A new Persister Enrichment Model in Mycobacterium tuberculosis links multidrug persistence to drug sequencing and maltokinase-dependent carbon distribution.

Prolonged combination chemotherapy is required to cure Tuberculosis, in part because Mycobacterium tuberculosis (Mtb) forms antibiotic-tolerant persister cells that survive exposure to bactericidal drugs. Experimental systems to study multidrug persistence in Mtb remain limited. Here, we describe a rapid and tractable Persister Enrichment Model (PEM) in which removal of glycerol markedly increases the frequency of multidrug-tolerant Mtb cells that survive exposure to high concentrations of bactericidal antibiotics with distinct mechanisms of action. Persister enrichment in PEM depends on carbon source identity rather than carbon abundance and enables direct interrogation at the transition from active growth into multidrug tolerance. Using PEM, we show that the temporal order of isoniazid (INH) and rifampicin (RIF) exposure shapes the magnitude of multidrug persistence. A transposon sequencing screen under combination drug pressure identified maltokinase (mak) as a major determinant of multidrug persistence. Deletion of mak enhanced susceptibility to INH+RIF in PEM and impaired bacterial survival during the persistent phase of infection in mice. Metabolic analyses reveal that loss of mak destabilizes carbon distribution during co-catabolism of glycolytic carbon sources, leading to defective growth and antibiotic persistence. This defect is ameliorated by inactivating mutations in glucose-1-phosphate adenylyltransferase (glgC), which alleviate the buildup of ADP-glucose. Together, these findings establish PEM as a practical platform to study multidrug persistence, reveal that drug sequencing can influence entry into multidrug tolerance, and identify maltokinase-dependent carbon distribution as a metabolic vulnerability that constrains Mtb survival. ImportanceTuberculosis treatment requires prolonged multidrug chemotherapy in part because Mycobacterium tuberculosis forms antibiotic-tolerant persister cells that survive bactericidal drug exposure. However, experimental systems to study multidrug persistence remain limited, and the metabolic mechanisms underlying this phenotype are poorly understood. Here, we establish a rapid and experimentally tractable Persister Enrichment Model (PEM) that enriches multidrug-tolerant M. tuberculosis populations by removing glycerol from culture medium. Using this model, we demonstrate that the order of antibiotic exposure influences multidrug persistence both in vitro and in vivo and identify maltokinase-dependent carbon distribution as a critical determinant of persistence and long-term bacterial survival. These findings provide a new platform for studying multidrug tolerance and reveal carbon buffering pathways as potential therapeutic vulnerabilities that could help shorten tuberculosis treatment.