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

Abstract

Tuberculous sputum contains multipleMycobacterium tuberculosispopulations with different requirements for isolationin vitro These include cells that form colonies on solid media (plateableM. tuberculosis), cells requiring standard liquid medium for growth (nonplateableM. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependentM. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of theseM. tuberculosispopulations 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 log10reductions), and SN-dependentM. tuberculosiswas more tolerant to streptomycin and isoniazid than the plateable and nonplateableM. tuberculosisstrains. Susceptibility of plateableM. tuberculosisto bactericidal drugs was significantly increased after passagein 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 simpleex vivosystem for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection.

FIG 1

Comparison of counts for mycobacterial populations obtained from sputum before and after freezing in glycerol. Samples were decontaminated and frozen in 10% glycerol. M. tuberculosis counts were assessed before (H37Rv, S5, S7, S8, S9, and S10) and after (H37RvF, S5F, S7F, S8F, S9F, and S10F) freezing and defrosting. Error bars indicate 95% limits of confidence for MPN counts and standard deviation values for CFU counts. Note that because the MPN counts in all samples apart from S5 were the same or less than the CFU counts, no nonplateable M. tuberculosis cells were detected in the baseline samples, while a significant excess of SN-dependent bacilli was present in each sample.

FIG 2

Characterization of mycobacterial populations in sputum from four samples selected for antimicrobial treatment experiments. Samples were decontaminated and stored as described in Materials and Methods. Error bars indicate 95% limits of confidence for MPN counts.

FIG 3

Investigation of drug effects on the viability of mycobacterial populations isolated from sputum. (A) Pyrazinamide; (B) ethambutol; (C) streptomycin; (D) isoniazid. The percent survival was calculated using the following formula: [(viable count_{after exposure})/(viable count_{before exposure})] ×100%. Mean values for 4 sputum samples (S6, S19, S20, and S21) are shown; error bars indicate standard deviations. *, MPN_SN survival values were statistically significantly different from CFU and MPN survival values (P < 0.05, one-way ANOVA); **, MPN_SN survival values were statistically significantly different from CFU and MPN survival values (P < 0.01, one-way ANOVA).

FIG 4

Rifampin has a variable effect on M. tuberculosis obtained from sputum. Mycobacteria were treated with 1 μg/ml (A) or 5 μg/ml (B) rifampin for 3 or 7 days. CFU, plateable M. tuberculosis; MPN, M. tuberculosis grown in liquid; MPN_SN, M. tuberculosis grown in the presence of culture supernatant.

FIG 5

Bactericidal effects of drugs on plateable M. tuberculosis H37Rv and in vitro-grown sputum isolates. Log-phase bacteria were inoculated in 7H9 supplemented medium and treated with drugs for 3 (A) and 7 (B) days. S, streptomycin, 20 μg/ml; E, ethambutol, 20 μg/ml; I, isoniazid, 10 μg/ml; R, rifampin, 5 μg/ml. The limit of detection was 0.00015%.

FIG 6

Plateable M. tuberculosis in sputum is more tolerant to drug treatment than corresponding isolates processed in vitro. The M. tuberculosis isolate from sputum sample S20 was grown in 7H9 medium to logarithmic phase. These mycobacteria were decontaminated and frozen in glycerol (orange bars); the same bacteria were spiked into nontuberculosis sputum, decontaminated, and frozen in glycerol (turquoise bars). After defrosting, mycobacteria were treated with drugs for 3 (A) and 7 (B) days before assessment of CFU counts. These counts were compared to those obtained in the direct sputum tolerance assay for sputum sample S20 (lilac bars). S, streptomycin, 20 μg/ml; E, ethambutol, 20 μg/ml; I, isoniazid, 10 μg/ml; R, rifampin, 5 μg/ml. **, survival values for M. tuberculosis treated directly in sputum were statistically significantly different from those of the treated sputum isolate or spiked sputum isolate (P < 0.01, one-way ANOVA).

FIG 7

Suggested transitions between M. tuberculosis populations in vivo and during the sputum drug tolerance assay. Note that there may be substantial overlap between the populations such that all the colony-forming cells are propagated in the MPN and MPN_SN assays.