Mycobacterium tuberculosis Subculture Results in Loss of Potentially Clinically Relevant Heteroresistance

Abstract

Multidrug-resistant tuberculosis (TB) presents a major public health dilemma. Heteroresistance, the coexistence of drug-resistant and drug-susceptible strains or of multiple drug-resistant strains with discrete haplotypes, may affect accurate diagnosis and the institution of effective treatment. Subculture, or passage of cells onto fresh growth medium, is utilized to preserve Mycobacterium tuberculosis cell lines and is universally employed in TB diagnostics. The impact of such passages, typically performed in the absence of drug, on drug-resistant subpopulations is hypothesized to vary according to the competitive costs of genotypic resistance-associated variants. We applied ultradeep next-generation sequencing to 61 phenotypically rifampin-monoresistant (n = 17) and preextensively (n = 41) and extensively (n = 3) drug-resistant isolates with presumptive heteroresistance at two time points in serial subculture. We found significant dynamic loss of minor-variant resistant subpopulations across all analyzed resistance-determining regions, including eight isolates (13%) whose antibiogram data would have transitioned from resistant to susceptible for at least one drug through subculture. Surprisingly, some resistance-associated variants appeared to be selected for in subculture.

Keywords: drug-resistant tuberculosis; gyrA; heteroresistance; rpoB; rrs.

Copyright © 2017 American Society for Microbiology.

Figures

FIG 1

Derivation of study samples. M. tuberculosis laboratory procedures and the rationale employed in derivation of analyzed specimens were as follows: primary drug susceptibility testing (DST) culture (Culture 2) and quaternary subculture (Culture 5). MGIT, Mycobacteria Growth Indicator Tube.

FIG 2

Dynamic changes in heteroresistant M. tuberculosis profiles with serial subculture. The color-coded map illustrates the overall loss of M. tuberculosis resistance-associated variant (RAV) subpopulations (empty circles or “−” label), while some RAVs (e.g., gyrA 94GGC) appear to be selected for in subculture (“N” or “+” label). Red boxes indicate fixed-resistance mutations (≥95% of the total M. tuberculosis population) in both samples (cultures 2 and 5); salmon-colored boxes indicate macroheteroresistance (5% to 95% of the total M. tuberculosis population) in both samples; light blue boxes indicate microheteroresistance (<5% of the total M. tuberculosis population) in both samples; dark blue boxes indicate microheteroresistance (<1% of the total M. tuberculosis population) in both samples. Also illustrated is RAV disappearance (empty circles, with the background color illustrating the subpopulation size in the original culture), appearance (“New”), and categorical decline or growth (− or +, respectively). Please note that data corresponding to growth or decline within categories (e.g., R_3206 gyrA 90GTG increasing from 25% to 44%, with both samples being “macroheteroresistant”) are not shown. Sample R_5491 was demonstrated to be wild type at all RDRs analyzed. ★, all early reads for sample R_2362 identified the 9-bp deletion at rpoB 516–525 in the original culture, though in subculture this deletion was strongly offset by a minority strain harboring the rpoB 526GAC mutation (please see Discussion for details). †, isolates transitioning from resistant to susceptible for at least one drug. XDR, extensively drug resistant (MDR, with additional resistance to both FQ and SLI).