Assessment of tuberculosis drug efficacy using preclinical animal models and in vitro predictive techniques

Abstract

Tuberculosis (TB) killed approximately 1.3 million people in 2022 and remains a leading cause of death from the bacteria Mycobacterium tuberculosis (M.tb); this number of deaths was surpassed only by COVID-19, caused by the SARS-CoV-2 virus. The alarming emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) M.tb strains presents an urgent need for effective new treatments. Our study aimed to determine the synergistic effects of antibiotic combinations against M.tb. Using a high-throughput in vitro checkerboard assay, we evaluated the interactions of Bedaquiline (BDQ) and other antibiotics including Capreomycin (CAP), Linezolid (LIN), and Sutezolid (SUT) against M.tb H37Rv. BDQ and CAP demonstrated in vitro enhanced effect, which prompted further investigation in vivo using the murine low dose aerosol (LDA) model. After aerosol challenge with M.tb, C57BL/6 mice were treated with BDQ, CAP, or their combination, starting 28 days post-infection. The antimicrobial treatment lasted four weeks, and the bacterial burden in lung and spleen tissues was assessed at the end of treatment. At 4 weeks post-treatment, a significant reduction in bacterial load was observed within the lungs and spleens of mice given BDQ alone or given as a BDQ/CAP combination compared to the untreated group. In contrast, CAP monotherapy led to an increase in bacterial load within the lung and no significant difference in bacterial burden in the spleen in comparison to the untreated mice. These results were confirmed in the guinea pig model of TB, where both BDQ and the BDQ/CAP combination treatment led to a decrease in bacterial burden in the lung and spleen, whereas CAP had no significant effect on bacterial burden at the 4-week post treatment timepoint. We next determined whether there may be differences in vitro with the BDQ/CAP combination against M.tb lineages 1, 2 and 4. We determined that in vitro enhanced effect was not observed in some representative strains of M.tb lineage 4, indicating variability in drug effectiveness across M.tb lineages. This research underscores the complexity of TB treatment and the critical need for innovative approaches to combat this global health threat.

Fig. 1. Bedaquiline (BDQ) and Capreomycin (CAP) synergize against M.tb H37Rv.

The M.tb H37Rv cultures were treated with escalating concentrations of BDQ and CAP in 96-well plates for 7 days. Cultures were stamped onto 7H10 agar plate supplemented with OADC agar for determination of bacterial killing.

Fig. 2. Effect of BDQ and CAP combination on bacterial killing of different M.tb lineages using an escalating checkerboard assay.

The cultures from M.tb strains representing Lineages 1 (M.tb T46, M.tb N0072), 2 (M.tb HN878), and 4 (M.tb Erdman and M.tb N1216) were treated with escalating concentrations of BDQ and CAP in 96-well plates for 7 days. Cultures were stamped onto 7H10 agar plates supplemented with OADC agar for determination of bacterial killing.

Fig. 3. Bacterial burden of BDQ/CAP single and combination 1 month treatment in the lungs and spleens of C57BL/6 mice infected with M.tb HN878.

C57BL/6 mice were infected with a LDA of M.tb HN878. Timeline of the in vivo experimental design (A). Bacterial burden was assessed by colony forming unit (CFU) in the lung (B), and the spleen (E) 4 weeks post-infection (prior to treatment). Bacterial burden in the lung (C), and the spleen (F) 6 weeks post- infection (2 weeks post-treatment); and 8 weeks post-infection (4 weeks post-treatment) in the lung (D), and spleen (G). CFU means were compared between each group using one-way ANOVA with Dunnett's multiple comparisons test. Black line and error bars show mean ± SEM, dots represent individual mice, n = 6–7/group. Asterisks indicate statistical significance, where ***p < 0.001 and ****p < 0.0001.

Fig. 4. Bacterial burden after 1 month of BDQ/CAP single and combination treatment in the lungs and spleens of Guinea pigs infected with M.tb HN878.

Guinea pigs were infected with M.tb HN878 by aerosol route. Timeline of antimicrobial therapy and M.tb HN878 aerosol challenge (A). Bacterial burden was assessed by colony forming unit (CFU) in lung (B) and spleen (D) before start of antimicrobial therapy, 3 weeks post challenge lung (C), Spleen (E), organ homogenates, 7 weeks post challenge. CFU means were compared between each group using one-way ANOVA with Dunnett's multiple comparisons test. Black line and error bars show mean ± SEM, dots represent individual guinea pigs, n = 6–7/group. Asterisks indicate statistical significance, where ***p < 0.001 and ****p < 0.0001.

Fig. 5. Lung lesion burden in the lungs of M.tb HN878 infected guinea pigs following one month of BDQ/CAP drug treatment.

Bars show mean ± SEM percent lesion area, dots represent individual animal, n = 12/group. Asterisks indicate statistical significance, where *p < 0.05, **p < 0.01 using one-way ANOVA with Dunnett's multiple comparisons test.