A water culture containing naturally occurring Legionella pneumophila and associated microbiota was maintained in the laboratory by serially transferring the culture in tap water which had been sterilized by membrane filtration. Successful maintenance of the water culture depended upon transferring the culture when the growth of L. pneumophila was in the late-exponential to early-stationary phase. The water culture was used as a source of naturally occurring bacteria to determine some of the parameters which affect the multiplication of L. pneumophila in tap water. Naturally occurring L. pneumophila multiplied at a temperature between 25 and 37°C, at pH levels of 5.5 to 9.2, and at concentrations of dissolved oxygen of 6.0 to 6.7 mg/liter. Multiplication did not occur in tap water which contained less than 2.2 mg of dissolved oxygen per liter. An association was observed between the multiplication of L. pneumophila and the non-Legionellaceae bacteria which were also present in the water culture. The method of preserving naturally occurring L. pneumophila and associated microbiota may facilitate studies on the symbiosis of L. pneumophila with other microorganisms.

An investigation of the chemical environment and growth of Legionella pneumophila in plumbing systems was conducted to gain a better understanding of its ecology in this habitat. Water samples were collected from hospital and institutional hot-water tanks known to have supported L. pneumophila and were analyzed for 23 chemical parameters. The chemical environment of these tanks was found to vary extensively, with the concentrations of certain metals reaching relatively high levels due to corrosion. The effect of various chemical conditions on L. pneumophila growth was then examined by observing its multiplication in a series of tap water samples artificially supplemented with various concentrations of metals. Additionally, growth of L. pneumophila was examined in a more natural setting by monitoring its multiplication in the chemically analyzed hot-water tank samples after sterilization and reinoculation with L. pneumophila. L. pneumophila and associated microbiota used in these experiments were obtained from a hot-water tank. These strains were maintained in tap water and had never been passaged on agar. The results of the growth studies indicate that although elevated concentrations of a number of metals are toxic, lower levels of certain metals such as iron, zinc, and potassium enhance growth of naturally occurring L. pneumophila. Parallel observations on accompanying non-Legionellaceae bacteria failed to show the same relationship. These findings suggest that metal plumbing components and associated corrosion products are important factors in the survival and growth of L. pneumophila in plumbing systems and may also be important in related habitats such as cooling towers and airconditioning systems.

Previous studies have shown that bacteria maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21°C, pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples from plumbing fixtures in a hospital yielded legionellae which were "super"-chorine resistant when assayed under natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been previously exposed to additional chlorination. These studies more closely approximate natural conditions than our previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already resistant agar medium-passaged counterpart.

Studies were conducted to investigate the survival and multiplication of Legionella spp. in public drinking water supplies. An attempt was made, over a period of several years, to isolate legionellae from a municipal system. Sampling sites included the river water supply, treatment plant, finished water reservoir system, mains, and distribution taps. Despite the use of several isolation techniques, Legionella spp. could not be detected in any of the samples other than those collected from the river. It was hypothesized that this was due to the maintenance of a chlorine residual throughout this system. To investigate the potential for Legionella growth, additional water samples, collected from throughout the system, were dechlorinated, pasteurized, and inoculated with Legionella pneumophila. Subsequent growth indicated that many of these samples, especially those collected from areas affected by an accumulation of algal materials, exhibited a much greater ability to support LegioneUa multiplication than did river water prior to treatment. Chemical analyses were also performed on these samples. Correlation of chemical data and experimental growth results indicated that the chemical environment significantly affects the ability of the water to support multiplication, with turbidity, organic carbon, and certain metals being of particular importance. These studies indicate that the potential exists for Legionella growth within municipal systems and support the hypothesis that public water supplies may contaminate the plumbing systems of hospitals and other large buildings. The results also suggest that useful methods to control this contamination include adequate treatment plant filtration, maintenance of a chlorine residual throughout the treatment and distribution network, and effective covering of open reservoirs.

Earlier field and laboratory studies have shown that Legionella species survive and multiply in the pH range 5.5 to 9.2. Additionally, the technical feasibility of operating cooling towers at elevated alkalinities and pH has previously been documented by published guidelines. The guidelines indicate that these conditions facilitate corrosion control and favor chlorine persistence which enhances the effectiveness of continuous chlorination in biofouling control. This information suggests that control of Legionella species in cooling towers can be accomplished by operating the towers under alkaline conditions. To test this possibility, we collected water samples over a period of months from a hospital cooling tower. The samples were analyzed for a variety of chemical parameters. Subsamples were pasteurized and inoculated with non-agar-passaged Legionella pneumophila which had been maintained in tap water. Correlation of subsequent Legionella growth with corresponding pH and alkalinity values revealed statistically significant inverse associations. These data support the hypothesis that operating cooling towers outside of the optimal conditions for Legionella growth (e.g., at elevated alkalinities and a pH greater than 9) may be a useful approach to controlling growth in this habitat.