Antibiotic susceptibility of intracellular Mtb.

<p>(A) Schematic outline of the experiments, with antibiotics being added either 1 h (B) after infection or after 3 days (C) when Mtb in MOI 10-infected cells already had replicated once. Intracellular bacteria were quantified 4 days later on day 4 or 7, respectively. Antibiotics were used at the following concentrations derived from human peak serum levels: 1 µg/ml ethambutol (EMB), 10 µg/ml isoniazid (INH) and 20 µg/ml pyrazinamide (PZA). ALU as a measure of bacterial numbers were normalized against untreated controls of the same donor. Significant changes were determined using 2-way ANOVA followed by Bonferroni’s post-hoc test comparing treated samples to untreated control. Differences between MOIs were not significant. (D) contains the data from (B) and (C). Groups were compared using 1-way ANOVA and Tukey’s post-hoc test. No significant differences were found. (E) Lower concentrations of INH and PZA were added than in B–D (0.01, 0.1 and 1 µg/ml INH and 0.2 and 2 µg/ml PZA), and intracellular bacteria were measured on day 4. Significant differences between treated samples and untreated control were determined using 2-way ANOVA followed by Bonferroni’s post-hoc test (indicated with asterisks). No significant difference between MOIs was found. (F) Antibiotics were added 1 h after infection as in (B) and (E), but infection was extended beyond day 4 with another measurement on day 6. n = 5–10 in (B–D), n = 3 in (E) and n = 3 in (F). Bars and error bars represent means and SEMs, respectively. *p<0.05, **p<0.01 and ***p<0.001.