Specificity and killing activity of mycobacterial phages against GD01. (A) Plaque assay. Tenfold serial dilutions of phages Gabriela and Muddy were spotted on a lawn of GD01 grown on 7H10^OADC/CaCl2. Lysis was observed after 5 days. PFU, plaque-forming units. (B) Killing assay. GD01 was grown in 7H9^OADC, incubated with the various phages for 5 days at 37°C and plated on LB agar prior to colony-forming unit (CFU) counting. D0, GD01 inoculum; CTRL, GD01 without phages. ****P<0.0001 (unpaired Student's t-test). Data shown are the mean of three independent experiments±s.d.

In vitro killing activity of Muddy and antibiotics against GD01. (A-H) GD01 was grown in CaMHB at 37°C and incubated with Muddy (MOI=0.0001) in the absence or presence of different antibiotics used in clinical settings. Three drug concentrations were used (in µg/ml) at sub-MIC doses. After 5 days of treatment, bacteria were plated on LB agar prior to CFU counting. D0, GD01 inoculum; CTRL, GD01 without phages. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (unpaired Student's t-test). Data shown are the mean of three independent experiments±s.d.

Impact of bacteriophage Muddy on GD01 infection in wild-type zebrafish. At 30 hpf, embryos were infected with 250-300 CFU of GD01 expressing tdTomato via caudal vein injection. At 1 dpi, embryos commenced phage therapy through caudal vein administration at an MOI of 50:1 (phage:bacteria) based on the initial bacterial infection inoculum. Embryos were treated daily from 1 dpi up to and including 5 dpi with either Muddy (M), Gabriela (G) or a phage cocktail containing both Muddy and Gabriela (M+G). (A) A generalised schematic showing the experimental design corresponding to the figure. (B) Embryo survival was monitored over a 12 day period, with embryos counted daily. Survival curves were analysed using the log-rank (Mantel–Cox) statistical test. The red bar across the x-axis indicates the duration of daily phage administration in the current experiment. (C) Bacterial burden [fluorescent pixel count (FPC)] was analysed at 6 dpi using fluorescent microscopy. Fluorescent images were analysed in ImageJ using the ‘Analyze particles’ function. Bacterial burden was analysed using a Kruskal–Wallis one-way ANOVA. (D,E) The proportion of embryos with cords (D) and abscesses (E) was enumerated at 2, 4 and 6 dpi using fluorescent microscopy. Abscess and cord quantification was analysed using a Kruskal–Wallis one-way ANOVA. Data shown are the mean of three independent experiments±s.d. (n=20-30 per group for each experiment). (F) Representative zebrafish images of untreated (GD01) or phage-treated embryos at 6 dpi, showing the presence of extracellular bacterial cords. Red overlay represents GD01 expressing TdTomato. Scale bars: 0.5 mm. n.s., not significant; *P<0.05; **P<0.01; ****P<0.0001.

Activity of Muddy in GD01-infected zebrafish lacking macrophages. At 24 hpf, embryos were micro-injected with either PBS liposomes (PBS) or lipoclodronate-filled liposomes (Clod) to achieve macrophage depletion. At 30 hpf, treated embryos were infected with 250-300 CFU of GD01 (PBS GD01 and Clod GD01) via caudal vein injection. At 1 dpi, embryos commenced phage therapy through caudal vein injection at an MOI of 50:1 (phage:bacteria) based on initial bacterial infection inoculum. Embryos were treated daily from 1 dpi, up to and including 5 dpi with Muddy (M), Gabriela (G) or a phage cocktail containing both Muddy and Gabriela (M+G). (A) A generalised schematic showing the experimental design corresponding to the figure. (B) Embryo survival was monitored over a 12 day period, with embryos counted daily. Survival curves were analysed using the log-rank (Mantel–Cox) statistical test. The red bar across the x-axis indicates the duration of daily phage administration in the current experiment. (C) Bacterial burden (FPC) was analysed at 6 dpi using fluorescent microscopy. Fluorescent images were analysed in ImageJ using the ‘Analyze particles’ function. Bacterial burden was analysed using a Kruskal–Wallis one-way ANOVA. (D,E) The proportion of embryos with cords (D) and abscesses (E) was enumerated at 2, 4 and 6 dpi using fluorescent microscopy. Abscess and cord quantification was analysed using a Kruskal–Wallis one-way ANOVA. Data shown are the mean of two independent experiments±s.d. (n=20-30 per group for each experiment). (F) Representative zebrafish images of untreated (GD01) or phage-treated embryos at 6 dpi, showing the presence of extracellular bacterial cords. Red overlay represents GD01 expressing TdTomato. Scale bars: 0.5 mm. *P<0.05; ****P<0.0001.

Effect of Muddy in GD01-infected cftr morphants. At the one- to four-cell stage, fertilised zebrafish eggs were injected in the nucleus with either control morpholino (control) or cftr morpholino (cftr). At 30 hpf, embryos were infected with 250-300 CFU of GD01 (control GD01 or cftr GD01) via caudal vein injection. At 1 dpi, embryos commenced phage therapy through caudal vein injection at an MOI of 50:1 (phage:bacteria) based on initial bacterial infection inoculum. Embryos were treated daily from 1 dpi, up to and including 5 dpi with Muddy (M), Gabriela (G) or a phage cocktail containing both Muddy and Gabriela (M+G). (A) A generalised schematic showing the experimental design corresponding to the figure. (B) Embryo survival was monitored over a 12 day period, with embryos counted daily. Survival curves were analysed using the log-rank (Mantel–Cox) statistical test. The red bar across the x-axis indicates the duration of daily phage administration in the current experiment. (C) Bacterial burden (FPC) was analysed at 6 dpi using fluorescent microscopy. Fluorescent images were analysed in ImageJ using the ‘Analyze particles’ function. Bacterial burden was analysed using a Kruskal–Wallis one-way ANOVA. (D,E) The proportion of embryos with cords (D) and abscesses (E) was enumerated at 2, 4 and 6 dpi using fluorescent microscopy. Abscess and cord quantification was analysed using a Kruskal–Wallis one-way ANOVA. Data shown are the mean of three independent experiments±s.d. (n=20-30 per group for each experiment). (F) Representative cftr morphant images of untreated (GD01) or phage-treated embryos at 6 dpi, showing the presence of extracellular bacterial cords or localised infection. Red overlay represents GD01 expressing TdTomato. Scale bars: 0.5 mm. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Activity of Muddy and rifabutin in GD01-infected cftr morphants. At the one- to four-cell stage, fertilised zebrafish eggs were injected in the nucleus with either control morpholino (control) or cftr morpholino via caudal vein injection. At 1 dpi, embryos commenced phage Muddy (M) and/or rifabutin (RFB) therapy through caudal vein injection at an MOI of 50:1 (phage:bacteria) based on initial bacterial infection inoculum. Embryos were treated daily from 1 dpi, up to and including 5 dpi with Muddy. RFB was added to zebrafish water daily at a final concentration of 50 µg/ml. (A) A generalised schematic showing the experimental design corresponding to the figure. (B) Embryo survival was monitored over a 12 day period, with embryos counted daily. Survival curves were analysed using the log-rank (Mantel–Cox) statistical test. The red bar across the x-axis indicates the duration of daily phage and RFB administration in the current experiment. (C) Bacterial burden (FPC) was analysed at 6 dpi using fluorescent microscopy. Fluorescent images were analysed in ImageJ using the ‘Analyze particles’ function. Bacterial burden was analysed using a Kruskal–Wallis one-way ANOVA. (D,E) The proportion of embryos with cords (D) and abscesses (E) was enumerated at 2, 4 and 6 dpi using fluorescent microscopy. Abscess and cord quantification was analysed using a Kruskal–Wallis one-way ANOVA. Data shown are the mean of three independent experiments±s.d. (n=20-30 per group for each experiment). (F) Representative cftr morphant images of embryos left untreated (GD01), treated with Muddy alone, RFB alone or with Muddy plus RFB at 6 dpi, showing the presence of extracellular bacterial cords or localised infection. Red overlay represents GD01 expressing TdTomato. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.