FIG 5.
RFB displays high bactericidal activity against M. abscessus in an embryonic zebrafish infection model. (A) Groups of uninfected embryos were immersed in water containing increasing concentrations of RFB (ranging from 3.125 to 250 μg/ml) for 4 days. The red bar indicates the duration of treatment. The graph shows the survival of the RFB-treated and untreated (DMSO) embryos over a 12-day period. (B) Zebrafish embryos at 30 h post-fertilization were intravenously infected with approximately 250 to 300 CFU of M. abscessus CIP104536T (R variant) expressing TdTomato (n = 20 to 25). A standard PBS injection control was included for each experiment. At 1 dpi, embryos were randomly split into equal groups of approximately 20 embryos per group, and various concentrations of RFB (5 to 50 μg/ml) were added to the water. DMSO was included as a positive control group. RFB was changed daily, after which, embryos were washed twice in fresh embryo water, maintained in embryo water, and monitored daily over a 12-day period. Each treatment group was compared against the untreated infected group with significant differences calculated using the log-rank (Mantel-Cox) statistical test for survival curves. Data shown are the merge of three independent experiments. (C) Bacterial burden was determined at 2, 4, and 6 days postinfection following treatment with either DMSO or 50 μg/ml RFB. Bacteria were quantified by fluorescent pixel count determination using ImageJ software, with each data point representing a single embryo. Error bars represent standard deviations. Statistical significance was determined using Student’s t test. The plots represent a pool of 2 independent experiments containing approximately 20 to 25 embryos per group. (D) Representative embryos from the untreated group (WT) (top) and from the treated group with 50 μg/ml RFB at 6 days postinfection. White arrowheads show TdTomato-expressing bacteria. Scale bars represent 1 mm. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001.