Supplementary Appendix

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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Bloemberg GV, Gagneux S, Böttger EC. Acquired resistance to bedaquiline and delamanid in therapy for tuberculosis. N Engl J Med 2015;373: DOI: /NEJMc

2 SUPPLEMENTAL APPENDIX: Acquired Resistance to Bedaquiline and Delamanid in Therapy for Tuberculosis Guido V. Bloemberg 1, a, Ph.D., Peter M. Keller 1,2, a, M.D., David Stucki 3, a, Ph.D, Andrej Trauner 3, Ph.D., Sonia Borrell 3, Ph.D., Tsogyal Latshang 4, M.D., Mireia Coscolla 3, Ph.D., Thomas Rothe 5, M.D., Rico Hömke 1,2, Claudia Ritter 1,2, Julia Feldmann 3, Bettina Schulthess 1, Ph.D., Sebastien Gagneux 3b, Ph.D., and Erik C. Böttger 1,2,b, M.D. Affiliations: 1 Institut für Medizinische Mikrobiologie, Universität Zürich, Zürich, Switzerland. 2 Nationales Referenzzentrum für Mykobakterien, Universität Zürich, Zürich, Switzerland. 3 Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute and University of Basel, Basel, Switzerland. 4 Pulmonary Division, University Hospital Zurich, Zurich, Switzerland. 5 Zürcher Höhenklinik Davos, Klinikstrasse 6, 7272 Davos Clavadel, Switzerland. a GVB, PMK and DS: equal contribution as first authors b SG and ECB: equal contribution as senior authors Content Page Case report:... 2 Supplementary Methods:... 5 Supplementary Results:... 6 Supplementary References... 9 Supplementary Tables Table S Table S Table S Table S Supplementary Figure Figure S

3 Case report: A 35-year old Tibetan male refugee, who arrived in Switzerland in December 2010, was admitted to a Swiss hospital the same month. The patient suffered from weight loss (10 kg), night sweats, and cough since 5 months. Acid-fast bacilli (AFB) were present in the sputum and the chest radiograph showed bilateral cavitary chest abnormalities. Further examination revealed that he was HIV uninfected and had chronic, active hepatitis B (HBs antigen positive, HBe antigen negative) with signs of fibrosis in a liver biopsy. On December 23 rd 2010 HBV viral load (VL) was IU/ml (=10 10 IU/ml). Tenofovir therapy was started on January Because of increasing liver enzymes the therapy was changed to entecavir on February 15 th 2011 although HBV VL decreased to IU/ml on February 4 th On March 30 th 2011 the HBV VL fell to a load of 426 IU/ml and the liver enzymes normalized. The patient reported no previous history of TB treatment. Sputum culture grew Mycobacterium tuberculosis (Table S1). The initial isolate was classified as pre-xdr based on phenotypic drug susceptibility testing (DST) results that showed resistance to isoniazid, rifampicin, pyrazinamide, ethionamide, linezolid, moxifloxacin, and streptomycin (Table S2). Genotypic DST based on line-probe assay and Sanger sequencing identified mutations associated with drug resistance (Table S2). 1 The patient assured that he was not treated with linezolid or quinolones for other infections. On February 9 th 2011, the patient s chest computer tomography (CT) images showed multiple cavities bronchiectasis and star-shaped consolidations in upper and lower lobes on both sides. After a delay due to acute hepatopathy, antituberculous therapy was initiated in March 2011 with cycloserine, capreomycin, para-aminosalicylic acid (PAS), and ethambutol. Bedaquiline was added on a compassionate basis starting in September 2011 and continued until February 2012; a period longer than 6 months is not recommended according the Sirturo TM (Janssen Therapeutics, Titusville, NJ, USA) FDA medication guide. In November 2011, the patient 2

4 became sputum culture-negative, with all subsequent respiratory samples remaining culturenegative. During the first months on antituberculous therapy the pulmonary consolidations and cavities partially responded and then remained stable suggesting post inflammatory scarving. Antibiotic therapy was stopped on March 30 th After an overall treatment period of 24 months, the patient was considered clinically cured in March Five months later, in August 2013, the patient was re-admitted with fever and cough. Sputum microscopy was positive with AFB. After consultation of a WHO-expert user group, antituberculous therapy was re-initiated with a six-drug regimen consisting of cycloserine, capreomycin, PAS, ethambutol, clofazimine, and inhaled amikacin (4 ml 0.9% NaCL containing 10 mg amikacin twice a day). Phenotypic DST of the relapse M. tuberculosis isolate from August 2013 revealed resistance to clofazimine and a 10-fold increase in the MIC of bedaquiline compared to the initial isolate from January Genetic analysis identified a mutation in the gene encoding the transcriptional regulator MmpR (Rv0678), which has recently been associated with cross-resistance to clofazimine and bedaquiline. 3 Based on these findings, antibiotic therapy was adapted by the end of August 2013 and included cycloserine, capreomycin, PAS, ethambutol, meropenem, clavulanic acid (in the form of amoxicillin/clavulanic acid), and inhaled amikacin. However, four subsequent sputum samples remained smear-positive and culture-positive. Newly acquired resistance to aminoglycosides/capreomycin was noted in January 2014 based on phenotypic DST. Therapy was adjusted in March 2014 to include cycloserine, PAS, ethambutol, levofloxacin, sulfamethoxazole/trimethoprim (cotrim), and delamanid on a compassionate basis. In June 2014, three consecutive sputum samples were smear-negative but remained culture-positive. In July 2014, the sputum became smearpositive indicating a further treatment failure. At that time, the patient developed serious depression due to isolation and associated psychological stress. On August 13 th 2014 the patient s chest CT images showed multiple cavities bronchiectasis, star-shaped consolidations and tree-in-bud pattern in upper and lower lobes as compared to CT images of Februray 9 th 3

5 2011 (Figure S1 Panels A and B). Finally, surgical lobectomies of both the right and left upper lobes and segment S6 of the right and left inferior were performed in August and September The patient suffered from hypoxemia and partial respiratory failure due to the combined chronic obstructive pulmonary disease and postsurgical restriction. Because delamanid was not made available for phenotypic DST by the manufacturer, we performed delamanid DST using a commercial tablet formulation. This test revealed that isolates starting from June 2014 onward showed a >30-fold increase in MIC for delamanid (Table S2), consistent with delamanid resistance. Genetic testing also suggested resistance to delamanid by revealing an acquired mutation in fbia (D49T), a gene previously linked to nitroimidazole resistance (Tables 1, S2 and S4). 4 Administration of delamanid was discontinued in December Since undergoing surgery in August and September 2014, the patient s sputum has remained smear-negative and culture-negative. Surgical therapy in this patient appears to have been decisive in controlling the disease. The patient was released from the hospital in February Cycloserine was stopped due to polyneuropathy and the patient is currently (March 2015) treated with a 7-drug regimen consisting of PAS, ethambutol, amikacin, levofloxacin, meropenem, clavulanic acid (in the form of amoxicillin/clavulanic acid) and clarithromycin. During the whole period of drug treatment every single dose intake was observed by employees of the hospital or employees of a specialized service (Spitex) in the ambulatory setting to ensure an optimal adherence for all orally taken medications. Additionally, the intravenous given medications were performed by specialized nurses on an everyday basis (directly observed treatment; DOT). After a few complications the patient recovered finally, he regained his strength and is mastering his everyday life. In June 2015, he developed symmetric hearing loss, presumably due to ototoxicity of amikacin, after which amikacin was stopped. 4

6 Supplementary Methods: Clinical specimens were decontaminated using the N-acetyl-L-cysteine-sodium hydroxide method. 5 Auramine-rhodamine fluorochrome staining was used for acid-fast bacteria (AFB) microscopic examination; AFB positive results were confirmed by using Ziehl-Neelsen staining. 6 Mycobacteria were recovered on standard culture media (7H11 plates and BBL MGIT [Becton, Dickinson and Company, Franklin Lakes, NJ, USA]) by incubation at 37 C for a maximum of 7 weeks. Positive MGIT tubes were subcultured on 7H10 plates. After three weeks of growth at 37 o C three loops of bacterial cells were scraped from the 7H10 plate and used for chromosomal DNA extraction using a phenol-chloroform extraction protocol as described before. 7 Six serial M. tuberculosis isolates of the patient (Figure 1, Table S1) were selected for whole genome sequencing (WGS). WGS was performed using the Illumina platform to a median sequencing depth of 154-fold. Sequencing reads were mapped to a reference genome and mutations annotated as previously reported. 8 Mutations in repetitive regions were removed. Additionally, the breseq-pipeline was used to compare mutation calls and obtain proportions for low-frequency alleles. 9 Raw sequencing data are available under study accession number PRJEB8800 (European Nucleotide Archive, runs ERR789235, ERR789234, ERR789238, ERR789236, ERR789237, ERR789239). WGS results were compared with genotypic results obtained with line-probe assays and Sanger amplicon sequencing (Table S2). 1,2 fbia PCR amplification followed by sequencing was performed using three primer pairs: pair 1 FbiAF1 5`-GGCCAACCGCGAAATGACACTGAT-3`/ FbiAR1 5`-GCGCACCCACCACTCCTGAAAAT-3`, pair 2 FbiAF2 5`-GGCCGGCTACCCCCTGTCAC-3/ FbiAR2 5`-CCGCACCGTCACCCCGTCAATCT-3` and pair 3 FbiAF3 5`-GCCGACGCCGACATCATCAT-3`/ FbiAR3 5`-CAGCCGGCCCTCGCATTTGGACA-3`. PCR conditions were 5 minutes at 95 C followed 5

7 by 40 cycles of 30 seconds at 95 C, 30 seconds at 50 C and 70 seconds at 72 C. After a final 7 minutes at 72 C the reactions were stopped by cooling to 4 C. Quantitative phenotypic DST was performed in the BACTEC MGIT 960 system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). 10 Supplementary Results: The WGS data revealed a total of 5 variable nucleotide positions and 0-3 single nucleotide polymorphisms (SNPs) between any two consecutive isolates (excluding drug resistance conferring mutations). Such a close phylogenetic relationship is consistent with intra-host evolution of a single M. tuberculosis strain, as opposed to reinfection or mixed infection with different strains. This strain belonged to the Lineage 2/Beijing family of M. tuberculosis, which has been associated with drug resistance in many parts of the world. 11 WGS identified 9 mutations associated with drug resistance shared by all six isolates, corroborating the phenotypic and genotypic DST results described above (Figure 1, Table S2 and S4). The detection of a D94Y substitution in GyrA, which confers resistance to fluoroquinolones in all isolates, indicates that the initial MDR strain isolated in January 2011 was already pre-xdr (Figure 1). Moreover, all strains shared a D485Y change in RpoC. Mutations in rpoc have been suggested to compensate for fitness costs associated with rifampicin resistance-conferring mutations in rpob. 12 The V630G mutation in GyrB has not been reported before, but some mutations in GyrB have been shown to confer resistance to fluoroquinolones. 13 Alternatively, given that the isolates described here also harbored the D94Y mutation in GyrA, the GyrB mutation might play a compensatory role. 14 More work is needed to differentiate between these possibilities. The genome data also confirmed that the patient developed resistance to bedaquiline and clofazimine due to the V1A mutation in MmpR (Rv0678) present in all isolates obtained after 6

8 the patient relapsed (Figure 1). MmpR is a transcriptional repressor of the multi-substrate efflux pump MmpL5. Non-synonymous and missense mutations in MmpR have been shown to confer cross-resistance to bedaquiline and clofazimine by inactivating MmpR. 3 Our finding supports the hypothesis that clinically relevant bedaquiline resistance is mainly driven by an efflux mediated mechanism. All patient isolates were wild type in the bedaquiline drug target ATP synthase AtpE. 2,15 Moreover, we found that the MmpR mutation persisted despite bedaquiline being discontinued in February 2012, suggesting it causes little fitness cost to the bacteria in absence of the drug. 14,16 In addition to the drug resistance mutations that were stable (i.e. fixed) across the isolates, several other drug resistance mutations fluctuated over time, changing in frequency from one isolate to the next. 17 Such mutational fluctuations give rise to heteroresistance, a phenomenon that can greatly affect the results of molecular and phenotypic DST. 18 One striking example we observed was the fluctuation of the G1491T mutation in rrs (Table S3), which causes high-level resistance to amikacin and capreomycin. Note that the resistant subpopulation carrying the corresponding mutation was found in <10% of the population (Table S3). The rrs G1491T mutation is associated with a substantial fitness cost, providing an explanation for the observed intra-patient diversity. 19, 20 Two fluctuating mutations were observed in rrl (Table S2). Mutations in rrl have been associated with resistance to linezolid. 21,22 This notion was supported here given that the initial isolate from January 2011 was phenotypically resistant to linezolid and showed 100% WGS reads supporting a mutation in rrl (Figure 1). Intriguingly, the linezolid resistance associated mutation decreased in frequency in the subsequent isolates. The WGS data supported increasing resistance to delamanid starting in June 2014 as the mutant form of fbia (Rv3261) accounted for 25% of sequencing reads in isolate from June 2014 and increased to 65% of reads in isolate from July 2014 (Figure 1, Table S4). This heterogeneity was also observed using standard Sanger sequencing. Mutations in fbia and other genes involved in the synthesis of the deazaflavin cofactor F420 7

9 have previously been shown to confer resistance to nitroimidazoles, including delamanid. 4,23 Because the D49T mutation was not observed in any of the isolates pre-dating delamanid treatment, our findings strongly suggest that this mutation was acquired de novo during treatment and thus likely causes resistance. In addition, a second mutation was observed that may explain an increase in delamanid resistance. WGS revealed a transient increase in a subpopulation with a frameshift insertion in Rv0407 (Figure 1). Rv0407 encodes a cofactor F420-dependent glucose 6-phosphate dehydrogenase Fgd1 and is believed to activate nitroimidazoles. Loss of this gene confers nitroimidazole resistance in M. tuberculosis. 24 A polymorphism in FbiA R175H was observed. However, this polymorphism was present in all isolates from both before and during delamanid therapy, thus representing a natural polymorphism unlikely to be involved in delamanid resistance. Comparison to a large collection of M. tuberculosis genome sequences (Table S4) indicates that this is a strain specific polymorphism, pointing to the necessity not only to consider lineage-specific polymorphisms, but also strain-specific polymorphisms in the identification of drug resistance mutations. 25 8

10 Supplementary References 1. Ritter C, Lucke K, Sirgel FA, Warren RW, van Helden PD, Böttger EC, Bloemberg GV Evaluation of the AID TB resistance line probe assay for rapid detection of genetic alterations associated with drug resistance in Mycobacterium tuberculosis strains. J Clin Microbiol 52: Somoskovi A, Bruderer V, Hömke R, et al. A mutation associated with clofazimine and bedaquiline cross-resistance in MDR-TB following bedaquiline treatment. Eur Respir J 2015;45: Hartkoorn RC, Uplekar S, Cole ST. Cross-resistance between clofazimine and bedaquiline through upregulation of MmpL5 in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2014;58: Choi KP, Bair TB, Bae YM, et al. Use of transposon Tn5367 mutagenesis and a nitroimidazopyran-based selection system to demonstrate a requirement for fbia and fbib in coenzyme F(420) biosynthesis by Mycobacterium bovis BCG. J Bacteriol 2001;183: Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory Centers for Disease Control and Prevention, Atlanta,GA. 6. IsenbergHD (ed) Clinical microbiology procedures handbook, vol 1. American Society for Microbiology, Washington, DC. 7. van Embden JD, Cave MD, Crawford JT et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993;31(2): Comas I, Chakravartti J, Small PM, et al. Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved. Nat Genet 2010;42:

11 9. Deatherage DE, Barrick JE. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol Biol 2014;1151: Springer B, Lucke K, Calligaris-Maibach R, et al. Quantitative drug susceptibility testing of Mycobacterium tuberculosis by use of MGIT 960 and EpiCenter instrumentation. J Clin Microbiol 2009;47: Borrell S, Gagneux S. Infectiousness, reproductive fitness and evolution of drug resistant Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2009; 13: Comas I, Borrell S, Roetzer A, et al. Whole-genome sequencing of rifampicinresistant Mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes. Nat Genet 2011;44: Maruri F, Sterling TR, Kaiga AW, et al. A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system. J Antimicrob Chemother 2012;67: Trauner A, Borrell S, Reither K, et al. Evolution of drug resistance in tuberculosis: recent progress and implications for diagnosis and therapy. Drugs 2014;74: Andries K, Villellas C, Coeck N, et al. Acquired resistance of Mycobacterium tuberculosis to bedaquiline. PLoS One 2014;9(7):e Böttger EC, Springer B, Pletschette M, Sander P. Fitness of antibiotic-resistant microorganisms and compensatory mutations. Nat Med 1998;4: Meier A, Heifets L, Wallace RJ Jr, et al. Molecular mechanisms of clarithromycin resistance in Mycobacterium avium: observation of multiple 23S rdna mutations in a clonal population. J Infect Dis 1996;174: Streicher EM, Bergval I, Dheda K, et al. Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing. Antimicrob Agents Chemother 2012;56:

12 19. Shcherbakov D, Akbergenov R, Matt T, et al. Directed mutagenesis of Mycobacterium smegmatis 16S rrna to reconstruct the in-vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis. Mol Microbiol. 2010;77: Akbergenov R, Shcherbakov D, Matt T, et al. Molecular basis for the selectivity of antituberculosis compounds capreomycin and viomycin. Antimicrob Agents Chemother 2011;55: Sander P, Belova L, Kidan YG, et al. Ribosomal and non-ribosomal resistance to oxazolidinones: species-specific idiosyncrasy of ribosomal alterations. Mol Microbiol 2002;46: Hillemann D, Rüsch-Gerdes S, Richter E. In vitro-selected linezolid-resistant Mycobacterium tuberculosis mutants. Antimicrob Agents Chemother 2008;52: Feuerriegel S, Köser CU, Baù D, et al. Impact of Fgd1 and ddn diversity in Mycobacterium tuberculosis complex on in vitro susceptibility to PA-824. Antimicrob Agents Chemother 2011;55: Stover CK, Warrener P, VanDevanter DR, et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 2000;405: Müller B, Borrell S, Rose G, et al. The heterogeneous evolution of multidrug-resistant Mycobacterium tuberculosis. Trends Genet. 2013;29:

13 Supplementary Tables Table S1. M. tuberculosis isolates studied. Date of clinical sample IMM internal number Specimen a Microscopy for acid-fast bacilli b Culture for M. tuberculosis Drug susceptibility testing 1 Comment 04 Jan , 2 culture c not applicable not applicable done MDR 30 Apr sputum ++ positive done 14 Jun sputum ++ positive done 21 Jun sputum - positive - 22 Jul sputum - positive done 12 Aug sputum - positive - 24 Aug BAL ++ positive done Sep 2011 Feb 2012: Bedaquilin treatment 19 Oct sputum - positive - 11 Nov sputum - negative - 16 Dec TS - negative - 05 Jan sputum - negative - 30 Aug ,2 sputum +++ positive done Bedaquiline resistance 30 Aug sputum +++ positive - 31 Aug sputum ++ positive - 14 Sep sputum +++ positive - 15 Sep sputum +++ positive - 14 Oct sputum +++ positive - 06 Dec , 2 sputum ++ positive done XDR 06 Jan sputum ++ positive - 03 Feb sputum +++ positive done 15 Mar , 2 sputum ++ positive done March 2014-Dec 2014: Delamanid treatment 30 May sputum - positive - 04 Jun sputum - positive done Delamanid resistance 11 Jun sputum (+) positive - 18 Jun sputum - positive - 10 Jul , 2 sputum +++ positive done 17 Jul TS + positive - 15 Aug culture c not applicable not applicable done 20 Oct sputum - negative - 27 Oct sputum - negative - 12

14 03 Nov sputum - negative - 15 Dec sputum - negative - a TS, tracheal secretion; BAL, bronchoalveolar lavage; b Microscopy. - negative, (+) doubtful, + low load AFB, ++ medium load AFB and +++ high load AFB. c Culture received from an external laboratory isolated from sputum 1 For detailed phenotypic and genotypic drug susceptibility testing of selected isolates (grey marked) see Table S2. 2 Whole genome sequencing performed, see Fig 1 and Table S4. 13

15 Table S2. Quantitative phenotypic drug susceptibility testing A results and resistance-associated mutations identified by line-probe assays and sequencing of amplified gene fragments. Isolate number Date 04 Jan Aug Dec Feb Mar Jun Jul Aug 2014 Remark 1st isolate (pre-xdr) relapse isolate 1st XDR isolate INH (mg/l) 0.1 R R R R R R R R 1 R R R R R R R R 3 R R R R R R R R 10 R R R R R R R R inha promoter WT WT B WT B WT WT WT WT WT katg S315T S315T S315T S315T S315T S315T S315T S315T ETH (mg/l) 5 R R R R R R R R 10 R R R R R R R R 25 R R R R R R R R F349 del nt1047 C F349 del etha nt1047 C nd nd F349 del nt1047 C F349 del nt1047 C F349 del nt1047 C nd RIF (mg/l) 1 R R R R R R R R 4 R R R R R R R R 20 R R R R R R R R RBT (mg/l) 0.1 R R R R R R R R 0.4 R R R R R R R R 2 S R R R I I I R rpob S531L/W S531L/W S531L/W S531L/W S531L/W S531L/W S531L/W S531L/W EMB (mg/l) 5 S S S S I S S S 12.5 S S S S S S S S 50 S S S S S S S S embb D328Y D D328Y D nd nd D328Y D D328Y D D328Y D nd 14

16 PZA (mg/l) 100 R R R R R R R R Pyrazinamidase negative negative negative negative negative negative negative negative Gly107Stop Gly107Stop Gly107Stop Gly107Stop Gly107Stop Gly107Stop Gly107Stop nd pnca (ntg319t) (ntg319t) (ntg319t) (ntg319t) (ntg319t) (ntg319t) (ntg319t) STR (mg/l) 1 R R R R R R R R 4 S S R R R R R R 20 S S R R R R R R rspl K88R K88R K88R K88R K88R K88R K88R K88R rrs (SM mutations) WT WT WT WT WT WT WT WT AMK (mg/l) 1 S S R S R S S S 4 S S R S I S S S 20 S S R S S S S S CAP (mg/l) 2.5 I S R R R R R R 5 S S R R R S S S 25 S S I S S S S S rrs(16s rrna) (heteroresistance) WT WT MGIT: WT AMK MIGT: G1491T CAP MGIT: G1491T MGIT: WT CAP MGIT: WT MGIT: WT AMK MGIT: G1491T CAP MGIT: WT MGIT: WT CAP MGIT: WT nd MGIT: WT CAP MGIT: WT tlya WT WT A17E A17E A17E A17E A17E A17E PAS (mg/l) 4 S I R I R S S S 16 S I I S R S S S 64 S S S S S S S S thya WT E WT E WT E WT E WT E WT E WT E WT E MOX (mg/l) 0.25 R R R R R R R R 0.5 R R R R R R R R 2.5 I R I I R S S S 7.5 nd S S S S S S S gyra D94Y D94Y D94Y D94Y D94Y D94Y D94Y D94Y 15

17 LIN (mg/l) 1 R R R R R R R R 4 R R R R R S R I 16 S S S S S S S S rrl (23S rrna) G2576T A2572C, G2576T nd A2572C, G2576T A2572C, G2576T MGIT: WT LIN MGIT: 2572WT, G2576T nd nd rplc WT WT nd WT WT WT WT nd DCS (mg/l) 50 S S S S S S S S CLO (mg/l) 0.25 R R R R R R R R 0.5 R R R R R R R R 1 S R R R R R R R 4 S R I S S S S S BDQ (mg/l) 0.4 R R nd nd nd nd R nd 0.8 S R nd nd nd nd R nd 1.6 S R nd nd nd nd R nd 3.2 S R nd nd nd nd R nd 6.4 S S nd nd nd nd S nd atpe WT WT WT WT WT WT WT nd Rv0678 (mmpr) WT V1A V1A nd nd V1A nd V1A DEL (mg/l) nd nd nd R R R R R 0.01 nd nd nd S S R R R 0.02 nd nd nd S S R R R 0.04 nd nd nd S S R R R 0.08 nd nd nd S S R R R 0.16 nd nd nd S S R R R 0.32 nd nd nd S S R R R fgd1, ddn, fbib, fbic WT WT WT WT WT WT WT WT fbia WT F WT F WT F WT F WT F D49T F D49T F D49T F 16

18 A Quantitative drug susceptibility testing was done using the BACTEC MGIT 960 / TB exist system (Becton-Dickinson Inc., East Rutherford, NJ, USA) Critical concentrations of first- and second-line antituberculosis drugs in the MGIT 960 system are highlighted in bold underlined. Critical concentrations are not established for para-aminosalicylic acid, cycloserine, clofazimine, bedaquiline and delamanid in the BACTEC MGIT 960 system. TB: tuberculosis. B inha sequencing: WT C polymorphism EthA S266R D polymorphism ThyA G204V E EmbB D328Y: mutation putatively affecting susceptibility to ethambutol. F polymorphism FbiA R175H Abbreviations: INH, isoniazid; ETH, ethionamid; RIF, rifampicin; RBT, rifabutin; EMB, ethambutol; PZA, pyrazinamide; STR, streptomycin; AMK, amikacin; CAP, capreomycin; PAS, para-aminosalicylic acid; MOX, moxifloxacin; LIN, linezolid; DCS, cycloserine; CLO, clofazimine; BDQ, bedaquiline; DEL, delamanid. MGIT: MGIT tube without drug, AMK MGIT: MGIT tube containing amikacin, CAP MGIT: MGIT tube containing capreomycin. 17

19 Table S3. Fluctuations in mutations associated with drug resistance in Mycobacterium tuberculosis heterogenous populations. drug affected amikacin linezolid mutation Isolate Date rrs G1491T rrl A2572C, rrl G2576T obtained Jan Aug Dec 2013 (+) nd nd Feb Mar 2014 (+) Jun (+) (+) Jul 2014 nd nd nd Aug nd nd - : wild-type +++ : homogenous mutation observed in original MGIT subculture tube (homogenous population) (+) : mutation observed only in drug containing MGIT tube, >1% and < 10% of the population contains the mutation (heteroresistance) nd, not determined 18

20 Table S4. Frequencies of observed mutations. Only mutations listed in TBDreamDB and considered to be involved in drug resistance. Unfixed mutations (frequency of <100%) whose frequency did not reach at least 10% of sequencing reads in at least one sample were not considered. (see attached Excel file) 19

21 Supplementary Figure Figure S1. Patient`s chest computer tomography (CT) images. Panels A1-2: Patient`s chest CT images at presentation before treatment on February 9 th 2011 show multiple cavities bronchiectasis and star-shaped consolidations in upper and lower lobes on both sides. Panels B1-2: Patient`s chest CT images prior to surgery on August 13 th 2014 show multiple cavities bronchiectasis, star-shaped consolidations and tree-in-bud pattern in upper and lower lobes. 20

22 February 9 th 2011 (at presentation) A1 A2 August 13 th 2014 (prior to surgery) B1 B2 21