Phenotypically adapted Mycobacterium tuberculosis populations from sputum are tolerant to first line drugs

Tuberculous sputum contains multiple Mycobacterium tuberculosis (Mtb) populations with different requirements for isolation in vitro. These include cells forming colonies on solid media (platable Mtb), cells requiring standard liquid medium for growth (non-platable Mtb), and cells requiring supplementation of liquid medium with culture supernatant for growth (SN-dependent Mtb). Here we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of these Mtb populations within sputum. Our results show that first line drugs achieved only modest cidal effects on all three populations over 7 days (1-2.5xlog10 reductions) and SN-dependent Mtb were more tolerant to streptomycin and isoniazid compared to platable and non-platable Mtb. Susceptibility of platable Mtb to bactericidal drugs was significantly increased after passage in vitro, thus tolerance observed in the sputum populations was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simple ex vivo system for testing drug efficacies against mycobacteria phenotypically adapted during tuberculosis infection