Independent bacterial colonies of serial dilutions were numbered after 5 days at 30°C. In the co-infection experiment, the same cells

amount of each strain was added to achieve a final MOI of 5. Extracellular bacteria (10-5 and 10-6 dilutions) were plated on BCYE agar, selleck compound 48 h post-infection. Independent bacterial colonies were picked-up after 3 days at 30°C to perform a PCR analysis. Cytotoxicity to Acanthamoeba castellani To quantify the viable A. castellanii cells remaining after infection with Legionellae (MOI 5), a monolayer of amoebae cells at the final concentration of 1 × 106 cells per ml in a 96 multiwell plate was washed (fourfold) with PY and then treated with 10% Alamar blue (Invitrogen). Cytotoxicity of each Legionella strain was tested in triplicate. After an overnight incubation at 30°C, measurements were performed at the optical density (OD) of 570 nm and corrected for background at OD600 nm with a μQuant microplate reader (Biotek Instruments Inc., Winooski, USA) The relative degree of amoeba mortality corresponds to the cytotoxicity and was expressed as the ratio of the OD value of infected monolayer to that of the uninfected one as following:

[1-(mean OD value of infected/mean OD value of uninfected)] × 100%. Acknowlegdments This study is supported by grants from the Centre National de la Recherche Scientifique and the Université Lyon 1. Z. Chaabna was the recipient of a fellowship from the Axelera Chemical Environmental competitiveness Cluster (LEGIOSECURE program). The authors CBL-0137 are grateful to Claire Andréa for skilful technical assistance. Electronic supplementary material Additional file 1: PFGE analysis of environmental and clinical Legionella P5091 pneumophila strains. Legionella DNA samples were digested with SfiI restriction enzyme

for 16 h at 50°C. Fragments of DNA were separated in a 0.8% agarose gel prepared and run in 0.5x Tris-borate-EDTA buffer (pH 8.3) in a contour-clamped homogeneous field apparatus 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 pulse times (35 to 60 s) at 10°C for 9 h. (PDF 1 MB) Additional file 2: Multiple alignment of mip sequences from environmental ( mip1 , mip2 and mip3 ) and clinical L. pneumophila sg1 strains. Clinical Amino acid strains: Lp1Corby (NC009494.2), Lp1 Lens (NC006369.1), Lp1 Paris (NC006368) and Lp1 Philadelphia (AE017354.1). (PDF 98 KB) References 1. McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR: Legionnaires’ Disease. N Engl J Med 1977,297(22):1197–1203.PubMedCrossRef 2. Nguyen TMN, Ilef D, Jarraud S, Rouil L, Campese C, Che D, Haeghebaert S, Ganiayre FO, Marcel F, Etienne J, et al.: A community-wide outbreak of legionnaires disease linked to industrial cooling towers – How far can contaminated aerosols spread? J Infect Dis 2006,193(1):102–111.PubMedCrossRef 3.