N AB I L A I S M AI L

Transcription

1 Evaluation of methods for generation of in vitro mutants resistant to bedaquiline, clofazimine and linezolid using Mycobacterium tuberculosis reference strains NABILA ISMAIL 1, SHAHEED V OMAR 2, NAZIR A ISMAIL 1,2 AND REMCO PH PETERS 1,3 (1) Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria (2) Centre for Tuberculosis, WHO Supranational TB Reference Laboratory, National Institute for Communicable Diseases, National Health Laboratory Service (3) Department of Medical Microbiology, CAPHRI School of Public Health and Primary Care, Maastricht University Medical Centre, Maastricht University 1

2 Introduction: Drug-resistant TB o 10.4 million new TB cases globally (WHO, 2016) o Approximately half a million are DR-TB o DR-TB treatment o Drug susceptibility testing (DST): expensive and requires infrastructure o Second-line drugs: more expensive and toxic o Cure rates low o Novel and repurposed drugs used in various regimens o FOCUS: bedaquiline (BDQ), clofazimine (CFZ) and linezolid (LZD) 2

3 Introduction: Novel and re-purposed drugs DRUG BEDAQUILINE CLOFAZIMINE LINEZOLID Type Novel Re-purposed Re-purposed Class Diarylquinoline Riminophenazine Oxazolidinone Genes associated with resistance atpe, rv0678 rv0678, rv1979c, rv2535 rplc, rrl DR-TB regimen incorporation WHO category 5, compassionate use, MDR-TB and XDR-TB WHO shorter MDR-TB regimen WHO category 5, reserved use for MDR-TB and XDR- TB 3

4 Problem Statement o Resistance to novel drugs is a constant threat o Drugs are being used at a limited scale o Regimens including BDQ, CFZ or LZD are still in clinical trials o Information around genetic basis of resistance in vivo: lack complete picture particularly for BDQ and CFZ 4

5 Aim To evaluate two approaches for generation or selection of in vitro mutants to BDQ, CFZ and LZD and to identify resistance associated variants (RAVs) for each drug 5

6 Objectives o Apply two approaches and compare mutants obtained o Levels of resistance generated o Difference in resistance profiles from reference strains oto identify RAVs associated with high MIC values for BDQ, CFZ and LZD 6

7 Methods: Study Design o Two different mutant generation approaches o Serial passaging approach-induction o Spontaneous approach- adapted from Luria-Delbrück fluctuation assay o ATCC reference strains o ATCC27294 Fully susceptible (WT) o ATCC35822 Isoniazid resistant (INH R ) o ATCC35827 Kanamycin resistant (KAN R ) o ATCC35828 Pyrazinamide resistant (PZA R ) o ATCC35838 Rifampicin resistant (RIF R ) o MGIT960 MIC performed using provisional critical concentrations 7

8 Methods: Serial passaging approach o Maximum 6 passages o All 5 ATCC reference strains o Concentrated inoculum o Increasing drug concentrations o 0.5x 8x proposed CC ophenotypic confirmation (MGIT960 MIC) o All mutants after completing 5 to 6 passages ogenotypic confirmation (WGS) o All mutants after phenotypic confirmation Plate 100 µl onto: Drug-free plates and plates containing 0.5X proposed CC of BDQ, CFZ or LZD 8 8

9 Methods: Isolation of spontaneous mutants o PZA R and WT ATCC strains o Best performing strain and control o12 replicate cultures odrug-naïve till one step selection (2x proposed CC) othree mutants selected based on size ophenotypic confirmation o >36 mutants per drug ogenotypic confirmation o 3 mutants with low, mid and high MIC values within the range 9

10 Number of mutations Results Distribution of RAVs for serial passage mutants G187C G183T A83C G187C A83G (3) G183T T461C A83C 201_206 A83G (3) del A63T T407C T131C (2) T407C C204A T2303G G74A T131C (2) 529_543del T460C (4) atpe rv0678 rv0678 mmpl5 rplc rplv BDQ C204A G74A CFZ T2303G T460C (4) LZD 529_543 del No. of strains No. of passages Pre-induction MIC (µg/ml) MGIT Post-induction MIC (µg/ml) MGIT960 >8 4->4 8->

11 Results Drug mutant BDQ CFZ LZD ATCC Strain Total no. of mutants Selection of spontaneous mutants Mutation frequency Confirmation of resistance in selected putative mutants Postselection MIC (µg/ml) RAV NT AA WT 1 ~6x 10-9 >8 atpe A83T Asp28Val PZA R 619 ~4x atpe A83G Asp28Gly 4 rv0678 C403G Arg135Gly >8 atpe G187C Ala63Pro 2 rv delG Ile67fs 4 rv delG Ile67fs WT 6937 ~5x 10-5 A65T Gln22Leu 4 rv0678 G61A Gly21Lys PZA R ~7x rv0678 C214T Arg72Trp 1 rv0678 T407C Leu136Pro 4 rv0678 A97G Thr33Ala >8 rplc T460C Cys154Arg WT 2 ~1x 10-8 >8 rplc T460C Cys154Arg 4 rrl G2270C - PZA R 13 ~1x rrl A2810C - 8 rplc T460C Cys154Arg >8 rplc T460C Cys154Arg 11 11

12 Discussion o RAVs correspond with: o Previously described RAVs for in vitro mutants (BDQ and CFZ) o Previously described RAVs for in vitro mutants and clinically resistant isolates (LZD) oserial passaging method mimics in vivo situation (drug penetration to granulomas) o number of passages can be altered to reflect the development of low level resistance 12

13 Limitations o Number of strains limited for the selection and analysis of spontaneous mutants o o Laborious and costly Cost of sequencing all mutants prohibitive o PZA R strain in vitro: faster onset of resistance to these three drugs o Complete the work for various resistance profiles 13

14 Conclusion Identification of RAVs useful for: o Learning about the development of drug resistance in vitro o e.g. efflux pumps vs targeted mutations o Understanding drug targets and pathways o Determining hotspots: o Designing molecular diagnostics o Regimen design o Surveillance of drug resistance: o Informing DST 14

15 Future perspectives o Perform cross-resistance studies on BDQ and CFZ mutants o Clinical sample set o Low passage o Varying resistance profiles and lineages o Use the serial passaging approach o Only use BDQ and CFZ 15

16 Acknowledgements Supervisors: Prof Remco PH Peters, Dr Shaheed Vally Omar and Dr Nazir Ismail Project funding Personal funding Staff at NICD-CTB Conference bursary

17 References World Health Organization. Global Tuberculosis Report Geneva, 2016 Diacon AH, Pym A, Grobusch M, Patientia R, Rustomjee R, Page-Shipp L, et al. The diarylquinoline TMC207 for multidrug-resistant tuberculosis. N Engl J Med Jun 4;360(23): Andries K, Verhasselt P, Guillemont J, Gohlmann HW, Neefs JM, Winkler H, et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science Jan 14;307(5707): WHO. The use of bedaquiline in the treatment of multidrug resistant tuberculosis. In: Report EGM, ed Andries K, Villellas C, Coeck N, Thys K, Gevers T, Vranckx L, et al. Acquired Resistance of Mycobacterium tuberculosis to Bedaquiline. PloS one. 2014;9(7):e Xavier AS, Lakshmanan M. Delamanid: A new armor in combating drug-resistant tuberculosis. Journal of Pharmacology & Pharmacotherapeutics Jul-Sep;5(3):222-4 WHO. The shorter MDR-TB regimen (Internet): WHO Press; 2016 [23 June 2016]. Available from: Hartkoorn RC, Uplekar S, Cole ST. Cross-Resistance between Clofazimine and Bedaquiline through Upregulation of MmpL5 in Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy. 2014;58(5): Huitric E, Verhasselt P, Koul A, Andries K, Hoffner S, Andersson DI. Rates and mechanisms of resistance development in Mycobacterium tuberculosis to a novel diarylquinoline ATP synthase inhibitor. Antimicrob Agents Chemother. 2010;54(3): Petrella S, Cambau E, Chauffour A, Andries K, Jarlier V, Sougakoff W. Genetic basis for natural and acquired resistance to the diarylquinoline R in mycobacteria. Antimicrob Agents Chemother. 2006;50(8): Zhang S, Chen J, Cui P, Shi W, Zhang W, Zhang Y. Identification of novel mutations associated with clofazimine resistance in Mycobacterium tuberculosis. J Antimicrob Chemother. 2015;70(9): Zhang S, Chen J, Cui P, Shi W, Shi X, Niu H, et al. Mycobacterium tuberculosis Mutations Associated with Reduced Susceptibility to Linezolid. Antimicrob Agents Chemother. 2016;60(4): Lee M, Lee J, Carroll MW, Choi H, Min S, Song T, et al. Linezolid for treatment of chronic extensively drug-resistant tuberculosis. N Engl J Med. 2012;367(16): Balasubramanian V, Solapure S, Iyer H, Ghosh A, Sharma S, Kaur P, et al. Bactericidal activity and mechanism of action of AZD5847, a novel oxazolidinone for treatment of tuberculosis. Antimicrob Agents Chemother. 2014;58(1): McNeil MB, Dennison DD, Shelton CD, Parish T. In Vitro Isolation and Characterization of Oxazolidinone-Resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2017;61(10). Richter E, Rusch-Gerdes S, Hillemann D. First linezolid-resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2007;51(4): Hillemann D, Rusch-Gerdes S, Richter E. In vitro-selected linezolid-resistant Mycobacterium tuberculosis mutants. Antimicrob Agents Chemother. 2008;52(2): Somoskovi A, Bruderer V, Homke R, Bloemberg GV, Bottger EC. A mutation associated with clofazimine and bedaquiline cross-resistance in MDR-TB following bedaquiline treatment. Eur Respir J. 2015;45(2): Hoffmann H, Kohl TA, Hofmann-Thiel S, Merker M, Beckert P, Jaton K, et al. Delamanid and Bedaquiline Resistance in Mycobacterium tuberculosis Ancestral Beijing Genotype Causing Extensively Drug-Resistant Tuberculosis in a Tibetan Refugee. Am J Respir Crit Care Med. 2016;193(3): Xu J, Wang B, Hu M, Huo F, Guo S, Jing W, et al. Primary Clofazimine and Bedaquiline Resistance among Isolates from Patients with Multidrug-Resistant Tuberculosis. Antimicrob Agents Chemother. 2017;61(6). Villellas C, Coeck N, Meehan CJ, Lounis N, de Jong B, Rigouts L, et al. Unexpected high prevalence of resistance-associated Rv0678 variants in MDR-TB patients without documented prior use of clofazimine or bedaquiline. J Antimicrob Chemother. 2017;72(3):

18 THANK YOU 18

19 Acquired Resistance of Mycobacterium tuberculosis to Bedaquiline Koen Andries, Cristina Villellas, Nele Coeck, Anil Koul et al. 19

20 Drug mutant BDQ CFZ LZD Mutants obtained through serial passaging approach. MIC values determined using MGIT 960 SYSTEM. RAVs identified with WGS. ATCC Preinduction No. of Confirmation of resistance in putative mutants Post- induction strain MIC (µg/ml) passages MIC (µg/ml) RAVs NT AA WT 1 5 >8 atpe G187C Ala63Pro INH R 1 5 >8 atpe A83G Asp28Gly rv0678 T461C Leu154Pro KAN R 1 5 >8 atpe A83C Asp28Ala rv _206del Ser68_Thr69del PZA R 1 5 >8 atpe A83G Asp28Gly atpe G183T Glu61Asp RIF R >8 atpe A83G Asp28Gly rv0678 A63T Glu21Asp WT mmpl5 T2303G Leu768Trp rv0678 G74A Gly25Asp INH R >4 rv0678 T131C Leu44Pro KAN R rv0678 T407C Leu136Pro PZA R 1 5 >4 rv0678 C204A Ser68Arg RIF R rv0678 T131C Leu44Pro WT Not available INH R 2 6 >8 rplc T460C Cys154Arg rplv 529_543del Lys181_Lys185del KAN R rplc T460C Cys154Arg PZA R 2 6 >8 rplc T460C Cys154Arg RIF R rplc T460C Cys154Arg 20 19

21 Comparison of RAV between mutants from both approaches and to literature ATCC Strain Approach Drug mutant Pre-induction/ selection MIC (µg/ml) Post-induction/ selection MIC (µg/ml) RAVs NT AA WT Serial Passage BDQ 1 >8 atpe G187C Ala63Pro PZA R Serial Passage BDQ 1 >8 atpe A83G Asp28Gly atpe G183T Glu61Asp WT Spontaneous BDQ 1 >8 atpe A83T Asp28Val PZA R Spontaneous BDQ 1 4 rv0678 C403G Arg135Gly PZA R Spontaneous BDQ 1 8 atpe A83G Asp28Gly PZA R Spontaneous BDQ 1 >8 atpe G187C Ala63Pro WT Serial Passage CFZ Mmpl5 T2303G Leu768Trp Rv0678 G74A Gly25Asp PZA R Serial Passage CFZ 1 >4 rv0678 C204A Ser68Arg PZA R Spontaneous CFZ 1 1 rv0678 T407C Leu136Pro PZA R Spontaneous CFZ 1 2 Rv0678 C214T Arg72Trp PZA R Spontaneous CFZ 1 4 Rv0678 A97G Thr33Ala WT Spontaneous CFZ rv delG Ile67fs WT Spontaneous CFZ rv delG Ile67fs WT Spontaneous CFZ rv0678 A65T Gln22Leu G61A Gly21Lys WT Serial Passage LZD 1 2 NOT AVAILABLE PZA R Serial Passage LZD 2 >8 rplc T460C Cys154Arg WT Spontaneous LZD 1 >8 rplc T460C Cys154Arg WT Spontaneous LZD 1 >8 rplc T460C Cys154Arg PZA R Spontaneous LZD 2 4 rrl G2270C - PZA R Spontaneous LZD 2 4 rrl A2810C - PZA R Spontaneous LZD 2 8 b rplc T460C Cys154Arg PZA R Spontaneous LZD 2 >8 c rplc T460C Cys154Arg 21 20

22 Literature highlights 22