ubiquinones with 10 or more isoprene units. All strains were isolated from potable or industrial water; none were associated with disease in humans or animals. The type strain of L. anisa is WA-316-C3 (ATCC 35292). It was isolated by one of us (G.W.G.) from tap water from the Veterans Administration Medical Center, Wadsworth Division, Los Angeles. The characteristics of the type strain are the same as those given for the species. TABLE 4. Ubiquinone content of L. anisa Amt of ubiquinone": Strain Q-9 Q-10 Q-11 Q-12 Q-13 WA-316-C T E327F-DP T CH-47-C CH-47-C T JA-19-JS-C T a Ubiquinones were determined by high-pressure liquid chromatography with a reverse-phase column. Numbers refer to the relative amounts of ubiquinones with the major component(s) designated by 4; T, Present but in amounts less than 10% of the major component. ACKNOWLEDGMENTS P.H.E. was supported by the Medical Research Service of the Veterans Administration. We thank William T. Martin and Hazel Wilkinson and her staff at CDC for providing the comparative DFA reactions. We thank Thomas 0. MacAdoo, Department of Foreign Languages and Literatures, Virginia Polytechnic Institute and State University, Blacksburg, Va., for his expert advice on the etymology of the new species name. We also thank Robert C. Good for his advice and Jane Cronic and Sandra Barnett for the preparation of this manuscript. LITERATURE CITED 1. Abe, K. K., M. 0. Ishibashi, K. Kawabe, and G. Katsui Determination of ubiquinone in serum and liver by high speed liquid chromatography. J. Nutr. Sci. Vitaminol. 24: Bibb, W. F., R. J. Sorg, B. M. Thomason, M. D. Hicklin, A. G. Steigerwalt, D. J. Brenner, and M. R. Wulf Recognition of a second serogroup of Legionella longbeachae. J. Clin. Microbiol. 14: Bopp, C. A., J. W. Sumner, G. K. Morris, and J. G. Wells Isolation of Legionella spp. from environmental water samples by low-ph treatment and use of a selective medium. J. Clin. Microbiol. 13: Brenner, D. J., A. G. Steigerwalt, G. W. Gorman, R. E. Weaver, J. C. Feeley, L. G. Cordes, H. W. Wilkinson, C. M. Patton, B. M. Thomason, and K. R. Lewallen Sasseville 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. Steigerwalt, and J. E. McDade Classification of the Legionnaires' disease bacterium: Legionella pneumophila, genus novum, species nova of the family Legionellaceae, familia nova. Ann. Intern. Med. 90: Campbell, J., W. F. Bibb, M. A. Lambert, S. Eng, A. G. Steigerwalt, J. Allard, C. W. Moss, and D. J. Brenner Legionella sainthelensi: a new species of Legionella isolated from water near Mt. St. Helens. Appl. Environ. Microbiol. 47: 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 Legionellajordanis: a new species of Legionella isolated from water and sewage. J. Clin. Microbiol. 15: Cherry, W. B., and R. M. McKinney Detection of Legionnaires' disease bacteria in clinical specimens by direct immunofluorescence, p In G. L. Jones and G. A. I-ebert (ed.), "Legionnaires"' disease, the bacterium and methodology. Centers for Disease Control, Atlanta. 9. Collins, M. D., and D. Jones Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol. Rev. 45: Edelstein, P. H Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and envirdrnental specimens. J. Clin. Microbiol. 14: Edelstein, P. H., D. J. Brenner, C. W. Moss, A. G. Steigerwalt, E. M. Francis, and W. L. George Legionella wadsworthii

5 VOL. 49, 1985 species nova: a cause of human pneumonia. Ann. Intern. Med. 97: Feeley, J. C., R. J. Gibson, G. W. Gorman, N. C. Langford, J. K. Rasheed, D. C. Mackel, and W. B. Baine Charcoalyeast extract agar: primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10: Hebert, 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: Herwaldt, L. A., G. W. Gorman, T. McGrath, S. Tome, B. Brake, A. W. Hightower, J. Jones, A. L. Reingold, P. A. Boxer, P. W. Tang, C. W. Moss, H. Wilkinson, D. J. Brenner, A. G. Steigerwalt, and C. V. Broome A new Legionella species, Legionella feeleii species nova, causes Pontiac Fever in an automobile plant. Ann. Intern. Med. 100: Karr, D. E., W. F. Bibb, and C. W. Moss Isoprenoid quinones of the genus Legionella. J. Clin. Microbiol. 15: McKinney, R. M., R. K. Porschen, P. H. Edelstein, M. L. Bissett, P. P. Harris, S. P. Bondell, A. G. Steigerwalt, R. E. Weaver, M. E. Ein, D. S. Lindquist, R. S. Kops, and D. J. Brenner Legionella longbeachae sp. nov., another etiologic agent of human pneumonia. Ann. Intern. Med. 94: Morris, G. K., A. Steigerwaldt, J. C. Feeley, E. S. Wong, W. T. Martin, C. M. Patton, and D. J. Brenner Legionella LEGIONELLA ANISA, A NEW LEGIONELLA SP. 309 gormanii sp. nov. J. Clin. Microbiol. 12: Moss, C. W Gas-liquid chromatography as an analytical tool in microbiology. J. Chromatogr. 203: Moss, C. W., W. F. Bibb, D. E. Karr, G. 0. Guerrant, and M. A. Lambert Cellular fatty acid composition and ubiquinone content of Legionella feeleii sp. nov. J. Clin. Microbiol. 18: Moss, C. W., and G. 0. Guerrant Separation of bacterial ubiquinones by reverse-phase high-pressure liquid chromatography. J. Clin. Microbiol. 18: 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., R. E. Weaver, S. B. Dees, and W. B. Cherry Cellular fatty acid composition of isolates from Legionnaires disease. J. Clin. Microbiol. 6: Orrison, L. H., W. B. Cherry, R. L. Tyndall, C. B. Fliermans, S. B. Gough, M. A. Lambert, W. F. Bibb, L. K. McDougal, and D. J. Brenner Legionella oakridgensis: unusual new species isolated from cooling tower water. Appl. Environ. Microbiol. 45: Pasculle, S. W., J. C. Feeley, R. J. Gibson, L. G. Cordes, R. L. Myerowitz, C. M. Patton, G. W. Gorman, C. L. Carmack, J. W. Ezzell, and J. N. Dowling Pittsburgh pneumonia agent: direct isolation from human lung tissue. J. Infect. Dis. 141: Downloaded from on January 27, 2019 by guest