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

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

Introduction:

Novel and re-purposed drugs

DRUG BEDAQUILINE CLOFAZIMINE LINEZOLID

Type Novel Re-purposed Re-purposed

Class Diarylquinoline Riminophenazine Oxazolidinone

Genes associated

with resistanceatpE, rv0678

rv0678, rv1979c,

rv2535rplC, 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

Problem Statement

o Resistance to novel drugs is a constant threat

o Drugs are being used at a limited scale

oRegimens 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

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

Objectives

o Apply two approaches and compare mutants obtained

o Levels of resistance generated

oDifference in resistance profiles from reference strains

oTo identify RAVs associated with high MIC values for BDQ, CFZ and LZD

6

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 (INHR)

o ATCC35827 Kanamycin resistant (KANR)

o ATCC35828 Pyrazinamide resistant (PZAR)

o ATCC35838 Rifampicin resistant (RIFR)

o MGIT960 MIC performed using provisional critical concentrations

7

Methods:

Serial passaging approacho 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)oAll mutants after completing 5 to 6 passages

oGenotypic confirmation (WGS)oAll 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

Methods:

Isolation of spontaneous mutants

o PZAR and WT ATCC strains oBest performing strain and control

o12 replicate cultures

oDrug-naïve till one step selection (2x proposed CC)

oThree mutants selected based on size

oPhenotypic confirmationo >36 mutants per drug

oGenotypic confirmationo 3 mutants with low, mid and high MIC values within the range

9

G187C

G183T

A83C

A83G (3)

T407C

T131C (2)

C204A

G74A

T2303G

529_543del

T460C (4)

No. of strains 5 5 4

No. of passages 5 5 6

Pre-induction MIC (µg/ml)

MGIT9600.5-1 0.5-1 1-2

Post-induction MIC (µg/ml)

MGIT960>8 4->4 8->8

Results

0

1

2

3

4

5

6

7

atpE rv0678 rv0678 mmpl5 rplC rplV

Nu

mb

er

of

mu

tati

on

s

BDQ

Distribution of RAVs for serial passage mutants

CFZ LZD

G187C

G183T

A83C

A83G (3)

T407C

T131C (2)

C204A

G74A 529_543

del

T460C (4)

T461C

201_206

del

A63T T2303G

10 10

Selection of spontaneous mutants

Drug

mutantATCC Strain

Total

no. of

mutants

Mutation

frequency

Confirmation of resistance in selected putative

mutants

Post-

selection

MIC (µg/ml)

RAV ∆NT ∆AA

BDQ

WT 1 ~6x 10-9 >8 atpE A83T Asp28Val

PZAR 619 ~4x 10-7

4 rv0678 C403G Arg135Gly

8 atpE A83G Asp28Gly

>8 atpE G187C Ala63Pro

CFZ

WT 6937 ~5x 10-5

2 rv0678 193delG Ile67fs

4 rv0678 193delG Ile67fs

4 rv0678A65T Gln22Leu

G61A Gly21Lys

PZAR 12371 ~7x 10-7

1 rv0678 T407C Leu136Pro

2 rv0678 C214T Arg72Trp

4 rv0678 A97G Thr33Ala

LZD

WT 2 ~1x 10-8>8 rplC T460C Cys154Arg

>8 rplC T460C Cys154Arg

PZAR 13 ~1x 10-7

4 rrl G2270C -

4 rrl A2810C -

8 rplC T460C Cys154Arg

>8 rplC T460C Cys154Arg

Results

11 11

Discussion

o RAVs correspond with:

oPreviously described RAVs for in vitro mutants (BDQ and CFZ)

oPreviously 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

Limitations

o Number of strains limited for the selection and analysis of spontaneous

mutants

o Laborious and costly

o Cost of sequencing all mutants prohibitive

o PZAR strain in vitro: faster onset of resistance to these three drugs

oComplete the work for various resistance profiles

13

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

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

Acknowledgements

Supervisors: Prof Remco PH Peters, Dr Shaheed Vally Omar and Dr Nazir Ismail

Project funding Personal funding

Staff at NICD-CTB

Conference bursary

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17

THANK YOU

18

Acquired Resistance of Mycobacterium tuberculosis to Bedaquiline

Koen Andries, Cristina Villellas, Nele Coeck, Anil Koul et al.

19

Mutants obtained through serial passaging approach. MIC values determined using MGIT 960 SYSTEM. RAVs identified with WGS.

Drug

mutant

ATCC

strain

Pre-

induction

MIC

(µg/ml)

No. of

passages

Confirmation of resistance in putative mutants

Post- induction

MIC

(µg/ml)

RAVs ∆ NT ∆ AA

BDQ

WT 1 5 >8 atpE G187C Ala63Pro

INHR 1 5 >8atpE A83G Asp28Gly

rv0678 T461C Leu154Pro

KANR 1 5 >8atpE A83C Asp28Ala

rv0678 201_206del Ser68_Thr69del

PZAR 1 5 >8atpE A83G Asp28Gly

atpE G183T Glu61Asp

RIFR 0.5 5 >8atpE A83G Asp28Gly

rv0678 A63T Glu21Asp

CFZ

WT 0.5 5 4mmpl5 T2303G Leu768Trp

rv0678 G74A Gly25Asp

INHR 0.5 5 >4 rv0678 T131C Leu44Pro

KANR 0.5 5 4 rv0678 T407C Leu136Pro

PZAR 1 5 >4 rv0678 C204A Ser68Arg

RIFR 0.5 5 4 rv0678 T131C Leu44Pro

LZD

WT 1 2 2 Not available

INHR 2 6 >8rplC T460C Cys154Arg

rplV 529_543del Lys181_Lys185del

KANR 1 6 8 rplC T460C Cys154Arg

PZAR 2 6 >8 rplC T460C Cys154Arg

RIFR 1 6 8 rplC T460C Cys154Arg20 19

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

PZAR Serial Passage BDQ 1 >8atpE A83G Asp28Gly

atpE G183T Glu61Asp

WT Spontaneous BDQ 1 >8 atpE A83T Asp28Val

PZAR Spontaneous BDQ 1 4 rv0678 C403G Arg135Gly

PZAR Spontaneous BDQ 1 8 atpE A83G Asp28Gly

PZAR Spontaneous BDQ 1 >8 atpE G187C Ala63Pro

WT Serial Passage CFZ 0.5 4Mmpl5 T2303G Leu768Trp

Rv0678 G74A Gly25Asp

PZAR Serial Passage CFZ 1 >4 rv0678 C204A Ser68Arg

PZAR Spontaneous CFZ 1 1 rv0678 T407C Leu136Pro

PZAR Spontaneous CFZ 1 2 Rv0678 C214T Arg72Trp

PZAR Spontaneous CFZ 1 4 Rv0678 A97G Thr33Ala

WT Spontaneous CFZ 0.5 2 rv0678 193delG Ile67fs

WT Spontaneous CFZ 0.5 4 rv0678 193delG Ile67fs

WT Spontaneous CFZ 0.5 4 rv0678A65T Gln22Leu

G61A Gly21Lys

WT Serial Passage LZD 1 2 NOT AVAILABLE

PZAR Serial Passage LZD 2 >8 rplC T460C Cys154Arg

WT Spontaneous LZD 1 >8 rplC T460C Cys154Arg

WT Spontaneous LZD 1 >8 rplC T460C Cys154Arg

PZAR Spontaneous LZD 2 4 rrl G2270C -

PZAR Spontaneous LZD 2 4 rrl A2810C -

PZAR Spontaneous LZD 2 8b rplC T460C Cys154Arg

PZAR Spontaneous LZD 2 >8c rplC T460C Cys154Arg

Comparison of RAV between mutants from both approaches and to literature

21 20

Literature highlights

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