Trehalose catalytic shift inherently enhances phenotypic heterogeneity and multidrug resistance in Mycobacterium tuberculosis.

Drug-resistance (DR) in bacteria often develops through the repetitive formation of drug-tolerant persisters, which survive antibiotics without genetic changes. It is unclear whether Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), undergoes a similar transitioning process. Recent studies highlight changes in trehalose metabolism as crucial for persister formation and drug resistance. Here, we observe that mutants lacking trehalose catalytic shift activity exhibited fewer DR mutants due to decreased persisters. This shift enhances Mtb survival during antibiotic treatment by increasing metabolic heterogeneity and drug tolerance, facilitating drug resistance. Rifampicin (RIF)-resistant bacilli display cross-resistance to other antibiotics linked to higher trehalose catalytic shift, explaining how multidrug resistance (MDR) can follow RIF-resistance. In particular, the HN878 W-Beijing strain exhibits higher trehalose catalytic shift, increasing MDR risk. Both genetic and pharmacological inactivation of this shift reduces persister formation and MDR development, suggesting trehalose catalytic shift as a potential therapeutic target to combat TB resistance.