INT J TUBERC LUNG DIS 19(11):1276–1289 STATE OF THE ART

Q 2015 The Union

http://dx.doi.org/10.5588/ijtld.15.0389

Mechanisms of drug resistance in Mycobacterium tuberculosis:

update 2015

Y. Zhang,* W-W. Yew†

*Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins
University, Baltimore, Maryland, USA; †Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University
of Hong Kong, Hong Kong SAR, China

SUMMARY

Drug-resistant tuberculosis (DR-TB), including multi-
and extensively drug-resistant TB, is posing a significant
challenge to effective treatment and TB control world-
wide. New progress has been made in our understanding
of the mechanisms of resistance to anti-tuberculosis
drugs. This review provides an update on the major
advances in drug resistance mechanisms since the
previous publication in 2009, as well as added informa-
tion on mechanisms of resistance to new drugs and
repurposed agents. The recent application of whole
genome sequencing technologies has provided new
insight into the mechanisms and complexity of drug
resistance. However, further research is needed to
address the significance of newly discovered gene
mutations in causing drug resistance. Improved knowl-
edge of drug resistance mechanisms will help understand
the mechanisms of action of the drugs, devise better
molecular diagnostic tests for more effective DR-TB
management (and for personalised treatment), and
facilitate the development of new drugs to improve the
treatment of this disease.
K E Y W O R D S : antibiotics; drug resistance; mechanisms;
molecular diagnostics; new drugs

The use of multiple-drug therapy, although defi-
nitely beneficial, is not an absolute guarantee
against the emergence of drug-resistant in-
fections. . . Consequently, we cannot have confi-
dence that drug-resistant tubercle bacilli will not
emerge simply because multidrug therapy is
employed.1
ACCORDING TO the World Health Organization’s
(WHO’s) 2014 global tuberculosis report,2 there were
about 9.0 million new tuberculosis (TB) patients and
1.5 million deaths in 2013; 3.5% of newly diagnosed
and 20.5% of previously treated patients had
multidrug-resistant TB (MDR-TB, defined as bacil-
lary resistance to at least rifampicin [RMP] and
isoniazid (INH]). The highest levels of MDR-TB were
found in Eastern Europe and Central Asia, with rates
reaching 20% and 50%, respectively. At least one
case of extensively drug-resistant TB (XDR-TB,
defined as MDR-TB with additional resistance to
fluoroquinolone[s] [FQs] and one or more of three
second-line injectable drugs [SLIDs], namely capreo-
mycin [CPM], kanamycin [KM] and amikacin
[AMK]) had been reported to the WHO from 92
countries by the end of 2012. An estimated 9% of
MDR-TB patients had XDR-TB. The worldwide
drug-resistant TB (DR-TB) epidemic thus remains
an alarming problem, and is further aggravated by
human immunodeficiency virus (HIV) coinfection.3
The present review is aimed at updating readers on
major advances in drug resistance mechanisms in
Mycobacterium tuberculosis since the publication of
the previous article in 2009.4 Additional information
pertaining to newly developed drugs and repurposed
agents have also been included.
BASIC CONCEPTS IN THE DEVELOPMENT OF
DRUG-RESISTANT TUBERCULOSIS
There are two types of drug resistance in M.
tuberculosis: genetic resistance and phenotypic resis-
tance. Genetic drug resistance is due to mutations in
chromosomal genes in growing bacteria, while
phenotypic resistance or drug tolerance is due to
epigenetic changes in gene expression and protein
modification that cause tolerance to drugs in non-
growing persister bacteria. The two types of resis-
tance have been responsible for a number of problems
in effective TB control, with genetic resistance (Yang
resistance), as present in MDR-/XDR-TB, causing
problems worldwide, while the more subtle pheno-

Correspondence to: Ying Zhang, Department of Molecular Microbiology and Immunology, Bloomberg School of Public
Health, Johns Hopkins University, 615 N Wolfe Street, Baltimore, MD 21205, USA. Tel: (þ1) 410 614 2975. e-mail:
yzhang@jhsph.edu
Article submitted 5 May 2015. Final version accepted 18 June 2015.
[A version in French of this article is available from the Editorial Office in Paris and from the Union website www.theunion.org]

typic drug resistance, or tolerance (Yin resistance), as
present in persisters, entails prolonged treatment and
risk of post-treatment relapse.5,6 The situation in vivo
appears more complex, and the two types of
resistance can overlap and interconvert. Prior stress
or subinhibitory concentration of drugs may induce
efflux pump expression,7,8 which causes phenotypic
resistance and may in turn facilitate the development
of more stable genetic drug resistance,8 while genetic
resistance in growing organisms can develop persis-
tence or phenotypic resistance. There is increasing
interest in understanding the biology of mycobacte-
rial persisters and developing anti-tuberculosis drugs
that target them.5
M. tuberculosis drug-resistant strains develop
largely through the selection of genetic mutants. This
is almost a wholly man-made phenomenon, resulting
from suboptimal physician prescription and/or poor
patient adherence. However, there is some recent
evidence that pharmacokinetic-pharmacodynamic
variability scenarios related to the induction of the
mycobacterial drug efflux pump may also facilitate
the development of genetic mutations in M. tubercu-
losis.9
The development of drug resistance as a result of
mutations in drug resistance genes in M. tuberculosis
may incur a cost in terms of fitness and virulence of
the organism. Acquired resistance can also be
compounded by transmitted resistance. Recent sys-
tematic reviews have alluded to an association
between primary resistance in M. tuberculosis and
HIV coinfection, suggesting transmitted DR-TB, as a
significant challenge to the management of this
patient population.10,11 Furthermore, recent studies
from China have indicated that a significant number
of MDR- and XDR-TB cases are due to the active
transmission of (mainly) the Beijing genotype,12 and
the same is true for Europe13 and Africa.14 This is a
worrying development that requires more studies to
better understand how such apparently virulent DR-
TB strains evolve and adapt in the host, and
highlights the need for more effective means to curtail
transmission.
Clinical relevance of anti-tuberculosis drug resistance
The clinical relevance of resistance to RMP and INH
has been amply discussed.4 Besides the known
negative impact of bacillary resistance to pyrazin-
amide (PZA) on treatment outcomes among MDR-
TB patients, including PZA in the treatment regimen
for MDR-TB as guided by drug susceptibility testing
(DST) might substantially improve early sputum
culture conversion and subsequent cure or treatment
completion.15,16
A prospective cohort study conducted in eight
countries that addressed the prevalence of and risk
factors for resistance to second-line drugs (SLDs) in
MDR-TB patients has yielded interesting and impor-
Mechanisms of drug resistance: update 2015 1277
tant findings.17 Among over 1200 patients, 43.7%
showed bacillary resistance to at least one SLD, 20%
to at least one SLID and 12.9% to at least one FQ,
with 6.7% of cases meeting the definition for XDR-
TB. Previous treatment with SLDs was consistently
the strongest risk factor for resistance to these drugs,
with a four-fold increased risk for XDR-TB. Bacillary
resistance to FQs and XDR-TB were found more
frequently in women than men. Unemployment,
alcohol abuse and smoking were associated with
mycobacterial resistance to SLIDs across countries. In
addition, in a study in the United States, the risk
factors for bacillary acquired resistance to SLIDs
included age 25–44 years, positive HIV status, MDR-
TB at treatment initiation and treatment with any
SLD. However, the only predictor for bacillary
acquired resistance to FQs was MDR-TB at treatment
initiation.18 A recent expanded analysis of these data
from the same group of researchers further showed
that mortality was significantly higher among TB
patients with bacillary acquired resistance to SLDs,
after controlling for age. MDR-TB at treatment
initiation, positive HIV status and extra-pulmonary
disease were also significantly associated with mor-
tality.19 A meta-analysis on treatment outcomes of
MDR-TB was undertaken in about 6700 patients
from 26 centres using individual patient data
analysis.20 Compared with treatment failure, relapse
and death, treatment success was higher in MDR-TB
patients carrying bacilli without additional resistance
or with additional resistance to SLIDs only than in
those with resistance to FQs alone or to FQs plus
SLIDs. In XDR-TB patients, treatment success was
highest if at least six drugs were used in the intensive
phase and four in the continuation phase (odds ratios
4.9 and 6.1, respectively). Likewise, in another
similar study focusing on XDR-TB, the odds of cure
were significantly lower in XDR-TB patients with
additional bacillary resistance to CPM and KM/AMK
than patients with only XDR-TB. The odds of failure
and death were found to be higher in all XDR-TB
patients with additional bacillary resistance, inclusive
of groups with bacillary resistance to oral bacterio-
static agents, with or without ethambutol (EMB) plus
PZA.21 It was recently noted that combined resis-
tance to FQ (high-level) and PZA was associated with
a poor treatment outcome in some MDR-TB patients
treated with the 9-month Bangladesh regimen.22
MECHANISMS OF RESISTANCE TO FIRST- AND
SECOND-LINE ANTI-TUBERCULOSIS DRUGS
The mechanisms of resistance of the various drugs are
discussed below. Table 1 summarises the genetic basis
and related information on resistance in M. tubercu-
losis to first-line drugs and SLDs. The resistance
mutation profile for each drug is not listed due to
space limitations. Readers may refer to drug resis-
 1278

Table 1 Mechanisms of resistance in M. tuberculosis against first- and second-line drugs

Drug MIC Gene involved Mutation frequency
(year of discovery) lg/ml in resistance Gene function Mechanism of action %
Isoniazid (1952) 0.02–0.2 katG, inhA Catalase-peroxidase, Inhibition of mycolic acid synthesis and other multiple effects 50–95
enoyl ACP reductase 8–43

Pyrazinamide (1952) 16–100 pncA, rpsA, panD Nicotinamidase/pyrazinamidase, ribosomal Depletion of membrane energy; inhibition of trans-translation; 72–99
S1 protein, aspartate decarboxylase inhibition of pantothenate and CoA synthesis
Rifampin (1966) 0.05–1 rpoB ß-subunit of RNA polymerase Inhibition of RNA synthesis 95
Ethambutol (1961) 1–5 embB Arabinosyl transferase, *DPPR synthase Inhibition of arabinogalactan synthesis 47–65
ubiA
Streptomycin (1943) 2–8 rpsL, rrs, gidB S12 ribosomal protein, Inhibition of protein synthesis 52–59
16S rRNA, 8–21
16S rRNA methyltransferase (G527 in
530 loop)
Amikacin/kanamycin (1957) 2–4 rrs, eis, whiB7 16S rRNA, Inhibition of protein synthesis 76
aminoglycoside acetyltransferase,
The International Journal of Tuberculosis and Lung Disease

transcriptional regulator
Capreomycin (1960) 2–4 rrs 16S rRNA, 2 0 -O-methyltransferase Inhibition of protein synthesis 85
tlyA
Quinolones (1963) 0.5–2.5 gyrA DNA gyrase subunit A, Inhibition of DNA synthesis 75–94
gyrB DNA gyrase subunit B
Ethionamide (1956) 2.5–10 etaA/ethA Flavin monooxygenase, Inhibition of mycolic acid synthesis 37
ethR transcription repressor, 56
inhA enoyl ACP reductase

PAS (1946) 1–8 thyA Thymidylate synthase, Inhibition of folic acid and thymine nucleotide metabolism 37
dfrA dihydrofolate reductase,
folC dihydrofolate synthase,
ribD enzyme in riboflavin biosynthesis
Cycloserine (1955) 10–40 alr Alanine racemase, Inhibition of cell wall peptidoglycan synthesis To be determined
ddl D-alanine-D-alanine ligase,
cycA D-serine proton symporter
MIC ¼ minimum inhibitory concentration; ACP ¼ acyl-carrier-protein; CoA ¼ coenzyme A; DPPR ¼ 5-phospho-a-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase.

tance mutation databases for major frontline and
SLDs at https://tbdreamdb.ki.se/Info/, https://
umr5558-bibiserv.univ-lyon1.fr/mubii/mubii-select.
cgi or http://pathogenseq.lshtm.ac.uk/rapiddrdata.
Isoniazid
INH, a pro-drug that is activated by the catalase-
peroxidase enzyme (KatG) encoded by the katG
gene23 to generate highly reactive species, is capable
of attacking multiple targets in M. tuberculosis,24 the
primary one being the InhA enzyme (enoyl acyl
carrier protein reductase). The active species (iso-
nicotinic acyl radical or anion) reacts with nicotin-
amide adenine dinucleotide (H), forming INH-NAD
adduct, which then inhibits InhA, causing inhibition
of cell wall mycolic acid synthesis.25 INH is active
only against growing M. tuberculosis but not against
non-growing bacilli (persisters). INH tolerance in
non-growing organisms may be caused by mycobac-
terial DNA-binding protein 1 (MDP1), a histone-like
protein, which downregulates katG transcription and
could lead to tolerance to INH.26
Mutations in katG are the major mechanism of
INH resistance (Table 1).4,23,27 The KatG S315
mutation is the most common mutation in INH-
resistant strains, accounting for 50–95% of INH-
resistant clinical isolates.4,24 KatG S315 mutations
usually do not completely eliminate catalase activity,
and such strains may still retain fitness and virulence,
which may explain its frequent occurrence among
clinical isolates. Mutations in the katG promoter
region furA-katG intergenic region that affect KatG
expression were occasionally found to cause INH
resistance in some strains.28,29 Resistance to INH can
also occur due to mutations in the promoter region of
mabA(fabG1)/inhA operon causing overexpression of
InhA or by mutations at the InhA active site.25,30 In
contrast to katG mutations, which usually cause
high-level resistance, mutations in inhA or its
promoter region are usually associated with low-
level resistance (minimum inhibitory concentration
[MIC] 0.2–1 lg/ml) and are less frequent than katG
mutations (Table 1).4,24 Mutations in inhA not only
cause INH resistance, they also confer cross-resis-
tance to the structurally related drug ethionamide
(ETH).30 A small percentage of low-level INH-
resistant strains do not have mutations in katG or
inhA,31–33 which may be due to new mechanism(s) of
resistance. However, an alternative explanation is
heteroresistance or mixed population, where purifi-
cation of single clones shows a small proportion of
resistant clones containing katG mutations among
sensitive clones with wild-type (wt) katG (Y Zhang,
unpublished observations). INH-resistant strains due
to katG deletion or mutations that lead to complete
loss of catalase activity causing high-level resistance
may cause loss of virulence. Strains with point
mutations in katG, such as KatG315, which do not
Mechanisms of drug resistance: update 2015 1279
completely lose catalase activity, have a limited effect
on fitness or virulence. In contrast, mutations in inhA,
which cause low-level resistance, do not cause loss of
virulence.34
Pyrazinamide
PZA is a paradoxical drug with unique sterilising
activity in anti-tuberculosis treatment.35 In striking
contrast to common antibiotics that kill or inhibit
growing bacteria, PZA only kills non-growing
persister bacteria and has poor activity against
growing bacteria.36 PZA is a pro-drug that has to
be converted to its active form, pyrazinoic acid
(POA), for activity by the pyrazinamidase/nicotina-
midase enzyme encoded by the pncA gene of M.
tuberculosis.37 PZA has multiple effects on M.
tuberculosis, including interference with membrane
energy production,38 and inhibition of RpsA (ribo-
somal protein S1), which is involved in trans-
translation,39 and PanD, which is involved in
pantothenate and co-enzyme A synthesis.40,41 Over-
expression of RpsA renders M. tuberculosis more
resistant to PZA.39 In addition, a low-level PZA-
resistant clinical strain, DHM444, without pncA
mutations,42 was found to contain a deletion of
alanine at the 438th residue (438 DA) due to a 3 base
pair (bp) GCC deletion in the C-terminus of RpsA.39
POA bound to the wt RpsA but not the mutant
RspADA438 from the PZA-resistant strain, and
specifically inhibited the trans-translation of M.
tuberculosis but not its canonical translation.39 More
recently, panD-encoding aspartate decarboxylase,
involved in the synthesis of ß-alanine, which is a
precursor for pantothenate and co-enzyme A biosyn-
thesis, has been shown to serve as a new target of
PZA.41
Mutations in the pncA gene are the main mecha-
nism of PZA resistance in M. tuberculosis.37,42–47
pncA mutations are highly diverse and scattered
along the gene, which is unique to PZA resistance.
Most PZA-resistant M. tuberculosis strains (72–99%,
average 85%) have mutations in pncA,42–46 but some
do not. Those that do not have mutations in the drug
target RpsA.39,48 RpsA target mutations are usually
associated with low-level PZA resistance (MIC 200–
300 lg/ml PZA). More recently, panD was found to
be involved in PZA resistance.40 panD mutations
were identified in naturally PZA-resistant M. canettii
strains and some PZA-resistant MDR-TB strains. M.
canettii, a member of the M. tuberculosis complex, is
intrinsically resistant to PZA,48,49 and has mutations
in both rpsA48 and panD.40
Culture-based DST of PZA is difficult and unreli-
able, with frequent problems of false resis-
tance.36,50,51 The very high correlation between
PZA resistance and pncA mutations provides the
opportunity to rapidly detect PZA resistance using
pncA sequencing.42,52–54 Dividing MDR-TB into
1280 The International Journal of Tuberculosis and Lung Disease
PZA-susceptible and PZA-resistant MDR-TB for
improved treatment of MDR-TB has thus been
proposed.15 However, some PZA-susceptible strains
were reported to have non-synonymous mutations in
pncA.31,53 These mutations could cause low-level
resistance close to the MIC cut-off for resistance that
is not easily detected by current DST for PZA, and
may be mistaken for PZA susceptibility. Further
studies are required to address this issue. PZA-
resistant strains with diverse pncA mutations do not
appear to have fitness cost or loss of virulence.43
Interestingly, a recent study found that patients
infected with PZA-monoresistant strains had worse
clinical outcomes than those with drug-susceptible
strains.55
Rifampicin
RMP is highly bactericidal and sterilising for M.
tuberculosis. Although RMP is known to interfere
with RNA synthesis by binding to the ß subunit of the
RNA polymerase, a recent study showed that RMP
binding to the RpoB target induces hydroxyl radical
formation in susceptible but not resistant bacilli, and
may contribute to the killing effect of RMP.56 RMP
resistance occurs at a frequency of 107/8, but a
recent study showed a much higher mutation
frequency, at 103, for the Beijing genotype strain
than with the EAI (East African Indian) genotype
strain, at 106 for RMP resistance, but no difference
for other drugs, including INH and moxifloxacin
(MFX).57 This high mutation frequency is intriguing
and may be related to the lower RMP concentration
used or previous exposure to RMP before selection
for RMP resistance.58 Mutations in a defined region
of the 81-bp region of rpoB are found in about 96%
of RMP-resistant M. tuberculosis isolates.59 Muta-
tions at positions 531, 526 and 516 are among the
most frequent mutations in RMP-resistant strains.
Mutations in rpoB generally result in cross-resistance
to all rifamycins, including rifabutin (RBT), but some
RMP-resistant strains are RBT-susceptible.60 Cross-
resistance to RMP and RBT seems to involve both the
common 531 and 526 sites and also the beginning of
the RpoB region61 and the D516A-R529Q double
mutations.60 Mutations at F514FF, D516V and
S522L are associated with resistance to RMP but
susceptibility to RBT.60 However, not all mutations in
rpoB are associated with RMP resistance.60,62 For
example, mutations at E510H, L511P, D516Y,
N518D, H526N and L533P are not associated with
RMP resistance and are found in RMP-susceptible
strains.60,63 In a separate study, about 10.5% (16/
133) of the strains with rpoB mutations identified by
Xpertw MTB/RIF (Cepheid, Sunnyvale, CA, USA) as
RMP-resistant were found to be RMP-susceptible on
phenotypic MGITe test (BD, Sparks, MD, USA).62
These findings caution us against relying solely on the
molecular test for detecting RMP resistance, and
suggest that the molecular test may not be able to
completely replace phenotypic DST. However, a
recent study showed that disputed rpoB mutations
may be responsible for RMP resistance among new
cases and may lead to adverse treatment outcomes
with first-line agents.64
Reports of RMP-dependent or enhanced strains of
M. tuberculosis in some MDR-TB strains65–67 is
potentially worrying and less appreciated. RMP-
dependent strains may be more prevalent than
currently realised, as the current diagnostic practice
uses only drug-free media and may simply discount or
discard strains that grow poorly in normal culture
media without RMP. A recent study from China
suggests that RMP-dependent/enhanced strains are
common, with as many as 39% (18/46) of MDR-TB
strains having RMP dependence or enhancement
phenomenon.67 The circumstances under which these
strains arise remain unclear, but they often occur as
MDR-TB, and seem to develop upon repeated
treatment with rifamycins in retreatment patients,66
and could be a result of overlapping genetic and
phenotypic resistance. RBT has been suggested for
treating RMP-resistant strains.68 However, this might
be a risky practice, as more powerful rifamycins such
as RBT could lead to the development of RMP
dependence or enhancement and potentially worsen
the disease,66 possibly as a result of genetic and
epigenetic changes that enhance the fitness and
virulence of the organism. The impact of rpoB
mutations on the fitness and pathogenesis of the
organism has recently been reviewed,69 and is worth
paying attention to, as RMP resistance may carry
more severe consequences, with enhanced virulence
and pathogenesis. It would be of interest to determine
the frequency of RMP dependence/enhancement, the
mechanisms involved, and the contribution of such
strains to treatment failure. RMP resistance due to
rpoB mutations could alter M. tuberculosis fitness;
however, compensatory mutations in rpoC or rpoA
could occur.70–72
Ethambutol
EMB interferes with the biosynthesis of cell wall
arabinogalactan.73 Arabinosyl transferase, encoded
by embB, an enzyme involved in the synthesis of
arabinogalactan, is the target of EMB in M.
tuberculosis.74 Mutations in embCAB operon, in
particular embB, and occasionally embC, are respon-
sible for resistance to EMB.74 embB codon 306
mutation is most frequent in clinical isolates resistant
to EMB, accounting for as much as 68% of resistant
strains.75,76 While mutations at EmbB306 leading to
certain amino acid changes caused EMB resistance,
other amino acid substitutions had little effect on
EMB resistance.77 However, about 35% of EMB-
resistant strains (MIC , 10 lg/ml) do not have embB
mutations,78 suggesting other mechanisms of resis-

tance. Mutations in ubiA, encoding the DPPR
synthase involved in cell-wall synthesis, have recently
been found to cause higher-level EMB resistance in
conjunction with embB mutations.79,80
Fluoroquinolones
The well-known dominant role of gyrA mutations in
FQ-resistant TB remains unchanged. In a systematic
review of gyr mutations,81 of 1220 FQ-resistant M.
tuberculosis isolates that were subjected to sequenc-
ing of the quinolone-resistance-determining region
(QRDR) of gyrA, 780 (64%) were found to have
mutations. The QRDR of gyrB was sequenced in 534
resistant isolates, only 17 (3%) of which had
mutations. Of the gyrA mutations, 81% were inside
the QRDR and 19% outside. Mutations at gyrA
codons 90, 91 and 94 were present in 54% of the FQ-
resistant isolates (substitutions at amino acid 94
accounted for 37%). Of the gyrB mutations, only
44% were inside the QRDR. Such findings may have
implications for the currently available genotypic
diagnostic tests for FQ resistance in M. tuberculo-
sis.82 Less common genetic mutations in gyrA are
associated with amino acid positions 7483 and 88.84
Those associated with amino acid 80 might not
confer FQ resistance,85 but may represent non-
functional polymorphisms similar to mutations in-
volving codon 95.4
Heteroresistance generally refers to the coexistence
of genetic populations/clones with differing nucleo-
tides at a drug resistance locus in a sample of
organisms. FQ-resistant M. tuberculosis isolates were
recently found to have a distinct proportion of
heteroresistance, and heteroresistant isolates fre-
quently demonstrated multiple mutations.86
On the whole, codons 94, 90 and 88 of gyrA were
demonstrated to confer high-level FQ resistance,87–89
as did multiple mutations. On the other hand, the
much less common gyrB mutations (up to a total of
10–15%) were generally, but not consistently, asso-
ciated with lower levels of FQ resistance;81,89
however, combined gyrA and gyrB mutations could
result in a much higher level of resistance.90 Some
notable examples included Asn538lle (GyrB)-As-
p94Ala (GyrA) and Ala543Val (GyrB)-Asp94Asn
(GyrA). Furthermore, mutations in gyrA and As-
n538Asp as well as Asp500His substitutions in gyrB
were linked to cross-resistance of M. tuberculosis to
FQs, whereas Arg485His in gyrB did not result in
such resistance.91
Some studies have been undertaken in recent years
to address the functional genetic analysis of gyrA and
gyrB mutations.83,92 In a study on the structural
modelling of the interaction between levofloxacin
(LVX) and the M. tuberculosis gyrase catalytic site,93
the loss of an acetyl group in the Asp94Gly mutation
removes the acid-base interaction with LVX neces-
Mechanisms of drug resistance: update 2015 1281
sary for FQ activity. These approaches underscore the
relevance of genetic mutations to FQ resistance.
Aside from the mycobacterial pentapeptide MfpA
and ATPase complex Rv2686c-Rv2687c-Rv2688c
operon discussed, other efflux pumps that might be
at play in FQ resistance include antiporters LfrA and
Tap.94 A recent study in FQ-monoresistant clinical
isolates of M. tuberculosis revealed high levels of
efflux pump pstB transcripts in a few of these
isolates,95 suggesting a contribution of the pump to
resistance. In the presence of unstable drug exposure
for the bacilli, facilitation in acquiring additional
genetic resistance through gyr mutations could
markedly worsen the scenario.9 Likewise, a recent
study has shown a worsening of resistance of M.
tuberculosis to FQ with the concomitant administra-
tion of RMP, perhaps related to the induction of the
efflux pump.96
Aminoglycosides (streptomycin, kanamycin/amikacin)
and capreomycin
Streptomycin (SM) inhibits protein synthesis by
binding to the 30S subunit of bacterial ribosome,
causing misreadings of the mRNA message. Resis-
tance to SM is caused by mutations in the S12 protein
encoded by the rpsL gene and 16S rRNA encoded by
the rrs gene.97 Mutations in rpsL and rrs are the
principal mechanism of SM resistance,97,98 account-
ing for respectively about 50% and 20% of SM-
resistant strains.97,98 However, about 20–30% of
SM-resistant strains with low-level resistance (MICs
, 32 lg/ml) do not have mutations in rpsL or rrs,99
and may have mutations in gidB encoding a
conserved 7-methylguanosine (m(7)G) methyltrans-
ferase specific for 16S rRNA.100 It has been shown
recently that mutations in the promoter region of
whiB7 contribute to cross-resistance to SM and KM
due to increased expression of the tap efflux gene and
eis controlled by whiB7.101 Mutations at the 16S
rRNA (rrs) position 1400 cause high-level resistance
to KM and AMK.102,103 SM-resistant strains usually
remain susceptible to KM and AMK. Mutations in
the promoter region of the eis gene, encoding
aminoglycoside acetyltransferase, cause low-level
resistance to KM but not to AMK.104
Mutations in tlyA encoding rRNA methyltransfer-
ase105 and the 23S rRNA gene rrs (A1401G and
G1484T) are involved in CPM resistance. rRNA
methyltransferase modifies the C1409 nucleotide in
helix 44 of 16S rRNA and the C1920 nucleotide in
helix 69 of 23S rRNA.106 Mutants with A1401G in
the rrs gene could cause resistance to KM and CPM
but not viomycin, while C1402T or G1484T could
cause resistance to CPM, KM or viomycin.107
Multiple mutations may occur in the rrs gene in one
strain, conferring cross-resistance among these
agents.107
1282 The International Journal of Tuberculosis and Lung Disease
Table 2 Some newly developed drugs and repurposed agents of focus
Drug Drug class
Bedaquiline (TMC207) Diarylquinoline
Pretomanid (PA-824) Nitroimidazopyran
Delamanid (OPC-67683) Nitrodihydroimidazooxazole
SQ109 Ethylenediamine
Linezolid Oxazolidinone
Clofazimine Riminophenazine
Ethionamide/prothionamide
Ethionamide (ETH) is a derivative of isonicotinic
acid, and is a bactericidal agent only against M.
tuberculosis. The MICs of ETH for M. tuberculosis
are 0.5–2 lg/ml in liquid media, 2.5–10 lg/ml in
7H11 agar and 5–20 lg/ml in Löwenstein-Jensen
medium. Like INH, ETH is also a prodrug that is
activated by EtaA/EthA (a mono-oxygenase)108,109
and inhibits the same target as INH, i.e., InhA of the
mycolic acid synthesis pathway.30 The structure and
activity of prothionamide are almost identical to
those of ETH. EthA is an FAD-containing enzyme
that oxidises ETH to the corresponding S-oxide,
which is further oxidised to 2-ethyl-4-amidopyridine,
presumably via the unstable oxidised sulfinic acid
intermediate.110 EthA also activates thioacetazone,
thiocarlide, thiobenzamide and perhaps other thio-
amide drugs,110 which explains the cross-resistance
between these agents (e.g., Isoxyl). In addition,
mutations in the target InhA confer resistance to
both ETH and INH.
D-cycloserine/terizidone
D-cycloserine (DCS) is a bacteriostatic agent used in
the treatment of MDR-TB. Terizidone is a combina-
tion of two molecules of DCS. The MIC of DCS for
M. tuberculosis ranges widely from 1.5 to 30 lg/ml,
depending on the medium of culture used. DCS
inhibits the synthesis of cell wall peptidoglycan by
blocking the action of D-alanine racemase (Alr) and
D-alanine:D-alanine ligase (Ddl).111 Alr is involved in
the conversion of L-alanine to D-alanine, which then
serves as a substrate for Ddl. Overexpression of alrA
encoding D-alanine racemase from M. smegmatis
causes resistance to DCS in M. bovis bacille Calmette-
Guérin (BCG).112 Overexpression of Alr confers
higher resistance to DCS than Ddl overexpression in
M. smegmatis, suggesting that Alr might be the
primary target of DCS.113 However, a recent study
suggests that the primary target of DCS in M.
tuberculosis is Ddl.114 It was recently reported that
cycA encoding D-serine, L- and D-alanine and glycine
transporter involved in the uptake of D-cycloserine
was defective in M. bovis BCG, which could be
related to its natural resistance to DCS.115 However,
the mechanism of DCS resistance in M. tuberculosis
remains to be established.
New Repurposed
drug agent
Yes
Yes
Yes
Yes
Yes
Yes
Para-aminosalicylic acid
Para-aminosalicylic acid (PAS) is a bacteriostatic
agent with an MIC of 0.5–2 lg/ml for M. tuberculo-
sis.4 Interference with folic acid biosynthesis116 and
inhibition of iron uptake117 have been proposed as
two possible mechanisms of action.118 PAS was
recently shown to be a prodrug that inhibits folic acid
metabolism by incorporation into the folate pathway
through activation by dihydropteroate synthase
(DHPS, FolP) and dihydrofolate synthase (DHFS,
FolC) to generate a toxic hydroxydihydrofolate
antimetabolite which inhibits dihydrofolate reductase
(DHFR, encoded by dfrA [Rv2763c]).119,120 Muta-
tions causing PAS resistance occur at a frequency of
105 to 109. The mechanism of PAS resistance is not
well understood. Mutations in thyA encoding thymi-
dylate synthase, which reduce the utilisation of
tetrahydrofolate, were responsible for resistance in
about 37% of PAS-resistant clinical isolates.116,121
Mutations in folC and dfrA have also been found in
PAS-resistant strains.121,122 Overexpression of the
PAS drug-activating enzyme DHFS (FolC) restored
sensitivity to PAS in the resistant strain,122 while
overexpression of the target DHFR caused PAS
resistance.119 More studies are needed to validate
the PAS resistance genes identified in clinical isolates.
RESISTANCE TO NEWLY DEVELOPED DRUGS
AND REPURPOSED AGENTS FOR TB
Table 2 depicts the drugs and repurposed agents for
TB discussed below. Table 3 summarises the genetic
basis and associated information regarding M.
tuberculosis resistance to these listed drugs.
Bedaquiline
Bedaquiline (TMC207), which is highly active
against M. tuberculosis (MIC 0.03 lg/ml),123 inhibits
mycobacterial F1F0 proton adenosine triphosphate
(ATP) synthase, a novel target,123 leading to ATP
depletion. Bedaquiline is active against both growing
and non-growing mycobacterial populations,124 and
is also active against MDR-TB strains in vitro and in
mice.125 Of particular interest is the synergy between
bedaquiline and PZA, and this combination provides
the highest sterilising effects in the mouse TB
model.123 The finding is consistent with the previous

Table 3 Mechanisms of resistance against newly developed drugs and repurposed agents in M. tuberculosis
Drug MIC Gene involved
(year of discovery) lg/ml in resistance
Pretomanid (PA-824) 0.015–0.25 ddn
(2000) fdg1
Delamanid (OPC-67683) 0.006–0.012 ddn
(2006) fdg1
Bedaquiline (TMC207) 0.06–0.12 atpE
(2005) rv0678
SQ109 0.5 mmpL3
(2005)
Clofazimine 0.1–0.25 rv0678
(1956)
Linezolid 0.25–0.5 rrn
(1996)
MIC ¼ minimum inhibitory concentration; ATP ¼ adenosine triphosphate.
observation pertaining to N,N’-dicyclohexyl carbo-
diimide (DCCD) with the same drug target, as
synergised with PZA.38 Resistance to bedaquiline is
caused by mutations in the subunit c encoded by atpE
in the F0 moiety of the mycobacterial F1F0 proton
ATP synthase, which is a key enzyme in ATP synthesis
and membrane potential generation.123 Mutations in
the transcriptional regulator Rv0678, leading to
upregulation of efflux pump MmpL5, were recently
found to cause cross-resistance involving both clo-
fazimine (CFZ) and bedaquiline.126,127
Pretomanid and delamanid
Pretomanid (PA-824) is highly active against both
growing and non-growing M. tuberculosis (MIC
0.015–0.25 lg/ml). The drug was initially thought
to inhibit cell-wall lipid biosynthesis.128 It is a
prodrug activated by a deazaflavin-dependent nitro-
reductase (Ddn)(Rv3547) to form three primary
metabolites, the main one being the des-nitro-
imidazole (des-nitro).129,130 Des-nitro compounds
generate reactive nitrogen species, including nitric
oxide (NO), and are responsible for the anaerobic
activity of these compounds and may synergise the
mycobacterial killing with the host-derived NO
produced by macrophages. Mutations in ddn
(Rv3547) encoding deazaflavin-dependent nitrore-
ductase and fgd1 (Rv0407) encoding F420-depen-
dent glucose-6-phosphate dehydrogenase, both of
which are involved in F420 coenzyme biosynthesis,
are found in mutants resistant to pretomanid and
complementation restored susceptibility.130 Muta-
tions in fgd1 and ddn found in 65 M. tuberculosis
complex strains representing the main phylogenetic
lineages do not appear to cause resistance to
pretomanid (MIC , 0.25 lg/ml). Interestingly, M.
canetti, a member of the M. tuberculosis complex,
was found to be intrinsically resistant to pretoma-
nid, with an MIC of 8 lg/ml;131 however, the basis
Mechanisms of drug resistance: update 2015 1283
Gene function Mechanism of action
Deazaflavin-dependent nitroreductase, Inhibition of mycolic acid synthesis,
F420-dependent glucose-6-phosphate production of reactive nitrogen
dehydrogenase species
Deazaflavin-dependent nitroreductase, Inhibition of mycolic acid synthesis,
F420-dependent glucose-6-phosphate production of reactive nitrogen
dehydrogenase species
ATP synthase c chain, transcription Inhibition of ATP production
repressor for transporter MmpL5
Membrane transporter Inhibition of mycolic acid synthesis
Transcription repressor for transporter Production of reactive oxygen
MmpL5 species, inhibition of energy
production, membrane
disruption
23S rRNA Inhibition of protein synthesis
of its resistance is unknown. Because of the intrinsic
resistance of M. canetti to both pretomanid131 and
PZA,48 the pretomanid-MFX-PZA regimen that is
currently in Phase 3 trial for shortening anti-
tuberculosis treatment may not work for disease
caused by M. canetti.
Delamanid (OPC-67683) is a new drug that has
recently received approval from the European Union
and Japan for the treatment of MDR-TB in combi-
nation with other drugs. Delamanid inhibits mycolic
acid synthesis of M. tuberculosis. It has an MIC of
0.006–0.024 lg/ml, and thus appears to be 20 times
more active than pretomanid. Delamanid has activity
against non-replicating persister bacteria.132 Similar
to pretomanid, it is a prodrug that is activated by
Ddn. Mutations in one of the five coenzyme F420
genes (fgd1, ddn, fbiA, fbiB and fbiC) are associated
with resistance to delamanid.
SQ109
SQ109 (N-geranyl-N‘-(2-adamantyl)ethane-1,2-di-
amine) is a new compound derived from high
throughput screening of EMB analogues; however,
its mode of action is distinct from that of EMB.133,134
SQ109 is active against M. tuberculosis, with an MIC
of 0.5 lg/ml. SQ109 inhibits cell-wall synthesis and is
active against drug-susceptible, EMB-resistant and
MDR-TB strains. SQ109 targets MmpL3, a trans-
porter of trehalose monomycolate involved in my-
colic acid incorporation to the cell-wall core.135 An
alternative mechanism of action of SQ109 is the
disruption of the membrane potential required for the
transport of lipids and other substances.136 The
activity on the membrane suggests that SQ109 is
active against both growing and non-replicating
bacilli.136 Mutations in the mmpL3 gene, encoding
the transmembrane transporter, are associated with
SQ109 resistance.135
1284 The International Journal of Tuberculosis and Lung Disease
Linezolid
Oxazolidinones are originally a class of antibiotics
approved for the treatment of drug-resistant Gram-
positive bacterial infections.137 Oxazolidinones have
significant activity against M. tuberculosis, with an
MIC of 0.125–0.5 lg/ml.138 The congeners inhibit an
early step of protein synthesis by binding to ribosom-
al 50S subunits, most likely within domain V of the
23S rRNA peptidyl transferase, and forming a
secondary interaction with the 30S subunit.137
Mutations at G2061T and G2576T in the 23S rRNA
of M. tuberculosis can cause high levels of resistance
to linezolid, an early member of the oxazolidinones,
with MICs of 32 lg/ml and 16 lg/ml, respectively, but
low-level resistance mutants (MIC 4–8 lg/ml) have
no mutations in 23S rRNA.139 Mutations in the rplC
encoding ribosomal protein L3 (T460C mutation)
were putatively involved in linezolid resistance in M.
tuberculosis,140 largely accounting for low-level
resistance phenotypes.141 A recent study from China
suggests that resistance to linezolid was already seen
in about 11% of MDR-TB strains; however, only
30% of these strains had mutations in the 23S rRNA
gene or the rplC gene,141 suggesting other, currently
unknown possible mechanisms. Linezolid has a
recognised role in the treatment of complicated
MDR- and XDR-TB.142,143 However, significant side
effects such as peripheral neuropathy, optic neurop-
athy and anaemia occur with high frequency.142,144
Clofazimine
CFZ, which has good activity against mycobacteria,
including M. tuberculosis (MIC 0.5–2 lg/ml),145 is
conventionally used for leprosy treatment;146 how-
ever, emergence of MDR-TB led to renewed interest
in the use of CFZ for the treatment of this disease.145
The Bangladesh regimen, a 9–12-month standardised
regimen for treating MDR-TB, comprising high-dose
gatifloxacin, alongside CFZ, EMB and PZA through-
out, supplemented by KM, PTH and medium–high
dose INH during the intensive phase of a minimum of
4 months, achieved a high relapse-free cure rate
(88%).147,148 The mechanisms of action of CFZ are
poorly understood, and may include the production
of reactive oxygen species,149 the inhibition of energy
production through inhibiting NDH-2 (NADH de-
hydrogenase) and membrane disruption, which could
lead to the inhibition of Kþ uptake and subsequent
reduction in ATP production.146 The molecular basis
of CFZ resistance is not clearly understood. Recent
studies showed that mutations in a transcriptional
regulator Rv0678 led to upregulation of efflux pump
MmpL5 and caused resistance to both CFZ and
bedaquiline,126,127 as well as azole drugs.150 Muta-
tions in rv0678 are the main mechanism of CFZ
resistance.151 Two new genes, rv1979c and rv2535c,
were found to be associated with CFZ resistance.151
Further studies are needed to identify CFZ targets.
WHOLE GENOME SEQUENCING AND DRUG
RESISTANCE GENE DISCOVERY
As whole genome sequencing (WGS) has become
more affordable in recent years, there has been
significant interest and effort in using WGS to
characterise drug-resistant M. tuberculosis isolates
in different parts of the world to shed light on the
molecular epidemiology and strain characteristics
associated with the development and evolution of
drug resistance and disease transmission.72,152,153 An
interesting observation that emerges from such
studies is that there are over 100 genetic loci that
seem to be associated with drug resistance.152,153 This
has led to the suggestion that drug resistance may be
more complex than previously realised. Besides the
conventional drug resistance gene mutations dis-
cussed above, there are many other mutations that
may collectively contribute to the drug resistance
phenotype. This is reminiscent of cancer-genomic
sequencing studies, where numerous mutations are
discovered as driver mutations and passenger muta-
tions, and where it is hard to assign a causative role.
Mutations associated with drug resistance are thus
expected to play varying roles, from causative to
compensatory or adaptive roles, to increase fitness
which is only indirectly associated with drug resis-
tance. A recent WGS study identified more than 40
genes whose mutations are associated with INH
resistance.33 In addition to MIC assessments and
correlation with clinical outcomes, the roles of the
mutations identified by the WGS studies need to be
carefully addressed by functional assays, structural
studies or allelic exchange experiments to better
delineate their relevance. Although such studies are
laborious and costly, they are essential to convinc-
ingly demonstrate whether the identified mutations
indeed cause resistance to the drugs.
CONCLUSION AND FUTURE PERSPECTIVES
Improved understanding of the drug resistance
mechanisms in M. tuberculosis will help develop
more reliable molecular tests and increase treatment
efficacy. Significant progress has been made in our
understanding of the molecular basis of drug resis-
tance in M. tuberculosis. This knowledge is beginning
to impact the diagnosis and treatment of drug-
resistant TB. However, how drug resistance really
develops in patients and the factors that facilitate
resistance development are still poorly understood
and merit further study. Despite ongoing advances in
molecular tools for diagnosing drug resistance in M.
tuberculosis, one crucial fact must be borne in mind.
Resistance-conferring mutations in bacteria can

evolve dynamically over time under antibiotic pres-
sure in patients,154 when both genetic and epigentic
(phenotypic) resistances can develop and overlap.
The relationship between drug resistance and fitness
and virulence of the MDR-/XDR-TB strains has to be
carefully studied using appropriate molecular epide-
miology and animal studies. The recent application of
WGS offers promise not only in the identification of
new drug resistance genes,40,126 but also in the
comprehensive molecular detection of drug resistance
mutations in clinical isolates for patient care.
However, the causative role of the genes identified
using WGS has to be verified by tedious genetic
studies to rule out phylogenetic polymorphisms that
are not involved in drug resistance.155 The current
commercial molecular tests for drug-resistant TB,
such as the Hain (GenoTypew, Hain Lifescience,
Nehren, Germany) and Xpert tests, although useful,
cannot readily provide information regarding resis-
tance to some drugs, and their utility is limited in
achieving the practice of personalised (and precision)
medicine for improved treatment. Rapid molecular
tests (using WGS or other platforms) capable of
detecting all drug resistances are likely needed to
guide the treatment of difficult bacillary resistance
scenarios. Finally, an understanding of the mecha-
nisms of drug action and resistance should also
facilitate the overall development of new drugs to
improve the treatment of TB.
Acknowledgements
This work was support by National Institutes of Health (Bethesda,
MD, USA) grants AI099512 and AI108535. An apology is owed
for incomplete citation of research work due to space limitations.
Conflicts of interest: WWY is a consultant with Otsuka
Pharmaceutical Co, Tokyo, Japan. No other conflicts declared.
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La tuberculose (TB) pharmacorésistante, incluant la TB
multi- et ultrarésistante, pose un défi majeur en termes
de traitement efficace et de lutte contre la TB dans le
monde. Des progrès ont été accomplis dans notre
compréhension des mécanismes de la résistance aux
médicaments antituberculeux. Cette revue offre une
mise à jour des avancées majeures dans la connaissance
des mécanismes de pharmacorésistance depuis la
publication préc édente en 2009, ainsi que des
informations supplémentaires sur les mécanismes de
résistance aux nouveaux m édicaments et à des
médicaments réadapt és. L’application r écente des
techniques de séquençage de l’ensemble du génome a
La tuberculosis (TB) farmacorresistente, incluida la
enfermedad multi- y extremadamente drogorresistente,
plantea un peligro considerable al tratamiento eficaz y el
control mundial de la TB. Se han logrado progresos
recientes en la comprensión de los mecanismos de
resistencia a los medicamentos antituberculosos. En el
presente artı́culo se ofrece una actualización de los
principales avances en este campo desde la publicación
anterior en el 2009; se analiza además la información
sobre los mecanismos de resistencia a los nuevos
medicamentos y a los fármacos con una nueva
asignaci ón de uso. La aplicación reciente de las
técnicas de secuenciación del genoma completo aportó
una mejor percepci ón de los mecanismos y la
Mechanisms of drug resistance: update 2015 i
RESUME
fourni de nouvelles connaissances des mécanismes et de
la complexité de la pharmacorésistance. Cependant,
d’autres recherches sont nécessaires pour aborder la
portée des mutations génétiques récemment découvertes
à l’origine de la pharmacorésistance. Une meilleure
connaissance des mécanismes de la pharmacorésistance
contribuera à comprendre les mécanismes d’action des
médicaments, à concevoir de meilleurs tests
diagnostiques pour une meilleure prise en charge de la
TB pharmacorésistante (et un traitement personnalisé)
et à faciliter l’élaboration de nouveaux médicaments
pour améliorer le traitement de cette maladie.
RESUMEN
complejidad de la resistencia a los medicamentos
antituberculosos. Aun ası́, se precisan nuevas
investigaciones que aborden la participación de las
mutaciones g énicas reci én descubiertas en la
farmacorresistencia. Un mayor conocimiento de los
mecanismos de resistencia a los medicamentos
contribuirá a comprender mejor los mecanismos de
acción de los fármacos, desarrollar pruebas moleculares
diagnósticas más eficaces a fin de optimizar la atención
de la TB farmacorresistente (y el tratamiento
personalizado) y facilitará el desarrollo de nuevas
mol éculas que mejoren el tratamiento de esta
enfermedad.