Rv0678 Mutations Research Articles

Mutations in Rv0678, Rv2535c, and Rv1979c Confer Resistance to Bedaquiline in Clinical Isolates of Mycobacterium Tuberculosis.

Reduced bedaquiline (BDQ) sensitivity to antimycobacterial drugs has been linked to mutations in the Rv0678, pepQ, and Rv1979c genes of Mycobacterium tuberculosis (MTB). Resistance-causing mutations in MTB strains under treatment may have an impact on novel BDQ-based medication regimens intended to reduce treatment time. Due to this, we investigated the genetic basis of BDQ resistance in Turkish TB patients with MTB clinical isolates. Furthermore, mutations in the genes linked to efflux pumps were examined as a backup resistance mechanism. We scrutinized 100 MTB clinical isolates from TB patients using convenience sampling. Eighty MDR and twenty pan-drug susceptible MTB strains were among these isolates. Sequencing was performed on all strains, and genomic analyses were performed to find mutations in BDQ resistance-associated genes, including Rv0678 and pepQ(Rv2535c), which correspond to a putative Xaa-Pro aminopeptidase, and Rv1979c. Of the 74 isolates with PepQ (Rv2535c) mutations, four isolates (2.96%) exhibited MGIT-BDQ susceptibility. Twenty-one (19.11%) of the ninety-one isolates carrying mutations, including Rv1979c, were MGIT-BDQ-sensitive. Nonetheless, out of the 39 isolates with Rv0678 mutations, four (2.96%) were sensitive to MGIT-BDQ. It was found that resistance-associated variants (RAVs) in Rv0678, pepQ, and Rv1979c are often linked to BDQ resistance. In order to include variations in efflux pump genes in genome-based diagnostics for drug-resistant MTB, further evidence about their involvement in resistance is needed.

Bactericidal and sterilizing activity of sudapyridine-clofazimine-TB47 combined with linezolid or pyrazinamide in a murine model of tuberculosis.

As an obligate aerobe, Mycobacterium tuberculosis relies on its branched electron transport chain (ETC) for energy production through oxidative phosphorylation. Regimens targeting ETC exhibit promising potential to enhance bactericidal activity against M. tuberculosis and hold the prospect of shortening treatment duration. Our previous research demonstrated that the bacteriostatic drug candidate TB47 (T) inhibited the growth of M. tuberculosis by targeting the cytochrome bc1 complex and exhibited synergistic activity with clofazimine (C). Here, we found synergistic activities between C and sudapyridine (S), a structural analog of bedaquiline (B). S has shown similar anti-tuberculosis efficacy and may share a mechanism of action with B, which inhibits ATP synthesis and the energy metabolism of bacteria. We evaluated the efficacy of SCT in combination with linezolid (L) or pyrazinamide (Z) using a well-established murine model of tuberculosis. Compared to the BPa(pretomanid)L regimen, SCT and SCTL demonstrated similar bactericidal and sterilizing activities. There was no significant difference in activity between SCT and SCTL. In contrast, SCZ and SCTZ showed much higher activities, with none of the 15 mice experiencing relapse after 2 months of treatment with either SCZ or SCTZ. However, T did not contribute to the activity of the SCZ. Our findings emphasize the efficacy and the potential clinical significance of combination therapy with ETC inhibitors. Additionally, cross-resistance exists not only between S and B but also between S/B and C. This is supported by our findings, as spontaneous S-resistant mutants exhibited mutations in Rv0678, which are associated with cross-resistance to B and C.

Investigation of genomic mutations and their association with phenotypic resistance to new and repurposed drugs in Mycobacterium tuberculosis complex clinical isolates.

WGS has the potential to detect resistance-associated mutations and guide treatment of MDR TB. However, the knowledge base to confidently interpret mutations associated with the new and repurposed drugs is sparse, and phenotypic drug susceptibility testing is required to detect resistance. We screened 900 Mycobacterium tuberculosis complex genomes from Ireland, a low TB incidence country, for mutations in 13 candidate genes and assessed their association with phenotypic resistance to bedaquiline, clofazimine, linezolid, delamanid and pretomanid. We identified a large diversity of mutations in the candidate genes of 195 clinical isolates, with very few isolates associated with phenotypic resistance to bedaquiline (n = 4), delamanid (n = 4) and pretomanid (n = 2). We identified bedaquiline resistance among two drug-susceptible TB isolates that harboured mutations in Rv0678. Bedaquiline resistance was also identified in two MDR-TB isolates harbouring Met146Thr in Rv0678, which dated back to 2007, prior to the introduction of bedaquiline. High-level delamanid resistance was observed in two isolates with deletions in ddn, which were also resistant to pretomanid. Delamanid resistance was detected in two further isolates that harboured mutations in fbiA, but did not show cross-resistance to pretomanid. All isolates were susceptible to linezolid and clofazimine, and no mutations found were associated with resistance. More studies that correlate genotypic and phenotypic drug susceptibility data are needed to increase the knowledge base of mutations associated with resistance, in particular for pretomanid. Overall, this study contributes to the development of future mutation catalogues for M. tuberculosis complex isolates.

Bedquiline Resistance Mutations: Correlations with Drug Exposures and Impact on the Proteome in M. tuberculosis.

Bedaquiline (BDQ) is an effective drug for the treatment of drug-resistant tuberculosis. Mutations in atpE, which encodes the target of BDQ, are associated with large increases in MICs. Mutations in Rv0678 that derepress the transcription of the MmpL5-MmpS5 efflux transporter are associated with smaller increases in MICs. However, Rv0678 mutations are the most common mutations that are associated with BDQ resistance in clinical isolates, and they also confer cross-resistance to clofazimine (CFZ). To investigate the mechanism of BDQ resistance and the correlation between Rv0678 mutations and target-based atpE mutations, M. tuberculosis strains were exposed to different concentrations of BDQ or CFZ to select Rv0678 mutations and atpE mutations. Gene overexpression strains were constructed to illustrate the roles of MmpL5 and MmpS5. A quantitative proteome analysis was performed to compare the BDQ-resistant mutants to the isogenic strain H37Rv. Here, we report that the Rv0678 mutations were more readily selected than were the atpE mutations at low concentrations of BDQ or CFZ. The atpE mutations were selected by high concentrations of BDQ exposure. The overexpression of both mmpL5 and mmpS5 reduced the susceptibility of Mycobacterium tuberculosis to BDQ and CFZ. Secreted immunogenic proteins and proteins involved in the biosynthesis and transport of phthiocerol dimycocerosates were associated with Rv0678 mutations conferring BDQ resistance in the proteome analysis. In conclusion, exposure to different bedaquiline concentrations resulted in the selection of different mutations. The coexpression of MmpL5 and MmpS5 contributed to drug resistance and upregulated pathogenic proteins in M. tuberculosis, suggesting MmpL5-MmpS5 as a new potential target for antituberculosis drug development. These results warrant further surveillance for the evolution of BDQ resistance during clinical usage.

Bedaquiline- and clofazimine- selected Mycobacterium tuberculosis mutants: further insights on resistance driven largely by Rv0678

Drug-resistant tuberculosis is a serious global health threat. Bedaquiline (BDQ) is a relatively new core drug, targeting the respiratory chain in Mycobacterium tuberculosis (Mtb). While mutations in the BDQ target gene, atpE, are rare in clinical isolates, mutations in the Rv0678 gene, a transcriptional repressor regulating the efflux pump MmpS5-MmpL5, are increasingly observed, and have been linked to worse treatment outcomes. Nevertheless, underlying mechanisms of (cross)-resistance remain incompletely resolved. Our study aims to distinguish resistance associated variants from other polymorphisms, by assessing the in vitro onset of mutations under drug pressure, combined with their impact on minimum inhibitory concentrations (MICs) and on protein stability. For this purpose, isolates were exposed in vitro to sub-lethal concentrations of BDQ or clofazimine (CFZ). Selected colonies had BDQ- and CFZ-MICs determined on 7H10 and 7H11 agar. Sanger sequencing and additional Deeplex Myc-TB and whole genome sequencing (WGS) for a subset of isolates were used to search for mutations in Rv0678, atpE and pepQ. In silico characterization of relevant mutations was performed using computational tools. We found that colonies that grew on BDQ medium had mutations in Rv0678, atpE or pepQ, while CFZ-exposed isolates presented mutations in Rv0678 and pepQ, but none in atpE. Twenty-eight Rv0678 mutations had previously been described among in vitro selected mutants or in patients’ isolates, while 85 were new. Mutations were scattered across the Rv0678 gene without apparent hotspot. While most Rv0678 mutations led to an increased BDQ- and/or CFZ-MIC, only a part of them surpassed the critical concentration (69.1% for BDQ and 87.9% for CFZ). Among the mutations leading to elevated MICs for BDQ and CFZ, we report a synonymous Val1Val mutation in the Rv0678 start codon. Finally, in silico characterization of Rv0678 mutations suggests that especially the C46R mutant may render Rv0678 less stable.

A Bayesian approach to estimate the probability of resistance to bedaquiline in the presence of a genomic variant.

Bedaquiline is a core drug for treatment of rifampicin-resistant tuberculosis. Few genomic variants have been statistically associated with bedaquiline resistance. Alternative approaches for determining the genotypic-phenotypic association are needed to guide clinical care. Using published phenotype data for variants in Rv0678, atpE, pepQ and Rv1979c genes in 756 Mycobacterium tuberculosis isolates and survey data of the opinion of 33 experts, we applied Bayesian methods to estimate the posterior probability of bedaquiline resistance and corresponding 95% credible intervals. Experts agreed on the role of Rv0678, and atpE, were uncertain about the role of pepQ and Rv1979c variants and overestimated the probability of bedaquiline resistance for most variant types, resulting in lower posterior probabilities compared to prior estimates. The posterior median probability of bedaquiline resistance was low for synonymous mutations in atpE (0.1%) and Rv0678 (3.3%), high for missense mutations in atpE (60.8%) and nonsense mutations in Rv0678 (55.1%), relatively low for missense (31.5%) mutations and frameshift (30.0%) in Rv0678 and low for missense mutations in pepQ (2.6%) and Rv1979c (2.9%), but 95% credible intervals were wide. Bayesian probability estimates of bedaquiline resistance given the presence of a specific mutation could be useful for clinical decision-making as it presents interpretable probabilities compared to standard odds ratios. For a newly emerging variant, the probability of resistance for the variant type and gene can still be used to guide clinical decision-making. Future studies should investigate the feasibility of using Bayesian probabilities for bedaquiline resistance in clinical practice.

Bedaquiline resistance pattern in clofazimine-resistant clinical isolates of tuberculosis patients

Bedaquiline (BDQ) is a potent drug for treating drug-resistant tuberculosis (TB). Here, we analysed the resistance profiles of BDQ in CFZ-resistant clinical isolates and investigated the clinical risk factors of BDQ and CFZ cross/co-resistance. The AlarmarBlue microplate assay was performed to determine the minimum inhibitory concentration (MIC) of the CFZ-resistant Mycobacterium tuberculosis (MTB) clinical isolates to CFZ and BDQ. The clinical characteristics of the respective patients were analysed to explore the possible risk factors of BDQ resistance. The drug-resistance-associated genes including Rv0678, Rv1979c, atpE, pepQ and Rv1453 were sequenced and analysed. A total of 72 clinical CFZ-resistant MTB isolates were collected; among these, half were identified as BDQ-resistant. The MIC value of BDQ closely correlated with CFZ (Spearman's q=0.766, P < 0.005). Among the isolates with a MIC of CFZ ≥4 mg/L, 92.31% (12/13) were resistant to BDQ. Pre-XDR and exposure to BDQ or CFZ are the major risk factors for concurrent BDQ resistance. Among the 36 cross/co-resistant isolates, 50% (18/36) had mutations in Rv0678, 8.3% (3/36) had mutations in Rv0678+Rv1453, 5.6% (2/36) had mutations in Rv0678+Rv1979c, 2.8% (1/36) had mutations in Rv0678+Rv1979c+Rv1453, 2.8% (1/36) had mutations in atpE+Rv0678+Rv1453, 2.8% (1/36) had mutations in Rv1979c, and 27.7% (10/36) had no variations in the target genes. Nearly half of the CFZ-resistant isolates were still sensitive to BDQ, whereas this rate dramatically decreased among patients with pre-XDR TB or those who had been exposed to BDQ or CFZ.

Investigation of bedaquiline resistance and genetic mutations in multi-drug resistant Mycobacterium tuberculosis clinical isolates in Chongqing, China

BackgroundTo investigate the prevalence and molecular characterization of bedaquiline resistance among MDR-TB isolates collected from Chongqing, China.MethodsA total of 205 MDR-TB isolates were collected from Chongqing Tuberculosis Control Institute between March 2019 and June 2020. The MICs of BDQ were determined by microplate alamarblue assay. All strains were genotyped by melting curve spoligotyping, and were subjected to WGS.ResultsAmong the 205 MDR isolates, the resistance rate of BDQ was 4.4% (9/205). The 55 (26.8%) were from male patients and 50 (24.4%) were new cases. Furthermore, 81 (39.5%) of these patients exhibited lung cavitation, 13 (6.3%) patients afflicted with diabetes mellitus, and 170 (82.9%) isolates belonged to Beijing family. However, the distribution of BDQ resistant isolates showed no significant difference among these characteristics. Of the 86 OFX resistant isolates, 8 isolates were XDR (9.3%, 8/86). Six BDQ resistant isolates (66.7%, 6/9) and two BDQ susceptible isolates (1.0%, 2/196) carried mutations in Rv0678. A total of 4 mutations types were identified in BDQ resistant isolates, including mutation in A152G (50%, 3/6), T56C (16.7%, 1/6), GA492 insertion (16.7%, 1/6), and A274 insertion (16.7%, 1/6). BDQ showed excellent activity against MDR-TB in Chongqing.ConclusionsBDQ showed excellent activity against MDR-TB in Chongqing. The resistance rate of BDQ was not related to demographic and clinical characteristics. Mutations in Rv0678 gene were the major mechanism to BDQ resistance, with A152G as the most common mutation type. WGS has a good popularize value and application prospect in the rapid detection of BDQ resistance.

Bedaquiline resistant Mycobacterium tuberculosis clinical isolates with and without rv0678 mutations have similar growth patterns under varying BDQ drug pressure

Resistance associated mutations have been reported to alter the growth of Mycobacterium tuberculosis (MTB) isolates under drug pressure. However, there is little information on the growth characteristics of bedaquiline (BDQ) resistant isolates in the presence of BDQ. To further understand the role of rv0678, we aimed to study whether the presence of rv0678 variants in BDQ resistant isolates alters the killing effect of BDQ. We, therefore, selected BDQ resistant clinical MTB isolates with (n = 6) and without (n = 3) variants in rv0678 gene. Using time kill assays, growth inhibition; taken as the relative change in log average colony forming unit (CFU)/ml at selected time points (24–96 h), was studied at Minimum Inhibitory Concentrations (MICs): 0x, 1x, 2.5x, 5x, 7.5x, 10x for these isolates. Growth inhibition was then analyzed using Kruskal Wallis and Kolmogorov Smirnov tests in PRISM vr.9. During the 24–96 h lag phase isolates with and without variants in rv0678 showed a similar growth inhibition pattern. No difference was noted in growth inhibition between BDQ resistant isolates and H37Rv. These findings suggest that role of alternate mechanisms in contributing to BDQ tolerance needs to be explored.

Whole Genome Sequencing Identifies Novel Mutations Associated With Bedaquiline Resistance in Mycobacterium tuberculosis.

Bedaquiline (BDQ), a new antitubercular agent, has been used to treat drug-resistant tuberculosis (TB). Although mutations in atpE, rv0678, and pepQ confer major resistance to BDQ, the mechanisms of resistance to BDQ in vitro and in clinical settings have not been fully elucidated. We selected BDQ-resistant mutants from 7H10 agar plates containing 0.5 mg/L BDQ (the critical concentration) and identified mutations associated with BDQ resistance through whole genome sequencing and Sanger sequencing. A total of 1,025 mutants were resistant to BDQ. We randomly selected 168 mutants for further analysis and discovered that 157/168 BDQ-resistant mutants harbored mutations in rv0678, which encodes a transcriptional regulator that represses the expression of the efflux pump, MmpS5–MmpL5. Moreover, we found two mutations with high frequency in rv0678 at nucleotide positions 286–287 (CG286–287 insertion; accounting for 26.8% [45/168]) and 198–199 (G198, G199 insertion, and G198 deletion; accounting for 14.3% [24/168]). The other mutations were dispersed covering the entire rv0678 gene. Moreover, we found that one new gene, glpK, harbors a G572 insertion; this mutation has a high prevalence (85.7%; 144/168) in the isolated mutants, and the minimum inhibitory concentration (MIC) assay demonstrated that it is closely associated with BDQ resistance. In summary, we characterized 168/1,025 mutants resistant to BDQ and found that mutations in rv0678 confer the primary mechanism of BDQ resistance. Moreover, we identified a new gene (glpK) involved in BDQ resistance. Our study offers new insights and valuable information that will contribute to rapid identification of BDQ-resistant isolates in clinical settings.

Assessment of epidemiological and genetic characteristics and clinical outcomes of resistance to bedaquiline in patients treated for rifampicin-resistant tuberculosis: a cross-sectional and longitudinal study

Bedaquiline improves outcomes of patients with rifampicin-resistant and multidrug-resistant (MDR) tuberculosis; however, emerging resistance threatens this success. We did a cross-sectional and longitudinal analysis evaluating the epidemiology, genetic basis, and treatment outcomes associated with bedaquiline resistance, using data from South Africa (2015-19). Patients with drug-resistant tuberculosis starting bedaquiline-based treatment had surveillance samples submitted at baseline, month 2, and month 6, along with demographic information. Culture-positive baseline and post-baseline isolates had phenotypic resistance determined. Eligible patients were aged 12 years or older with a positive culture sample at baseline or, if the sample was invalid or negative, a sample within 30 days of the baseline sample submitted for bedaquiline drug susceptibility testing. For the longitudinal study, the first surveillance sample had to be phenotypically susceptible to bedaquiline for inclusion. Whole-genome sequencing was done on bedaquiline-resistant isolates and a subset of bedaquiline-susceptible isolates. The National Institute for Communicable Diseases tuberculosis reference laboratory, and national tuberculosis surveillance databases were matched to the Electronic Drug-Resistant Tuberculosis Register. We assessed baseline resistance prevalence, mutations, transmission, cumulative resistance incidence, and odds ratios (ORs) associating risk factors for resistance with patient outcomes. Between Jan 1, 2015, and July 31, 2019, 8041 patients had surveillance samples submitted, of whom 2023 were included in the cross-sectional analysis and 695 in the longitudinal analysis. Baseline bedaquiline resistance prevalence was 3·8% (76 of 2023 patients; 95% CI 2·9-4·6), and it was associated with previous exposure to bedaquiline or clofazimine (OR 7·1, 95% CI 2·3-21·9) and with rifampicin-resistant or MDR tuberculosis with additional resistance to either fluoroquinolones or injectable drugs (pre-extensively-drug resistant [XDR] tuberculosis: 4·2, 1·7-10·5) or to both (XDR tuberculosis: 4·8, 2·0-11·7). Rv0678 mutations were the sole genetic basis of phenotypic resistance. Baseline resistance could be attributed to previous bedaquiline or clofazimine exposure in four (5·3%) of 76 patients and to primary transmission in six (7·9%). Odds of successful treatment outcomes were lower in patients with baseline bedaquiline resistance (0·5, 0·3-1). Resistance during treatment developed in 16 (2·3%) of 695 patients, at a median of 90 days (IQR 62-195), with 12 of these 16 having pre-XDR or XDR. Bedaquiline resistance was associated with poorer treatment outcomes. Rapid assessment of bedaquiline resistance, especially when patients were previously exposed to bedaquiline or clofazimine, should be prioritised at baseline or if patients remain culture-positive after 2 months of treatment. Preventing resistance by use of novel combination therapies, current treatment optimisation, and patient support is essential. National Institute for Communicable Diseases of South Africa.

Primary Bedaquiline Resistance Among Cases of Drug-Resistant Tuberculosis in Taiwan.

Bedaquiline (BDQ), which is recommended for the treatment of drug-resistant tuberculosis (DR-TB), was introduced in Taiwan in 2014. Due to the alarming emergence of BDQ resistance, we conducted BDQ resistance analyses to strengthen our DR-TB management program. This retrospective population-based study included initial Mycobacterium tuberculosis isolates from 898 rifampicin-resistant (RR) or multidrug-resistant (MDR) TB cases never exposed to BDQ during 2008–2019. We randomly selected 65 isolates and identified 28 isolates with BDQ MIC<0.25μg/ml and MIC≥0.25μg/ml as the control and study groups, respectively. BDQ drug susceptibility testing (DST) using the MGIT960 system and Sanger sequencing of the atpE, Rv0678, and pepQ genes was conducted. Notably, 18 isolates with BDQ MIC=0.25μg/ml, 38.9% (7/18), and 61.1% (11/18) isolates were MGIT-BDQ resistant and susceptible, respectively. Consequently, we recommended redefining MIC=0.25μg/ml as an intermediate-susceptible category to resolve discordance between different DST methods. Of the 93 isolates, 22 isolates were MGIT-BDQ-resistant and 77.3% (17/22) of MGIT-BDQ-resistant isolates harbored Rv0678 mutations. After excluding 2 MGIT-BDQ-resistant isolates with borderline resistance (GU400growth control-GU100BDQ≤1day), 100% (15/15) harbored Rv0678 gene mutations, including seven novel mutations [g-14a, Ile80Ser (N=2), Phe100Tyr, Ala102Val, Ins g 181–182 frameshift mutation (N=2), Del 11–63 frameshift mutation, and whole gene deletion (N=2)]. Since the other 22.7% (5/22) MGIT-BDQ-resistant isolates with borderline resistance (GU400growth control-GU100BDQ≤1day) had no mutation in three analyzed genes. For isolates with phenotypic MGIT-BDQ borderline resistance, checking for GU differences or conducting genotypic analyses are suggested for ruling out BDQ resistance. In addition, we observed favorable outcomes among patients with BDQ-resistant isolates who received BDQ-containing regimens regardless of Rv0678 mutations. We concluded that based on MIC≥0.25μg/ml, 3.1% (28/898) of drug-resistant TB cases without BDQ exposure showed BDQ resistance, Rv0678 was not a robust marker of BDQ resistance, and its mutations were not associated with treatment outcomes.

Mycobacterium tuberculosis resistance to novel anti-tuberculosis drugs in russia

Aim and objectives: Emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains requires use of novel compounds. New drugs require long-term treatment regimen and, consequently, long-term drug selective pressure gives more time to mycobacteria to develop resistance. At the same time, new compounds are expensive and, albeit efficient, they are not routinely used in the Russian Federation which situation with tuberculosis is characterized by high and increasing prevalence of multidrug resistant strains. Methods: M. tuberculosis isolates were recovered from sputum at the 1-3 months intervals. M. tuberculosis drug susceptibility testing for first- and second-line drugs was performed using modified proportion method on Middlebrook 7H9 liquid culture and Bactec MGIT 960 system (Becton Dickinson, Sparks, Md.) according to the manufacturer's instructions. The laboratory is externally quality assured by the System for External Quality Assessment “Center for External Quality Control of Clinical Laboratory Research” (Moscow, Russia). Whole genome sequencing was performed at the MiSeq platform (Illumina). The fastq files were aligned to the complete genome of the reference strain H37Rv (NC_00962.3) using Geneious 9 program (Biomatters, New Zealand) and additionally checked with PhyReSE online tool (https://bioinf.fz-borstel.de/mchips/phyresse/). Results: Mutations in Rv0678 (mmpR) were most frequent in BDQ resistant strains and different types of mutations were found (non-synonymous substitutions, frameshift, multi-codon indels). In some cases, different mutations coexisted and heteroresistance was observed in all cases, i.e., simultaneous presence of wild type and mutants alleles (which correlates with previous studies). Only in one patient, atpE mutation emerged in M. tuberculosis isolate during treatment. Analysis of genes associated with PCZ resistance (ethA, ethR, and hadABC) identified three mutations in ethA and one mutation in hadA. The ethA frameshift mutation (genome position 4327363 CT&gt;C) was detected in isolates from patients who did not receive previous PCZ treatment but were all phenotypically resistant to PTH or ETH. Thus we assumed that this mutation emerged as PTH/ETH resistance mutation. Conclusions: To conclude, mutations in Rv0678 present a main mechanism of bedaquiline resistance in the Russian setting; development of resistance during treatment is not rare as bedaquiline resistance mutations emerged in 40% of patients. Furthermore, development of BDQ resistance 2-3 months after stop of treatment is possible, due to long half-life of the drug. With regard to PCZ resistance, presence of cross-resistance mutations to both PCZ and ETH/PTH suggests that these mutations should be assessed before inclusion of PCZ in the treatment regimen. A large prospective study of the more diverse M. tuberculosis collection is warranted to elucidate the development of resistance to these novel drugs in the Russian setting taking into consideration the population structure of M. tuberculosis population.

Emergence of resistance to bedaquiline

Resistance to first-line anti-tuberculosis (TB) drugs continues to threaten global TB control with over 500 000 cases of rifampicin resistant (RR)-TB being reported in 2018. Treatment of RR-TB requires the use of toxic drugs often leading to adverse events, poor adherence and poor treatment outcomes. In order to address the toxicity of treatment and to improve treatment outcomes the World Health Organization has promoted the use of injection free regimens which include new and repurposed drugs: bedaquiline (BDQ), linezolid (LZD) and clofazimine (CFZ). In 2017, South Africa rolled out the inclusion of BDQ in the treatment of all pre-XDR- and XDR-TB cases as well as patients experiencing adverse events from kanamycin. In late 2018, South Africa included BDQ into three injection free regimens for treating RR-TB. This was done before routine drug susceptibility testing for BDQ could be implemented. Resistance to BDQ occurs primarily through mutations in Rv0678 (mmpR), pepQ and atpE. Understanding which mutations confer resistance and why they arise in a patient will be critical for early diagnosis as well as for preventing the emergence of resistance thereby ensuring successful treatment. Collation of the mutations identified in Rv0678 shows that BDQ resistance is largely driven by small insertions/deletions which disrupt the function of the repressor thereby leading to over expression of the efflux pump mmpL5 responsible for extruding BDQ from the cell. Understanding the association between gene mutation and phenotypic resistance remains critical for the development of new molecular diagnostics to rapidly diagnose resistance thereby affording the clinician the opportunity to adjust the regimen. Analysis of serial isolates collected from patients receiving BDQ (Access program pre-2017) has shown the emergence of mutations in Rv0678, atpE and pepQ during treatment in patients who remain culture positive after three months of treatment. Using targeted deep sequencing it is now possible to track the emergence of resistant sub-populations at the sub-phenotypic level (i.e., micro-heteroresistance) thereby affording the opportunity to study the association of the emergence of resistance with drug regimens, MIC distributions, PK and PD data and treatment outcomes. Our analysis showed that mutations conferring resistance occurred soon after the introduction of BDQ into the regimen and that clones harbouring these mutations were subsequently selected. Most surprising was the observation that different resistant sub-populations emerged during the period after the cessation of BDQ in the regimen. This demonstrates the continuing selective pressure as a result of the long half-life of BDQ. Although the origin of the distinct resistant clones is unknown, we hypothesize that resistance evolved independently in spatially separated lesions subsequently rupturing into the airways. A surprising observation was that concomitant emergence of different variants of the rpoC gene suggesting a compensatory mechanism to overcome the fitness cost of Rv0678 mutations.

Acquisition of Cross-Resistance to Bedaquiline and Clofazimine following Treatment for Tuberculosis in Pakistan

Aims and objectives: In the present study, we report on the first seven cases of acquired bedaquiline and clofazimine resistance identified in Pakistan. The study was conducted within the framework of a retrospective surveillance project to monitor the acquisition of resistance to bedaquiline implemented by the National TB Reference Laboratory in Pakistan and TB Supranational Reference Laboratory. Methods: Seventy three sequential isolates from 31 drug-resistant-tuberculosis patients on bedaquiline-containing regimens were retrospectively tested for bedaquiline resistance by MIC testing in 7H11 medium, and for all isolates showing an increased MIC compared to the baseline isolate, further MIC testing for bedaquiline and clofazimine was conducted using the Bactec MGIT960 (BD, Franklin Lakes, NJ, USA). The H37Rv (ATCC 27294) strain was used as a susceptible control in MIC testing. Bedaquiline dry powder was supplied by Janssen-Pharmaceutica (Beerse, Belgium). WGS was carried out with the Illumina Nextera-XT DNA sample preparation kit to prepare paired-end libraries of 150-bp read length to sequence on an Illumina NextSeq platform. Data analysis and single nucleotide polymorphism (SNP) calling were performed using the MTBseq-Pipeline on low-frequency detection mode. Genes associated with resistance to bedaquiline and/or clofazimine (atpE, Rv0678, pepQ, and Rv1979c) were screened for mutations. Results: We studied 73 isolates from patients (n=31; 8 MDR, 16 pre-XDR, and 7 XDR) enrolled in bedaquiline-containing regimens. The sequential isolates tested included two strains from 22 patients, three from 7 patients, and four from 2 patients. All baseline strains included in the study were sensitive to bedaquiline. Seven patients developed an increase in bedaquiline MICs in 7H11 medium (range, 0.125 to &gt;0.5mg·liter−1) during therapy, and six of them became resistant to bedaquiline according to the current critical concentration proposed for the drug. In all seven patients we detected genetic variants in Rv0678 that were not present at baseline isolates. Moreover in five out of seven patients the treatment was ultimately failed. Conclusions: We documented cases failing therapy that developed specific mutations in Rv0678 and had increased MICs associated with cross-resistance to clofazimine during treatment. This study underlines the relevance of surveillance programs following the introduction of new drugs.

Population-level emergence of bedaquiline and clofazimine resistance-associated variants among patients with drug-resistant tuberculosis in southern Africa: a phenotypic and phylogenetic analysis

SummaryBackgroundBedaquiline and clofazimine are important drugs in the treatment of drug-resistant tuberculosis and are commonly used across southern Africa, although drug susceptibility testing is not routinely performed. In this study, we did a genotypic and phenotypic analysis of drug-resistant Mycobacterium tuberculosis isolates from cohort studies in hospitals in KwaZulu-Natal, South Africa, to identify resistance-associated variants (RAVs) and assess the extent of clofazimine and bedaquiline cross-resistance. We also used a comprehensive dataset of whole-genome sequences to investigate the phylogenetic and geographical distribution of bedaquiline and clofazimine RAVs in southern Africa.MethodsIn this study, we included M tuberculosis isolates reported from the PRAXIS study of patients with drug-resistant tuberculosis treated with bedaquiline (King Dinuzulu Hospital, Durban) and three other cohort studies of drug-resistant tuberculosis in other KwaZulu-Natal hospitals, and sequential isolates from six persistently culture-positive patients with extensively drug-resistant tuberculosis at the KwaZulu-Natal provincial referral laboratory. Samples were collected between 2013 and 2019. Microbiological cultures were done as part of all parent studies. We sequenced whole genomes of included isolates and measured bedaquiline and clofazimine minimum inhibitory concentrations (MICs) for isolates identified as carrying any Rv0678 variant or previously published atpE, pepQ, and Rv1979c RAVs, which were the subject of the phenotypic study. We combined all whole-genome sequences of M tuberculosis obtained in this study with publicly available sequence data from other tuberculosis studies in southern Africa (defined as the countries of the Southern African Development Community), including isolates with Rv0678 variants identified by screening public genomic databases. We used this extended dataset to reconstruct phylogenetic relationships across lineage 2 and 4 M tuberculosis isolates.FindingsWe sequenced the whole genome of 648 isolates from 385 patients with drug-resistant tuberculosis recruited into cohort studies in KwaZulu-Natal, and 28 isolates from six patients from the KwaZulu-Natal referral laboratory. We identified 30 isolates with Rv0678 RAVs from 16 (4%) of 391 patients. We did not identify any atpE, pepQ, or Rv1979c RAVs. MICs were measured for 21 isolates with Rv0678 RAVs. MICs were above the critical concentration for bedaquiline resistance in nine (43%) of 21 isolates, in the intermediate category in nine (43%) isolates, and within the wild-type range in three (14%) isolates. Clofazimine MICs in genetically wild-type isolates ranged from 0·12–0·5 μg/mL, and in isolates with RAVs from 0·25–4·0 μg/mL. Phylogenetic analysis of the extended dataset including M tuberculosis isolates from southern Africa resolved multiple emergences of Rv0678 variants in lineages 2 and 4, documented two likely nosocomial transmission events, and identified the spread of a possibly bedaquiline and clofazimine cross-resistant clone in eSwatini. We also identified four patients with pepQ frameshift mutations that may confer resistance.InterpretationBedaquiline and clofazimine cross-resistance in southern Africa is emerging repeatedly, with evidence of onward transmission largely due to Rv0678 mutations in M tuberculosis. Roll-out of bedaquiline and clofazimine treatment in the setting of limited drug susceptibility testing could allow further spread of resistance. Designing strong regimens would help reduce the emergence of resistance. Drug susceptibility testing is required to identify where resistance does emerge.FundingWellcome Trust, National Institute of Allergy and Infectious Diseases and National Center for Advancing Translational Sciences of the National Institutes of Health.

Molecular characteristics and in vitro susceptibility to bedaquiline of Mycobacterium tuberculosis isolates circulating in Shaanxi, China

ObjectiveThe aim of this study was to investigate the molecular characteristics and in vitro susceptibility to bedaquiline of Mycobacterium tuberculosis (MTB) isolates from Shaanxi, China. MethodsThe minimum inhibitory concentration (MIC) of bedaquiline was determined using the microplate alamarBlue assay for 518 MTB isolates from Shaanxi. Isolates with MIC values of bedaquiline ≥0.12 μg/mL were sequenced for the atpE, Rv0678, and pepQ genes. Drug susceptibility testing and spoligotyping were also conducted for all strains. ResultsTen (1.93%) bedaquiline-resistant strains were isolated from 518 tuberculosis patients. The resistance rate of bedaquiline was not correlated to sex, age, treatment history, region, or genotype. Five bedaquiline-resistant isolates and one bedaquiline-susceptible isolate were found to carry Rv0678 mutations; six mutation types were identified, including G5T, A263G, C185T, G19deletion, C265T, and T323C. No mutations within the atpE and pepQ genes were observed. ConclusionsBedaquiline showed strong in vitro antibacterial activity against MTB isolates, and the Rv0678 gene serves as the major mechanism contributing to bedaquiline resistance among MTB isolates from Shaanxi, China. Two novel mutation types (C265 T and T323 C) of the Rv0678 gene were associated with resistance to bedaquiline. Furthermore, in addition to the current three resistance-associated genes (atpE, Rv0678, and pepQ), other mechanisms of resistance to bedaquiline may exist that need further study.

Reduced Susceptibility of Mycobacterium tuberculosis to Bedaquiline During Antituberculosis Treatment and Its Correlation With Clinical Outcomes in China.

We aimed to assess the proportion of multidrug-resistant tuberculosis (MDR-TB) cases with initial bedaquiline (BDQ) resistance, monitor the dynamics of BDQ susceptibility of Mycobacterium tuberculosis isolates during therapy, and correlate susceptibility with MDR-TB patient clinical outcomes in China. A retrospective, cohort study of MDR-TB patients was conducted, with positive cultures collected from cases at 13 sites. Patients with nontuberculous mycobacterial infection during anti-TB therapy were excluded. BDQ minimal inhibitory concentrations (MICs) were determined using a 7H9 Middlebrook broth-based microdilution method. Mutations that conferred BDQ resistance were detected via Sanger sequencing. A total of 277 patients receiving BDQ treatment were studied, with BDQ resistance noted in isolates from 2.2% (6/277) of MDR-TB cases, sputum conversion observed in 5 cases, and culture conversion observed in 138 cases within 2 weeks. Another 15 and 30 isolates were excluded from final analysis due to failures in obtaining subcultures and serial isolates, respectively. Of 94 cases that yielded serial isolates, 11 exhibited reduced BDQ susceptibility, while 3 of 5 cases with acquired resistance failed to culture-convert. Sequence analysis revealed that 6 of 11 BDQ-resistant isolates harbored Rv0678 mutations; no mutations were detected in 3 other BDQ resistance-associated genes. No significant intergroup difference in culture conversion time was observed. MDR-TB patients in China exhibited a low initial BDQ resistance rate. MDR-TB cases with acquired BDQ resistance were at greater risk of treatment failure relative to initially BDQ-resistant cases. Rv0678 mutations accounted for BDQ resistance in this cohort.

In Vitro Study of Stepwise Acquisition of rv0678 and atpE Mutations Conferring Bedaquiline Resistance.

Bedaquiline resistance within Mycobacterium tuberculosis may arise through efflux-based (rv0678) or target-based (atpE) pathway mutations. M. tuberculosis mutant populations from each of five sequential steps in a passaging approach, using a pyrazinamide-resistant ATCC strain, were subjected to MIC determinations and whole-genome sequencing. Exposure to increasing bedaquiline concentrations resulted in increasing phenotypic resistance (up to >2 μg/ml) through MIC determination on solid medium (Middlebrook 7H10). rv0678 mutations were dynamic, while atpE mutations were fixed, once occurring. We present the following hypothesis for in vitro emergence of bedaquiline resistance: rv0678 mutations may be the first transient step in low-level resistance acquisition, followed by high-level resistance due to fixed atpE mutations.

Multicentre study to establish interpretive criteria for clofazimine drug susceptibility testing.

<sec id="st1"> <title>OBJECTIVE</title> To conduct a multicentre study to establish the critical concentration (CC) for clofazimine (CFZ) for drug susceptibility testing (DST) of Mycobacterium tuberculosis on the MGIT™960™ system using the distribution of minimum inhibitory concentrations (MIC) and genotypic analyses of Rv0678 mutations. </sec> <sec id="st2"> <title>DESIGN</title> In phase I of the study, the MIC distribution of laboratory strains (H37Rv and in vitro-selected Rv0678 mutants) and clinical pan-susceptible isolates were determined (n = 70). In phase II, a tentative CC for CFZ (n = 55) was proposed. In phase III, the proposed CC was validated using clinical drug-resistant tuberculosis (DR-TB) isolates stratified by Rv0678 mutation (n = 85). </sec> <sec id="st3"> <title>RESULTS AND CONCLUSION</title> The MIC distribution of CFZ for laboratory and clinical pan-susceptible strains ranged between 0.125 μg/ml and 0.5 μg/ml. As the MIC values of DR-TB isolates used for phase II ranged between 0.25 μg/ml and 1 μg/ml, a CC of 1 μg/ml was proposed. Validation of the CC in phase III showed that probably susceptible and probably resistant Rv0678 mutants overlapped at 1 μg/ml. We therefore recommend a CC of 1 μg/ml, with additional testing at 0.5 μg/ml to define an intermediate category. This was the first comprehensive study to establish a CC for routine phenotypic DST of CFZ using the MGIT960 system to guide therapeutic decisions. </sec>.