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, Maslennikova N., Leontev D., Zhuravlev V.,

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Russia during long-term treatment. Clinical Microbiology and Infection.

Supplementary material

Table S1. Summary on 11 patients who received bedaquiline treatment and their M. tuberculosis isolates.

N Diagnosis, year, HIV Phenotypic resistance prior to BDQ Treatment Efficiency of treatment Strain BDQ resistance mutations
treatment genotype

1 Disseminated TB, 2018, HIV HRSEthCsPAS / MDR ZELfxAmLzdBq Efficient, completed B0/W148 wt

2 FCT, 1999, HIV HRSЕKmCmEthOfxCsAmPAS /XDR ZMfxLzdPczBq Defaulted treatment, died of HIV B0/W148 Pre-existed, Rv0678

3 Infiltrative PTB, 2016 HRSEOfxEthPAS / pre-XDR CmZMfxTrdPczBq Treatment failure B0/W148 Emerged, Rv0678

4 FCT, 2010, HIV HSREKmCmEthOfxAmPAS / XDR ZCsCmMfxLfxPczBq Treatment failure, died of HIV B0/W148 wt

5 FCT, 2016 HRSZEKmCmOfxCsPth / XDR AmZLfxPASPczBq Defaulted treatment B0/W148 Emerged, atpE

6 FCT, 2007 HRSEKmOfx / XDR ZCmMfxTrdPthBq Efficient, completed B0/W148 wt

7 Bronchial TB, 2007 HRSEKmCmAmPthOfxPASCs / XDR ZLfxTrdPczBq Efficient, completed CAO wt

8 FCT, HIV HRSZKmCmAmOfxPth / XDR EСsLfxLzdPczBq Treatment failure CAO Emerged, Rv0678

9 Lung tuberculoma, 2005, HIV HSREKmEthOfxAmZPASCs / XDR ZLfxClzPczBq Treatment failure B0/W148 Emerged, Rv0678

10 FCT, 2009 HSREKmEtoOfxCs / XDR AmZLfxPASBq Treatment failure, died of TB B0/W148 Possibly pre-existed,
Rv0678

11 Infiltrative PTB, 2015 HRSZEEthOfxCmPAS / XDR AmCsLfxPczBq Efficient, completed B0/W148 wt

1
Abbreviations. FCT – fibrocavernous TB, pulmonary TB – PTB.

Antibiotics: H - isoniazid, R - rifampin, S - streptomycin, Е - ethambutol, Lfx - levofloxacin, Mfx - moxifloxacin, Ofx - ofloxacin, Cs - cycloserine, Am - amikacin,
Km – kanamycin, Cm - capreomycin, PAS - Para-aminosalicylic acid, Z - pyrazinamide, Pcz - perchlozone, Bq - bedaquiline, Clz – clofazimine, Lz – linezolid, Trd
– Terizidone, Eth – ethionamide, Pth – prothionamide

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 Table S2. Summary of mutations in bedaquiline resistance genes identified in M. tuberculosis isolates

Patient Gene Mutation, position in genome Codon and amino acid PAM1 value, in silico predicted Previously described (see
and gene change or frameshift effect, and comment/analysis References below this table)

2 Rv0678 779117 T>C Codon 43 CTG>CCG / PAM1=2; -

Leu>Pro Significant change, likely beneficial to
128 T>C
mycobacterial survival.
Leu43Pro substitution potentially
affects Rv0678: electrostatic potential
locally increased around nitrogen of
proline, and accessible surface area
increased by 102 Å2 (Fig. 1b).
Moreover, substitution to proline also
changes the folding of Rv0678: due to
rotationally blocked structure of the
side chain, proline restricts protein
folding (Bhagavan and Ha, 2015).
Since proline poorly adopts alpha-
helical structure, substitution of
leucine with proline in position 43
(alpha-helix) additionally disrupts the
protein (Betts and Russell, 2003).
3 Rv0678 779215 C>A Codon 76 CAA>AAA / PAM1=12; -
Gln>Lys Significant change, likely beneficial to
226 C>A
mycobacterial survival.
Gln76Lys substitution potentially
affects Rv0678: although accessible
surface area only minorly increased
(+22 Å2), electrostatic potential
dramatically changed from negative to

3
 positive (Fig. 1b).
3 Rv0678 779411-779419 Frameshift, changed protein Significant change (loss of function): -
ATCTGTTGG del (wild type amino acids 1-140, permanent derepression of mmpS5-
changed amino acids 141- mmpL5 system, leading to the
422-430 ATCTGTTGG del
163). bedaquiline efflux.
8 Rv0678 779096 C>T Codon 36 GCG>GTG PAM1=13; Clinical isolates from BDQ
Ala>Val Non-significant change of the clinical trial with BDQ resistance
107 C>T evolving spontaneously.
conformation.
MIC x8. Not accompanied by
The Rv0678 Ala36Val substitution
other Rv0678 or atpE mutations.
only modestly affects Rv0678, since it (Andries et al., 2014)
yields a minor change of surface area
(-28 Å2) and no change of electrostatic
potential.
8 Rv0678 779136 G>GC ins Frameshift, premature stop Significant change (loss of function): -
codon and truncated protein permanent derepression of mmpS5-
147 G>GC ins
(80 amino acid) mmpL5 system, leading to the
bedaquiline efflux.
8 Rv0678 779055 G>GGAACAG ins Codons 21-Glu and 22-Gln Non-significant change of the -
duplicated conformation.
66 G>GGAACAG ins
Duplication Glu-21-Gln-22 did not
change the conformation of Rv0678
(not shown) and has likely little effect
on the function of the mutant protein.
8 Rv0678 779187 G del Frameshift, premature stop Significant change (loss of function): 5 clinical isolates with mutation
codon - protein permanent derepression of mmpS5- emerged during treatment.
198 G del MIC x2-8. Heteroresistance.
changed/truncated from amino mmpL5 system, leading to the
(Zimenkov et al., 2017)
acid 65 onwards bedaquiline efflux.

4
9 Rv0678 779174-779183 Deletion of 3 amino acids and Significant change (loss of function): -
CCAGCAGCGG del frameshift, premature stop permanent derepression of mmpS5-
codon- protein mmpL5 system, leading to the
185-194 CCAGCAGCGG del
changed/truncated from amino bedaquiline efflux.
acid 62 onwards.
10 Rv0678 779187 G>GC ins Frameshift, premature stop Significant change (loss of function): -
codon- protein permanent derepression of mmpS5-
198 G>GC ins
changed/truncated from amino mmpL5 system, leading to the
acid 65 onwards bedaquiline efflux.
5 atpE 1461242 C>G Codon 66 ATC>ATG / PAM1=5; BDQ-resistant mutants derived
Ile>Met The AtpE Ile66Met substitution was from H37Rv and clinical isolates
198 C>G in vitro;
previously suggested to weaken the
MIC x4-30. Not accompanied by
bedaquiline. binding to AtpE [Salifu et
Rv0678 or atpE mutation in 5 of 6
al. 2020]. However this mutation has studies.
low impact on accessible surface area (Petrella et al., 2006; Huitric et
(+12Å2) and electrostatic potential al., 2010; Segala et al., 2012;
(Fig. 1b), and we consider its effect to Tantry et al., 2016, 2017; Andries
be uncertain. et al., 2014; Martinez et al., 2018)

Notes:
Rv0678: positions in H37Rv genome 778990-779487. The Rv0678 protein length - 165 amino acids.
atpE (Rv1305): positions in H37Rv genome 1461045-1461290. The AtpE protein length - 81 amino acids.
PAM1 - Point Accepted Mutation 1; it gives the probability (multiplied with 10,000) for the particular aa exchange to occur, given that 1% of the aa are changed.
A recent systematic review demonstrated that Rv0678 frameshifts at nucleotides 192–198 were among the most frequent mutations for bedaquiline resistance
although they were also identified in 4% of the isolates without prior exposure to bedaquiline or clofazimine (Kadura et al., 2020). Hypothetically, mutations in this
efflux-related gene could be selected as a complementary mechanism of resistance to other drugs during previous treatment. In our study, indels in this region at
nucleotide position 198 were found in two patients and seem emerged during bedaquiline treatment at least in one of them (Patient 8 in Fig. 1a).

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