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A novel Legionella species was identified based on analysis of 16S rRNA and mip (macrophage infectivity potentiator) gene sequences, cellular fatty acids, isoprenoid quinones, biochemical reactions, antigens and quantitative DNA-DNA hybridization. Strain CDC-1796-JAP-E(T) was isolated from well water at the Nagasaki Municipal Medical Center, Japan. Two strains, CDC-3041-AUS-E and CDC-3558-AUS-E, were isolated from water samples during an outbreak of legionellosis in South Australia. Strain CDC-5427-OH-H was isolated from a 66-year-old female patient diagnosed with Legionnaires' disease in the US. Cells from these four strains were gram-negative, non-fluorescent, rod-shaped, and positive for alkaline phosphatase, esterase, leucine arylamidase, catalase, gelatinase, β-lactamase and tyrosine browning assay. Phylogenetic analysis of 16S rRNA and mip genes revealed that the four strains formed a distinct cluster within the genus Legionella. The bacteria contained branched-chain fatty acids and quinones that are typical of members of the genus Legionella. Slide agglutination tests demonstrated no cross-reaction with 52 previously described members of the Legionellaceae. DNA-DNA hybridization studies indicated that DNAs from the four strains were highly related (78-84 %) but they showed 29 % relatedness to Legionella oakridgensis ATCC 33761(T) and less than 10 % to strains of other Legionella species tested. These characterizations suggest that the isolates represent a novel species, for which the name Legionella nagasakiensis sp. nov. is proposed; the type strain is CDC-1796-JAP-E(T) ( = ATCC BAA-1557(T) = JCM 15315(T)).

Two Legionella-like organisms were isolated from water samples obtained in Adelaide, Australia. One organisms was isolated from a drinking water distribution system, and the other was isolated from a cooling tower at a sewage treatment plant. Both strains required L-cysteine for growth and contained cellular branched-chain fatty acids and ubiquinones typical of the genus Legionella. These strains were serologically distinct from each other as determined by a slide agglutination test. STrain 2074-AUS-ET (T = type strain) was serologically distinct from all previously described Legionella species and serotypes. Strain 2055-AUS-E could not be differentiated biochemically or serologically from Legionella quinlivanii. Both strains were shown by DNA hybridization studies (Hydroxyapatite method) to be members of new Legionella species. Legionella waltersii sp. nov. is the name proposed for strain 2074-AUS-ET (= ATCC 51914T). L. waltersii was less than 10% related to other Legionella species. Strain 2055-AUS-E (= ATCC 51913) was informally named Legionella genomospecies 1, since it could not be phenotypically distinguished from L. quinlivanii. Legionella genomospecies 1 was closely related to L. quinlivanii strains (53 to 69% related with 4.5 to 6.5% divergence at 60 degrees C and 31 to 52% related at 75 degrees C).

Fourteen Legionella-like strains isolated from aquatic sources have been characterized serologically, biochemically, and in terms of DNA relatedness. The strains grew on buffered charcoal-yeast extract agar but not on blood agar and displayed phenotypic characteristics typical of the family Legionellaceae, including a requirement for cysteine, cellular fatty acid compositions in which branched-chain acids predominate, and the possession of isoprenoid quinones of the ubiquinone series with more than 10 isoprene units in their side chains. All were nonfermentative, lacked urease, were incapable of nitrate reduction, and reacted positively with a DNA probe specific for the Legionellaceae. DNA hybridization studies in which the hydroxyapatite method was used demonstrated that the strains represented five new species of the genus Legionella. Nine of the strains were more than 90% interrelated, and the name Legionella londiniensis sp. nov. is proposed for this group. Two strains formed a second hybridization group, for which the name Legionella nautarum sp. nov. is proposed, while the three remaining species, Legionella geestiana sp. nov., Legionella quateirensis sp. nov., and Legionella worsleiensis sp. nov., are each represented by a single strain. The levels of relatedness of the new species to each other are 23% or less, and the levels of relatedness to other members of the genus ranged from 0 to 36%. L. geestiana, L. nautarum, and L. londiniensis are serologically unrelated to all other known Legionella species. L. worsleiensis cannot be separated from Legionella pneumophila serogroup 4 by serological methods and is also serologically indistinguishable from L. quateirensis; distinctions may be made on the basis of fatty acid composition and biochemical reactions.

A Legionella-like organism, strain 1677-MI-H, was isolated from the bronchoscopy washings of a patient with pneumonia who had a 2-year history of progressive, chronic lymphocytic leukemia. The growth characteristics, cellular fatty acids, and ubiquinone content of the isolate were consistent with those for Legionella spp. The isolate was serologically distinct in the slide agglutination test with absorbed antisera. DNA hybridization studies showed that strain 1677-MI-H (ATCC 49751) represents a new Legionella species which is named Legionella lansingensis.

A Legionella-like organism (strain 214T [T = type strain]) was isolated from a cooling tower in Stratford-upon-Avon, England. This strain required L-cysteine and contained cellular branched-chain fatty acids that are typical of the genus Legionella. Strain 214T produced pink colonies on buffered charcoal-yeast extract agar. Ubiquinone Q-12 was the major quinone. Strain 214T was serologically distinct from other legionellae as determined by a slide agglutination test. The results of DNA hybridization studies showed that strain 214T (= ATCC 49655T) is a member of a new Legionella species, Legionella shakespearei.

A Legionella-like organism (strain 1762-AUS-E) was isolated from a cooling tower of an air-conditioning system in Adelaide, South Australia, Australia. The isolate was presumptively identified as a Legionella strain by its growth requirement for L-cysteine and its cellular branched-chain fatty acids. Strain 1762-AUS-E was serologically distinct in the slide agglutination test with absorbed antisera. DNA hybridization confirmed that it is a new Legionella species for which the name Legionella adelaidensis is proposed.

Three Legionella-like organisms were isolated from water from the cooling towers of two Australian institutions. The strains grew on buffered charcoal-yeast extract (BCYE) agar but not on BCYE agar in the absence of L-cysteine. Gas-liquid chromatography profiles of the isolates were consistent with those for Legionella spp. They were serologically distinct from other legionellae in a slide agglutination test. DNA hybridization studies showed that the three isolates belong to a new species of Legionella, Legionella fairfieldensis (ATCC 49588).

Three Legionella-like organisms isolated from patients with pneumonia are shown to belong to the species Legionella sainthelensi by DNA hybridization studies and to a new serogroup, serogroup 2, by serological studies (ATCC 49322). L. sainthelensi serogroup 2 and L. santicrucis are indistinguishable by slide agglutination, but are separable on the basis of their cell wall fatty acid profiles.

Legionella anisa, previously found only in environmental specimens, was isolated from a bronchial lavage specimen of an immunocompromised patient with pneumonia. Growth, physiologic, gas-liquid chromatographic, serologic, and DNA characteristics were consistent with those of the type strain of L. anisa, WA-316-C3 (ATCC 35292).

A Legionella-like organism (strain 570-CO-H [= ATCC 43290]) isolated from the lung tissue of a patient with pneumonia was shown by growth, as well as physiological, serological, and genetic characteristics, to belong to a new Legionella pneumophila serogroup, serogroup 12. Two additional strains were detected with antiserum specific for strain 570-CO-H. These strains were isolated from environmental sources.

During an epidemiologic survey, an unidentified strain of Legionella was isolated from water of a thermal spa in France. The strain (Lyon 8420412) had the cultural and biochemical characteristics typical of the genus Legionella. In direct immunofluorescence tests, the strain reacted weakly with fluorescein-conjugated antisera prepared against L. bozemanii serogroups 1 and 2, L. longbeachae serogroups 1 and 2 and L. anisa, and failed to react with sera prepared against 36 other species or serogroups. A fluorescein-conjugated antiserum prepared against strain Lyon 8420412 reacted strongly with the homologous strain and only weakly with the above-mentioned species. The cell-wall fatty acid profile, with a predominance of hexadecenoic (16:1) and hexadecanoic (16:0) acids, ubiquinone Q10 as the major quinone and a characteristic protein electrophoresis profile suggested that the isolate was different from other Legionella species. In DNA-DNA hybridization experiments, the strain was distinct from all named Legionella species, and from all unnamed species currently under study at the Centers for Disease Control. The name Legionella gratiana is proposed for the new species (type strain Lyon 8420412; CDC 1242). A serologic survey of antibodies reacting against L. gratiana indicated that personnel or patients at the spa therapy centre where the organism was isolated had higher antibody titres than a control population.

Two Legionella-like organisms were isolated from cooling-tower water samples in Czechoslovakia. They were presumptively identified as legionellae by their growth on buffered charcoal-yeast extract agar (BCYE) containing L-cysteine and their absence of growth on BCYE without L-cysteine. Both strains contained predominately branch-chained cellular fatty acids and were therefore definitively placed in the genus Legionella. They were serologically distinct from other described Legionella species and were shown by DNA studies to constitute two new Legionella species, Legionella moravica (type strain 316-36; ATCC 43877) and Legionella brunensis (type strain 441-1; ATCC 43878).

Three strains of Legionella feeleii from patients with pneumonia (425-MI-H, 691-WI-H, and 693-WI-H) and one environmental strain (713-MI-E) received at the Centers for Disease Control for reference diagnostic testing were compared with the type strain WO-44C-C3 (ATCC 35072) by DNA hybridization, chemical analysis of cellular fatty acids and ubiquinones, biochemical tests, and serological characteristics. All four isolates were assigned to the L. feeleii species on the basis of DNA hybridization results. However, strains 691-WI-H and 693-WI-H were serologically distinct from strain WO-44C-C3, as shown by their minimal reactivity (1 to 2+) with a direct immunofluorescence conjugate prepared against L. feeleii serogroup 1 (strain WO-44C-C3). Therefore, strains 691-WI-H and 693-WI-H were placed in a new L. feeleii serogroup (serogroup 2). The reference strain of L. feeleii serogroup 2 is 691-WI-H (ATCC 35849).

It is technically impractical for many clinical laboratories to use the direct immunofluorescence assay for identifying and serogrouping clinical isolates of Legionella. We compared the results obtained with the direct immunofluorescence assay with the results of a simple and less-demanding slide agglutination test for identifying 15 serogroups representing seven Legionella species. The slide agglutination test was in complete agreement with the direct immunofluorescence assay, and the serogroup to which 64 clinical isolates of Legionella belonged was correctly identified. With polyvalent, pooled antisera and absorbed, serogroup-specific antisera, the slide agglutination test is a useful alternative to the direct immunofluorescence assay in the diagnosis of Legionella infections and for studying the serological relationships of Legionella-like organisms.

While preparing slide agglutination test antisera and immunofluorescence conjugates for the identification of Legionella species and serogroups, we found that several of the reagents cross-reacted with Bordetella pertussis strains. To determine the extent of this problem and to estimate the specificity of Legionella reagents, we tested slide agglutination test antisera against 22 species and 35 serogroups with 92 bacterial strains representing 19 genera. The only cross-reactions observed were with Legionella pneumophila serogroup 10, L. maceachernii, L. gormanii, and L. feeleii serogroup 1 antisera and 4 of 10 B. pertussis strains. Nineteen conjugates, previously available from the Centers for Disease Control but no longer distributed as reference reagents, were tested with the four cross-reactive B. pertussis strains. Two conjugates, L. micdadei and L. wadsworthii, stained three of the B. pertussis strains at a fluorescence intensity of greater than or equal to 3+. All cross-reactions were removed from the antisera and conjugates by absorption with the cross-reacting strain without diminishing the homologous reaction. Special emphasis should be placed on the identification and removal of cross-reactions in Legionella reagents with strains that have similar morphologic and growth characteristics.

Two Legionella-like organisms, one isolated from postmortem lung tissue and the other from a bronchial aspirate, were shown by growth, physiologic, and genetic characteristics to belong to the species Legionella pneumophila. Subsequent serologic testing indicated that both strains belonged to a new serogroup, serogroup 14.

The susceptibility of the Legionnaires disease bacterium to various antimicrobial agents was determined by inoculation of embryonated eggs via the yolk sac. When administered prophylactically, the minimal dose of drug preventing all deaths due to the infection was as follows: rifampin, 0.02 mg; gentamicin, 0.25 mg; streptomycin, 0.39 mg; erythromycin, 0.62 mg; sulfadiazine, 1.56 mg; chloramphenicol, 2.50 mg; and cephalothin, 20.0 mg. Smaller amounts delayed deaths, and larger or equal amounts rendered the embryos free of infection. Oxytetracycline in the largest tested amount, 5.0 mg, protected 80% of the embryos from death, and as little as 0.31 mg delayed death. Chlortetracycline (0.50 mg) and ampicillin (10.0 mg) were ineffective. The six most effective drugs were studied in an experiment in which they were administered at various times after infection in doses that were twice the minimal prophylactic dose preventing all deaths. In this therapeutic experiment, rifampin, and erythromycin allowed 100% survival when given even 72 h after infection; gentamicin, streptomycin, sulfadiazine, and chloramphenicol did so when given 48 h after infection. All six drugs increased mean survival time when administered 72 h after infection.

A new serogroup of Legionella pneumophila was isolated from bronchoscopic washings and an open-lung biopsy specimen of a patient from California with pneumonia. A serologically identical isolate was obtained from tap water of a hospital ward in the Netherlands, and a fatal case of pneumonia in a patient from Virginia was shown retrospectively to have been caused by this new organism. The type strain of what is now serogroup 9 of L. pneumophila is IN-23-G1-C2 (American Type Culture Collection no. 35289). Disease caused by L. pneumophila serogroup 9 is not apparently different from that caused by other L. pneumophila serogroups.

A Legionella-like organism was isolated from an open-lung biopsy of a patient with fatal pneumonia. The isolate was shown by growth, physiologic, serologic, and genetic characteristics to belong to the species Legionella jordanis, which had previously been isolated only from the environment.

A Legionella-like organism (strain 82A3105; ATCC 43736) was isolated from a lung aspirate taken from a patient with pneumonia. Results of physiologic, gas-liquid chromatographic, genetic, and serologic tests showed that strain 82A3105 and four additional clinical isolates belong to a new Legionella pneumophila serogroup, serogroup 13.

Between 11 November 1986 and 28 February 1987, legionellosis was diagnosed in 23 patients at one hospital with a recently marketed Legionella-specific DNA probe for respiratory secretions. Only 10 of the 23 probe-positive patients showed findings typical of Legionella pneumonia, including a temperature of greater than or equal to 100.5 degrees F (approximately 38.1 degrees C) and radiographic evidence of pneumonia. No differences were found in the results of laboratory studies, demographic features, or underlying risk factors for these 10 probe-positive patients when compared with the 13 probe-positive patients with nonpneumonic illnesses. A case-control study comparing probe-positive and -negative patients failed to identify any different features of disease or epidemiologic characteristics. Probes of repeat specimens of sputum were still positive 2 to 13 weeks after the initial test in 5 (50%) of the 10 probe-positive patients. The clinical features in most patients were atypical for legionellosis, and the diagnosis could not be confirmed by traditional laboratory tests performed on duplicate specimens processed at the Centers for Disease Control. This report emphasizes the need for clinical microbiology laboratories to confirm test results from new procedures by accepted diagnostic methods.

A Legionella-like organism (strain 797-PA-H; ATCC 43130) was isolated from a specimen taken from an endotracheal tube of a patient 4 days before death and from the left lung at autopsy. Growth characteristics were consistent with those for Legionella species. Strain 797-PA-H gave negative test results with available direct immunofluorescence assay conjugates and with slide agglutination test antisera prepared against the 22 Legionella species and 35 serogroups now recognized. Minimal reactivity (1 to 2 +) was observed with both tests by using reagents prepared against the Legionella-like organism Lansing 3. Reciprocal absorption studies, however, showed that the cross-reactive antibodies could be removed easily. Physiologic, gas-liquid chromatographic, and DNA hybridization tests revealed that the strain belonged to the species Legionella pneumophila. Therefore, strain 797-PA-H was designated as the type strain of a new L. pneumophila serogroup, serogroup 11.

A Legionella-like organism, strain 1407-AL-H, was isolated from a transbronchial lung biopsy specimen from a cardiac transplant recipient undergoing immunosuppressive therapy. The strain grew on buffered charcoal-yeast extract agar (BCYE) but not on BCYE in the absence of cysteine, and it showed gas-liquid chromatographic fatty acid profiles that were predominantly branch chained. Strain 1407-AL-H was antigenically distinct in slide agglutination tests from the 23 Legionella species and 39 serogroups previously described. DNA hybridization studies placed it in a new Legionella species, Legionella birminghamensis (ATCC 43702).

A Legionella-like organism (strain 798-PA-H, ATCC 35999) isolated from a lung aspirate was shown by DNA hybridization to belong to the species Legionella hackeliae. Growth, gas-liquid chromatographic, and physiologic characteristics were consistent with those of the type strain of L. hackeliae, Lansing 2 (ATCC 35250). However, 798-PA-H was minimally reactive with L. hackeliae direct immunofluorescence assay conjugate or slide agglutination test antiserum. Cross-absorption studies with 798-PA-H and Lansing 2 antisera supported placing 798-PA-H in a second serogroup of L. hackeliae.

We used the slide agglutination test to determine the serologic relationships of 22 Legionella spp. representing 33 serogroups. Antisera prepared against 14 of the Legionella spp. contained cross-reactive antibodies (1+ or greater) at their working dilutions. Numerous cross-reactions were observed for the blue-white fluorescing Legionella spp. With only three exceptions in the latter group, cross-reactive antibodies were removed by absorption, thereby producing serogroup-specific antisera. For screening tests or for identification only to the genus level, nine polyvalent antiserum pools were prepared. Routine use of slide agglutination test reagents should expand the number of Legionella spp. that can be identified in the clinical laboratory and, at the same time, provide a simpler, less costly test procedure.

Legionella gormanii, previously isolated only from the environment, was grown from the bronchial brush specimen of a patient with pneumonia. The organism was characterized by serologic, biochemical, and DNA hybridization studies.

Legionella maceachernii, previously isolated only from the environment, was shown to be a cause of fatal pneumonia in an immunocompromised patient.

A Legionella-like organism (strain 72-OH-H [= ATCC 43753]) was isolated from an open-lung biopsy specimen from a hemodialysis patient with end-stage renal disease and bronchopneumonia. Growth characteristics and gas-liquid chromatographic profiles of the isolate were consistent with those for Legionella spp. The isolate was presumptively identified as a Legionella longbeachae serogroup 1 strain by direct immunofluorescence staining. However, the organism was serologically distinct in the slide agglutination test with absorbed antisera. DNA hybridization studies showed that strain 72-OH-H constitutes a new Legionella species, which is named Legionella cincinnatiensis (ATCC 43753).

A Legionella-like organism (strain 1087-AZ-H) was isolated from a pleural-fluid specimen from a renal transplant patient undergoing immunosuppressive therapy. Growth characteristics and gas-liquid chromatography profiles of the isolate were consistent with those for Legionella spp. The isolate fluoresced blue-white under long-wave UV light. Strain 1087-AZ-H was serologically distinct in the slide agglutination test with absorbed antisera. DNA hybridization studies placed it in a new Legionella species, Legionella tucsonensis (ATCC 49180).