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°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°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.     

Isolation of Legionella species from drinking water

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.     

Isolation of Legionella species from drinking water.

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.     

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 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.     

Growth-supporting activity for Legionella pneumophila in tap water cultures and implication of hartmannellid amoebae as growth factors.

Photosynthetic cyanobacteria, heterotrophic bacteria, free-living amoebae, and ciliated protozoa may support growth of Legionella pneumophila. Studies were done with two tap water cultures (WS1 and WS2) containing L. pneumophila and associated microbiota to characterize growth-supporting activity and assess the relative importance of the microbiota in supporting multiplication of L. pneumophila. The water cultures were incubated in the dark at 35 degrees C. The growth-supporting factor(s) was separated from each culture by filtration through 1-micron-pore-size membrane filters. The retentate was then suspended in sterile tap water. Multiplication of L. pneumophila occurred when both the retentate suspension and the filtrate from either culture were inoculated into sterile tap water. L. pneumophila did not multiply in tap water inoculated with only the filtrate, even though filtration did not reduce the concentration of L. pneumophila or heterotrophic bacteria in either culture. Growth-supporting activity of the retentate suspension from WS1 was inactivated at 60 degrees C but unaffected at 0, 25, and 45 degrees C after 30-min incubations. Filtration experiments indicated that the growth-supporting factor(s) in WS1 was 2 to 5 micron in diameter. Ciliated protozoa were not detected in either culture. Hartmannellid amoebae were conclusively demonstrated in WS2 but not in WS1. L. pneumophila multiplied in tap water inoculated with the amoebae (10(3)/ml) and the 1-micron filtrate of WS2. No multiplication occurred in tap water inoculated with the filtrate only. Growth-supporting activity for L. pneumophila may be present in plumbing systems; hartmannellid amoebae appear to be important determinants of multiplication of L. pneumophila in some tap water cultures.     

Influence of native microbiota on survival of Ralstonia solanacearum phylotype II in river water microcosms

Ralstonia solanacearum phylotype II biovar 2 causes bacterial wilt in solanaceous hosts, producing severe economic losses worldwide. Waterways can be major dissemination routes of this pathogen, which is able to survive for long periods in sterilized water. However, little is known about its survival in natural water when other microorganisms, such as bacteriophages, other bacteria, and protozoa, are present. This study looks into the fate of a Spanish strain of R. solanacearum inoculated in water microcosms from a Spanish river, containing different microbiota fractions, at 24 degrees C and 14 degrees C, for a month. At both temperatures, R. solanacearum densities remained constant at the initial levels in control microcosms of sterile river water while, by contrast, declines in the populations of the introduced strain were observed in the nonsterile microcosms. These decreases were less marked at 14 degrees C. Lytic bacteriophages present in this river water were involved in the declines of the pathogen populations, but indigenous protozoa and bacteria also contributed to the reduced persistence in water. R. solanacearum variants displaying resistance to phage infection were observed, but only in microcosms without protozoa and native bacteria. In water microcosms, the temperature of 14 degrees C was more favorable for the survival of this pathogen than 24 degrees C, since biotic interactions were slower at the lower temperature. Similar trends were observed in microcosms inoculated with a Dutch strain. This is the first study demonstrating the influence of different fractions of water microorganisms on the survival of R. solanacearum phylotype II released into river water microcosms.     

Growth-supporting activity for Legionella pneumophila in tap water cultures and implication of hartmannellid amoebae as growth factors

Photosynthetic cyanobacteria, heterotrophic bacteria, free-living amoebae, and ciliated protozoa may support growth of Legionella pneumophila. Studies were done with two tap water cultures (WS1 and WS2) containing L. pneumophila and associated microbiota to characterize growth-supporting activity and assess the relative importance of the microbiota in supporting multiplication of L. pneumophila. The water cultures were incubated in the dark at 35 degrees C. The growth-supporting factor(s) was separated from each culture by filtration through 1-micron-pore-size membrane filters. The retentate was then suspended in sterile tap water. Multiplication of L. pneumophila occurred when both the retentate suspension and the filtrate from either culture were inoculated into sterile tap water. L. pneumophila did not multiply in tap water inoculated with only the filtrate, even though filtration did not reduce the concentration of L. pneumophila or heterotrophic bacteria in either culture. Growth-supporting activity of the retentate suspension from WS1 was inactivated at 60 degrees C but unaffected at 0, 25, and 45 degrees C after 30-min incubations. Filtration experiments indicated that the growth-supporting factor(s) in WS1 was 2 to 5 micron in diameter. Ciliated protozoa were not detected in either culture. Hartmannellid amoebae were conclusively demonstrated in WS2 but not in WS1. L. pneumophila multiplied in tap water inoculated with the amoebae (10(3)/ml) and the 1-micron filtrate of WS2. No multiplication occurred in tap water inoculated with the filtrate only. Growth-supporting activity for L. pneumophila may be present in plumbing systems; hartmannellid amoebae appear to be important determinants of multiplication of L. pneumophila in some tap water cultures.     

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.     

Long-term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes

Abstract Extended survival of Legionella pneumophila , using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 10⁸ cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 10⁶ cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10² (cfu/ml) over the same period. After incubation for 1.5 years at 15°C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEα. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.     

A note on the survival of Legionella pneumophila in stagnant tap water

Tap water, from an experimental hot water system, containing a known virulent strain of Legionella pneumophila was stored in screw-capped bottles for 14 months. Viable counts showed survival of L. pneumophila and at least three other bacterial species. This reinforces the view that L. pneumophila can survive in stagnant water foatively long periods of time.     

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.     

On the reproducibility of microcosm experiments - different community composition in parallel phototrophic biofilm microcosms

Phototrophic biofilms were cultivated simultaneously using the same inoculum in three identical flow-lane microcosms located in different laboratories. The growth rates of the biofilms were similar in the different microcosms, but denaturing gradient gel electrophoresis (DGGE) analysis of both 16S and 18S rRNA gene fragments showed that the communities developed differently in terms of species richness and community composition. One microcosm was dominated by Microcoleus and Phormidium species, the second microcosm was dominated by Synechocystis and Phormidium species, and the third microcosm was dominated by Microcoleus- and Planktothrix- affiliated species. No clear effect of light intensity on the cyanobacterial community composition was observed. In addition, DGGE profiles obtained from the cultivated biofilms showed a low resemblance with the profiles derived from the inoculum. These findings demonstrate that validation of reproducibility is essential for the use of microcosm systems in microbial ecology studies.     

A note on the survival of Legionella pneumophila in stagnant tap water

Tap water, from an experimental hot water system, containing a known virulent strain of Legionella pneumophila was stored in screw-capped bottles for 14 months. Viable counts showed survival of L. pneumophila and at least three other bacterial species. This reinforces the view that L. pneumophila can survive in stagnant water for relatively long periods of time.     

Influence of Solid Surface, Adhesive Ability, and Inoculum Size on Bacterial Colonization in Microcosm Studies

Microcosm studies were performed to evaluate the effect of solid surfaces, bacterial adhesive ability, and inoculum size on colonization success and persistence of Pseudomonas fluorescens and Xanthomonas maltophilia, each with a Tn5 insertion that conferred resistance to kanamycin and streptomycin. Two types of microcosms were used: (i) a simple system that was colonized by Aeromonas hydrophila and a coryneform and (ii) a complex system produced from lake water enrichment cultures. Simple microcosms contained 100 ml of peptone- and yeast extract-supplemented artificial lake water or 60 ml of peptone- and yeast extract-supplemented artificial lake water with 70 g of 3-mm glass beads. Complex microcosms contained 100 ml of lake water with no nutrient additions or 100 ml of lake water with 70 g of glass beads. The microcosms were incubated for 35 days at 20°C. In lake water enrichment microcosms, the presence of beads increased the abilities of P. fluorescens or X. maltophilia to colonize, but their numbers decreased with time in microcosms both with and without beads. The adhesiveness of the bacteria, measured in an in vitro assay, did not relate to colonization success. In simple microcosms, the inoculum size (10, 10², or 10³) of P. fluorescens did not influence colonization success. However, in complex microcosms, an inoculum of 10³ cells was insufficient to ensure colonization by P. fluorescens, while 10⁶ cells resulted in colonization of liquid and beads. Simple microcosm studies, utilizing only a few species, were poor models for complex natural systems. In complex enrichment systems, colonization of surfaces resulted in higher numbers of organisms but did not noticeably promote persistence. Adhesiveness of a particular organism may be a relatively minor factor influencing its ability to colonize solid surfaces in complex natural environments.     

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.     

Intracellular proliferation of Legionella pneumophila in Hartmannella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride

The need for protozoa for the proliferation of Legionella pneumophila in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of L. pneumophila in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous L. pneumophila originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-microm pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable L. pneumophila, and for their concentrations of protozoa. L. pneumophila grew to high numbers (6.3 log CFU/cm2) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of Hartmannella vermiformis in all flasks in which L. pneumophila multiplied and also when cycloheximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of L. pneumophila in trophozoites of H. vermiformis, with 25.9% +/- 10.5% of the trophozoites containing L. pneumophila on day 10 and >90% containing L. pneumophila on day 14. Calculations confirmed that intracellular growth was most likely the only way for L. pneumophila to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher L. pneumophila concentrations, and therefore the growth of L. pneumophila within engineered water systems can be limited by controlling biofilm formation.     

微生物在水环境中的存活和生长

在灭菌自来水模拟水体中,研究了7种细菌的存活和生长规律。Klebsiella pneumo-niae,Enterobacter aerogenes,Agrobacterium tumefatciens,在7天内平板计数降至0,而水体中镜检细菌总数(AODC)和活菌直接计数(DVC)结果无大变化,说明细菌已变成活的非可培养状态。Micrococcus,flavus 和 Streptococcus faecalis 的可培养菌数也可降至0。Pseudomonas sp.在48小时内由10~5降至10~2cfu/ml,随即升至10~6 cfu/ml 并持续到实验终了(41天)。Bacillus subtilis 在48小时平板计数降至10~2cfu/ml 并维持在该水平至实验结束(38天)。研究结果表明仅用涂布平板法检测多种细菌在水环境中的生存和分布是不合适的。     

Validation of Microcosms for Examining the Survival of Pseudomonas aureofaciens (lacZY) in Soil

Evaluating the safety and efficacy of a recombinant bacterium prior to its release into the terrestrial environment requires that risk assessment data be collected in the laboratory. Much of this information is obtained with the use of microcosms. The design of the microcosm significantly affects the ability of the recombinant microorganism to survive in soil and, thus, complicates the risk assessment process. To standardize microcosms for future use, we evaluated the survival of Pseudomonas aureofaciens 3732 RN-L11 (lacZY Rif(supr) Nal(supr)) in intact soil cores (5.0 by 15 cm; polyvinyl chloride core) and disturbed soil microcosms (50 g of fresh, sieved soil). Survival data were compared with those obtained during a field release. The intact soil core microcosm was shown to closely simulate results obtained in the field. The intact soil core microcosm closely predicts survival in bulk soil and in the rhizosphere of wheat. Data obtained with the microcosm were also similar when evaluated in separate studies in two different years. In 1993, P. aureofaciens survived for approximately 63 days in bulk soil and 96 days in the rhizosphere. The disturbed soil microcosm exhibited a much more rapid decline in population size (34 days to zero) than the intact core microcosm. We speculate that drying and sieving of soil for the disturbed soil microcosm affected the ability of the soil to support the survival of P. aureofaciens. These results demonstrate that a small, inexpensive, and simple intact soil core microcosm may be appropriate for risk assessment.     

Effects of metals on Legionella pneumophila growth in drinking water plumbing systems.

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 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 stains 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 air-conditioning systems.