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Charcoal-yeast extract agar is a new bacteriological medium that supports excellent growth of the Legionella pneumophila. It results from modifications made in an existing L. pneumophila medium, F-G agar. Yeast extract, instead of an acid hydrolysate of casein, serves as the protein source. Beef extractives and starch are not added. Activated charcoal (Norit A or Norit SG) is included at 0.20% (wt/vol). Comparison of charcoal-yeast extract and F-G agars showed that a greater number of colony-forming units of L. pneumophila was recovered from a standardized tissue inoculum on charcoal-yeast extract agar (4.35 x 10(6) colony-forming units) than on F-G agar (4.85 x 10(4) colony-forming units). Macroscopic colonies of L. pneumophila were visible on the new medium within 3 days, whereas 4 days of growth was required on F-G agar.

Outbreaks of legionnaires' disease (LD) in tourists visiting Italian and Spanish resorts have been recently reported. An unusual number of reports of LD in tourists visiting the US Virgin Islands prompted an investigation of risk factors for development of LD in this area. Twenty-seven cases of LD were identified between 1979 and 1982 through press reports, personal communication, the national LD surveillance system, a review of hospital records, and a mail survey. Twenty-four of 27 persons with the disease had visited St Croix and 12 of them had stayed at a single hotel in 1981. Available evidence suggested that infection was due to Legionella pneumophila serogroup 1; L pneumophila serogroups 1 and 3 and several new Legionella species were isolated from the potable water system at the hotel. Following hyperchlorination of the potable water system, no further cases of LD in hotel visitors have been identified to date.

Yolk sac suspensions infected with the Legionnaires disease bacterium (LDB) were plated onto 17 different bacteriological agar media. The LDB grew only on Mueller-Hinton agar supplemented with 1% Iso Vitale X and 1% hemoglobin (MH-IH). This medium was subsequently analyzed to determine the components required to support growth of the LDB. L-Cysteine hydrochloride can replace the Iso Vitale X reagent, and soluble ferric pyrophosphate can replace hemoglobin. A new medium, F-G agar, was formulated incorporating these chemicals. Different cultures conditions (oxygen tension, temperature, and pH) were also evaluated. The LDB grew optimally at 35 degrees C under 2.5% CO2 on the F-G agar adjusted to pH 6.9. When infected tissues were inoculated onto both F-G agar and MH-IH, the F-G agar produced colonies of the LDB more rapidly and in greater numbers than did MH-IH.

Sets of sera (444) submitted for diagnostic testing for legionellosis were tested against 29 indirect immunofluorescence assay (IFA) antigens prepared from the characterized Legionella species and Legionella-like organisms to determine the prevalence of antibodies to Legionella organisms. Reciprocal titers of 15% of the serum sets rose fourfold or more to greater than or equal to 128 (indicating seroconversion) against one or more Legionella antigens. The specificity of the test was 96% when evaluated in patients with pneumonia due to non-Legionella organisms. Antibodies were of the IgG, IgM, and (infrequently) IgA classes and were either specific for a single species (as defined by a difference in titer of fourfold or more) or reacted with common Legionella antigens (30 [45%] vs. 36 [55%] of 66 seroconversions, respectively). No single antigen detected half of the positive sera. Elevated IFA titers (of greater than or equal to 256) against single or multiple Legionella antigens occurred in 12% of 184 normal control sera. Therefore, only seroconversions to titers of greater than or equal to 128 should be considered indicative of recent Legionella infection.

At the site of a legionellosis outbreak, amoebae and two ciliates, Tetrahymena sp. and Cyclidium sp., were isolated from cooling-tower water containing Legionella pneumophila. The Tetrahymena sp. and the amoebae repeatedly showed the ability to support intracellular multiplication of L. pneumophila. Both were isolated from cooling towers specifically implicated as the source for the spread of legionellosis. These protozoa may be reservoirs supporting the survival and multiplication of virulent legionellae in cooling-tower water.

Legionella micdadei (Pittsburgh pneumonia agent) was identified by direct fluorescent-antibody (DFA) examination of lung tissue in six of seven persons diagnosed previously as having L. micdadei pneumonia only by histopathology and in four persons who also had positive cultures of the organism. No cross-reactions occurred with monospecific DFA conjugates prepared against Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, and Legionella gormanii. One person had L. pneumophila serogroup 6 identified by DFA examination of lung tissue and subsequent culture of stored pulmonary secretions. Characterization of the four strains of L. micdadei revealed specific DFA reactions, bacteriological behavior, and cellular fatty acid composition that allow identification of the organism. DFA testing appears to be a sensitive method for identifying L. micdadei prescent in human lung tissue or cultured on artificial media.

We analyzed 24 environmental samples collected in or near the Indiana Memorial Union, where an epidemic of Legionnaires' disease occurred in early 1978. We conducted fluorescent antibody analyses and culture on F-G and charcoal yeast extract agars of each sample directly; splenic tissue of guinea pigs inoculated with the sample; and yolk sacs from embryonated eggs inoculated with splenic tissue of guinea pigs injected with the sample. Legionnaires' disease (LD) bacterium was isolated from seven of the 24 samples: one water sample from the air-conditioner cooling tower of the Union; three water samples from a stream near the Union; and three mud samples from the same stream. The LD bacterium strains were of three different serotypes. These findings indicate that LD bacteria may be widespread in nature.

An investigation of an outbreak of nosocomial legionella pneumonia revealed that the potable water of the hospital was contaminated with a distinctive strain of Legionella pneumophila (designated the Le-1 strain), which was by culture and serology linked with human illness. To examine the possible role of this water in this outbreak, eight healthy guinea pigs were exposed to an aerosol of concentrated potable water. Seven animals developed pneumonia due to the Le-1 strain of L. pneumophila. In one of these guinea pigs, the Le-1 strain was also isolated from the blood and spleen. These results provide support to the theory that exposure of humans to aerosols of potable water contaminated with L. pneumophila may cause legionella pneumonia.

A protocol for sampling environmental sites was developed and used to identify possible sources of Legionella species in support of epidemiologic investigations at two hospitals. In hospital A, legionellae were isolated from 43 of 106 (40%) different sites. Three separate Legionella pneumophila serotypes and a previously unrecognized species were present in different combinations in the positive samples. Two of five cooling towers contained the same L. pneumophila serogroup 1 monoclonal type (1,2,4,5) as was isolated from patients. The same monoclonal type was also isolated from make-up water for the two cooling towers, a hot water tank, water separators in four main air compressor systems for respiratory therapy, and cold and hot water faucets. In hospital B, 13 of 37 (38%) sample sites contained legionellae, all of which were L. pneumophila serogroup 1. The monoclonal type matching isolates from patients (1,2,4,5) was found at the highest concentration in a hot water tank, but it was also present at four other sample sites. Since legionellae not related to disease may be found in many of the sites sampled, an epidemiologic association with the probable source should be established before intervention methods, such as disinfection, are undertaken.

Pittsburgh pneumonia agent (PPA) was recently cultivated from infected egg yold on charcoal yeast extract agar. PPA has now been isolated both from infected egg yolk and human lung tissue on charcoal yeast extract agar and on a new medium, buffered charcoal yeast extract agar. PPA resembles Legionella pneumophila and other Legionella-like organisms in requirements for growth and composition of fatty acids. It differs in genetic relatedness, antigenic composition, and colonial morphology and has distinctive characteristics that allow it to be identified. The name Legionella pittsburgensis species nova is proposed for this organism.

Legionella pneumophila serogroup 6 was isolated from nine of 16 shower heads in a Chicago hospital ward where three patients had contracted Legionnaires' disease caused by serogroup 6 L. pneumophila. Each patient had showered there 2 to 10 days before the onset of disease symptoms. We also isolated the bacteria in two other hospitals, and found the same serogroups as had been causing Legionnaires' disease in those hospitals: serogroup 1 in Pittsburgh and serogroups 1 and 4 in Los Angeles. However, showers from hospital wards where no patients had contracted Legionnaires' disease also yielded L. pneumophila. Shower heads at the Chicago hospital were sterilized with ethylene oxide but rapidly became recontaminated, suggesting that the potable water at these hospitals may have contained the organism. The question of whether aerosols of shower water or other exposures to potable water containing L. pneumophila may cause nosocomial Legionnaires' disease has not been resolved but deserves further study.

A three-year epidemic of legionnaires' disease in a hospital was dramatically curtailed following hyperchlorination of the potable water supply. The hypothesis that potable water was the source for the outbreak was further supported by isolation of Legionella pneumophila (the agent of legionnaires' disease) from the hospital water supply, observation that a sudden upsurge had occurred in the number of cases following a peculiar manipulation of the hospital water system, and documentation of a 30-fold increase in concentration of organisms in the water when this manipulation was artificially recreated. Thus, potable water may be an important source of epidemic legionnaires' disease and continuous hyperchlorination a method of control.

During a five-week period in 1981, six cases of legionellosis due to Legionella pneumophila serogroup 1 were recognized in a hospital in Paris, France. Four cases were clearly nosocomial in origin. There was a direct association between development of disease and exposure to potable hot water (p = 0.003). The entire hot water system was contaminated with L. pneumophila serogroup 1; monoclonal antibody testing demonstrated that the case isolate and the potable water isolates belonged to the same subgroup. Although serogroup 1 was isolated from both the cooling tower and its drift, the cooling tower isolate was antigenically distant from the case isolate. In other nosocomial outbreaks of legionellosis, multiple sources have been found within the hospital environment, but an epidemiologic association of disease with potable water had not been shown. The significant association of cases with exposure to the potable hot water supply, and the identification of case and potable water isolates of the same subtype, suggest that the potable hot water was responsible for transmission of disease in this outbreak.