INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1986, p. 368-371 Vol. 36, No. 3 OO20-7713/86/030368-04$02.OO/O Copyright 0 1986, International Union of Microbiological Societies

Isolation of Legionellae from Oxidation Ponds and Fishponds in Israel and Description of israelensis sp. nov. HERVE BERCOVIER,' ARNOLD G. STEIGERWALT,~MICHELLE DERHI-COCHIN,~ c. WAYNE MOSS,^ HAZEL W. WILKINSON,3 ROBERT F. BENSON,3 AND DON J. BRENNER2* Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel' and Meningitis and Special Pathogens Branch' and Respiratory Diseases Bran~h,~Division of Bacterial Diseases, Centers for Disease Control, Atlanta, Georgia 30333

Strains of serogroup 4 and Legionella bozemanii and three strains of a new species, for which the name Legionella israelensis is proposed, were isolated from an oxidation pond in Israel. L. isruelensis exhibited the following biochemical reactions typical of legionellae: growth on buffered cysteine- yeast extract agar but not on chocolate agar or on buffered yeast extract agar without cysteine; nitrate negative; urease negative; nonfermentative; positive; and motile. It did not hydrolyze hippurate, autofluoresce, or produce a brown pigment on tyrosine-containingmedia, and it had weakly positive gelatinase activity. It had predominantly branched-chain cellular fatty acids, similar to those described for other legionellae, and had major amounts of ubiquinones with more than 10 isoprene units in their side chains. It could be distinguished from all previously described Legionella species and serogroups by using antisera in slide agglutination tests. As determined by the hydroxyapatite method at 60°C, deoxyribonucleic acids from the three L. israelensis strains were 95% or more related, and the levels of relatedness of the type strain to other legionellae were 12% or less. The type strain of L. israelensis is strain Bercovier 4 (= ATCC 43119).

Legionella pneumophila, the causative agent in a large the isolation of legionellae, 100-ml water samples were outbreak of at the American Legion Convention centrifuged at 5,000 rpm for 15 min at room temperature. in Philadelphia, Pa., in 1976 (8) was isolated by McDade et The sedimented material was suspended in 5 ml of the al. in 1977 (14) and was named in 1979 (5). It soon became supernatant water. Portions of the concentrated water Sam- clear that L. pneumophila and related species had been ples were further treated at low pH (3), by exposure to isolated as early as 1943 (9, 13), that there is serologic antibiotics (25), and by exposure to both treatments simul- diversity within strains of L. pneumophila (15), that legionel- taneously. Both samples treated as described above and lae cause two types of disease, Legionnaires disease and untreated samples were plated undiluted and at lo-* dilu- Pontiac fever (ll), and that water is the reservoir for tions on buffered charcoal-yeast extract (BCYE) agar sup- legionellae (7, 21, 26). There are now 22 named Legionella plemented with alpha-ketoglutaric acid (6), on MWY me- species that contain 35 serogroups (4, 16, 23, 24, 29), and dium (which is BCYE agar supplemented with glycine and preliminary data indicate the existence of at least nine antibiotics [27]), and on both chocolate agar and BCYE agar additional species and 10 additional serogroups within exist- without cysteine, neither of which support the growth of ing species (Brenner and Wilkinson, unpublished data). legionellae. Plates were incubated for 7 days at 35°C in a Eleven species (L. pneumophila, L. bozemanii, L. mic- humid atmosphere without added carbon dioxide. Suspected dadei, L. dumofii, L. longbeachae, L. jordanis, L. oakrid- legionellae were picked after 3 to 7 days of growth. gensis, L. wadsworthii, L. feeleii, L. maceachernii L. and Cultural and biochemical tests. Phenotypic characteriza- hackeliae) either have been isolated from or have been tion of legionellae was done as previously described (4), determined to be positive by the direct immunofluorescence except as noted below. Acidification of carbohydrates was assay in patients with legionellosis (4, 22, 24, 28). The assayed on deep-butt sugar agar incubated for 7 days at remaining 11 species have thus far been isolated solely from the environment (4, 24). 35"C, and gelatinase activity was determined by the film In this report we describe the isolation of legionellae from method, in which Kodak 3X film was heavily inoculated. oxidation ponds containing water used for irrigation and The film gelatin test was read after 24 h of incubation at from ponds used to grow fish in Israel. During this investi- 35°C. gation a new species, Legionella israelensis, was isolated. Cellular fatty acids and isoprenoid quinones. The cellular fatty acids and isoprenoid quinones were determined as previously described (18-20). Tentative identifications from MATERIALS AND METHODS chromatographic analyses were confirmed by mass spec- Strains. The Legionella strains isolated in Israel, including trometry (9, 17). the three strains of L. israelensis sp. nov., are listed in Table Slide agglutination test. Antisera were prepared to strain 1. The type and reference Legionella strains used in deoxy- Bercovier 4T (T = type strain) and absorbed to remove ribonucleic acid (DNA) hybridization studies (see Table 2) cross-reactions, and the slide agglutination test was done have been described previously (4). The strain designations with all known Legionella species and serogroup strains and used at the Centers for Disease Control and the American their corresponding antisera as described previously (23). Type Culture Collection are given for all type strains. For DNA studies. The isolation of DNA and all of the methods used to determine guanine-plus-cytosine content and DNA relatedness based on hydroxyapatite content were done * Corresponding author. exactly as previously described (4).

368 VOL.36, 1986 LEGIONELLA ISRAELENSIS SP. NOV. 369

TABLE 1. Source, identification, and biochemical characteristics of legionellae isolated from water in Israela Source Bluish white Browning on Strain Identification Autofluorescence tyrosine-containing hHyiz:iFE Gelatinase Town Pond type medium Bercovier 2 L. bozemunii serogroup 1 Gaash Oxidation +b + - + Bercovier 3 L. bozemunii serogroup 1 Gaash Oxidation + + - + Bercovier L. isruelensis sp. nov. Gaash Oxidation - - - + 5 isruelensis - (w) Bercovier L. sp. nov. Gaash Oxidation - - + (w) Bercovier 6 C. pneumophila serogroup 4 Gaash Oxidation - + - + Bercovier 7 L. isruelensis sp. nov. Gaash Oxidation - - - + (w) Bercovier 8 L. pneumophila serogroup 1 Fish - + + + Bercovier 9 L. pneumophila serogroup 2 Fish - + + + Bercovier 10 L. pneurnophila serogroup 3 Fish - + + + Bercovier 11 L. pneumophila serogroup 3 Fish - + + + ' All strains were gram-negative, oxidase-negative rods or coccobacilli that grew on BCYE agar and MWY agar, but not on chocolate agar or BCYE agar without cysteine. All strains were motile and catalase and B-lactamase positive and failed to ferment D-glucose, maltose, D-mannose, raffinose, or sucrose. They did not reduce nitrates or exhibit urease activity. +, Positive; -, negative; +(w), weak reaction.

RESULTS was 95 to 100% related to DNAs from the other two L. israelensis strains and 12% or less related to DNAs from Between February and June 1984, 70 water samples were type strains of 17 Legionella species and to additional collected from various locations in Israel. Among these were reference strains of L. pneumophila. 28 potable water samples, 28 samples from oxidation ponds Description of Legionella israelensis sp. nov. Legionella used for irrigation, and 14 samples from fishponds. Legionel- israelensis (is. ra. el. en'sis. N.L. fem. adj. israelensis lae were isolated from two irrigation water samples and from coming from Israel) is a gram-negative, oxidase-negative, two fishpond samples (Table 1). The four water samples motile rod or coccobaccillus. It has the following character- yielded 10 legionellae after culturing. Seven of these were istics that are typical of members of the genus Legionella: confirmed to be strains belonging to L. pneumophila growth on BCYE agar but not on media lacking cysteine; serogroups 1,2,3 (two strains), and 4 and L. bozemanii nitrate negative; urease negative; nonfermentative, catalase serogroup 1 (two strains). Three other Legionella-like positive; predominantly branched-chained cellular fatty ac- strains, all isolated from an oxidation pond in Gaash, did not ids; major amounts of ubiquinones with more than 10 produce a brown pigment on tyrosine-containing yeast ex- isoprene units in the side chains. Other biochemical reac- tract agar (Table 1). Further analyses of these strains showed tions include lack of bluish white or red autofluorescence, that they constituted a new species, L. israelensis. absence of a brown pigment on tyrosine-containing media, Slide agglutination test. The strain Bercovier 4T antiserum weak gelatinase activity, and a negative hippurate hydrolysis at a dilution of 1:8 gave 4+ agglutination with slide aggluti- reaction (Table 1). L. israelensis is the 23rd described nation test antigens prepared from the three L. israelensis Legionella species. The three known strains were isolated strains, and 3+ to 4+ agglutination with the L. wadsworthii from an oxidation pond in Gaash, Israel, in 1984. The strain 81-716 antigen. None of the remaining 34 Legionella guanine-plus-cystosine content of DNAs from the three species or serogroup antigens agglutinated with the L. strains is 40.9 mol%. The type strain, strain Bercovier 4 (= israelensis antiserum. None of the antisera prepared to ATCC 43119), has a guanine-plus cytosine content of 40.8 k previously described Legionella antigens, including L. 0.6 mol%. wadsworthii antigens, agglutinated the L. israelensis anti- gens. The cross-reaction in the L. israelensis antiserum was DISCUSSION completely removed by absorption with L. wadsworthii strain 81-716. Legionellae have been isolated from the environment and Cellular fatty acids and ubiquinones. The cellular fatty acid from humans in many parts of the world. Surprisingly, there compositions of the three strains of L. israelensis were has been only one confirmed case of legionellosis in Israel essentially identical; like other Legionella spp. strains, these (l), and only two isolates of L. pneurnophila serogroup 1 strains contained relatively large amounts (70%) of have been found in Israel previously (1, 2). Boldur et al. branched-chained acids and only traces of hydroxy acids isolated one strain from a pail of water in a hospital garden in (20). The major components were an anteiso-branched-chain Ramat-Gan during an investigation of high Legionella titers 15-carbon acid (a-15:O acid), which constituted 30% of the in the sera of some hospital patients (2). Bercovier et al. total acids, an iso-branched 16-carbon acid (i-16:0 acid) isolated the second strain at Hadassah University Hospital (21%), and an anteiso-branched 17-carbon acid (a-17:O acid) in Jerusalem from a lung biopsy of a 72-year-old man who (17%). Also present were palmitic acid (16:O acid) (6%), died with legionellosis (1). palmitoleic acid (16:l acid) (7%), arachidic acid (20:O acid) We now know that strains of L. pneumophila serogroups (4%), and a 17-carbon cyclopropane acid (17:O cyc acid) 1through 4, L. bozemanii serogroup 1, and L. isruelensis are (4%). The ubiquinone patterns of the three strains of L. present in freshwater sources in Israel. In all probability israelensis were similar, as each contained approximately additional study will reveal the presence of additional Le- equal amounts of ubiquinones Q12 and Q13 and small gionella species. amounts of ubiquinone Q11. L. israelensis was placed in the genus Legionella on the The guanine-plus-cytosine content of the three L. bases of its overall cultural and biochemical characteristics; israelensis strains was 40.9 mol%. DNA relatedness studies its predominantly branched-chain cellular fatty acid compo- (Table 2) showed that DNA from L. israelensis Bercovier 4T sition, and the presence of major amounts of ubiquinones 370 BERCOVIER ET AL. INT. J. SYST. BACTERIOL.

TABLE 2. DNA relatedness of L. isruelensis sp. nov.a impossible, to identify L. israelensis solely on the basis of its % Relatedness to biochemical reactions. This is because most species give labeled DNA from very similar reactions and because, in most cases, too few Source of unlabeled DNA L. israelensis strains have been tested to determine whether there is Bercovier 4T at variability in their biochemical profiles. L. israelensis fails to 6OoCb produce a brown pigment on tyrosine-containing yeast ex- L. isruelensis Bercovier 4T (= ATCC 43119T) 100 tract agar. This characteristic is shared only by L. wads- L. israelensis Bercovier 5 100 worthii, L. micdadei, and one known strain of L. dumofii, L. israelensis Bercovier 7 95 although L.feeleii gives a weak, delayed browning reaction. L. pneumophila Philadelphia lT(= ATCC 12 These species might be separated on the bases of their 33152T) gelatinase reactions (weakly positive in L. israelensis, posi- L. dumofii NY-23T (= ATCC 33279T) 10 L. sainthelensi Mount Saint Helens 4T 10 tive in L. wadsworthii and L. dumufii, and negative in L. (= ATCC 352MT) micdadei and L. feeleii), but this distinction would be L. micadadei TATLOCKT (= ATCC 332MT) 7 presumptive at best. A combination of cellular fatty acid and L. bozemanii WIGAT (= ATCC 33217T) 6 quinone analyses would distinguish among these species, as L. jordanis BL-540T (= ATCC 336239 6 would serologic typing. Antisera produced to the L. L. oakridgensis Oak Ridge loT (= ATCC 6 isruelensis type strain agglutinated only the homologous 33761T) antigens after absorption, and antisera produced to the other L. feeleii WO-44C-C3T (= ATCC 35072T) 6 35 Legionella serogroups did not agglutinate L. israelensis L. maceachernii PX-1-G2-E2T (= ATCC 6 353009 antigens. L. spiritensis Mount Saint Helens 9T (= ATCC 6 35249T) ACKNOWLEDGMENT L. gormanii LS-13T (= ATCC 33297T) 5 L. erythra SE-32A-CST (= ATCC 35303T) 5 We are indebted to Thomas Ozro MacAdoo, Department of L. longbeachae Long Beach 4T (= ATCC 4 Foreign Languages and Literatures, Virginia Polytechnic Institute 33462T) and State University, Blacksburg, for his expert advice on the L. wadsworthii 81-716T (= ATCC 33877T) 3 latinization of the species name. L.jamestowniensis JA-26-G1-E2T (= ATCC 3 35298T) LITERATURE CITED L. hackeliae Lansing 2T (= ATCC 35250T) 3 L. parisiensis PF-209C-C2T (= ATCC 35299T) 3 1. Bercovier, H., A. Goodman, A. Israeli, and M. Shapiro. 1984. Isolation of Legionella pneumophila serogroup 1 from a fatal Single-stranded, sheared, ''PO.,-labeled DNA from L. isruelensis Berco- case of pneumonia in Israel. Isr. J. Med. Sci. 20:1109-1111. vier 4T was reacted with similarly prepared, unlabeled DNAs from the same 2. Boldur, I., M. Ergaz, and D. Sompolinsky. 1984. The first strain (homologous reaction) and from a number of other legionellae (heterolo- isolation of Legionella pneumophila in Israel. Isr. J. Med. Sci. gous reactions). Each reaction was done at least twice. Reassociation values 20:74-75. in homologous reactions averaged 64% before normalization. Control reac- tions in which labeled DNA was incubated in the absence of unlabeled DNA 3. Bopp, C. A., J. W. Sumner, G. K. Morris, and J. G. Wells. 1981. showed 1.0 to 2.5% binding to hydroxyapatite. These control values were Isolation of Legionella from environmental water samples by subtracted before normalization. low pH treatment and use of selective medium. J. Clin. Micro- ' Percent relatedness (or relative binding ratio) was calculated as follows: biol. 13:714-719. percent relatedness = (percentage of DNA bound to hydroxyapatite in 4. Brenner, D. J., A. G. Steigerwalt, G. W. Gorman, H. W. heterdogous reactiodpercentage of DNA bound to hydroxyapatite in ho- Wilkinson, W. F. Bibb, M. Hackel, R. L. Tyndall, J. Campbell, mologous reaction) x 100. J. C. Feeley, W. L. Thacker, P. Skaliy, W. T. Martin, B. J. Brake, B. S. Fields, H. V. McEachern, and L. K. Corcoran. with more than 10 isoprenoid units in the side chains (lo), all 1985. Ten new species of Legionella. Int. J. Syst. Bacteriol. 35: 50-59. of which are typical properties of the genus. Further evi- 5. Brenner, D. J., A. G. Steigerwalt, and J. E. McDade. 1979. dence for the inclusion of the three L. isruelensis strains in Classification of the Legionnaires' disease bacterium: Legion- the genus Legionella was gained from their low, but signif- ella pneumophila genus novum, species nova of the family icant levels of relatedness to previously described Legion- Legionellaceae, familia nova. Ann. Intern. Med. 90:656-658. ellu species (Table 2) and their positive reactions (86% 6. Edelstein, P. H. 1981. Improved semiselective medium for relatedness, as determined by our standard hybridization isolation of Legionella pneumophila from contaminated clinical assay [4]) to a genus-specific Legionella ribosomal and environmental specimens. J. Clin. Microbiol. 14:29&303. ribonucleic acid gene probe (12) provided by D. E. Kohne of 7. Fliermans, C. B., W. B. Cherry, L. H. Orrison, S. J. Smith, Gen-Probe, Inc., San Diego, Calif. (data not shown). The D. L. Tison, and D. H. Pope. 1981. Ecological distribution of Legionella pneumophila. Appl. Environ. Microbiol. 41:9-16. three L. israelensis strains had identical phenotypic charac- 8. Fraser, D. W., T. F. Tsai, W. Orenstein, W. E. Parkin, H. J. teristics, and their DNAs were 95 to 100% related, leaving no Beecham, R. G. Sharrer, J. Harris, G. F. Mallison, S. M. Mar- doubt that they represent a single species. That L. tin, J. E. McDade, c. c. Shepard, P. s. Brachman, and the Field isruelensis represents a new Legionella species was con- Investigation Team. 1977. Legionnaires' disease: description of firmed by its less-than-species-level of relatedness to DNAs an epidemic. N. Engl. J. Med. 297:1189-1197. from 17 type strains of legionellae (strains of a species almost 9. Hebert, G. A., C. W. Moss, L. K. McDougal, F. M. Bozeman, always exhibit 70% or greater DNA relatedness [4]). Type R. M. McKinney, and D. J. Brenner. 1980. The rickettsia-like strains of five Legionella species were purposely not in- organisms TATLOCK (1943) and HEBA (1959) as cluded in the DNA relatedness studies because they were 35 phenotypically similar to, but genetically distinct from Legion- ella pneumophila and the WIGA bacterium. Ann. Intern. Med. to 65% related to other type strains that were included. The 92~45-52. low levels of relatedness of L. isruelensis to the species 10. Karr, D. E., W. F. Bibb, and C. W. Moss. 1982. Isoprenoid tested preclude the possibility of species level relatedness to quinones of the genus Legionella. J. Clin. Microbiol. 15: the species that were not tested. 1044-1048. As with most Legionella species, it is difficult, if not 11. Kaufmann, A. F., J. E. McDade, C. M. Patton, J. V. Bennett, P. VOL. 36, 1986 LEGIONELLA ISRAELENSIS SP. NOV. 371

Skaliy, J. C. Feeley, D. C. Anderson, M. E. Potter, V. F. New- 19. Moss, C. W., and G. 0. Guerrant. 1983. Separation of bacterial house, M. B. Gregg, and P. s. Brachman. 1981. Pontiac fever: ubiquinones using reverse-phase high-performance liquid chro- isolation of the etiologic agent (Legionella pneumophila) and matography. J. Clin. Microbiol. 18:15-17. demonstration of its mode of transmission. Am. J. Epidemiol. 20. MOSS,C. W., R. E. Weaver, S. B. Dees, and W. B. Cherry. 1977. 114:337-347. Cellular fatty acid composition of isolates from Legionnaires’ 12. Kohne, D. E., A. G. Steigerwalt, and D. J. Brenner. 1984. disease. J. Clin. Microbiol. 6:14&143. Nucleic acid probe specific for members of the genus Legion- 21. Orrison, L. H., W. B. Cherry, and D. Milan. 1981. Isolation of ella, p. 107-108. In C. Thornsberry, A. Balows, J. C. Feeley, Legionella from cooling tower water by filtration. Appl. and W. Jakubowski (ed.), Legionella. Proceedings of the Sec- Environ. Microbiol. 41:1202-1205. ond International Symposium. American Society for Microbiol- 22. Tang, P. W., S. Toma, and L. G. MacMillan. 1985. Legionella ogy, Washington, D. C . oakridgensis: laboratory diagnosis of a human infection. J. Clin. 13. McDade, J. E., D. J. Brenner, and F. M. Bozeman. 1979. Microbiol. 21:462463. Legionnaires’ disease bacterium isolated in 1947. Ann. Intern. 23. Thacker, W. L., B. B. Plikaytis, and H. W. Wilkinson. 1985. Med . !M:659-661. Identification of 22 Legionella species and 33 serogroups with 14. McDade, J. E., C. C. Shepard, D. W. Fraser, T. R. Tsai, M. A. the slide agglutination test. J. Clin. Microbiol. 21:779-782. Redus, W. R. Dowdle, and the Laboratory Investigation Team. 24. Thacker, W. L., H. W. Wilkinson, B. B. Plikaytis, A. G. 1977. Legionnaires’ disease: isolation of a bacterium and dem- Steigerwalt, W. R. Mayberry, C. W. Moss, and D. J. Brenner. onstration of its role in other respiratory disease. N. Engl. J. 1985. Second serogroup of Legionella feeleii strains isolated Med. 297:1197-1203. from humans. J. Clin. Microbiol. 22:14. 15. McKinney, R. M., L. Thacker, P. P. Harris, K. R. Lewallen, 25. Thorpe, T. C., and R. D. Miller. 1980. Negative enrichment G. A. HCbert, P. H. Edelstein, and B. M. Thomason. 1979. Four procedure for isolation of Legionella pneumophila from seeded serogroups of Legionnaires’ disease bacteria defined by immu- cooling tower water. Appl. Environ. Microbiol. 40:849-851. nofluorescence. Ann. Intern. Med. 90:621424. 26. Tobin, J. O’H., R. A. Swann, and C. L. R. Bartlett. 1981. 16. Meenhorst, P. L., A. L. Reingold, D. G. Groothius, G. W. Isolation of Legionella pneumophila from water systems: meth- Gorman, H. W. Wilkinson, R. M. McKinney, J. C. Feeley, D. J. ods and preliminary results. Br. Med. J. 282515-517. Brenner, and R. van Furth. 1985. Water-related nosocomial 27. Wadowsky, R. M., and R. B. Yee. 1981. A glycine-containing pneumonia caused by Legionella pneumophila serogroups 1and selective medium for isolation of Legionellaceae from environ- 10. J. Infect. Dis. 152:356364. mental specimens. Appl. Environ. Microbiol. 42:768-772. 17. Moss, C. W., W. F. Bibb, D. E. Karr, and G. 0. Guerrant. 1983. 28. Wilkinson, H. W., W. L. Thacker, D. J. Brenner, and K. Ryan. Chemical analysis of the genus Legionella: fatty acids and 1985. Fatal Legionella maceachernii pneumonia. J. Clin. Micro- isoprenoid quinones. INSERM 114:375-380. biol. 22:1055. 18. MOSS, C. W., W. F. Bibb, D. E. Karr, G. 0. Guerrant, and 29. Wilkinson, H. W., W. L. Thacker, A. G. Steigerwalt, D. J. M. A. Lambert. 1983. Cellular fatty acid composition and Brenner, N. M. Ampel, and E. J. Wing. 1985. Second serogroup ubiquinone content of Legionella feeleii sp. nov. J. Clin. Micro- of Legionella hackeliae isolated from a patient with pneumonia. biol. 18:917-919. J. Clin. Microbiol. 22:488-489.