RESUMEN
The aim of this study was to evaluate the frequency of Legionella pneumophila in three hot spring water recreation areas by conventional culture on GVPC and by Real Time-PCR. No legionellae were isolated from the spring water upstream the distribution system suggesting strongly that the thermal area was the probable source of these pathogens. L. pneumophila was present at high level in some samples, reaching values of 8.2 × 10(3) CFU/l at the site C. We observed a low diversity among the 18 isolated Legionella strains according to 16S DNA analysis, since only L. pneumophila and Legionella londiniensis were identified. All of the L. pneumophila strains belong to five serogroups (1, 4, 5, 6 and 8). Pulsed-field gel electrophoresis analysis identified various genotypes among these serogroups and showed that these strains had pulsotypes distinct from L. pneumophila present in the CNRL Legionella-database. These environmental strains showed higher tolerance to heat shock stress at 48 °C than L. pneumophila clinical strain, which may explain their presence in warm waters. Our data suggested that potential pathogen legionellae are present in Tunisian medicalized thermal spas and emphasized the importance of adopting control measures to prevent infection.