Reactions mixtures were then held at 10°C. 8 μL of the PCR amplification mixture was analyzed by gel electrophoresis in a 0.8% agarose gel stained with ethidium bromide (1.0 μg/mL) and photographed under U.V.

transillumination. Purification and sequencing of PCR mip products PCR mip products were analyzed by gel electrophoresis in a 0.8% agarose gel (50 mL) stained with 3 μL SYBR Safe DNA gel strain (Invitrogen). DNA products were visualized under blue U.V. transillumination and picked up with a band of agarose gel. Then PCR products were purified using GeneCleanR Turbo Kit (MP Biomedicals) according to the manufacturer’s instructions. Finally, the purified PCR products were suspended in 10 μL sterile water and then stored at −20°C. Sequencing was performed by GATC Biotech SARL Deforolimus (Mulhouse, France). PFGE subtyping Legionella isolates

were subtyped by pulsed field gel electrophoresis (PFGE) method as described previously [26]. Briefly, legionellae were treated with proteinase K (50 mg/mL) in TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 8) for 24 h at 55°C, and DNA was digested with 20 IU of SfiI restriction enzyme (Boehringer Mannheim, Meylan, France) for 16 h at 50°C. Fragments of DNA were separated in a 0.8% agarose gel prepared and run in 0.5× Tris-borate-EDTA buffer (pH 8.3) in a contour-clamped homogeneous field apparatus (CHEF DRII system; Bio-Rad, Ivry sur Seine, France) with a constant voltage of 150 V. Runs were carried out with increasing pulse times (2 to 25 s) at 10°C for 11 h and increasing FK228 pulse times (35 to 60 s) at 10°C for 9 h. Then, the gels were stained for 30 min with a ethidium bomide solution and PFGE patterns were analyzed with GelComparII software (Applied Maths, Saint-Martens-Latem, Belgium). Quantification of Legionella virulence towards the amoeba Acanthamoeba castellanii Legionellae

were grown on BCYE agar and A. castellanii cells in PYG Adenosine medium (Moffat and Tompkins, 1992) for five days at 30°C prior to infection. A. castellanii cells were first seeded in plates of 24 multiwell to a final concentration of 5 × 106 cells per ml in PY medium (PYG without glucose. Plates were incubated during two hours at 30°C to allow amoeba adhesion. Then, Legionellae were added to an MOI (“multiplicity of infection”) of 5 (in duplicate). In order to induce the adhesion of bacterial cells to the monolayer of amoeba cells, plates were spun at 2000 × g for 10 min and incubated for 1 h at 30°C. Non-adherent bacteria were removed by four successive washings of PY medium. This point was considered as the initial point of infection (T0) and the plates were incubated at 30°C. Extracellular cultivable bacteria released from amoebae were quantified at 1 day and 2 days post-infection as follows. Aliquots (100 μL) of the supernatants were taken and diluted in sterile water to the final 10-6 dilution.