Legionella Infection Risk From Domestic Hot Water

Paola Borella; M. Teresa Montagna; Vincenzo Romano-Spica; Serena Stampi; Giovanna Stancanelli; Maria Triassi; Rachele Neglia; Isabella Marchesi; Guglielmina Fantuzzi; Daniela Tatò; Christian Napoli; Gianluigi Quaranta; Patrizia Laurenti; Erica Leoni; Giovanna De Luca; Cristina Ossi; Matteo Moro; Gabriella Ribera D'Alcalà


Emerging Infectious Diseases. 2004;10(3) 

In This Article


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.

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% CO2 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-mm-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.

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% HNO3) concentrated by boiling.

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.

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 log10 (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).