Differential Impact of the rpoB Mutant on Rifampin and Rifabutin Resistance Signatures of Mycobacterium tuberculosis Is Revealed Using a Whole-Genome Sequencing Assay

Abstract

Drug resistance in Mycobacterium tuberculosis (MTB) has long been a serious health issue worldwide. Most drug-resistant MTB isolates were identified due to treatment failure or in clinical examinations 3~6 months postinfection. In this study, we propose a whole-genome sequencing (WGS) pipeline via the Nanopore MinION platform to facilitate the efficacy of phenotypic identification of clinical isolates. We used the Nanopore MinION platform to perform WGS of clinical MTB isolates, including susceptible (n = 30) and rifampin- (RIF) or rifabutin (RFB)-resistant isolates (n = 20) according to results of a susceptibility test. Nonsynonymous variants within the rpoB gene associated with RIF resistance were identified using the WGS analytical pipeline. In total, 131 variants within the rpoB gene in RIF-resistant isolates were identified. The presence of the emergent Asp531Gly or His445Gln was first identified to be associated with the rifampin and rifabutin resistance signatures of clinical isolates. The results of the minimum inhibitory concentration (MIC) test further indicated that the Ser450Leu or the mutant within the rifampin resistance-determining region (RRDR)-associated rifabutin-resistant signature was diminished in the presence of novel mutants, including Phe669Val, Leu206Ile, or Met148Leu, identified in this study. IMPORTANCE Current approaches to diagnose drug-resistant MTB are time-consuming, consequently leading to inefficient intervention or further disease transmission. In this study, we curated lists of coding variants associated with differential rifampin and rifabutin resistant signatures using a single molecule real-time (SMRT) sequencing platform with a shorter hands-on time. Accordingly, the emerging WGS pipeline constitutes a potential platform for efficacious and accurate diagnosis of drug-resistant MTB isolates.

Keywords: MinION; Mycobacterium tuberculosis; rifabutin; rifampin; rpoB.

FIG 1

Diagram presenting coverage rates of sequenced reads aligned to the entire Mycobacterium tuberculosis (MTB) genome in each group.

FIG 2

Predictive utility of whole-genome sequencing (WGS) assay results on the drug-resistant signatures of enrolled isolates estimated with statistical analyses. The utility of the presence of Ser450Leu or rifampin-resistance-determining region (RRDR)-variants for predicting (A) high RIF-resistant or (B) high RFB-resistant signatures of M. tuberculosis is evaluated using a receiver operating characteristic (ROC) analysis. MIC, minimum inhibitory concentration; AUC, area under the ROC curve.