Reduced Permeability to Rifampicin by Capsular Thickening as a Mechanism of Antibiotic Persistence in Mycobacterium tuberculosis

ABSTRACT Persisters constitute a subpopulation of bacteria that can tolerate lethal concentrations of antibiotics. Multiple mechanisms have been suggested for bacterial persistence against antibiotics. With mycobacteria being no exception to this behaviour, we had reported the de novo emergence of genetically antibiotic-resistant Mycobacterium tuberculosis from persister cells upon prolonged exposure to microbicidal concentrations of the anti-tuberculosis drugs, rifampicin and moxifloxacin. Here, we present evidence for reduced permeability to rifampicin as a mechanism for persistence of Mycobacterium tuberculosis in vitro . We observed that rifampicin persistent M. tuberculosis cells developed a thick outer layer (TOL) capsule. The TOL restricted the entry of fluorochrome-conjugated rifampicin, 5-carboxyfluorescein-rifampicin (5-FAM-rifampicin), which retained only 2.5% of its original bactericidal activity, but high levels of permeability, on actively growing mid-log phase cells. Gentle mechanical removal of TOL significantly enhanced 5-FAM-rifampicin entry into the persister cells. The level of 5-FAM-rifampicin in the persister cells was not affected by the pre-incubation of the cells with verapamil, a drug efflux pump inhibitor, ruling out the involvement of efflux pumps in the reduced intracellular concentration of 5-FAM-rifampicin. GC-MS analysis of TOL showed the presence of ∼7-fold, ∼5-fold and ∼2- fold higher levels of α-D-glucopyranoside, 1,2,5-linked-mannitol, and 3,4-linked mannose, respectively, among ∼2-fold higher levels of derivatives of several other types of sugars such as arabinose and galactose. Taken together, the present study reveals that rifampicin-persistent M. tuberculosis cells develop TOL that enables the bacilli to restrict entry of rifampicin and thereby remain tolerant to the antibiotic in vitro .