The molecular epidemiology of clinical and environmental Legionella species isolates was studied in seven hospitals from 1989 to 2006. The number of environmental pulsed field gel electrophoresis (PFGE) patterns ranged from one to nine according to the hospital. Genomic PFGE pattern persistence was observed in 71% of the hospitals, even after 17 years in some hospitals, and the relationship between environmental and clinical isolates was established. The isolates associated with hospital-acquired Legionnaires' disease corresponded to the persistent environmental PFGE patterns of Legionella pneumophila in potable water supplies.

The concentrations of Legionella pneumophila in cooling towers may vary considerably over short periods of time, producing significant fluctuations throughout the year. Despite genetic variability, in small geographical areas the same indistinguishable pulsed-field gel electrophoresis patterns may be shared among different cooling towers and persist over time.The involvement of cooling towers in some community outbreaks of legionellosis has been demonstrated (3,6,8,9,11), and it has been suggested that there may be an association between high Legionella counts in cooling towers and the occurrence of an outbreak of legionellosis (12). Although not all environmental isolates are associated with human disease, the present guidelines aimed at preventing legionellosis have proposed ranges of risk involving different environmental insults based on Legionella counts (4). However, the fluctuations in Legionella counts over short periods of time have led some authors to question these risk ranges (1).Since the recovery of a high inoculum of Legionella in a cooling tower is not necessarily related to an outbreak or sporadic cases of legionellosis, genetic studies are necessary to establish the link between environmental and clinical isolates. The "gold standard" technique for genotyping Legionella is the analysis of chromosomal restriction patterns by pulsed-field gel electrophoresis (PFGE). Isolation of Legionella strains from a cooling tower which are genetically indistinguishable from clinical isolates is considered to be clear evidence of the implication of the tower in an outbreak (1, 9). However, little is known regarding the genetic variability of Legionella within the same tower and in cooling towers from areas in the vicinity. Likewise, to our knowledge, no study has determined the persistence of molecular patterns in isolates in cooling towers. All these circumstances have epidemiological and legal consequences when assigning responsibility for an outbreak of legionellosis to a particular installation.The main objectives of this study were to establish the fluctuations in Legionella counts over time and to determine the genetic variability and persistence of PFGE patterns in cooling towers.Fifteen cooling towers (A to O) were selected within a radius of 3 km. Four of them (J, K, M, and N) shared the same main line for water distribution. The other cooling towers had a different domestic distribution system but were supplied by the same municipal water network. All the cooling towers followed the controls and hygiene measures required by Spanish regulations (RD 865/2003), and we were able to verify that the levels of disinfectant (sodium hypochlorite [0.2 to 0.5 ppm residual] in conjunction with the discontinuous dosage of nonoxidant biocide [dibromonitrilopropionamide]) were in accordance with legislation at the time of sampling. Cooling water samples were cultured for Legionella fortnightly over a 1-year period. The samples were concentrated by filtration, and four plates of GVPC agar (Oxoid, Wesel, Germany) we...

Genotypic variability and clonal persistence are important concepts in molecular epidemiology as they facilitate the search for the source of sporadic cases or outbreaks of legionellosis. We studied the genotypic variability and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns over time (period > 6 months) in 34 positive cooling towers from two different areas. In area A, radius of 70 km, 52 indistinguishable PFGE patterns were differentiated among the 27 cooling towers. In 13 cooling towers we observed >or= 2 PFGE patterns. Each cooling tower had its own indistinguishable Legionella PFGE pattern which was not shared with any other cooling tower. In area B, radius of 1 km, 10 indistinguishable PFGE patterns were obtained from the seven cooling towers. In four, we observed >or= 2 PFGE patterns. Three of these 10 indistinguishable PFGE patterns were shared by more than one cooling tower. In 27 of 34 cooling towers the same PFGE pattern was recovered after 6 months to up to 5 years of follow-up. The large genotypic diversity of Legionella observed in the cooling towers aids in the investigation of community outbreaks of Legionnaires' disease. However, shared patterns in small areas may confound the epidemiological investigation. The persistence of some PFGE patterns in cooling towers makes the recovery of the Legionella isolate causing the outbreak possible over time.

The cytopathogenicity of 22 Legionella pneumophila isolates from 17 hospitals was determined by assessing the dose of bacteria necessary to produce 50% cytopathic effect (CPED50) in U937 human-derived macrophages. All isolates were able to infect and grow in macrophage-like cells (range log10 CPED50: 2.67-6.73 c.f.u./ml). Five groups were established and related to the serogroup, the number of PFGE patterns coexisting in the same hospital water distribution system, and the possible reporting of hospital-acquired Legionnaires' disease cases. L. pneumophila serogroup 1 isolates had the highest cytopathogenicity (P=0.003). Moreover, a trend to more cytopathogenic groups (groups 1-3) in hospitals with more than one PFGE pattern of L. pneumophila in the water distribution system (60% vs. 17%) and in hospitals reporting cases of hospital-acquired Legionnaires' disease (36.3% vs. 16.6%) was observed. We conclude that the cytopathogenicty of environmental L. pneumophila should be taken into account in evaluating the risk of a contaminated water reservoir in a hospital and hospital acquisition of Legionnaires' disease.