Legionella Infection Risk from Domestic Hot Water

We investigated Legionella and Pseudomonas contamination of hot water in a cross-sectional multicentric survey in Italy. Chemical parameters (hardness, free chlorine, and trace elements) were determined. Legionella spp. were detected in 33 (22.6%) and Pseudomonas spp. in 56 (38.4%) of 146 samples. Some factors associated with Legionella contamination were heater type, tank distance and capacity, water plant age, and mineral content. Pseudomonas presence was influenced by water source, hardness, free chlorine, and temperature. Legionella contamination was associated with a centralized heater, distance from the heater point >10 m, and a water plant >10 years old. Furthermore, zinc levels of <20 μg/L and copper levels of >50 μg/L appeared to be protective against Legionella colonization. Legionella species and serogroups were differently distributed according to heater type, water temperature, and free chlorine, suggesting that Legionella strains may have a different sensibility and resistance to environmental factors and different ecologic niches.

L egionnaires' disease is normally acquired by inhalation or aspiration of legionellae from a contaminated environmental source. The first evidence of the association between potable water from shower and nosocomial legionellosis was reported approximately 20 years ago (1), and the hot water system is thought to be the most frequent source of cases or outbreaks within a hospital (2,3), where patients may be at a higher risk for a severe infection (4)(5)(6). Relatively little is known about sporadically occurring cases of community-acquired legionellosis, which accounts for most infections (7,8), although correlation analyses suggest that a substantial proportion of these cases may be residentially acquired and associated with bacteria in hot water distribution systems (9).
Legionella spp. have been isolated from water with a temperature as high as 63°C, and the contamination is associated with other bacteria and protozoa (10,11). Biofilm formation can provide a means for survival and dissemination of L. pneumophila (12,13), interfering with efforts to eradicate bacteria from water systems (14,15). The accumulation of microorganisms on the pipeline surfaces and the formation of biofilms are influenced by many factors, such as surface materials, concentration and quality of nutrients and disinfectants, temperature and hydraulics of the system, and pipe surface roughness (16).
To assess the potential public health impact of Legionella colonization at a domestic level, a descriptive multicentric study was undertaken to identify and quantify the levels of the microorganism in a substantial number of Italian domestic hot water samples. Pseudomonas spp. are part of the natural population of the water, but some species should be considered as opportunistic pathogens. Furthermore, Pseudomonas may compete with Legionella to grow in the aquatic environment (17,18); thus we also evaluated Pseudomonas colonization.
We addressed three specific aims: 1) to estimate the frequency of Legionella colonization and severity of contamination at the domestic level; 2) to identify potential risk factors for contamination relative to distribution systems and water characteristics; 3) and to define the relative role of each risk factor and suggest possible remediation. Lastly, risk for legionellosis was retrospectively evaluated by collecting information about pneumonia symptoms recorded by residents at home.

Sample Collection
From May through June 2002, a total of 146 water samples were collected from private homes of six towns (Milan, Modena, Bologna, Rome, Naples, Bari) representative of different Italian regions (Northern, Central, and Southern Italy). A similar number of samples were taken from each town; selection was made on the basis of the water distribution systems inside the town and building and heater types in each area. After we identified each building, we asked a random family (in case of a condominium) to participate in the study, i.e., to complete our questionnaire and give informed consensus for water collection. Laboratory examinations were free, and at the end of the study each participating family received a letter with results of Legionella analysis.
Hot water samples were drawn from the bathroom outlets (shower heads or bathroom tap) in three sterile 1-L glass bottles after a brief flow time (to eliminate cold water inside the tap or flexible shower pipe). To neutralize residual free chlorine, sodium thiosulphate was added in sterile bottles for bacteriologic analysis, whereas acid-preserved glass bottles were used for chemical determinations. Collection bottles were returned to the laboratory immediately after sampling for bacteriologic and chemical-physical examination; if analyses would not begin within 24 hours, samples were kept at >4°C and processed within 48 hours of collection.

Microbiologic Analysis
To detect Legionella spp., 2-L water samples were concentrated by membrane filtration (0.2-µm-pore-sized polyamide filter, Millipore, Billerica, Massachusetts, USA). The filter membrane was resuspended in 10 mL of original sample water and vortex-mixed for 10 min. To reduce contamination by other microorganisms, 5 mL of this suspension was heat-treated (50°C for 30 min in a water bath) (19). Two aliquots of 0.1 mL of the original and concentrated specimens (heat-treated and untreated, 1:10 diluted and undiluted) were each spread on duplicate plates of modified Wadowsky-Yee selective medium (Oxoid Ltd., Basingstoke, Hampshire, UK). The plates were incubated at 36°C in a humidified environment with at least 2.5% CO 2 for 10 days and read from day 5 at the dissecting microscope. Suspected colonies with a mottled surface or an iridescent and faceted cut-glass appearance, were counted from each sampling. All colonies from plates with <10 and 10-20 random colonies were subcultured on buffered charcoal yeast extract (BCYE) agar (with cysteine) and charcoal yeast extract agar (cysteine-free) media (Oxoid) for >2 days. Only colonies grown on BCYE were subsequently identified by an agglutination test (Legionella Latex Test, Oxoid). The test allows a separate identification of L. pneumophila serogroup 1 and serogroups 2-14 and detection of seven Legionella (polyvalent) species (other than L. pneumophila), which have been implicated in human disease: L. longbeachae, L. bozemanii 1 and 2, L. dumoffii, L. gormanii, L. jordanis, L. micdadei, L. anisa. Legionella-like bacteria serologically nonidentifiable (in total 4 colonies) are excluded from the account, awaiting a different confirmation by DNA sequence (polymerase chain reaction [PCR]-method). The results are expressed as CFU/L and the detection limit of the procedure was 25 CFU/L (mean value of two plates). All the research units participated in a quality control for Legionella detection in water that was organized by the National Health Institute, through a periodic distribution of water samples added with unknown Legionella species and concentration. The total microbial counts at 36°C and 22°C were obtained twice by the pour-plate method on plate count agar (Oxoid). The plates were incubated at 36°C for 48 h or at 22°C for 72 h.
To isolate Pseudomonas spp., 100-mL and 10-mL water samples were filtered through a 0.45-µm-pore-size membrane (Millipore). If the number of bacteria was high, suitable dilutions were made. The membranes were placed on Pseudomonas cetrimide fucidin cephalosporin (CFC) agar (Oxoid) and incubated at 30°C for 48 h. Each type of oxidase-positive colony was counted.

Physical and Chemical Analyses
Water temperature and residual free chlorine (DPD method, colorimetric) were determined at the time of sample collection. Standard techniques were used to measure oxidizability and water hardness. Concentrations of calcium, magnesium, iron, manganese, copper, and zinc were measured by flame atomic absorption spectrophotometer (Perkin-Elmer, Wellesley, MA, mod 5000) on acidified samples (1% HNO 3 ) concentrated by boiling.

Risk Factors
A detailed standardized questionnaire was developed to evaluate risk factors possibly associated with colonization. The first part collected information on family characteristics (number of components, age and sex, length of stay in residence) and on pneumonia events during their stay in the home. The second part was devoted to home data: type (flat, single house, villa), flats in the building, home floor, home rooms and bathrooms, building age, type of water supply, and disinfection systems used. The third part collected information on the heating system (central or independent, electric or gas heater), distance of the sample site from the water distribution point, existence of a tank and its volume, age of the system, service frequency, and existence and characteristics of a softening and water recycling systems. Water operating temperature (temperature at the distribution site) was also recorded.

Statistical Analysis
All statistical calculations were made with SPSS/pc (SPSS Inc, Chicago, IL). Logarithmic transformations were used in statistical analyses to normalize the nonnormal distributions, and results are presented as geometric means. The bacteriologic data were converted into log 10 (x+1). When possible, variables were categorized into dichotomous ones. The results were analyzed by correlation analysis, t test, one-way analysis of variance (ANOVA), and by chi-square test. Odds ratios (OR) and 95% confidence intervals (CI) were calculated to assess categorical risk variables associated with microbial contamination. Variables that were significant in the univariate analysis were entered in a multiple logistic regression model. By using conditional logistic regression models, independent predictors of colonization were established. Variables were retained in the model if the likelihood ratio test was significant (p < 0.05). Table 1 shows general characteristics of the examined water in terms of supply and distribution systems. Five heating systems were recognized, corresponding to those more frequently used at the domestic level, although with geographic differences, as the centralized systems were mainly adopted in northern Italy. Table 2 shows chemical and microbiologic qualities of hot water samples. When samples were grouped according to their origin (mixture or groundwater), groundwater had significantly higher levels of calcium (105.7 ± 32.1 mg/L vs. 68.8 ± 31.3 mg/L, p < 0.001) and magnesium (21.5 ± 16.0 mg/L vs. 14.9 ± 6.0 mg/L, p < 0.01) and was harder (35.4 ± 13.0 vs. 22.1 ± 8.8°F, p < 0.001). Pseudomonas spp. were isolated from 56 of 146 (38.4%) samples, with levels ranging from 1 to 6.4 x 10 4 CFU/100 mL; 85.7% of positive samples contained fewer than 10 3 CFU/100 mL.

Descriptive Data
A total of 33 (22.6%) samples of 146 were contaminated by Legionella spp., and L. pneumophila (Table 3) was the most frequently isolated species (75.8% of isolates). In the positive samples, the mean number of legionellae was 1.17 x 10 3 CFU/L (range 25 to 8.7 x 10 4 CFU/L); three samples (9.2%) contained >10 4 CFU/L, none of which were L. pneumophila serogroup 1. Although we examined colonies with different morphologic traits, the agglutination test did not reveal multiple species or serotypes in a single water sample.

Univariate Examination of Risk Factors
The risk for microbial contamination according to the system characteristics was evaluated by applying a univariate logistic regression (Table 4). A central warm water system and distance of the water from the heating point >10 m were strongly associated with the risk for Legionella contamination (OR 9.24 and 8.10, respectively, p < 0.001). Other significant and positive associations were observed with tank volume, plant age, flooring in the home, and total number of flats in the building.
Pseudomonas contamination was positively associated with heating system age and negatively with tank distance and water operating temperature. Pseudomonas was also associated with the particular water source, with 65.3% of groundwater colonized versus 12.3% of mixture water (OR 13.44; 95% CI 5.02 to 36.03, p < 0.001).
The univariate regression was then applied to study the association between microbiologic data and water chemical parameters (Table 5). Seven factors were independently protective against Legionella colonization: high levels of copper, hardness, oxidizability, and free chlorine and low concentrations of zinc, iron, and manganese. Lower levels of zinc and manganese were also associated with lower total count at 36°C and 22°C. Pseudomonas was positively associated with total hardness and iron <20 µg/L, whereas residual free chlorine significantly inhibited Pseudomonas. When water samples were grouped according to their trace element levels, water samples (n = 12) characterized by concentrations of zinc <100 µg/L, iron <20 µg/L, and copper >50 µg/L were all negative for Legionella. No other system or water parameters were associated with bacterial contamination of the examined samples. The data were reanalyzed by means of multivariate conditional logistic regression models. A central heating system, distance from heating point >10 m, and a system >10 years old were each independently associated with higher risk of Legionella colonization, whereas water with levels of copper >50 µg/L and zinc <100 µg/L were predictive of no contamination (Table 6). For Pseudomonas, only groundwater remained highly predictive of colonization (OR 12.69; 95% CI 2.66 to 44.00, p < 0.001), whereas an operating temperature >50°C was predictive of noncontaminated samples (OR 0.25; 95% CI 0.11 to 0.98, p < 0.05).

Risk Factors and Legionella Species
The percentage distribution of Legionella species differed significantly according to the heater system (chisquare = 14.00, p < 0.05). Electric heaters were legionellae-free; the gas-heated independent systems had little contamination (10.0% of those with tank and 16.4% of those without) and were mainly colonized by either L. pneumophila serogroup 1 or non-pneumophila Legionella species. In the centralized heating systems of both single buildings and neighborhoods, Legionella colonization was higher (52.8% and 66.7%, respectively), and L. pneu-mophila serogroups 2-14 were the most frequently isolated serotypes. Germ concentration did not differ according to the heater type. Table 7 shows that water temperature, level of free chlorine, and Pseudomonas contamination differed according to Legionella species (Table 7). Water samples contaminated by L. pneumophila serogroup 1 were characterized by significantly lower operating temperature compared to that of the other groups, whereas water samples positive for L. pneumophila serogroups 2-14 had lower residual chlorine and higher Pseudomonas count.

Risk Assessment
The reported frequency of pneumonia symptoms was double among persons living in the legionellae-positive homes compared to those living in legionellae-free buildings (8 cases in 95 residents vs. 15 cases of 333 residents), but the difference was not significant (OR 1.95; 95% CI 0.80 to 4.75). Results did not change by correcting for the duration of residence of each person in the examined house.

Discussion
In our study, Legionella spp. were isolated in 22.6% of domestic hot water samples, with a mean number of   In previous studies in Finland and Germany, the occurrence of legionellae was similar (30% and 26%, respectively) as well as the contaminating concentration (20,21). In an Italian study of hot water samples taken from swimming pool showers, 27% were positive for Legionella spp. and 46% for P. aeruginosa (18), findings in line with results of our study on domestic water plants. According to a survey in Germany (22), L. pneumophila is by far the most abundant species in potable and environmental water samples, as >75% of positive samples were contaminated by L. pneumophila.
We could not verify seasonal variability in the contamination, because all samples were taken in the spring. Recent studies, however, found that contamination was consistent throughout the year, both in terms of the species of legionellae isolated and in the concentration of organisms (18), suggesting that the occurrence of Legionnaires' disease most frequently in the summer is not necessarily linked to a higher water contamination.
By comparing the environmental factors associated with Legionella and Pseudomonas occurrence, substantial differences in the microbes' sensitivity to these factors were observed. Pseudomonas was not influenced by system characteristics but strongly affected by water parameters. Thus, free chlorine and operating temperature appeared to inhibit these microbes, whereas groundwater origin, which influences higher degree of hardness, was found to favor Pseudomonas occurrence. The negative effect of chlorine and the positive influence of hardness, particularly higher calcium level, have been already observed in other studies on Pseudomonas water contamination (17,23).
Conversely, system and building characteristics were the main predictors for Legionella in domestic hot water. Thus, residing at higher floors of large buildings with many apartments and with older, centralized water heating systems increased the risk for Legionella contamination compared to living in apartments with independent water heater systems and a short distance from the sampling point to the hot water distribution site. Among independent heaters, electric ones appeared to be most protective against contamination, whereas the opposite was observed in previous studies in Quebec City, where temperature of electric heaters was significantly lower than that of fossil-fuel   heaters (24). In a representative sample of Wellington, New Zealand, domestic residences with electrically heated hot water systems, no Legionella spp. were isolated by culture, but PCR tests were positive for Legionella in 12 homes, some with hot water temperatures >60°C, suggesting that the bacteria are killed during passage through the hot water tank (11). In our study, Legionella presence was not affected by the origin of water (groundwater vs. mixture), pipe materials, water temperature, or concentration of chlorine, and the negative association of Legionella with hardness and oxidizability disappeared in the multilogistic regression analysis. When the potable system was adopted, Legionella was found in both chlorinated and untreated water, confirming the low efficacy of this disinfecting system on microbe eradication (25). In addition, bacteria from a chlorinated water system may be more resistant to combined and free chlorine than bacteria from unchlorinated systems (26).
The examined domestic water samples were not colonized by multiple serotypes or strains, a common finding in hospitals, hotels, and spas (27)(28)(29). This result could depend on different distribution systems and frequency of water use between private and public buildings.
Because the contaminating organism (L. pneumophila serogroup 1, L. pneumophila serogroups 2-14, or nonpneumophila Legionella spp.) was specific to a system, we could examine differences in distribution of species according to the system and water characteristics. These differences have been insufficiently evaluated in previous studies, but recent studies demonstrated that intracellular replication, cytopathogenicity, and infectivity to mammalian and protozoan cells also vary with Legionella species (30,31).
Our hypothesis is that Legionella strains substantially differ in their sensitivity to environmental risk factors and, as a consequence, may have different ecologic niches. L. pneumophila serogroup 1, responsible for approximately 80%-90% of Legionnaires' disease cases (32), was predominantly isolated from independent water heating systems, despite the fact that they were less frequently contaminated. Furthermore, compared with the other legionellae, serogroup 1 was found in water with a lower temperature, less Pseudomonas contamination, and a relatively higher residual chlorine concentration. Taking results together, L. pneumophila serogroup 1 appears to survive and grow in systems with a short distance between the hot water distribution site and the distal outlets. In agreement with our findings, a recent study on contaminated dental units recovered L. pneumophila serogroup 1 in nearly all sites positive for Legionella species (33). In these conditions, the possibility of contaminated aerosol inhalation might be more frequent for L. pneumophila serogroup 1, despite the fact that this serogroup is not the most frequently isolated in hot water systems. If our hypothesis is correct, most probably simple hygienic procedures, like good cleaning practice and periodically replacing shower heads, would be effective in reducing the number of infections. From our experience with epidemic clusters of nosocomial legionellosis in a hospital mainly contaminated by L. pneumophila serogroups 2-14 with rare isolates of L. pneumophila serogroup 1, we observed that introducing adequate cleaning procedures in the bathroom and surveillance by health personnel was sufficient to avoid further cases, even when the central hot water distribution systems were not decontaminated (34).
Our findings show the possible effect of trace elements on Legionella in hot water samples. Experimental studies have shown that Legionella spp. are affected by osmolarity (35) and metal concentration (36) and that iron limitation in vitro reduces bacteria growth and expression of the zinc-metalloprotease that is an important pathogenicity   factor (37). We show that hot water samples low in iron, zinc, and manganese, but rich in copper, predicted the absence of Legionella colonization, confirming their roles as growth promoters or inhibitors.
Of particular interest is the inverse relationship between copper levels and Legionella presence. In the examined water, the risk of Legionella contamination was approximately six times lower when copper levels exceeded 50 µg/L, without influencing Pseudomonas contamination. In other studies, copper concentrations low enough to be commonly found in drinking water reduced numbers of coliform bacteria (13). Thus, we emphasize that this trace element influences some, but not all, bacterial growth (33). To control Legionella in hot water systems, methods that release copper and silver ions electrolytically in water may represent a promising solution (38)(39)(40)(41). Although both metals play a role in limiting bacterial colonization, copper seems to better penetrate biofilm. Amoebae, the natural hosts of legionellae, have not been controlled successfully in vitro by adding metal (42), suggesting that legionellae survive inside protozoa and are destroyed by metal ions when released into free water.
The risk of getting pneumonia was 1.95 higher among residents in the legionellae-positive homes than in residents of the legionellae-negative buildings, but the difference was not significant and was similar to that found in previous studies (19). Legionellae concentrations of 3-7,000 CFU/L could be sufficient to produce one case per year in a susceptible population (43), and these contamination levels correspond to those found in our study at the domestic level. In a recent epidemiologic survey on seropositivity in residents of homes with and without Legionella in the water systems, the prevalence of anti-Legionella antibodies was twice as high in persons in homes with legionellae as in those persons whose homes did not have legionellae (44). The antibodies were most likely the result of asymptomatic infections caused by exposure in their home water supply, as no cases of pneumonia in the exposed population were reported. Most cases of sporadic legionellosis are not reported to health authorities in Italy as well as in other countries, and finding an association with a specific source of infection such as domestic contamination is rare (45).
Our observations suggest that Legionella species should be considered when examining environmental contamination, which is essential to better evaluate environmental risk factors and select the most appropriate prevention and control measures (46). To limit Legionella colonization at the domestic level, we suggest simple and general measures: 1) use independent domestic water heaters, 2) maintain high cleaning standards, 3) periodically replace components of the system which could favor presence or dissemination of bacteria, and 4) have a water copper content >50 µg/L. We do not believe disinfecting measures at the domestic level are needed, considering that our retrospective study on pneumonia in residents did not show a relevant evidence of risk in colonized buildings.