Figure 6. Bacterial efflux pumps confer tolerance within macrophages.

(A and B) THP-1 macrophages were infected with Mm and lysed at two or 96 hpi. The released bacteria were treated for an additional 48 hours with 174 µM INH, 1.21 µM RIF or left untreated, in the presence or absence of 81.4 µM verapamil (A) or 65.7 µM reserpine (B), prior to enumeration of CFU.

(C) THP-1 macrophages were infected with Mtb strain CDC1551 and lysed at two, 96 or 144 hpi. The released bacteria were then treated with antibiotics for 48 hours as described in panel (A), except that the concentration of INH was 4.4 µM, as described for Figure 4E.

(D and E) THP-1 macrophages were infected with Mtb strains JHU1258c-715 (“M1”), JHU1258c-833 (“M2”) and the isogenic wild-type control, CDC 1551, for two or 96 hours prior to lysis and enumeration of CFU. (D) Released bacteria were treated as described in panel (C).

(F) THP1 cells were infected with Mm for 48 hours, prior to addition of 0 (UNT), 40.7 or 81.4 µM VER for an additional 48 hours. P<0.001 using one-way ANOVA, with Dunnett’s post-test comparing each treatment group to the untreated control after 48 hours of VER treatment.

(G) THP1 cells infected with Mm for two hours or 96 hours were incubated for an additional 48 hours with 174 µM INH, 1.21 µM RIF or both, and in the presence or absence of 40.7µM VER. Cells were lysed and CFU were enumerated at the end of the 48 hour treatment. Percent survival was calculated relative to the mean intracellular counts present at the start of antibiotic exposure. For all panels, error bars represent SEM. Significance testing was performed using one-way ANOVA with Dunnett’s (A,B, D and E) or Bonferroni (C and D) post-tests.