Phenotypically Adapted Mycobacterium tuberculosis Populations from Sputum Are Tolerant to First-Line Drugs

Abstract

Tuberculous sputum contains multiple Mycobacterium tuberculosis populations with different requirements for isolation in vitro. These include cells that form colonies on solid media (plateable M. tuberculosis), cells requiring standard liquid medium for growth (nonplateable M. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependent M. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of these M. tuberculosis populations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10 reductions), and SN-dependent M. tuberculosis was more tolerant to streptomycin and isoniazid than the plateable and nonplateable M. tuberculosis strains. Susceptibility of plateable M. tuberculosis to bactericidal drugs was significantly increased after passage in vitro; thus, tolerance observed in the sputum samples from the population groups 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 that have phenotypically adapted during tuberculosis infection.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

“…DD Mtb in patient sputum tested ex vivo were found to be phenotypically tolerant to INH and STREP (58). The PBS-starved Mtb studied here (not to be confused with the DD population generated from them) displayed class IIa phenotypic tolerance to RIF, INH, MOXI, SM, PA-824, CFZ, NTZ, TRZ, and the four-drug combination of RIF, INH, PZA and ETH used to treat drugsensitive TB.…”

Resuscitation-promoting factors are important determinants of the pathophysiology inMycobacterium tuberculosisinfection

“…Currently, there is no human data to confirm these findings. Of late, studies on sputum have demonstrated populations of Rpfdependent Mtb bacilli that are tolerant to the action of rifampicin, isoniazid, and streptomycin and are low to clear from sputum (Mukamolova et al, 2010;Turapov et al, 2016). It may be that clearance is required for cure although further studies are needed to verify this.…”

Early diagnosis and effective treatment regimens are the keys to tackle antimicrobial resistance in tuberculosis (TB): A report from Euroscicon's international TB Summit 2016

“…53 As it is more difficult to get rid of Rpf-dependent mycobacteria they are found to be enriched in treated TB patients. As persisters are often a cause of treatment failure and post-treatment relapses it is essential that evaluation of TB treatment regimens accounts for elimination/induction of this group of cells.…”

“…Treatment with rifampicin, isoniazid and ethambutol was shown to induce Rpf-dependency, whereas no such effect was observed in the case of treatment with streptomycin and amikacin. 53 Thereby it is critical that drugs be tested for their propensity to induce Rpf-dependency. Toward this end, Mukamolova and coworkers strived to design a simple assay to determine the potential of drugs to induce Rpf-dependency.…”