Expression of Mtb blaC in Mmar. (a) Amplification of blaC and housekeeping genes sigA and rrs during one-step RT-qPCR using RNA isolated from Mmar expressing wild-type Mtb blaC as template. (b) Relative expression of blaC variants as determined by RT-qPCR. Data represent three biological replicates, each with two technical replicates, and the blaC transcripts were normalized to sigA. The error bars represent one standard deviation. One-way ANOVA with Dunnett’s multiple comparison test indicates a significant difference between blaC WT and blaC D172N (p < 0.05).

Activity of Mtb BlaC mutants produced in Mmar. Cultures of Mmar chromosomally expressing wild-type BlaC or variants S70A (negative control), I105F, G132N, G132S, D172N, D179N, and K234R were incubated for 8 days at 30 °C on plates containing 20 or 80 μg mL⁻¹ ampicillin, or 8 μg mL⁻¹ sulbactam or 12 μg mL⁻¹ avibactam in the presence of 15 μg mL⁻¹ ampicillin. The complete plates are shown in Figs. S2–S4.

Effect of combination treatment on zebrafish embryos infected with mWasabi-labelled Mmar producing Mtb BlaC at 4 dpi. (a) Representative larvae showing systemic infection from the control group and treated with ampicillin, avibactam, or both. Arrows indicate collections of bacteria indicative of granuloma formation, and percentages indicate the percentage of the mean of the control. The control represents a picture of a larva just below the average value of 100% intensity (72%). (b) Bacterial load of Mmar producing wild-type Mtb BlaC as represented by fluorescence intensity after being given the indicated treatment 1 dpi. Each dot represents a larva. Larvae were injected with 1 nL of 29 mg mL⁻¹ ampicillin in PBS (estimated concentration 100 μg mL⁻¹ in the embryo, n = 39), 65 mg mL⁻¹ avibactam in PBS (225 μg mL⁻¹, n = 43), ampicillin and avibactam in PBS (same concentrations, n = 67), or PBS only (ctrl, n = 69). Data for the groups treated with both ampicillin and avibactam or the control were accumulated in three, and the groups treated with only ampicillin or avibactam in two independent experiments. Data were normalized by setting the mean of the control to 100%. Error bars represent the mean and standard error. Mood’s median test with Holm-Bonferroni post hoc test for multiple comparisons was used to compare groups with the control: ns = not significant; **** = p < 0.0001.

Effect of combination treatment on zebrafish embryos infected with mWasabi-labelled Mmar producing Mtb BlaC variants at 4 dpi. (a) Representative larvae showing systemic infection from the control group and treated with ampicillin, avibactam, or both. Arrows indicate collections of bacteria indicative of granuloma formation, and percentages indicate the percentage of the mean of the control. (b) Bacterial load of Mmar producing either wild-type (black dots) or K234R Mtb BlaC (blue dots) as represented by fluorescence intensity after being given the indicated treatment 1 dpi. Each dot represents a larva. Larvae were injected with 1 nL of 29 mg mL⁻¹ ampicillin and 22 mg mL⁻¹ avibactam in PBS (estimated concentration 100 μg mL⁻¹ ampicillin and 75 μg mL⁻¹ avibactam in the embryo, n = 69 for WT and n = 62 for K234R) or PBS only (ctrl, n = 66 for WT and n = 63 for K234R). Data were accumulated in three independent experiments and normalized by setting the mean of the control to 100%. Error bars represent the mean and standard error. Mood’s median test was used to compare treated groups: **** = p < 0.0001.