Six in vitro clofazimine-resistant spontaneous mutants obtained from a wild-type or pyrazinamide-resistant ATCC reference strain were selected to evaluate bedaquiline cross-resistance. The reverse was conducted for bedaquiline mutants.
KEYWORDS: bedaquiline, clofazimine, cross-resistance, efflux pump mutants
ABSTRACT
Six in vitro clofazimine-resistant spontaneous mutants obtained from a wild-type or pyrazinamide-resistant ATCC reference strain were selected to evaluate bedaquiline cross-resistance. The reverse was conducted for bedaquiline mutants. All clofazimine mutants harboring an rv0678 mutation displayed phenotypic cross-resistance. We observed the same for rv0678 bedaquiline mutants; however, atpE bedaquiline mutants showed no phenotypic cross-resistance. This confirms that upfront clofazimine usage may impact subsequent bedaquiline use due to a shared efflux resistance pathway.
INTRODUCTION
Clofazimine was recently incorporated into the WHO shortened drug-resistant tuberculosis (DR-TB) regimens due to recent evidence of good efficacy (1, 2). Patients with rifampin-resistant or multidrug-resistant TB can be placed on the shortened DR-TB regimen containing clofazimine or on a regimen comprising both bedaquiline (WHO group A) and clofazimine (WHO group B), if possible (3). The rv0678 gene mutation was identified in mutants and clinical isolates with increased clofazimine MIC values (4, 5). Although bedaquiline’s primary drug target is ATP synthase (encoded by the atpE gene), an enzyme involved in the synthesis of ATP (6), mutations in the rv0678 gene have also been found to result in bedaquiline resistance. This rv0678 gene encodes the mmpR5 repressor protein, with rv0678 mutations resulting in mmpL5-mmpS5 efflux pump overexpression (7). Mutations in the rv1979c and rv2535c (pepQ) genes have also been found in clofazimine-resistant mutants (4). In clinical isolates with prior bedaquiline exposure, rv0678 mutations were causative factors for increased clofazimine MIC values (8).
Because cross-resistance between clofazimine and bedaquiline may reduce available treatment options, we aimed to determine whether upfront clofazimine use could result in increased bedaquiline MIC values due to mutations in a shared efflux pathway.
We isolated spontaneous mutant colonies using 4× the proposed critical concentration (CC) of clofazimine (1 µg/ml [9]) with a Luria-Delbrück fluctuation assay as described previously (10). Two Mycobacterium tuberculosis ATCC reference strains were used: ATCC 27294 (H37Rv), a wild-type/fully susceptible [WT] strain; and ATCC 35828, a pyrazinamide-resistant [PZAr] strain. The latter was used for its potentially higher propensity for mutation development, as shown in our previous experiments (10), and because preexistent PZA resistance in rifampin-resistant TB and DR-TB is common (11, 12). Putative mutant colonies were subcultured for a single drug-free passage in preparation for clofazimine MIC testing (range, 0.06 to 4 µg/ml) using the MGIT960 platform (Becton, Dickinson Diagnostic Systems [BD Biosciences], Sparks, MD) (13). Cross-resistance to bedaquiline was evaluated in triplicate with MGIT960 (range, 0.12 to 8 µg/ml). Using the same methodology as described above, we evaluated clofazimine cross-resistance using six bedaquiline-resistant spontaneous mutants derived from the same ATCC strains and a bedaquiline CC of 1 µg/ml (9).
DNA extraction was performed with the generic protocol on the NucliSENS easyMAG (bioMérieux), and whole-genome sequencing was carried out on the Illumina MiSeq platform (Illumina, San Diego, CA) with the Nextera XT DNA library kit. Single nucleotide polymorphisms or insertions/deletions (indels) were detected at a frequency of >30% (21) and a Phred score of ≥Q20 (≥99% accuracy), exploring the rv0678, atpE, rv1979c, and rv2535c gene targets only.
From a multitude of putative clofazimine-resistant spontaneous mutants, three from the PZAr strain and three from the WT strain, with MIC values ranging from 1 to 4 µg/ml, were randomly selected (Table 1). All of these mutants harbored rv0678 mutations and phenotypic bedaquiline cross-resistance (MIC, 4 to 8 µg/ml).
TABLE 1.
Clofazimine-resistant spontaneous mutants with associated bedaquiline phenotypic data
| Mutant no. | ATCC straina | MICb (µg/ml) in: |
Genec |
||||
|---|---|---|---|---|---|---|---|
| CFZ mutant | BDQ |
||||||
| 1 | 2 | 3 | atpE | rv0678 | |||
| 1 | WT | 2 | 8 | 8 | 8 | WT | 193delG (Ile67fs) |
| 2 | WT | 4 | 8 | 8 | 8 | WT | 193delG (Ile67fs) |
| 3 | WT | 4 | 8 | 8 | 8 | WT | A65T (Gln22Leu) |
| 4 | PZAr | 1 | 4 | 8 | 8 | WT | T407C (Leu136Pro) |
| 5 | PZAr | 2 | 8 | 8 | 8 | WT | C214T (Arg72Trp) |
| 6 | PZAr | 4 | 4 | 8 | 8 | WT | A97G (Thr33Ala) |
WT, wild type/fully susceptible; PZAr, pyrazinamide resistant.
Mutant and cross-resistance MGIT960 MIC values.
Novel mutations (not previously identified in literature) are in boldface.
Six randomly selected putative bedaquiline mutant colonies (five from the PZAr strain and one from the WT strain) were confirmed as phenotypically bedaquiline resistant (MIC range, 4 to >8 µg/ml) (Table 2). Three bedaquiline-resistant mutants (atpE mutations only) showed no clofazimine cross-resistance (MIC range, 0.25 to 0.5 µg/ml). Three mutants with both atpE and rv0678 mutations displayed clofazimine cross-resistance (MIC range, 2 to 4 µg/ml). From these, one mutant had no detectable rv0678 mutations at the 30% or 10% frequency threshold for variant calling (Table 2). Rv0678 Sanger sequencing (4) of DNA extracted from these mutants revealed a Ser63Gly mutation, previously implicated in bedaquiline resistance (14–17).
TABLE 2.
Bedaquiline-resistant spontaneous mutants with associated clofazimine phenotypic data
| Mutant no. | ATCC straina | MICb (µg/ml) in: |
Genec |
||||
|---|---|---|---|---|---|---|---|
| BDQ mutant | CFZ |
||||||
| 1 | 2 | 3 | atpE | rv0678 | |||
| 1 | WT | >8 | 0.25 | 0.25 | 0.5 | A83T (Asp28Val) | WT |
| 2 | PZAr | 4 | 4 | >4 | >4 | WT | C403G (Arg135Gly) |
| 3 | PZAr | 8 | 2 | 4 | 4 | A83G (Asp28Gly) | A187G(Ser63Gly)d |
| 4 | PZAr | >8 | 0.5 | 0.5 | 0.5 | G187C (Ala63Pro) | WT |
| 5 | PZAr | 4 | 0.25 | 0.5 | 0.5 | A83G (Asp28Gly) | WT |
| 6 | PZAr | 4 | 4 | 4 | 4 | WT | C189A(Ser63Arg) |
WT, wild type/fully susceptible; PZAr, pyrazinamide resistant.
Mutant and cross-resistance MGIT960 MIC values.
Novel mutations (not previously identified in literature) are in boldface.
Identified in triplicate clofazimine cross-resistance tubes using Sanger sequencing.
In this study, cross-resistance resulted from four novel (Gln22Leu, Leu136Pro, Arg72Trp, Arg135Gly) and three previously described (Ile67fs [4], Ser63Arg [7], and Thr33Ala [8]) rv0678 mutations, all scattered across the rv0678 gene (Fig. 1). The Thr33Ala mutation was previously reported in a bedaquiline-treated clinical isolate with resultant clofazimine cross-resistance (8). Here, we show that a WT strain exposed to clofazimine obtained this mutation and displayed bedaquiline cross-resistance, confirming the key role of rv0678 mutations in cross-resistance. The atpE mutations observed (Asp28Gly, Asp28Val, and Ala63Pro) were previously described and occur in hot spot regions. No rv1979c or rv2535c (pepQ) mutations were identified. The occurrence of rv0678 mutations due to clofazimine exposure appears to be the determining factor for resultant bedaquiline cross-resistance and vice versa. Therefore, irrespective of the drug used for resistance selection, the common efflux-based pathway used by these drugs is highlighted through this study (18).
FIG 1.
Mutations in rv0678 gene resulting in bedaquiline and clofazimine cross-resistance. Ellipses indicate regions of the gene not shown. Single nucleotide polymorphisms are indicated in red as a change from the wild-type sequence (rv0678_WT) to the mutated sequence (rv0678_MUT).
Utmost care was taken to select single colonies, but smaller colonies may have combined, resulting in heterogeneous populations with both atpE and rv0678 mutations. However, these populations further confirmed the role of rv0678 mutations and, notably, were previously described in a single clinical isolate (19). We obtained only a single bedaquiline-resistant mutant from the WT strain, but we assume that obtaining further bedaquiline-resistant mutants from the WT strain would not impact our findings.
The replacement of injectable drugs for bedaquiline supports outcome improvement (20), while combination therapy with bedaquiline and clofazimine mitigates against emergent cross-resistance and enables benefits of both drugs to be realized early on. With the current rollout of the shorter DR-TB regimen, possible cross-resistance may occur after exposure to a single drug (clofazimine), resulting in subsequent bedaquiline loss or vice versa. It remains enigmatic whether efflux pump inhibitors can overcome the presence of an efflux-based resistance pathway (rv0678 mutations).
In this study, we show that bedaquiline-naive reference strains can accumulate resistance to bedaquiline after clofazimine exposure due to mutation of the rv0678 gene. Clofazimine cross-resistance can develop only in the presence of rv0678 but not atpE mutations. Because rv0678 mutations play a role in resistance to both of these drugs, there is an important emergent need to catalogue rv0678 mutations with associated phenotypic data, which may steer usage. Phenotypic drug susceptibility testing and targeted rv0678 sequencing (where feasible) should be considered as part of diagnostic algorithms to rule out cross-resistance and ensure optimal treatment outcomes.
(This research was presented at the 39th Annual Congress of the European Society of Mycobacteriology, Dresden, Germany.)
Data availability.
Raw sequence data are accessible on the NCBI platform under accession no. PRJNA494324.
ACKNOWLEDGMENTS
N.I. was supported by the National Research Fund (SFH150723130071) and the University of Pretoria.
This project was funded by the WHO Supranational Reference TB Laboratory, National Institute for Communicable Diseases, Johannesburg, South Africa, and by the National Health Laboratory Services (NHLS) Research Trust (grant 004_94640).
We have no conflicts of interest to declare for this study.
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Associated Data
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Data Availability Statement
Raw sequence data are accessible on the NCBI platform under accession no. PRJNA494324.