Fig 4

(A) Zebrafish were injected with 250–300 CFU of M. abscessus strains by tail vein injection and monitored daily for a 12-day infection period to determine relative embryo survival (B) At 6 dpi, zebrafish embryos were imaged under a fluorescent microscope and fluorescent pixel counts (FPC) calculated for each group using ImageJ. Each dot represents an individual embryo. (C) At 2, 4 and 6 dpi, infected zebrafish embryos were imaged using fluorescence microscopy to calculate the number of embryos with granulomas. Granulomas were identified on the basis of colocalisation of several fluorescent macrophages at an infection foci. (D) On days 2, 4, and 6 dpi, infected zebrafish embryos were imaged using fluorescence microscopy to calculate the proportion of embryos with abscesses. Abscesses were identified based on the relative size of infection foci far exceeding the surrounding recruited immune cells, representing uncontrolled bacterial growth. (E) Representative images of zebrafish infected with M. abscessus strains at day 6 post-infection. Red represents bacterial infection foci. Scale bar = 1mm. (F) Survival curves of C3HeB/FeJ mice after tail vein infection with 10⁸ CFU/mouse of WT (black line) and ΔesxUT (red line). (G) CFU counts of WT (black dots) and ΔesxUT (red dots) in lungs, liver, kidneys, and spleen at 1, 7, 14, and 20 dpi. Five mice were used per group and experiment was performed twice. P values were determined by ANOVA with Tukey’s test using GraphPad prism program (B, C and D) and unpaired t test (G); ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The log-rank (Mantel-Cox) test for Kaplan-Meier survival curves was used to assess survival statistics significance.