High prevalence, genetic diversity and intracellular growth ability of Legionella in hot spring environments.

BACKGROUND: Legionella is the causative agent of Legionnaires' disease, and hot springs are a major source of outbreaks of this disease. It is important from a public health perspective to survey hot spring environments for the presence of Legionella. METHODS: Prospective surveillance of the extent of Legionella pollution was conducted at three hot spring recreational areas in Beijing, China in 2011. Pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) were used to describe the genetic polymorphism of isolates. The intracellular growth ability of the isolates was determined by interacting with J774 cells and plating the dilutions onto BCYE agar plates. RESULTS: Overall, 51.9% of spring water samples showed Legionella-positive, and their concentrations ranged from 1 CFU/liter to 2,218 CFU/liter. The positive rates of Legionella were significantly associated with a free chlorine concentration of ≥0.2 mg/L, urea concentration of ≥0.05 mg/L, total microbial counts of ≥400 CFU/ml and total coliform of ≥3 MPN/L (p<0.01). The Legionella concentrations were significantly associated with sample temperature, pH, total microbial counts and total coliform (p<0.01). Legionella pneumophila was the most frequently isolated species (98.9%), and the isolated serogroups included serogroups 3 (25.3%), 6 (23.4%), 5 (19.2%), 1 (18.5%), 2 (10.2%), 8 (0.4%), 10 (0.8%), 9 (1.9%) and 12 (0.4%). Two hundred and twenty-eight isolates were analyzed by PFGE and 62 different patterns were obtained. Fifty-seven L. pneumophila isolates were selected for SBT analysis and divided into 35 different sequence types with 5 main clonal groups. All the 57 isolates had high intracellular growth ability. CONCLUSIONS: Our results demonstrated high prevalence and genetic polymorphism of Legionella in springs in Beijing, China, and the SBT and intracellular growth assay results suggested that the Legionella isolates of hot spring environments were pathogenic. Improved control and prevention strategies are urgently needed

Relationship between the monthly average number of <i>Legionella</i> and monthly average number of passenger flow.

<p>The curve represents the monthly average number of <i>Legionella</i> (CFU/L), and the histogram represents the monthly average number of passenger flow.</p

Clustering results of patterns obtained by PFGE analysis of 228 <i>Legionella</i> strains.

<p>These strains were isolated from three hot spring recreational areas in Beijing, China during the one-year study period.</p

Intracellular growth of <i>Legionella</i> isolates within J774 cells.

<p>The isolated <i>Legionella</i> strains were grown in BYE broth until they reached early stationary phase. Approximately 2×10⁹ bacteria diluted (1∶1,000) in RPMI 1640 tissue culture medium were added to J774 cells (2×10⁵ per well) with a multiplicity of infection (MOI) of approximately 10. The infected cells were incubated at 37 °C under 5% CO₂-air for 1.5 h and washed three times with PBS to remove extracellular bacteria. The CFUs were determined by plating dilutions on BCYE agar plates. The <i>L. pneumophila</i> philadelphila-1 strain and its <i>dotA</i> mutant were used as positive and negative controls, respectively.</p

Distribution of <i>Legionella</i> species and serogroups from springs in Beijing, China.

<p>Distribution of <i>Legionella</i> species and serogroups from springs in Beijing, China.</p

Distribution of clonal groups from 35 SBT profiles for <i>L. pneumophila</i> isolates (n = 57) in hot springs in Beijing, China.

<p>Distribution of clonal groups from 35 SBT profiles for <i>L. pneumophila</i> isolates (n = 57) in hot springs in Beijing, China.</p

Clustering results of patterns obtained by PFGE analysis of 55 <i>Legionella</i> strains.

<p>These strains were isolated from one sample of hot spring recreational area A, which was collected in April.</p

Minimum spanning tree analysis of 57 <i>Legionella</i> isolates from hot springs in Beijing, China.

<p>In the minimum spanning tree, the STs are displayed as circles. The size of each circle indicates the number of isolates within this particular type, and the STs and the number of isolates in each ST are shown in the circles. The colors of the halo surrounding the STs denote types that belong to the same clonal group.</p

The number of spa water samples, spa pools and months contaminated by different <i>Legionella</i> species and serogroups.

<p>The number of spa water samples, spa pools and months contaminated by different <i>Legionella</i> species and serogroups.</p

Association of physical, chemical, and microbiological water characteristics with <i>Legionella</i> spp. concentration of spa water samples.

<p>Association of physical, chemical, and microbiological water characteristics with <i>Legionella</i> spp. concentration of spa water samples.</p