Detection of flagella in 278 Legionella strains by latex reagent sensitized with antiflagellum immunoglobulins.

To determine whether all Legionella species show common flagellum antigen properties, we developed a reagent using latex beads sensitized with flagellin-specific immunoglobulins that could be used in a simple and rapid agglutination reaction to identify Legionella colonies. A total of 278 strains (68 Legionella reference strains and 210 patient and environmental isolates) were tested. The results were compared with those obtained by a direct immunofluorescence assay using an antiflagellum serum and by morphological observations by electron microscopy. The immunological methods based on the use of a flagellum-specific serum have confirmed the presence of a common flagellum antigen for all Legionella species described to date. Flagella were detected for all the legionellae studied except four species: L. oakridgensis, confirmed as a nonflagellate species; L. brunensis; L. cincinnatiensis; and L. longbeachae serogroup 1. However, we noted a remarkable variability in flagellum expression, of greater or lesser degree, according to the species and their origin. A combination of all three methods of flagellum detection revealed that 86.3% of Legionella strains studied were flagellate. The latex test identified 89.6% of these strains, 97.5% of L. pneumophila, and 100% of L. pneumophila serogroup 1.

Detection of hippurate hydrolysis by Legionella spp. by using a rapid high-performance liquid chromatography method.

A rapid high-performance liquid chromatography method was developed to determine hippurate hydrolysis by Legionella spp. Benzoic acid, an end product of enzymatic activity, was directly detected by high-performance liquid chromatography after 1 and 24 h of incubation in 1% sodium hippurate. Because of its sensitivity, this procedure offers more precise identification of some Legionella spp.

Relationship between free amoeba and Legionella: studies in vitro and in vivo.

In 1980, Robowtham demonstrated that Legionella multiplies in free amoeba cytoplasm and hypothesized that the amoeba could act as a reservoir of virulent bacteria. In this paper we report various aspects of the relationship between amoeba and Legionella. A liquid medium co-culture method was applied to Acanthamoeba sp. and Legionella pneumophila serogroup 1. Within 4 days, Legionella growth increased by 2 log s CFU/ml. Using a direct immunofluorescence assay and electron microscopy, Legionella was shown to grow abundantly inside phagosomes, and bacteria and/or antigen were present on the cytoplasmic membrane of the amoeba. These aspects are very similar to those observed with Legionella-infected alveolar macrophages. The morphology and structure of Legionella cells were modified after 20 days of co-culture: - viable bacteria showed large fatty cytoplasmic inclusions, - gas liquid chromatography analysis demonstrated a decrease in the i16:0 fatty acid ratio. Cystic forms of amoeba were abundant but none contained viable Legionella. In an in-vivo study using a guinea-pig aerosol infection model, we compared the virulence of Legionella in co-culture with Legionella grown on charcoal dialysed yeast extract (CDYE) agar medium. The Legionella obtained by co-culture had an LD 50 (50% lethal dose) similar to that obtained for those grown on CDYE, showing that bacterial virulence is preserved in the cellular model.

Legionella gratiana sp. nov. isolated from French spa water.

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.

Pleural infection caused by Legionella anisa.

The first case of infection caused by Legionella anisa with isolation of the organism is reported here. The presence of L. anisa in the water supply of the hospital and the isolation of this species in the pleural fluid of a patient suffering from nosocomial pleurisy confirm the potential pathogenicity of this Legionella species.

Exposure to Legionellaceae at a hot spring spa: a prospective clinical and serological study.

Following the occurrence of five cases of Legionnaires' disease among patients and therapists at a French hot spring spa, a series of cleansing procedures and an epidemiological study were undertaken. During a 3-month period, the spring water was repeatedly sampled. Serum samples were taken from 689 randomly selected patients, 230 therapists, 134 administrative staff and a control group of 904 blood donors. Legionellaceae were present in the spring water at concentrations of 10(3)-10(5) colony forming units/l. Fifteen different species or serogroups were isolated with Legionella pneumophila serogroups 3 and 1 predominating. No clinical cases of Legionnaires disease were observed during the study. However, 11% of the therapists and 5% of the patients either had a high titre of antibody (greater than or equal to 256) to at least one species or serogroup or seroconverted during the study. Mean antibody titres in the three study groups were significantly higher than those in the blood donors against 11 of the 32 legionella antigens tested. Nine of these 11 antigens corresponded to species or serogroups isolated from the spring water. The highest mean antibody titres in all three study groups were against L. pneumophila serogroup 3, the most common legionella in the spring water. These findings have important implications for the maintenance of adequate standards of hygiene, bacteriological sampling and clinical surveillance in this and similar establishments.

[Use of cultured epidermis of human origin for demonstrating the acantholytic action of staphylococcal exfoliatin A]. / Emploi d'épidermes de culture d'origine humaine pour mettre en évidence l'action acantholytique de l'exfoliatine A staphylococcique.

Cultured human epithelia obtained from epidermal cells in vitro were used to assay the activity of staphylococcal epidermolytic toxin and develop an in vitro experimental model for the staphylococcal scalded skin syndrome. Human epidermal cells were grown from single epidermal cell suspensions obtained through trypsinization of adult normal skin into multilayered epithelia (with a basal cell layer, several intermediate and one or two upper layers) on mouse 3T3 feeder cells. First passage cultures were incubated with exfoliative toxin A from phage Group II staphylococci at various concentrations in DMEM. They were examined at various time intervals by direct microscopic and histological examination of respectively the culture plates or the epidermal sheets after their detachment from the plates with dispase grad II. A total exfoliation could be obtained at 24 hour at concentrations of Img and 500 micrograms/ml, only local areas of epidermolysis noted at 100 micrograms/ml. The intraepithelial separation was noted to occur between the basal layer and the lowest intermediate layer. No exfoliation could be observed at lower concentrations. Up to 4-5 hours few changes were evident, but at this time small areas of epidermolysis developed. With exfoliatin 100 micrograms/ml, intraepidermal blisters were clearly visible, occurring either between the basal cells and the lowest intermediate layer or between the first two intermediate cell layers. At the ultrastructural level, desmosomes were sparse and altered, with enlargement of the intercellular spaces and condensation of tonofilaments. These data indicate that human epidermal cell cultures, although their differentiation in culture only mimics what occurs in vivo, can be used as an in vitro model of the staphylococcal TEN to further investigate the site of action of such a toxin and the cellular mechanism responsible for the syndrome.

[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).

Isolation of Legionella anisa from a hospital hot water system.

Several cases of Legionnaires' disease occurred in a French hospital in 1982. Thirteen strains of a legionella-like organism with several unusual characteristics were subsequently isolated from the hospital hot water system. The various features of these strains show that they are identical to the new species 'Legionella anisa' described by the Centers for Disease Control. The possible pathogenicity of these strains to man and their relationship with the recently described Legionella bozemanii serogroup 2 are discussed.

Nosocomial colonization and infection by Achromobacter xylosoxidans.

Achromobacter xylosoxidans, a bacterial species named in 1971, is often isolated from aqueous environments, but little has been reported about its pathogenicity in humans, its epidemiological pattern, and its susceptibility to antibiotics and antiseptics. We were faced with an epidemic caused by this microorganism for 18 months in an intensive care unit. Two patients had fatal infections and 37 others were colonized. The source was the deionized water of the hemodialysis system. The 46 isolates were identified by comparison with the reference strain A. xylosoxidans ATCC 27061. The characteristic cellular fatty acids of this species were demonstrated by gas-liquid chromatography. The minimal inhibitory concentrations of 27 antibiotics were determined. The isolates were susceptible to only two: moxalactam at 4 micrograms/ml and ceftazidime at 8 micrograms/ml. The minimal bactericidal concentrations of one disinfectant and three antiseptics were: sodium hypochloride, 109 micrograms/ml; chlorhexidine digluconate in ethanol solution, 15 to 125 micrograms/ml; polyvinylpyrrolidone iodine, 750 micrograms/ml; and iodine ethanol, 312 to 625 micrograms/ml.

[Isolation and characterization of the three first strains of "Legionella pneumophila" found in France (author's transl)]. / Isolement et caractérisation des throis premières souches de Legionella pneumophila isolées en France.

The technical methods of culture and identification of the three first strains isolated in France of Legionella pneumophila serogroup 1, are described. In two patients, bacilli were seen by the direct fluorescent antibody (DFA) method, either during the course of the disease (case 1) or at postmortem examination (case 3). The causal strain was cultivated after guinea-pig inoculation or directly from either bronchial aspirate or postmortem lung. Identification of L. pneumophila required the determination of metabolic characteristics, DFA characterization and cellular-fatty acids analysis (by gas-liquid chromatography). In the three cases, a significant seroconversion was detected by indirect fluorescent antibody test. The relative value of the differnt methods of diagnostic methos is discussed and the importance of strain isolation is emphasized.