Effect of temperature, pH, and oxygen level on the multiplication of naturally occurring Legionella pneumophila in potable water

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 degrees 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.     

Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water.

A naturally occurring suspension of Legionella pneumophila and associated microbiota contained three unidentified non-Legionellaceae bacteria which supported satellite growth of a subculture of L. pneumophila on an L-cysteine-deficient medium and another bacterium which did not support growth of the subculture. Washed suspensions containing 10(3), 10(5), 10(7), or 10(8) CFU of a mixture of isolates of these non-Legionellaceae bacteria failed to support the multiplication of an isolate of agar-grown L. pneumophila which had been washed and seeded into the suspensions. The suspensions which contained 10(3), 10(5), or 10(7) CFU of the non-Legionellaceae bacteria per ml appeared to enhance survival or cryptic growth of agar-grown L. pneumophila. A decline of 1.3 log CFU of L. pneumophila per ml occurred within the first week of incubation in the sample which contained 10(8) CFU of the non-Legionellaceae bacteria per ml. In contrast to these results, naturally occurring L. pneumophila multiplied in the presence of associated microbiota. The necessity to subculture L. pneumophila and the non-Legionellaceae bacteria on artificial medium to obtain pure cultures may have affected the multiplication of L. pneumophila in tap water. Alternatively, other microorganisms may be present in the naturally occurring suspension which support the growth of this bacterium.     

Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water

A naturally occurring suspension of Legionella pneumophila and associated microbiota contained three unidentified non-Legionellaceae bacteria which supported satellite growth of a subculture of L. pneumophila on an L-cysteine-deficient medium and another bacterium which did not support growth of the subculture. Washed suspensions containing 10(3), 10(5), 10(7), or 10(8) CFU of a mixture of isolates of these non-Legionellaceae bacteria failed to support the multiplication of an isolate of agar-grown L. pneumophila which had been washed and seeded into the suspensions. The suspensions which contained 10(3), 10(5), or 10(7) CFU of the non-Legionellaceae bacteria per ml appeared to enhance survival or cryptic growth of agar-grown L. pneumophila. A decline of 1.3 log CFU of L. pneumophila per ml occurred within the first week of incubation in the sample which contained 10(8) CFU of the non-Legionellaceae bacteria per ml. In contrast to these results, naturally occurring L. pneumophila multiplied in the presence of associated microbiota. The necessity to subculture L. pneumophila and the non-Legionellaceae bacteria on artificial medium to obtain pure cultures may have affected the multiplication of L. pneumophila in tap water. Alternatively, other microorganisms may be present in the naturally occurring suspension which support the growth of this bacterium.     

Survival and multiplication of Legionella pneumophila in municipal drinking water systems.

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 the 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 Legionella 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.     

Survival and multiplication of Legionella pneumophila in municipal drinking water systems

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 the 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 Legionella 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.     

Influence of temperature on the number of Legionella pneumophila in hot water systems

The occurrence of legionella in the hot water systems of two buildings (A and B) was investigated in relation to the water temperature. The peripheral parts of both hot water systems were found to be colonized by these organisms. A temperature of 60 degrees C in the hot water mains returning from the building eliminated legionellas from the mains as well as from the peripheral taps and showers. Legionellas could be isolated from taps, showers and the mains when the temperature in the return mains was kept at 54 degrees C. The hot water systems could not be completely decontaminated by raising the hot water temperature in the return mains to 70 degrees C combined with flushing all the taps and showers. It is suggested that failure to decontaminate the systems is due to dead ends in the pipeline network, which are not reached by the hot water and that these dead ends are the source for recolonization of the systems.     

Influence of temperature on the number of Legionella pneumophila in hot water systems

The occurrence of legionella in the hot water systems of two buildings (A and B) was investigated in relation to the water temperature. The peripheral parts of both hot water systems were found to be colonized by these organisms. A temperature of 60C in the hot water mains returning from the building eliminated legionellas from the mains as well as from the peripheral taps and showers. Legionellas could be isolated from taps, showers and the mains when the temperature in the return mains was kept at 54C. The hot water systems could not be completely decontaminated by raising the hot water temperature in the return mains to 70C combined with flushing all the taps and showers. It is suggested that failure to decontaminate the systems is due to dead ends in the pipeline network, which are not reached by the hot water and that these dead ends are the source for recolonization of the systems.     

Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora.

Survival and growth of Legionella pneumophila in both biofilm and planktonic phases were determined with a two-stage model system. The model used filter-sterilized tap water as the sole source of nutrient to culture a naturally occurring mixed population of microorganisms including virulent L. pneumophila. At 20 degrees C, L. pneumophila accounted for a low proportion of biofilm flora on polybutylene and chlorinated polyvinyl chloride, but was absent from copper surfaces. The pathogen was most abundant on biofilms on plastics at 40 degrees C, where it accounted for up to 50% of the total biofilm flora. Copper surfaces were inhibitory to total biofouling and included only low numbers of L. pneumophila organisms. The pathogen was able to survive in biofilms on the surface of the plastic materials at 50 degrees C, but was absent from the copper surfaces at the same temperature. L. pneumophila could not be detected in the model system at 60 degrees C. In the presence of copper surfaces, biofilms forming on adjacent control glass surfaces were found to incorporate copper ions which subsequently inhibited colonization of their surfaces. This work suggests that the use of copper tubing in water systems may help to limit the colonization of water systems by L. pneumophila.     

Effect of salt concentration and temperature on survival of Legionella pneumophila

The effects of various concentrations of sodium chloride solutions (0·1%–3%) and different temperatures (4, 10, 20, 30 and 37 °C) on survival of Legionella pneumophila were investigated. It was found that at temperatures between 4 °C and 20 °C, Legionella organisms survived in salt solutions up to 3% NaCl. Only the combination of high temperatures, i. e. 30 °C and 37 °C, with NaCl concentrations over 1·5%, reduced cell numbers significantly. It was interesting to note that the addition of small amounts of NaCl (0·1%–0·5%) enhanced survival of Leg. pneumophila , suggesting a protective effect of NaCl. In order to obtain information about conditions encountered in the environment, the survival experiments were repeated in sterile sea water from the Baltic Sea and the North Sea. The marked bacterial die-off, especially at higher temperatures, was not observed in natural sea water. All these results indicate that Leg. pneumophila can survive in the marine environment.     

Legionella incidence and density in potable drinking water supplies.

The incidence and density of Legionella spp. in raw water, water at various stages of treatment, and in potable distribution water were determined by direct immunofluorescence. The number of cells reacting with Legionella-specific fluorescent antibody conjugates in raw waters ranged from about 10(4) to 10(5) cells/liter, whereas the concentrations of fluorescent antibody-positive cells in the distribution waters were generally 10- to 100-fold lower than in the raw source waters. No viable or virulent Legionella strains were isolated from either the source or distribution waters. However, Legionella spp. are infrequently isolated from water at temperatures below 15 degrees C as was the case in the system surveyed in this study.     

Interferon-gamma-activated human monocytes inhibit the intracellular multiplication of Legionella pneumophila

We have examined the interaction between interferon-gamma (IFN-gamma)-activated human monocytes and Legionella pneumophila, the agent of Legionnaires' disease. Human monocytes activated with human recombinant IFN-gamma inhibit the intracellular multiplication of L. pneumophila. The degree of inhibition is proportional to the concentration of IFN-gamma, and maximal inhibition consistently occurs with greater than or equal to 2 micrograms/ml. Monoclonal anti-IFN-gamma antibody completely neutralizes the capacity of IFN-gamma to activate monocytes. Monocytes infected 24 hr after explantation maximally inhibit L. pneumophila multiplication if treated with IFN-gamma before infection or up to 2 hr after infection; treatment 6 hr or more after infection results in submaximal inhibition. Monocytes infected 48 hr after explantation inhibit L. pneumophila multiplication maximally if treated with IFN-gamma up to 12 hr before infection, but submaximally if treated at the time of infection. Once activated, monocytes inhibit L. pneumophila multiplication in the absence of IFN-gamma in the culture. Strikingly, monocytes maximally inhibit L. pneumophila multiplication after treatment with IFN-gamma for as briefly as 1 hr before infection. In the absence of anti-L. pneumophila antibody, neither IFN-gamma-activated monocytes nor nonactivated monocytes kill L. pneumophila. In the presence of specific antibody and complement, IFN-gamma-activated monocytes kill a proportion (0.5 log) of an inoculum but not more than nonactivated monocytes. L. pneumophila forms a specialized phagosome in IFN-gamma-activated monocytes that does not differ ultrastructurally from the L. pneumophila phagosome in nonactivated monocytes. These results demonstrate that IFN-gamma can activate human monocytes to exert a potent antimicrobial effect against a highly virulent intracellular bacterial pathogen. These findings extend previous observations on interactions between activated mononuclear phagocytes and L. pneumophila, and additionally support the hypothesis that cell-mediated immunity plays a major role in host defense against L. pneumophila.     

Multiplication of Legionella pneumophila in unsterilized tap water.

Naturally occurring Legionella pneumophila, an environmental isolate which had not been grown on artificial medium, was tested for the ability to multiply in tap water. A showerhead containing L. pneumophila and non-Legionellaceae bacteria was immersed in nonsterile tap water supplying this fixture. Also L. pneumophila and non-Legionellaceae bacteria were sedimented from tap water from a surgical intensive care unit. This bacterial suspension was inoculated into tap water from our laboratory. The legionellae in both suspensions multiplied in the tap water at 32, 37, and 42 degrees C. The non-Legionellaceae bacteria multiplied at 25, 32, and 37 degrees C. A water sample which was collected from the bottom of a hot water tank was found to contain L. pneumophila and non-Legionellaceae bacteria. These legionellae also multiplied when the water sample was incubated at 37 degrees C. These results indicate that L. pneumophila may multiply in warm water environments such as hot water plumbing fixtures, hot water tanks, and cooling towers.     

IFN-gamma-activated human alveolar macrophages inhibit the intracellular multiplication of Legionella pneumophila

Human alveolar macrophages activated by human rIFN-gamma inhibit the intracellular multiplication of Legionella pneumophila, an intracellular bacterial pathogen and the agent of Legionnaires' disease. Activation of alveolar macrophages with IFN-gamma is dose dependent; significant inhibition of L. pneumophila multiplication (mean 1.60 +/- 0.20 logs) is achieved consistently with concentrations of IFN-gamma of greater than or equal to 2 x 10(-2) micrograms/ml (220 U/ml). Activation of alveolar macrophages is also time dependent. In macrophages treated continuously after explantation, macrophages infected at 48 to 96 h after explantation are more inhibitory than macrophages infected at 24 h after explantation. In macrophages not treated continuously after explantation but treated for various lengths of time before infection, the longer their exposure to IFN-gamma before infection, the greater the inhibition of L. pneumophila multiplication (96 greater than 72 greater than 48 greater than 24 h). IFN-gamma-activated alveolar macrophages exhibit morphologic signs of activation, including increased size, spreading, and aggregation. This paper demonstrates that a human resident macrophage can be activated with IFN-gamma such that it exhibits enhanced antimicrobial activity against a relevant pathogen.     

Incorporation of natural uncultivable Legionella pneumophila into potable water biofilms provides a protective niche against chlorination stress

Legionella pneumophila is a waterborne pathogen that has been isolated sporadically from drinking water distribution systems (DWDS). Resistance to disinfectants is mainly attributed to the association of cells with amoebae, but biofilms are also thought to provide some degree of protection. In the present work, a two-stage chemostat was used to form heterotrophic biofilms from drinking water to study the influence of chlorine on the presence of naturally occurring L. pneumophila. The pathogen was tracked in planktonic and sessile biofilm phases using standard culture recovery techniques for cultivable cells and a peptide nucleic acid fluorescence in situ hybridisation assay for total cells. The results showed that the total number of L. pneumophila cells in biofilms was not affected by the concentrations of chlorine tested, and the presence of L. pneumophila could not be detected by culturing. To restrict the outbreaks of disease caused by this bacterium, efforts need to be concentrated on preventing L. pneumophila from re-entering an infectious state by maintaining residual disinfectant levels through the entire DWDS network so that the resuscitation of cells via contact with amoebae is prevented.     

Metal complexes of lactoferrin and their effect on the intracellular multiplication of Legionella pneumophila

The action of bovine lactoferrin saturated with iron, zinc and manganese on the intracellular multiplication of Legionella pneumophila in HeLa cells has been tested. The results obtained showed that lactoferrin did not influence the invasive efficiency of Legionella. The intracellular multiplication of the bacterium was inhibited by apo-lactoferrin and by lactoferrin saturated with manganese and zinc, whereas lactoferrin saturated with iron enhanced the intracellular growth. Experiments in parallel were performed with iron, manganese and zinc citrate to test the effect due to the metal ions alone. Even in this condition the addition of an iron chelate enhanced the multiplication of Legionella while the manganese chelate produced a certain inhibition.