Fig. 2. Phage DS6A eliminates Mtb in infected primary human macrophages.

A Experimental procedures of the assay. B, C: Half million macrophages were infected with H37Rv at an MOI of 1. After 4 h of Mtb infection, three different phages (5 × 10⁶, MOI of 10) were applied to the Mtb-infected primary human macrophages, and cultured at 37 °C. The cells were sampled at days 0, 5 and 10 to determine the bacillary load for the evaluation the Mtb-killing ability of the phages by plating the ultrasound-broken cells on 7H10 plates (supplemented with 10% OADC) and the CFUs were counted on Day 8. B shows the statistics of the CFUs of all phages, and (C) shows the representative pictures (duplicated) of the 7H10 plates taken at day 5 and day 10. D, E: Testing the Mtb-killing ability of phage DS6A in Mtb-infected macrophages derived from four different healthy donors, and phage D29 was used as a negative control. The infected macrophages were sampled at different time points, sonicated, and plated on 7H10 agar plates using different dilutions to titer the bacillary load. D shows the statistics of five donors, and (E) shows the representative pictures taken from 10x diluted plates for days 4 and 7 cultures. F: Confocal macroscopy images show the Mtb-killing efficacy of phage DS6A in H37Rv-GFP infected macrophages. Cellmask plasma membrane stain (red) was used to localize the cell membrane, GFP (green) was used for tracking the Mtb bacilli, and DAPI (blue) was used to stain the nuclei of the macrophages. The red arrows show the Mtb in the macrophages. Unpaired student T-tests were used to analyze the differences between groups in Figure D. n = 5 independent donors. All statistical data are represented as mean ± SEM. Statistical significance was defined as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.