Genetic determinants of rifabutin susceptibility in multidrug-resistant tuberculosis: insights from whole-genome sequencing

Multidrug-resistant tuberculosis (MDR-TB) is a critical global health threat. Rifabutin (RFB) is active against some rifampicin (RIF)-resistant Mycobacterium tuberculosis strains, but genetic markers guiding RFB use remain unclear. This study aimed to identify determinants of RFB susceptibility using whole-genome sequencing (WGS) and Minimum inhibitory concentrations (MICs) testing. MICs for RFB were determined by broth microdilution according to CLSI M24–A2 guidelines (susceptibility defined as MIC < 0.5 µg/mL) for 183 MDR-TB isolates from Shenzhen, China. Associations of rpoB mutations, lineage, and compensatory mutations with log 2 transformed RFB MIC were assessed using Kruskal-Wallis tests, Fisher’s exact tests, and multivariable linear regression models. rpoB mutation type was the primary determinant of RFB susceptibility. D435V was consistently associated with preserved RFB susceptibility, with 66.7% (4/6; 95% CI: 22.3%–95.7%) of isolates susceptible, whereas S450L and H445Y conferred high‑level RFB resistance (26.4% and 25.0% susceptible, respectively). Multiple rpoB mutations yielded intermediate susceptibility (48.8% susceptible). In the multivariable model adjusting for rpoB genotype, the Non-L2 lineage had a modest independent effect (adjusted β = −0.90, 95% CI: −1.77 to −0.04, p = 0.041), suggesting L2 has higher RIF-RFB log2 ratios. Compensatory mutations were not independently linked to log₂ RFB MIC (adjusted β = −0.34, 95% CI: −1.02 to 0.33, p = 0.323), indicating that the rpoB mutation type primarily determines RFB susceptibility.RFB resistance level was not significantly associated with molecular transmission clustering (OR = 1.95, 95% CI: 0.84–4.80, p = 0.129). rpoB genotype, particularly D435V, is a robust biomarker for RFB susceptibility in MDR-TB. WGS can enable precision prescribing of RFB for MDR-TB and strengthen global antimicrobial resistance surveillance.