Colonization of components of a model hot water system by Legionella pneumophila

A model hot water distribution network was seeded with a virulent strain of Legionella pneumophila serotype 1. Ten weeks after inoculation, components of the system, which include aluminium discs, copper, stainless steel, silicone tubing, rubber and glass beads, were examined for colonization by L. pneumophila. The samples were stained with fluorescein-labelled antibodies to the strain and were examined with scanning electron microscopy. Colonization, which was accompanied by copious quantities of a slime-like debris, was heaviest on the rubber and least on the copper. Adherence to silicone tubing and stainless steel was observed.     

Survival of Legionella pneumophila in a model hot water distribution system

A virulent strain of Legionella pneumophila was inoculated into an enclosed system supplied with unsterilized water from a domestic hot water supply. Growth of bacteria was monitored over 10 weeks. An increase in the number of organisms other than legionellas occurred but few amoebae were observed and none could be cultured. Viable counts of L. pneumophila in the circulation fluid decreased slightly. However, particles of debris which accumulated in the apparatus and which were stained by the indirect fluorescent antibody technique were found to be almost totally composed of L. pneumophila. On dismantling the apparatus Legionella was isolated in moderately high numbers from several different types of surfaces, particularly natural rubber and silicone.     

Rapid detection and enumeration of Legionella pneumophila in hot water systems by solid-phase cytometry

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water 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 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.     

Factors influencing survival of Legionella pneumophila serotype 1 in hot spring water and tap water

The factors involved in the survival of Legionella pneumophila in the microcosms of both hot spring water and tap water were studied by examining cultivability and metabolic activity. L. pneumophila could survive by maintaining metabolic activity but was noncultivable in all microcosms at 42 degrees C, except for one microcosm with a pH of <2.0. Lower temperatures supported survival without loss of cultivability. The cultivability declined with increasing temperature, although metabolic activity was observed at temperatures of up to 45 degrees C. The optimal range of pH for survival was between 6.0 and 8. The metabolic activity could be maintained for long periods even in microcosms with high concentrations of salt. The cultivability of organisms in the post-exponential phase in a tap water microcosm with a low inoculum size was more rapidly reduced than that of organisms in the exponential phase. In contrast, the loss of cultivability in microcosms of a high inoculum size was significant in the exponential phase. Random(ly) amplified polymorphic DNA analysis of microcosms where cultivability was lost but metabolic activity was retained showed no change compared to cells grown freshly, although an effect on the amplified DNA band pattern by production of stress proteins was expected. Resuscitation by the addition of Acanthamoeba castellanii to the microcosm in which cultivability was completely lost but metabolic activity was maintained was observed only in part of the cell population. Our results suggest that L. pneumophila cell populations can potentially survive as free organisms for long periods by maintaining metabolic activity but temporarily losing cultivability under strict environments and requiring resuscitation by ingestion by amoebas.     

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.     

Relationship between colonization of hospital building with Legionella pneumophila and hot water temperatures.

Legionella pneumophila was isolated from four hospital buildings that maintained hot water storage temperatures at 43 to 45 degrees C. Two adjacent hospital buildings with negative cultures maintained temperatures at 58 to 60 degrees C.     

Ecological behaviour of three serogroups of Legionella pneumophila within a model plumbing system

Three Legionella pneumophila strains isolated from water samples and belonging to serogroups (sgs) 1, 6 and 9 were analysed for their capacity to colonise an experimental model simulating a domestic hot water distribution system. Ecological factors that could influence the persistence of the sgs such as intracellular life within protozoan hosts and bacterial interference by the production of antagonistic compounds were also studied. Viable counts of L. pneumophila increased both in the planktonic and in the sessile phases. Sg 6 showed a marked prevalence during the whole experiment and exhibited the highest host infection efficiency. Sg 1 was significantly less represented, but showed the highest capacity to reproduce in the protozoan hosts. Sg 9 was poorly represented and less adapted to intracellular life. Among the 14 bacteria constantly isolated in the system, five (35.7%) produced antagonistic substances against Legionella, with differences according to the bacterial strain and L. pneumophila sgs.     

Ecological behaviour of three serogroups of Legionella pneumophila within a model plumbing system

Three Legionella pneumophila strains isolated from water samples and belonging to serogroups (sgs) 1, 6 and 9 were analysed for their capacity to colonise an experimental model simulating a domestic hot water distribution system. Ecological factors that could influence the persistence of the sgs such as intracellular life within protozoan hosts and bacterial interference by the production of antagonistic compounds were also studied. Viable counts of L. pneumophila increased both in the planktonic and in the sessile phases. Sg 6 showed a marked prevalence during the whole experiment and exhibited the highest host infection efficiency. Sg 1 was significantly less represented, but showed the highest capacity to reproduce in the protozoan hosts. Sg 9 was poorly represented and less adapted to intracellular life. Among the 14 bacteria constantly isolated in the system, five (35.7%) produced antagonistic substances against Legionella, with differences according to the bacterial strain and L. pneumophila sgs.     

Legionella pneumophila in commercial bottled mineral water

Sixty-eight commercial bottled mineral waters (64 brands, 68 different 'best-before dates') were tested for the presence of bacteria and fungi. Six samples were Legionella antigen positive and six were Legionella pneumophila PCR positive. Two samples were both Legionella antigen and L. pneumophila PCR positive. Legionella cultures were negative. Although the PCR might have detected only dead Legionella cells, the PCR has been described to detect specifically viable but not culturable (VBNC) L. pneumophila cells as well. Whether VBNC bacteria may be present in bottled mineral waters and the risk for infection this may pose for severely immunocompromised patients should be investigated.     

Iron Acquisition by Legionella pneumophila

For nearly 20 years, it was believed that Legionella pneumophila does not produce siderophores. Yet, we have now determined that L. pneumophila secretes a siderophore (legiobactin) that is detectable by the CAS assay. We have optimized conditions for legiobactin expression, shown its biological activity, and found genes (lbtAB) involved in its production and secretion. LbtA is homologous with siderophore synthetases from E. coli (aerobactin), Sinorhizobium (rhizobactin), and Bordetella (alcaligin), while LbtB is a member of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtAB produce 40–70% less CAS reactivity. The lbtA mutant is also defective for growth in deferrated media containing citrate, indicating that legiobactin is required in conditions of severe iron limitation. lbtAB mutants grow normally in macrophages and amoebae host cells as well as within the lungs of mice. L. pneumophila does express lbtA in macrophages, suggesting that legiobactin has a dispensable role in infection. Legiobactin is iron repressed and does not react in the Csáky and Arnow assays. Anion-exchange HPLC has been used to purify legiobactin, and thus far, structural analysis suggests that the molecule is similar but not identical to rhizobactin, rhizoferrin, and alcaligin. The residual CAS reactivity present in supernatants of the lbtAB mutants suggests that L. pneumophila might produce a second siderophore. Besides siderophores, we have determined that ferrous iron transport, encoded by feoB, is critical for L. pneumophila growth in low-iron conditions, in host cells, and in the mammalian lung. Some of our other studies have discovered a critical, yet undefined, role for the L. pneumophila cytochrome c maturation locus in low-iron growth, intracellular infection, and virulence.     

Ecology of Legionella pneumophila within water distribution systems

The reservoir for hospital-acquired Legionnaires disease has been shown to be the potable water distribution system. We investigated the influence of the natural microbial population and sediment (scale and organic particulates) found in water systems as growth-promoting factors for Legionella pneumophila. Our in vitro experiments showed that: (i) water from hot-water storage tank readily supported the survival of L. pneumophila, (ii) the concentration of sediment was directly related to the survival of L. pneumophila, (iii) the presence of environmental bacteria improved the survival of L. pneumophila via nutritional symbiosis, (iv) the combination of sediment and environmental bacteria acted synergistically to improve the survival of L. pneumophila, and (v) the role of sediment in this synergistic effect was determined to be nutritional. Sediment was found to stimulate the growth of environmental microflora, which in turn stimulated the growth of L. pneumophila. These findings confirm the empiric observations of the predilection of L. pneumophila for growth in hot-water tanks and its localization to sediment. L. pneumophila occupies an ecological niche within the potable water system, with interrelationships between microflora, sediment, and temperature.     

Introduction of a boost of Legionella pneumophila into a stagnant-water model by heat treatment

An environmentally representative stagnant-water model was developed to monitor the growth dynamics of Legionella pneumophila. This model was evaluated for three distinct water treatments: untreated tap water, heat-treated tap water, and heat-treated tap water supplemented with Pseudomonas putida, a known biofilm-forming bacterium. Bringing heat-treated tap water after subsequent cooling into contact with a densely formed untreated biofilm was found to promote the number of L. pneumophila by 4 log units within the biofilm, while the use of untreated water only sustained the L. pneumophila levels. Subsequent colonization of the water phase by L. pneumophila was noticed in the heat-treated stagnant-water models, with concentrations as high as 1 x 10(10) mip gene copies L(-1) stagnant water. Denaturing gradient gel electrophoresis in combination with clustering analysis of the prokaryotic community in the water phase and in the biofilm phase suggests that the different water treatments induced different communities. Moreover, boosts of L. pneumophila arising from heat treatment of water were accompanied by shifts to a more diverse eukaryotic community. Stimulated growth of L. pneumophila after heating of the water may explain the rapid recolonization of L. pneumophila in water systems. These results highlight the need for additional or alternative measures to heat treatment of water in order to prevent or abate potential outbreaks of L. pneumophila.     

Inactivation of Legionella pneumophila by monochloramine

Chloramination which is used in South Australia to control the growth of Naegleria fowleri , was investigated to see if it would also control that of Legionella pneumophila . It was found that L. pneumophila was more sensitive than Escherichia coli to monochloramine. At 1.0 mg/l, a 99% kill of L. pneumophila was achieved in 15 min compared with 37 min for a 99% kill of E. coli. Combined with the stability of monochloramine, even at elevated temperatures, the results suggest that this disinfectant would control the growth of L. pneumophila in water distribution systems.     

Inactivation of Legionella pneumophila by monochloramine

Chloramination which is used in South Australia to control the growth of Naegleria fowleri, was investigated to see if it would also control that of Legionella pneumophila. It was found that L. pneumophila was more sensitive than Escherichia coli to monochloramine. At 1.0 mg/l, a 99% kill of L. pneumophila was achieved in 15 min compared with 37 min for a 99% kill of E. coli. Combined with the stability of monochloramine, even at elevated temperatures, the results suggest that this disinfectant would control the growth of L. pneumophila in water distribution systems.     

Susceptibility of Legionella pneumophila to chlorine in tap water

A study was conducted to compare the susceptibility of legionellae and coliforms to disinfection by chlorine. The chlorine residuals used were similar to concentrations that might be found in the distribution systems of large public potable water supplies. The effects of various chlorine concentrations, temperatures, and pH levels were considered. A number of different Legionella strains, both environmental and clinical, were tested. The results indicate that legionellae are much more resistant to chlorine than are coliform bacteria. At 21 degrees C, pH 7.6, and 0.1 mg of free chlorine residual per liter, a 99% kill of L. pneumophila was achieved within 40 min, compared with less than 1 min for Escherichia coli. The observed resistance is enhanced as conditions for disinfection become less optimal. The required contact time for the removal of L. pneumophilia was twice as long at 4 degrees C than it was at 21 degrees C. These data suggest that legionellae can survive low levels of chlorine for relatively long periods of time.     

Antibodies isolated by using cloned surface antigens recognize antigenically related components of Legionella pneumophila and other Legionella species

We studied the antigenic cross-reactivity of surface proteins among various strains of Legionella pneumophila and other Legionella species by using a novel method of antibody purification. Anti-bacterial antibodies in hyperimmune sera were adsorbed to and eluted from the surface of recombinant E. coli cells that express individual L. pneumophila antigens on their surface. These affinity-purified antibodies were then used to probe protein immunoblots prepared from the test strains to detect cross-reactive domains. We found that antigenic proteins are generally conserved in all L. pneumophila serogroups. Although some of these antigenic domains are shared with members of other Legionella species, they are associated with proteins of different molecular mass. Our approach to the study of antigenic cross-reactivity has potential advantages over similar studies that use either monoclonal antibodies or monospecific antibodies prepared by immunization with purified antigens.