Variants in Bedaquiline-Candidate-Resistance Genes: Prevalence in Bedaquiline-Naive Patients, Effect on MIC, and Association with Mycobacterium tuberculosis Lineage

Abstract

Studies have shown that variants in bedaquiline-resistance genes can occur in isolates from bedaquiline-naive patients. We assessed the prevalence of variants in all bedaquiline-candidate-resistance genes in bedaquiline-naive patients, investigated the association between these variants and lineage, and the effect on phenotype. We used whole-genome sequencing to identify variants in bedaquiline-resistance genes in isolates from 509 bedaquiline treatment naive South African tuberculosis patients. A phylogenetic tree was constructed to investigate the association with the isolate lineage background. Bedaquiline MIC was determined using the UKMYC6 microtiter assay. Variants were identified in 502 of 509 isolates (98.6%), with the highest (85%) prevalence of variants in the Rv0676c (mmpL5) gene. We identified 36 unique variants, including 19 variants not reported previously. Only four isolates had a bedaquiline MIC equal to or above the epidemiological cut-off value of 0.25 μg/mL. Phylogenetic analysis showed that 14 of the 15 variants observed more than once occurred monophyletically in one Mycobacterium tuberculosis (sub)lineage. The bedaquiline MIC differed between isolates belonging to lineage 2 and 4 (Fisher's exact test, P = 0.0004). The prevalence of variants in bedaquiline-resistance genes in isolates from bedaquiline-naive patients is high, but very few (<2%) isolates were phenotypically resistant. We found an association between variants in bedaquiline resistance genes and Mycobacterium tuberculosis (sub)lineage, resulting in a lineage-dependent difference in bedaquiline phenotype. Future studies should investigate the impact of the presence of variants on bedaquiline-resistance acquisition and treatment outcome.

Keywords: antibiotic resistance; antimicrobial resistance; bedaquiline; drug-resistant tuberculosis; epidemiology; phenotypic drug susceptibility testing; phylogeny; tuberculosis; whole-genome sequencing.

FIG 1

Variants identified in bedaquiline candidate resistance genes Rv0676c, Rv0677c, Rv0678, Rv1979c, and Rv2535c, and flanking promoter regions. The single variant in Rv1304 (upstream of atpE) is not shown. Nucleotide positions are used for frameshift indels and intergenic variants, codon positions are used for synonymous, nonsense and missense variants. ^$-11 position is upstream relative to the Rv0678 gene.

FIG 2

Maximum likelihood phylogenetic tree of the 509 Mycobacterium tuberculosis samples collected from South African patients. Each concentric circle represents a unique variant (corresponding number in legend table) that was observed more than once (variants observed only once are listed in Table 1). Variants occurring exclusively in one clade are shown in gray. Homoplastic variants (occurring in independent samples) are shown in vibrant green (number 12). ^$Variants were observed independently in the phylogenetic tree; ^# Synonymous SNPs.

FIG 3

Observed bedaquiline (BDQ) MIC values for each combination of variants in the data set for which phenotypic data were available. MIC reading was done after 2 weeks of incubation.

FIG 4

Bedaquiline (BDQ) MIC stratified by lineage. (A) BDQ MIC using the CRyPTIC (UKMYC6) plates for lineage 1 isolates. (B) BDQ MIC using the CRyPTIC (UKMYC6) plates for lineage 2 isolates. (C) BDQ MIC using the CRyPTIC (UKMYC6) plates for lineage 4 isolates. (D) Kernel density estimation (KDE) of BDQ MIC of lineage 1, 2, and 4 isolates. KDE and corresponding confidence intervals were calculated using the prop.test function in the R stats package (version 4.0.0). MIC reading was done after 2 weeks of incubation.