Antimicrobial Chemotherapy | Short Form
Loss-of-function mutations in ndh do not confer delamanid,
ethionamide, isoniazid, or pretomanid resistance in
Mycobacterium tuberculosis
Sushil Pandey,1 Catherine Vilchèze,2 Jim Werngren,3 Arnold Bainomugisa,1 Mikael Mansjö,3 Ramona Groenheit,3 Paolo Miotto,4
Daniela M. Cirillo,4 Christopher Coulter,1 Alain R. Baulard,5 Thomas Schön,6,7,8 William R. Jacobs Jr,2 Kamel Djaout,5 Claudio U. Köser9
AUTHOR AFFILIATIONS See affiliation list on p. 4.
ABSTRACT Results from clinical strains and knockouts of the H37Rv and CDC1551
laboratory strains demonstrated that ndh (Rv1854c) is not a resistance-conferring gene
for isoniazid, ethionamide, delamanid, or pretomanid in Mycobacterium tuberculosis.
This difference in the susceptibility to NAD-adduct-forming drugs compared with
other mycobacteria may be driven by differences in the absolute intrabacterial NADH
concentration.
Mycobacterium tuberculosis, delamanid, ethionamide, isoniazid, pretoma­
H
ayashi et al. demonstrated that ndh mutations in Mycobacterium smegmatis are
necessary and sufficient to confer MIC increases not only to isoniazid (INH) and
ethionamide (ETH), as previously known, but also to delamanid (DLM), a World Health
Organization (WHO) group C drug recommended for treating multidrug-resistant (MDR)
and rifampicin-resistant tuberculosis (1–3). Based on these findings, Gómez-González
et al. predicted that DLM resistance may be widespread among MDR Mycobacterium
tuberculosis Beijing (lineage 2) strains from Daru Island, Papua New Guinea, as these
harbor a deletion at nucleotide 304 of ndh (Rv1854c) with a likely loss-of-function
phenotype (Fig. 1) (4–6).
We tested this hypothesis by phenotypic antimicrobial susceptibility testing for DLM
at the Queensland Mycobacterium Reference Laboratory using the current WHO critical
concentration of 0.06 µg/mL for the BACTEC Mycobacterial Growth Indicator Tube
(MGIT) system (7). DLM was dissolved and diluted in DMSO before adding to MGIT tubes
(DLM is poorly soluble in water and should not be used as recommended by the WHO
manual) (8). All seven Beijing strains from Papua New Guinea with the aforementioned
ndh frameshift deletion tested phenotypically susceptible as did four wild-type ndh
strains from the same phylogenetic group (clade A) and three further wild-type ndh
strains from clade B (Fig. 1).
Given that all ndh mutants tested shared the inhA c-15t promoter mutation that
confers cross-resistance to INH and ETH, these strains from Papua New Guinea did not
provide any insight regarding the consequence of the ndh frameshift for INH and ETH
(Fig. 1). Moreover, the results for the INH mono-resistant SEA201800149 strain from the
Public Health Agency of Sweden were inconclusive regarding the role of the in-frame
ndh deletion in this strain (see Supplementary Results and Table S2). Therefore, we
carried out broth microdilution testing at the Albert Einstein College of Medicine for the
lineage 4 H37Rv and CDC1551 reference strains and their respective ndh knockouts (see
Supplementary Methods). All MICs for INH, ETH, DLM, and pretomanid (PMD) were either
January 2024 Volume 68
Issue 1
Editor Jared A. Silverman, Bill & Melinda
Gates Medical Research Institute, Cambridge,
Massachusetts, USA
Address correspondence to Claudio U. Köser,
cuk21@cam.ac.uk.
Sushil Pandey, Catherine Vilchèze, and Jim Werngren
contributed equally to this article. Author order was
based on the amount of novel data contributed.
Kamel Djaout and Claudio U. Köser contributed
equally to this article.
D.M.C. is the co-chair of the Working Group of
the Stop TB Partnership New Diagnostics and is
an unpaid member of EUCAST subcommittee for
antimicrobial susceptibility testing of mycobacteria,
the CLSI mycobacterial committee, and the
WHO Strategic and Technical Advisory Group
for diagnostics. C.U.K. is a consultant for Becton
Dickinson, the Foundation for Innovative New
Diagnostics, the TB Alliance, and the WHO Global TB
Programme. C.U.K.'s consulting for Becton Dickinson
involves a collaboration with Janssen and Thermo
Fisher Scientific. C.U.K. is collaborating with PZA
Innovation and is an unpaid advisor to Cepheid and
GenoScreen. C.U.K. worked as a consultant for the
Stop TB Partnership and the WHO Regional Office
for Europe. C.U.K. gave a paid educational talk for
Oxford Immunotec. C.U.K. was an unpaid advisor to
BioVersys.
See the funding table on p. 5.
Received 23 August 2023
Accepted 13 October 2023
Published 1 December 2023
Copyright © 2023 American Society for
Microbiology. All Rights Reserved.
10.1128/aac.01096-23 1
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KEYWORDS
nid
Antimicrobial Agents and Chemotherapy
FIG 1 Phylogenetic tree of Beijing (lineage 2) strains from Papua New Guinea provinces and associated DLM AST results (6). As shown by the purple triangle,
the ndh frameshift mutation arose in 1992 (95% highest posterior density, 1985–1996) and is shared by most strains from the Daru-dominant clade A, whereas
all strains from the National Capital District clade B lacked this mutation (grey tip labels correspond to Daru, red to National Capital District, and turquoise to
other provinces, respectively). The first 14 columns depict different resistance mutations for first- and second-line drugs (with multiple colors where more than
one mutation was involved). The last two columns show the distribution of the ndh frameshift (shown in purple), followed by the phenotypic DLM AST results for
14 strains, all of which tested susceptible, as indicated in green (Table S1). AG, aminoglycosides; AST, antimicrobial susceptibility testing; BDQ, bedaquiline; CAP,
capreomycin; DLM, delamanid; E, ethambutol; ETH, ethionamide; FQ, fluoroquinolone; INH, isoniazid; R, rifampicin; p, promoter; PAS, para-aminosalicyclic acid; S,
streptomycin; Z, pyrazinamide.
identical or within one doubling dilution, demonstrating that loss of ndh does not result
in a substantial effect on the MIC for these drugs in M. tuberculosis (Table 1).
These results contrasted with the shift in the INH, ETH, and DLM MICs observed in M.
smegmatis and Mycobacterium bovis BCG mutated in ndh (1, 2). To better understand why
TABLE 1 Broth microdilution MICs for INH, ETH, DLM, and PMD of parental and Δndh laboratory reference
strains
Strain
H37Rv
H37Rv Δndh
CDC1551
CDC1551 Δndh
January 2024 Volume 68
MIC (μg/mL)
INH
ETH
DLM
PMD
0.03
0.06
0.03
0.03–0.06
3.13
3.13
3.13
3.13
0.016
0.016
0.016
0.008
0.12
0.06
0.12
0.06
Issue 1
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Short Form
Short Form
Antimicrobial Agents and Chemotherapy
B
1000
10
M. smeg
1
BCG ndh D366G
0.1 CDC1551 Δndh BCG
CDC1551
MIC ETH (µg/ml)
M. smeg ndh L100P
100
100
M. smeg ndh L100P
BCG ndh D366G
10
1
M. smeg
CDC1551 Δndh
BCG
CDC1551
0.1
50
10
0
20
0
40
0
80
0
16
00
32
00
0.01
1000
50
10
0
20
0
40
0
80
0
16
00
32
00
MIC INH (µg/ml)
A
NADH (nM)
NADH (nM)
FIG 2 Correlation between intrabacterial NADH concentration and the sensitivity to INH (A) and ETH (B) for M. smegmatis, M. bovis BCG Pasteur, M. tuberculosis
CDC1551, and their respective ndh mutants. Detailed references for source data can be found in Table S3 (1, 9).
January 2024 Volume 68
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these phenotypes were not transposable to M. tuberculosis, we reanalyzed data from two
of our earlier publications (1, 9). M. tuberculosis, M. bovis BCG, and M. smegmatis innately
differ in their sensitivity to INH. For example, the INH MIC of wild-type M. smegmatis is
50-fold higher than that of wild-type M. bovis BCG (Fig. 2A). However, their respective
NADH/NAD+ ratios are nearly identical (Table S3). The ndh mutants of both species show
an increased NADH/NAD+ ratio of two- to threefold relative to their wild-type parental
strain, resulting in a similar shift in the intrabacterial redox balance. Yet, the INH MIC
increases 20-fold for the M. smegmatis ndh mutant compared with only threefold for the
M. bovis BCG mutant. This inconsistency prompted us to look at other factors that could
explain the innate differences in drug susceptibility between these species as well as the
differences caused by ndh loss-of-function.
We found that NADH concentration, more than NADH/NAD+ ratios, correlated with
the MICs of INH and ETH for wild-type and ndh mutants of all three species considered
(Fig. 2). This observation suggested that NADH concentration may be the key factor
driving the MICs of INH and ETH across these mycobacterial species and their respec­
tive ndh mutants. This is in line with our previous observation that increasing NADH
concentrations compete with INH-NAD and ETH-NAD adducts for the binding site of
their target InhA, leading to resistance to these drugs (1).
In M. smegmatis, NAD-drug adducts innately face strong competition with high NADH
concentration, explaining the high INH and ETH MICs for this species. Loss-of-function
in ndh increases NADH concentration by two- to threefold and thus further exacerbates
this competition, resulting in decreased target inhibition and increased MICs (Fig. 2). By
contrast, the NADH concentration in M. tuberculosis is comparatively lower, as are INH
and ETH MICs. When ndh is mutated, the NADH concentration increases by the same
two- to threefold, but the resulting NADH concentration is likely still not sufficient to
effectively compete with NAD-drug adducts for the binding site of InhA. This would
explain why ndh mutations do not result in significant INH and ETH resistance in M.
tuberculosis. M. bovis BCG fits well in this model, harboring an intermediate NADH
concentration level leading to a moderate phenotype between the latter two mycobac­
terial species (Fig. 2).
Antimicrobial Agents and Chemotherapy
Short Form
DLM and PMD are also proposed to act through NAD adducts (2, 10). As such, ndh
mutations were shown to have impact on DLM resistance in M. smegmatis. Nevertheless,
we showed here that ndh loss-of-function does not significantly affect PMD and DLM
MICs in M. tuberculosis (Table 1). Consistent with our proposed model, DLM and PMD
were recently shown to target the NADH-dependent DprE2 subunit of decaprenyl-phos­
phoryl-ribose 2’-epimerase that is essential for arabinogalactan synthesis (11).
In summary, our results indicated that ndh mutations should not be regarded as in
vitro resistance determinants for INH, ETH, DLM, or PMD in M. tuberculosis. Moreover,
our study emphasized that the absolute NADH concentration, rather than NADH/NAD+
ratio, is likely the pivotal determinant of the susceptibility to NAD-adduct-forming drugs
in mycobacteria despite subtleties in their NAD metabolism and how they resolve
NAD-drug adducts (10, 12). Additional MIC results from clinical strains belonging to
different M. tuberculosis lineages are needed to exclude the possibility that the genetic
background may play a role within M. tuberculosis (13, 14). Determining the potential
role of ndh under other in vitro conditions or during patient treatment, including drug
tolerance, was beyond the scope of this work (15).
ACKNOWLEDGMENTS
This publication includes further work performed on M. tuberculosis strains from Papua
New Guinea which were described in previous publications. The work and support of
the PNG National Tuberculosis Programme are once again acknowledged. A.R.B. and
K.D. were supported by the Programme d’Investissements d’Avenir “Mustart” (ANR-20PAMR-0005). W.R.J. was funded by the National Institute of Health Grant AI26170. C.U.K.
is a visiting scientist at the Department of Genetics, University of Cambridge, and a
research associate at Wolfson College, University of Cambridge.
AUTHOR AFFILIATIONS
Queensland Mycobacterium Reference Laboratory, Pathology Queensland, Brisbane,
Queensland, Australia
2
Department of Microbiology and Immunology, Albert Einstein College of Medicine,
Bronx, New York, USA
3
Public Health Agency of Sweden, Solna, Sweden
4
Emerging Bacterial Pathogens Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
5
Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR9017 - CIIL Center for Infection and Immunity of Lille, Lille, France
6
Department of Infectious Diseases, Linköping University Hospital, Linköping, Sweden
7
Division of Infection and Inflammation, Institute of Biomedical and Clinical Sciences,
Linköping University, Linköping, Sweden
8
Department of Infectious Diseases, Region Östergötland and Kalmar County Hospital,
Linköping University, Linköping, Sweden
9
Department of Genetics, University of Cambridge, Cambridge, United Kingdom
AUTHOR ORCIDs
Sushil Pandey http://orcid.org/0000-0002-7585-1040
Catherine Vilchèze http://orcid.org/0000-0001-5960-6670
Jim Werngren http://orcid.org/0000-0003-2500-9792
Arnold Bainomugisa http://orcid.org/0000-0003-4180-6249
Mikael Mansjö http://orcid.org/0000-0001-9289-351X
Ramona Groenheit http://orcid.org/0000-0003-2696-437X
Paolo Miotto http://orcid.org/0000-0003-4610-2427
Daniela M. Cirillo http://orcid.org/0000-0001-6415-1535
Christopher Coulter http://orcid.org/0000-0003-2221-9946
Alain R. Baulard http://orcid.org/0000-0002-0150-5241
January 2024 Volume 68
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1
Antimicrobial Agents and Chemotherapy
Short Form
William R. Jacobs Jr http://orcid.org/0000-0003-3321-3080
Kamel Djaout http://orcid.org/0000-0003-1218-8031
Claudio U. Köser http://orcid.org/0000-0002-0232-846X
FUNDING
Funder
Grant(s)
Author(s)
National Institute of Health Grant
AI26170
William R. Jacobs Jr
Programme d'Investissements d'Avenir
"Mustart"
ANR-20-PAMR-0005 Alain R. Baulard
Kamel Djaout
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