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, were determined at 0, 1 and 3 dpi. Data are mean values ± SD for three independent experiments

, Data were analysed using a one-way ANOVA Kruskal-Wallis test, p.1

, macrophages at 0, 1 and 3 days post-infection after infection with M. abscessus S or (D) M. abscessus 766

R. , Data are mean values ± SD for three independent experiments. Data were analysed using a one-767 way ANOVA Kruskal-Wallis test. (E) Percentage of S-infected macrophage categories and (F) 768 percentage of R-infected macrophage categories infected with different numbers of bacilli

, The categories were counted at 0 or at 1 and 3 days post-infection in the 770 absence of antibiotics or in the presence of RIF or AMK at 50 µg/mL, or RFB at 12.5 µg/mL. Values are 771 means ± SD from three independent experiments performed in triplicate. (G) Four immuno-772 fluorescent fields were taken at 1 day post-infection showing macrophages infected with M. 773 abscessus expressing Tdtomato (red). The surface and the endolysosomal system of the macrophages 774 were detected using anti-CD63 antibodies (green). The nuclei were stained with DAPI (blue), 769 5-10 bacilli and >10 bacilli)

, E-G) clinical strains belonging to S or R morphotypes at MOI of 2:1 for 3 hrs 780 prior to treatment with 250 µg/mL AMK for 2 hrs to kill extracellular bacteria. Following extensive 781 PBS washes, cells were exposed to 50 µg/mL RIF, 50 µg/mL AMK, 25 or 12.5 µg/mL RFB . CFU were determined at 0, 1 and 3 days post-infection. Data are mean values ± SD for two independent 783 experiments, Figure 3. Intracellular activity of RFB on S and R clinical isolates. CFU counts of clinical isolates 778 exposed to 25 and 12.5 µg/mL RFB. Macrophages were infected with M. abscessus (A-B) M. bolletii 779 (C-D) or M. massiliense

, A) Total number of cords 786 displayed in 20 fields at 3 days post-infection after infection of macrophages with M. abscessus R 787 variant. Data are mean values ± SD for three independent experiments performed in triplicate. Data 788 were analysed using one tailed Mann Whitney's t-test. (B) Percentage of cords formed either 789 extracellularly or intracellularly. The two categories were counted at 3 days post-infection in the 790 absence of antibiotics or in the presence of 50 µg/mL RIF

, Extracellular or intracellular cords are highlighted using the indicated colour codes. Values are means 792

±. Sd-for, C) Four immuno-793 fluorescent fields were taken at 3 days post-infection showing the cords formed extracellularly or 794 within macrophages infected with M. abscessus R variant expressing Tdtomato (red). Macrophages 795 were infected for 3 days in the presence of DMSO, RIF (50 µg/mL)

, White arrows indicate intracellular cords, while red arrows indicate 798 extracellular cords