Legionella adelaidensis, a new species isolated from cooling tower water.

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.

[Molecular epidemiological markers of Legionella pneumophila in the Valencian Community (III). Peptide profiles]. / Marcadores epidemiológicos moleculares de Legionella pneumophila en la Comunidad Valenciana (III). Perfiles peptídicos.

Electrophoretic profiles using soluble peptides obtained from Legionella pneumophila serogroup 1 have been studied to be used as epidemiological markers. Using 8.5% polyacrylamide gels (SDS-PAGE) a unique profile common to environmental and clinical isolates has been detected showing 2 differential bands: a 150 kD and a 230 kD band present in only one clinical isolate. In order to achieve better resolution in the peptide profile 10 to 20% gradient SDS-PAGE has been used, confirming the existence of a common profile and 2 subtypes as well as another profile present in a smaller number of strains.

Isolation and sequence of an FK506-binding protein from N. crassa which catalyses protein folding.

Slow protein-folding reactions are accelerated by a prolyl cis/trans isomerase isolated from porcine kidney which is identical to cyclophilin, a protein that is probably the cellular receptor for the immunosuppressant cyclosporin A. Catalysis probably involves the isomerization of prolyl peptide bonds in the folding protein chains. Cyclosporin A inhibits folding catalysis by cyclophilin. Here we report the isolation, cloning, sequencing and expression of another protein with prolyl isomerase activity from Neurospora crassa which is unrelated to cyclophilin and which also catalyses slow steps in protein folding. This protein does, however, show sequence similarity to a human protein that binds to another, recently discovered immunosuppressive drug, FK506. Moreover, it shares 39% identity with the carboxy-terminal 114 residues of a cell-surface protein from the bacterium Legionella pneumophila, the causative agent of Legionnaires' disease. Catalysis of folding by the FK506-binding protein from N. crassa is inhibited by FK506, but not by cyclosporin A. Thus, at least two different classes of conformationally active enzymes (conformases) exist that catalyse slow steps in protein folding. Both occur in a wide variety of cells and are inhibited by immunosuppressive drugs.

Chemotaxonomic differentiation of legionellae by detection and characterization of aminodideoxyhexoses and other unique sugars using gas chromatography-mass spectrometry.

Legionellae have been differentiated previously by analyzing their carbohydrate contents by gas chromatography with flame ionization detection. In the present study, total ion mode gas chromatography-mass spectrometry (GC-MS) was used to detect a number of unusual sugars, including one that is structurally related to O-methyldideoxyheptoses. Increased sensitivity and selectivity for carbohydrate detection was achieved by selected ion-monitoring GC-MS. Two of the uncommon sugars previously discovered in the legionellae (X1 and X2) were identified as quinovosamine and fucosamine, respectively. Legionella pneumophila contained rhamnose and quinovosamine but not the quinovosamine isomer fucosamine. Tatlockia micdadei and Legionella maceachernii contained large amounts of rhamnose, fucose, and fucosamine but not quinovosamine. These two species were the only legionellae studied that contained another unusual sugar that is referred to as X3, pending determination of its structure. Fluoribacter dumoffi, Fluoribacter bozemanae, and Legionella anisa were varied in their carbohydrate contents, both within and between species, but could be distinguished from L. pneumophila and the T. micdadei and L. maceachernii group. Fluoribacter gormanii was unique among the legionellae in that it lacked both quinovosamine and fucosamine. Legionella jordanis contained other unusual carbohydrates in addition to quinovosamine. GC-MS may have wide application in the differentiation of bacterial species.

Problems associated with identification of Legionella species from the environment and isolation of six possible new species.

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.

Reevaluation of the cellular fatty acid composition of Legionella micdadei Bari 2/158.

The cellular fatty acid composition of Legionella micdadei Bari 2/158 was reevaluated because of its purported differences from other L. micdadei strains and its similarity to L. bozemanii. We found the fatty acid content of this strain to be consistent with that of 11 other strains of L. micdadei, including the presence of an anteiso branched-chain, monounsaturated, 17-carbon acid (Ca17:1) which is characteristic of this species. The double-bond position of Ca17:1 was established at the omega 7 (or delta 9) position by combined gas chromatographic-mass spectrometric analysis of dimethyl disulfide derivatives. The Ca17:1 omega 7 acid was absent in each of 14 strains of L. bozemanii.

Characterization of a Legionella anisa strain isolated from a patient with pneumonia.

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).

Legionella tucsonensis sp. nov. isolated from a renal transplant recipient.

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).

Cellular fatty acid compositions and isoprenoid quinone contents of 23 Legionella species.

The cellular fatty acid compositions and ubiquinone contents of 182 Legionella strains representing 23 species were determined by capillary gas-liquid chromatography and reverse-phase high-performance liquid chromatography, respectively. Except for the type strain of Legionella erythra (ATCC 35303T), all Legionella species contained large (40 to 90%) amounts of branched-chain fatty acids and only trace to small (less than 0.5 to 5%) amounts of ester-linked hydroxy acids. The 23 species were placed in three major fatty acid groups on the basis of differences in the relative amounts of 14-methylpentadecanoic (Ci16:0), hexadecanoic (C16:1), and 12-methyltetradecanoic (Ca15:0) acids. All Legionella species contained ubiquinones with 9 to 14 isoprene units in the side chains and were divided into five different ubiquinone groups. The species were further differentiated into 16 groups on the basis of qualitative and quantitative differences in their fatty acid compositions and ubiquinone contents. Both of these chemical characteristics can be used to distinguish Legionella species from other gram-negative bacteria and rapidly and accurately identify suspected isolates before serologic and other tests are done.

[Methods and problems of Legionella diagnosis]. / Methoden und Probleme der Legionellendiagnostik.

33 species of legionellae with 50 different serotypes are known. Phenotypical and biochemical similarities as well as immunologic cross-reactions impede their identification. Chemical analysis of structural features such as isoprenoidquinones, fatty acids or membrane proteins may facilitate the species classification of new isolates. A simple analytical procedure exists for ubiquinone determination. The microbiological diagnostics of legionellosis remains complex, however. Culture, direct immunofluorescence, and antigen detection in urine should be used complementary. The development of nucleic acid probes is promising but has not yet reached the stage for wide application. Serological diagnostic assays are still useful; at least in the diagnostics of non-pneumophila legionellosis the result of enzyme immunoassay and immunofluorescence should be completed by immunoblotting with distinct membrane antigens.

An unusual strain of Legionella micdadei.

A microorganism antigenically identified as Legionella micdadei but showing a cellular fatty acid profile distinct from that described previously for this species and more similar to that of L. bozemanii has been studied by phenotypic characterization, crossed immunoelectrophoresis, gas-liquid chromatography, and transmission electron microscopy. Although the phenotypic characters, the crossed immunoelectrophoresis, and the ultrastructural features were similar to those of L. micdadei, the fatty acid composition differed significantly from this species; moreover it differed also from L. bozemanii, even though it was quantitatively more similar.

Chemical composition of a lipopolysaccharide from Legionella pneumophila.

Lipopolysaccharide isolated from Legionella pneumophila (Phil. 1) was examined for chemical composition. The polysaccharide split off by mild acid hydrolysis contained rhamnose, mannose, glucose, quinovosamine, glucosamine and 2-keto-3-deoxyoctonate, in molar proportions 1.6:1.8:1.0:1.5:4.1:2.7. Heptoses were absent and glucose was probably mainly phosphorylated. The carbohydrate backbone of the lipid A part consisted of glucosamine, quinovosamine and glycerol, in the molar ratios 3.9:1.0:3.4, with glycerol as a phosphorylated moiety. A complex fatty acid substitution pattern comprising eight O-ester-linked, exclusively nonhydroxylated acids, and nineteen amide-linked, exclusively 3-hydroxylated acids was revealed. Both straight- and branched (iso and anteiso) carbon chains occurred. The major hydroxy fatty acid was 3-hydroxy-12-methyltridecanoic acid and six others were of a chain-length above 20 carbon atoms, with 3-hydroxy-20-methyldocosanoic acid as the longest. Two dihydroxy fatty acids, 2,3-dihydroxy-12-methyltridecanoic and 2,3-dihydroxytetradecanoic acids, were also detected. These results suggest that L. pneumophila contains a rather complex and unusual lipopolysaccharide structure of considerable biological and chemotaxonomic interest.

Differences in lipopolysaccharide profiles of serologically identical Legionella pneumophila serogroup 6 strains.

Over five years 18 strains of Legionella pneumophila serogroup 6 were isolated in Amsterdam from the hot water supply in three hospitals and from one patient. Immunodiffusion and immunoblot procedures showed that these strains were identical. Profiles of isolated lipopolysaccharides from the 18 strains and the reference serogroup 6 strain were visualised in polyacrylamide gels stained with silver. Four strains from hospital A, isolated in 1982, 1984, and 1985 displayed similar lipopolysaccharide profiles which were different in relative mobility from those of hospitals B and C. Those from hospital B (12 strains isolated in 1983 and 1986) and C (one strain) were similar in relative mobility but different in colour. The strain from a patient with acquired immune deficiency syndrome (AIDS) in hospital A displayed a lipopolysaccharide profile characteristic of hospital A. These reproducible profiles were all different in relative mobility from the reference serogroup 6 strain. They can be used as a marker system in epidemiological surveys of serologically identical serogroup 6 strains. Lipopolysaccharide patterns from strains isolated throughout the years in the same hospital were similar. This suggests an outgrowth from organisms inhabiting the plumbing system rather than reseeding from the Amsterdam mains supply.

Legionella moravica sp. nov. and Legionella brunensis sp. nov. isolated from cooling-tower water.

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).

Serological examinations for antibodies against Legionella species in dental personnel.

Serum samples from 107 dentists, dental assistants, and dental technicians were examined with an indirect immunofluorescence test for antibodies to Legionella pneumophila SG1-SG6, L. micdadei, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, and L. longbeachae SG1 + 2. Thirty-six (34%) employees from dental personnel from 13 practices showed a positive reaction for antibodies to Legionella pneumophila. Only five samples (5%) from a control group (non-medical workers) were positive. Of the 36 positive serum samples, 13 (36%) reacted with Serogroup 6, 12 with SG 1 (33%), 12 with SG 5 (33%), and three with SG 4 (8%), and eight samples were positive for antibodies to other Legionella species. Dentists had the highest prevalence (50%) of L. pneumophila antibodies, followed by assistants (38%) and technicians (20%). These results indicate that dental personnel are at an increased risk of legionella infection.

[Identification of Legionella by gas phase chromatography of fatty acids and high performance liquid chromatography of ubiquinones]. / Identification des Legionella par analyse des acides gras en chromatographie phase gazeuse (CPG) et des ubiquinones en chromatographie liquide haute performance (CLHP).

The fatty acids and ubiquinones of 44 reference strains corresponding to species of Legionella and 3 strains of Legionella isolated in the environment have been evaluated. The analysis of fatty acids profiles allows a classification of 22 species into 4 groups depending on the predominance of some branched-chain fatty acids of linear fatty acids. As for ubiquinones our results for the 22 species corroborate the classification in 5 groups established by Moss in 1983 based on the study of 10 species of Legionella. The analysis of fatty acids composition combined with ubiquinones content has allowed us to identify the antigenically similar strains which cannot be differentiated by direct immunofluorescence on one hand (strain 1) and on the other a more precise approach to the identification, before DNA-ADN hybridization of strains of new species (strains 2).

Limitations of flow cytometry for the specific detection of bacteria in mixed populations.

Flow immunofluorescence (FIF) techniques were established for the specific detection of the bacteria Escherichia coli, Legionella pneumophila and Bacillus anthracis spores after staining with fluorescein-conjugated antibacterial antibody. For each bacterial type, a comparison was made of gating on narrow forward angle (NFA) light scatter and on the red fluorescence (Red Flu) signal available from staining with the nucleic acid dye propidium iodide. No universal gating method was found, since Bacillus spores did not take up propidium iodide and only a part of the Legionella population gave detectable NFA scatter signals. The efficiency of detecting bacteria stained with antibody remained constant with differing concentrations of the specific bacterium, and the estimate of the count for specific bacteria expressed as a fraction of the total cytometer count fell sharply with bacterial concentration. This effect was apparently due to cytometer noise inherent in the high sensitivity of detection needed for particles as small as these bacteria. The noise did not originate in the photomultipliers and was evidently the result either of light scatter from sub-micron particles in the sheath fluid or scatter from optical components. Part of the noise could be removed by selective gating, but there remained a noise component overlapping with the NFA scatter and Red Flu signals from the heterologous bacteria, i.e., those not stained with specific antibody. In consequence, at the low bacterial concentrations used no meaningful cytometer count could be obtained for the excess of the unstained bacteria and the proportion of specific bacteria in the mixed population could not, therefore, be calculated.

Diagnostico laboratorial de legionella em amostras clinicas / The diagnosis of legionella infections

Os autores revisam as alternativas laboratoriais para o diagnostico de infecçoes por Legionella. E enfatizado o emprego de amostras clinicas acessiveis, como escarro e analisados metodos bacteriologicos e sorologicos