Tuberculosis 128 (2021) 102083

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Tuberculosis
journal homepage: www.elsevier.com/locate/tube

Mechanisms and detection methods of Mycobacterium tuberculosis

rifampicin resistance: The phenomenon of drug resistance is complex
Ge Xu a, Hangchi Liu a, Xudong Jia a, Xiaomin Wang b, **, Peng Xu a, *
a
Key Laboratory of Characteristic Infectious Disease & Bio-safety Development of Guizhou Province Education Department, Institute of Life Sciences, Zunyi Medical
University, No.6 West Xuefu Road, Xinpu District, Zunyi, Guizhou Province, 563000, China
b
Department of Microbiology, Zunyi Medical University, No.6 West Xuefu Road, Xinpu District, Zunyi, Guizhou Province, 563000, China

A R T I C L E I N F O A B S T R A C T

Keywords: Tuberculosis (TB) is an infectious disease that poses a serious threat to human health. Rifampin (RIF) is an
Mycobacterium tuberculosis important first-line anti-TB drug, and rifampin resistance (RIF-R) is a key factor in formulating treatment
Rifampin regimen and evaluating the prognosis of TB. Compared with other drugs resistance, the RIF-R mechanism of
Resistance
Mycobacterium tuberculosis (M. tuberculosis) is one of the clearest, which is mainly caused by RIF resistance-
Susceptibility
Detection
related mutations in the rpoB gene. This provides a convenient condition for developing rapid detection
methods, and also an ideal object for studying the general drug resistance mechanisms of M. tuberculosis. This
review focuses on the mechanisms that influence the RIF resistance of M. tuberculosis and related detection
methods. Besides the mutations in rpoB, M. tuberculosis can decrease the amount of drugs entering the cells,
enhance the drugs efflux, and be heterogeneous RIF susceptibility to resist drug pressure. Based on the results of
current researches, many genes participate in influencing the susceptibility to RIF, which indicates the phe­
nomenon of M. tuberculosis drug resistance is very complex.

1. Introduction
Tuberculosis (TB) is a major infectious disease caused by Mycobac­
terium tuberculosis (M. tuberculosis) which seriously threatens human
health. According to the global tuberculosis report 2020 by World
Health Organization (WHO) [1], approximately one quarter of people
worldwide are infected with M. tuberculosis, and 1.4 million people died
from this disease in 2019. The number of TB deaths has exceeded that of
AIDS, making M. tuberculosis the leading pathogen that undermines
human health.
Drug-resistant TB has been a challenge in TB treatment and disease
control. Rifampin (RIF), a drug that has been used to treat TB for more
than half a century [2], remains an important first-line drug against TB.
Although several RIF analogues or derivatives have been discovered,
such as rifapentine, rifabutin, rifalazil [3], and rifacinna [4], the RIF is
still the most widely used rifamycins in the TB treatment, which resis­
tance mechanisms are also paid more attention in research fields. Its
ability to bind with RNA polymerase (RNAP) of M. tuberculosis interferes
with protein synthesis, thereby achieving bactericidal effect [5]. RIF
combined with other anti-TB drugs can reduce the duration of treatment
and lead to improved cure rates [6]. However, with the long-term of RIF
treatment, rifampin resistance (RIF-R) has become a major problem in
TB control. Nearly half a million people developed RIF-R TB in 2019 [1].
RIF-R can cause serious adverse consequences such as TB treatment
failure, a prolonged course of treatment, and increased retreatment
rates. Because there are few RIF mono-resistant strains and most RIF-R
strains are also resistant to isoniazid (INH) [7], RIF-R becomes a
marker for multidrug-resistant (MDR) TB (resistance to both RIF and
INH). Since 2014, the WHO has combined the data of RIF-R TB with
MDR-TB and made the term MDR/RR-TB for better assessing regional
drug resistance levels [8].
In order to control and treat drug-resistant TB effectively, it is
important to investigate the mechanism of drug resistance and develop
rapid and effective detection methods. Unfortunately, the mechanism
related to RIF resistance is still not fully clear. The phenomena of
tolerance (an increased survival times under a bactericidal drug con­
centration without any changed minimal inhibitory concentration) and
persistence (a small subset of bacterial population with enhanced drug
resistance but unchanged genomes) to drugs were discovered [9–11],
which indicates that the responses of M. tuberculosis to drugs are more

* Corresponding author.
** Corresponding author.
E-mail addresses: WXM_ZMU@163.com (X. Wang), PengXu_ZMU@163.com (P. Xu).

https://doi.org/10.1016/j.tube.2021.102083
Received 25 December 2020; Received in revised form 30 March 2021; Accepted 25 April 2021
Available online 8 May 2021
1472-9792/© 2021 Elsevier Ltd. All rights reserved.
G. Xu et al. Tuberculosis 128 (2021) 102083

complicated than our expectation. In addition, RIF resistance-related
mutations are mostly located in the rifampin resistance-determining
region (RRDR) of rpoB gene [12], which makes genotypic based
drug-susceptibility testing (DST) of RIF more advantageous over other
drugs. The GeneXpert MTB/RIF (Cepheid Inc. USA) [13] and Genotype
MTBDRplus (Hain Lifescience Inc. Germany) [14] have been endorsed
by WHO as rapid genotypic RIF susceptibility tests [15,16]. However,
these tests have their own drawbacks, for instance, the RIF
resistance-related mutations outside the RRDR would be easily ignored
[17], which would be a potential risk of under diagnosis of RIF-R TB, and
would lead to treatment failure, further drug resistance and
drug-resistant TB transmission. Therefore, this review will focus on the
mechanisms related to RIF-R and the detection methods.
1.1. Primary RIF-R mechanism
Due to the absence of plasmids and low frequency of recombination
in M. tuberculosis, its heritable resistance is mainly generated by genetic
mutations [18–20]. RIF inhibits RNA synthesis through binding to the
RNAP of bacterium [21], thus exerting bactericidal activity. The RNAP
consists of five subunits, α (two copies), β, β′ and σ [22], which are
encoded by the rpoA, rpoB, rpoC and rpoZ gene, respectively. Mutations
in the rpoB gene are the primary mechanism of RIF-R, which was first
discovered in Escherichia coli [23,24].
Mutations in the gene may result in structural changes of β subunit
and make RIF difficult or unable to bind to the β subunit of RNAP, thus
causing RIF resistance [25] (Fig. 1-A). Studies have shown that about
90–100% of RIF-R M. tuberculosis isolates were due to rpoB mutations
[26,27]. Insertions, deletions, and point mutations could occur in the
rpoB [28], of which point mutations are the most common [29,30]. Most
of the RIF-R mutations concentrated in the 81bp RRDR of rpoB (Fig. 2),
of which the most common mutations were S531L, H526Y, H526D and
D516V that account for 41–74%, 6–24%, 2–30% and 5–18% of RIF-R
strains, respectively [31]. There are other RIF-R mutations beyond
RRDR with low frequency. For these mutations, Stephanie et al. devel­
oped structure-based machine learning approaches and provided web
server (https://biosig.unimelb.edu.au/suspect_rif/.) to predict their
resistance [32]. Some studies found that the proportion of these muta­
tions varies in different M. tuberculosis strains with different genetic
backgrounds, for example, S531L is mainly found in lineage 2 (Beijing
genotype) in Germany [33], while D516V has a higher proportion in
lineage 4 (LAM family) in Russia [34]. However, whether the strains
with different genetic backgrounds tend to have specific mutation in the
rpoB, or these mutations have contributions to the transmission of spe­
cific strains, is still unknown.
Using both statistical and machine-learning approaches, Asma et al.
[35] studied the impacts of wildtype and mutant residues on the
protein-RIF interactions, and found that the wildtype residue 531
formed a hydrogen bond with RIF, while the residue 526 formed hy­
drophobic interactions with residue 516 and both were involved in
forming hydrophobic interactions with RFP, and the mutants of any
these residues could cause steric clashes with RIF. The different

Fig. 1. The mechanisms and genes related to RIF resistance in M. tuberculosis. There are three major mechanisms related to RIF resistance of M. tuberculosis:
rpoB gene mutations (A), permeability of cell wall (B) and efflux pumps (C). For each mechanism, both statuses of RIF susceptible and RIF resistant (less susceptible)
are illustrated, and the related genes are listed in the right column. In the first row (A), rpoB gene without drug resistance-related mutations, translates wild type of β
subunit of RNA polymerase (green), which can be bound with RIF and lose its activity (grey). If rpoB gene with drug resistance-related mutation, it translates
structurally changed β subunit of RNA polymerase (red) which cannot be bound with RIF and keeps its activity. The second row (B) illustrates how the permeability
of cell wall affects the RIF susceptibility. When the permeability is reduced, less RIF enters into the M. tuberculosis, the actual intracellular concentration of RIF is also
reduced, therefore the susceptibility is elevated. The third row (C) shows the function of efflux pumps to RIF susceptibility. Compared with the unprompted efflux
pump (green), the prompted efflux pump (red) can excrete more RIF to reduce the intracellular concentration, thus increasing the level of RIF resistance. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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G. Xu et al. Tuberculosis 128 (2021) 102083

Fig. 2. Distribution of rpoB gene mutations in M. tuberculosis. The rpoB gene mutation data are retrieved from the M. tuberculosis resistance mutation database
(Tuberculosis drug resistance mutation database, http://www.tbdreamdb.com [19]), and the sequence of strain H37Rv (GenBank accession no. AL123456.3) is used
as the reference. The mutations with an orange background indicate that the mutation is highly correlated with RIF-R (the resistance information of I572F is obtained
from the previous literature not the database [39]); Del indicates the deletion; Ins indicates the insertion, and Stop indicates the stop codon. (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web version of this article.)

interactions among residues and mutation types can lead to diverse
RIF-binding stability, and consequently result in the different level of
RIF resistance, such as mutations L511P, H526L, H526 N, L533P and
I572F, which is often associated with low-level resistance to RIF [36,37].
Up to now, there is no research that comprehensively studies the rela­
tionship between the mutations in the rpoB and RIF resistant level in
M. tuberculosis or other species of mycobacterium strains with identical
genetic background. Perharps, with the help of deep sequencing tech­
nology, this kind of studies would be easier, since similar study has
already been conducted on Staphylococcus aureus at high throughput
from mixed bacterial populations [38]. The information of this rela­
tionship would be very useful to develop accurate detection methods
and treat clinical patients.
2. Other mechanisms related to RIF resistance
2.1. The permeability of cell wall
In addition to directly altering the structure of the RIF drug target,
M. tuberculosis can also prevent drugs from entering into cells to reduce
its susceptibility (Fig. 1-B) [40]. Many genes and proteins involved in
the cell wall synthesis are related to RIF resistance. The PE11
(LipX/Rv1169c) protein of the PE/PPE family of M. tuberculosis plays an
important role in maintaining M. tuberculosis cell wall [41]. The
expression of M. tuberculosis PE11 protein in M. smegmatis can cause
elevated resistance level to various antibiotics including RIF, and it is
speculated that the up-regulation of PE11 protein expression can reduce
the amount of antibiotic entering into M. tuberculosis [42]. Similarly, the
monooxygenase operon which is encoded by the mymA (Rv3083) gene
and regulated by the virS (Rv3082c) gene, is also necessary to maintain
the mycolic acid and the integrity of the cell wall [43]. The mutations in
either virS or mymA can increase the permeability of the M. tuberculosis
cell wall, thereby accelerating the spread of drugs and changing the
susceptibility to antibiotics such as RIF and INH, indicating that both
virS and mymA genes can affect the ability of drug entrance into
M. tuberculosis [44]. In both M. tuberculosis and M. bovis, CpnT
(Rv3903c) is an outer membrane channel protein that allows nutrients
into cells [45]. Danilchanka et al. found that [46] mutant CpnT protein
could prevent drugs from entering cells, increasing the minimum
inhibitory concentration (MIC) of RIF by 8–32 fold. However, the in­
fluence of cell permeability on RIF-R is not well evaluated in clinic. To
evaluate this influence in clinical isolations, both genetic poly­
morphisms and expression levels of these genes need to be investigated,
since this it may be affected by gene mutations and quantity of protein
expression.
2.2. Efflux pumps to RIF
M. tuberculosis can also excrete drugs out of bacteria through efflux
pumps to reduce the drug concentration in the intracellular environ­
ment, thus increasing the ability of drug resistance (Fig. 1-C) [47,48].
Studies have shown that [49,50] the efflux pump inhibitors, such as
carbonyl cyanide m-chlorophenyl hydrazine (CCCP), verapamil (VP),
thioridazine (TZ) and chlorpromazine (CPZ), can decrease the resistance
of M. tuberculosis to RIF. Many efflux pump genes or putative efflux
pump genes have been found to have potential associations with RIF
resistance, including Rv0842, pstB (Rv0933), mmpL13a (Rv1145),
mmpL13b (Rv1146), bacA (Rv1819c), stp (Rv2333), drrA (Rv2936), drrB
(Rv2937), drrC (Rv2938) and efpA (Rv2846c) [49,51]. In addition, bio­
informatics analysis found that [52] mmpL3 (Rv0206c), Rv1258c and
Rv1634 might also have the function of RIF excretion. As a member of
major facilitator superfamily efflux pumps, Rv1258c can mediate
macrophage-induced RIF tolerance [53], this phenomenon of RIF
tolerance has been confirmed in lineage 1, 3, and 4 M. tuberculosis
clinical isolates, but not lineage 2 (Beijing genotype) isolates, which may
harbor a loss-of-function mutation in Rv1258c [54]. Some studies also
showed that [51,55] there was a relationship between RIF-R and the
expression levels of efflux pump genes Rv0876c, Rv1217c, Rv1218c and
Rv1250. Although the efflux pumps, as a major mode of drug excretion
in M. tuberculosis, the over-expression of their coding genes is related to
the increased RIF resistance, while the loss-of-function mutations may
lead to opposite result. However, their contributions to RIF resistance
still need further investigation in clinics.

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G. Xu et al. Tuberculosis 128 (2021) 102083

3. Different RIF susceptibility of subpopulations
At genotypic level, the heterogeneous M. tuberculosis population with
different drug susceptibility has been found in vivo by several studies
[56,57]. These drug resistant subpopulations are considered as the
origin of acquired drug resistance, and dynamic according to treatment
regimens. Besides genotypic heterogeneity, phenotypic heterogeneity
can also affect the RIF susceptibility within bacterial population. Errors
during the process of protein translation is a common phenomenon in
bacteria, which helps them to adapt to changeable environment [58,59].
Due to the protein sequence error caused by mistranslation, genotypic
RIF sensitive (no RIF-R mutation in rpoB) M. smegmatis also has a certain
proportion of RIF-R RNAP β subunit within the population, resulting in
phenotypic RIF-R (Fig. 3) [60]. Meanwhile, the RIF drug environment
can also induce rpoB gene expression of M. smegmatis by different pro­
moters (compared with no RIF environment), which would trend to
more phenotypic RIF-R bacteria caused by rpoB translation errors [61].
Asymmetric cell division is another reason for phenotypic hetero­
geneity of RIF susceptibility. After elongation of the poles, the division
mode of mycobacteria is split into two independent bacteria
asymmetrically [62]. Therefore, there are two poles of each bacterium,
one is from the parent bacteria, and the other is the newly formed pole.
Studies have shown that the growth rate of the old pole is faster than the
new one, and bacteria with older poles are more sensitive to drugs tar­
geting cell wall synthesis, but less sensitive to RIF (Fig. 4) [63,64]. These
phenomena indicate that within a Mycobacterium population, different
subpopulations could have diverse susceptibility to RIF even without
genetic mutation.
3.1. RIF-R fitness cost and compensatory mutations
Antibiotics usually exert bactericidal or antibacterial activity by
inhibiting key pathways of bacterial growth, reproduction, or meta­
bolism [65]. Most of the drug resistance-related mutations in the genes
of these key pathways are non-synonymous mutations that cause
structural or functional changes in drug targets, or mutations in the
promoter region that affect the gene expression. Mutations in these
genes may have adverse impacts that affect the normal physiological
state of bacteria, and this negative effect is called fitness cost [66]. For
example, compared with wild type, the relative fitness of S531W, H526Y

Fig. 3. The difference of RIF-R caused by mistranslation and mutation of rpoB. The descendant of RIF susceptible bacterium could be heterogeneous, which
consists of RIF susceptible (green) and RIF-R (red) subpopulation. The RIF susceptible bacteria produce susceptible β subunit of RNA polymerase encoded by wild
type rpoB (A). There are two types of bacteria among the RIF-R subpopulation. One is phenotypic RIF-R with wild type rpoB but mistranlated resistant β subunit of
RNA polymerase (B). Another is genotypic RIF-R with resistant β subunit of RNA polymerase encoded by mutant rpoB (C). Because no genetic mutation occurred in
the rpoB, the offspring of susceptible and phenotypic RIF-R bacteria have the similar subpopulations of different RIF susceptibility to their susceptible ancestor (A, B),
while the offspring of genotypic RIF-R bacteria are still resistant (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)

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G. Xu et al.
and H526D mutations of rpoB are 0.67–0.88, 0.81–0.89 and 0.78–0.81
respectively, by the experiment of competition [67,68]. Gagneux et al.
found that [67] both H526D and S531L RIF-R mutations in rpoB could
reduce the adaptability in M. tuberculosis, but the effect of the H526D
varied between M. tuberculosis lineage 2 and lineage 4, which might be
caused by the different genetic background. Although the adaptability of
resistant strains is lower, it has been found that bacteria will continue to
evolve in order to increase its adaptability. Compensatory mutations can
occur in drug resistance genes or related genes to compensate for the loss
of adaptability [69,70].
After RIF-R mutation occurred in the rpoB gene, the compensatory
mutation could be found in the rpoB as well as the rpoA and rpoC gene so
as to potentially restore the activity of RNAP [71]. Meftahi et al. found
[69] that in the rpoB S531L mutant RIF-R strain, additional V615 M
mutation could change the structure of RNAP, thereby increasing the
elongation rate of transcription to compensate for the enzyme activity
defect caused by the rpoB S531L mutation. In addition to compensating
for RNAP function, compensatory mutations in rpoA and rpoC which
were found in up to 30% of MDR cases in countries with high MDR
burden, are speculated to promote MDR transmission [72]. Studies have
shown that about 27%–70% of strains with rpoB resistance-related
mutations have compensatory mutations in rpoA or rpoC [31], but the
specific compensatory effects of these mutations are rarely reported and
need to be further studied.
3.2. Driving factors to RIF-R mutation rate
Acquired drug resistance in M. tuberculosis is attributed primarily to
the accumulation of genetic mutations [18,19]. For RIF-R, the mutation
rate in M. tuberculosis is about 10− 8 mutations per cell division under 2
μg/ml of RIF selection in vitro experiment [73]. The drug resistant
mutation rates may vary according to each drug and its
resistance-related genes. One contributing factor is the number of sites
5
Tuberculosis 128 (2021) 102083
Fig. 4. The RIF susceptibility of poles in popula­
tion. The division mode of mycobacteria is elonga­
tion of poles, then mycobacteria split into two
independent bacteria. The characters of old and new
poles (from red to green) are not same. Older poles
are trend to grow faster and less susceptible to RIF,
but newer ones grow slower and more susceptible to
RIF. This phenomenon can lead to heterogeneous RIF
susceptibility of the population. (For interpretation of
the references to colour in this figure legend, the
reader is referred to the Web version of this article.)
related to drug resistance in the gene, and another is the fitness cost of
drug resistant mutation. For example, the pyrazinamide (PZA) resistant
mutations are widely distributed in the pncA gene (561 bp), and most
non-synonymous and frameshift mutations can lead to PZA resistance
[74]. Moreover, since the pncA is a non-essential gene, the fitness cost of
pncA mutations is relatively low, and thereby, each mutation has an
opportunity to be fixed in the bacterial population. Compared with the
pncA gene, although the rpoB is much longer (3519 bp), the RIF-R mu­
tations are mainly concentrated in the 81 bp RRDR. And because the
rpoB is crucial for M. tuberculosis survival, mutations with high fitness
cost are rarely observed in the population or even fatal. Therefore, it is
reasonable that the RIF-R mutation rate is much lower than that of PZA.
For RIF, this mutation rate is also not fixed. It may be influenced by
the genetic backgrounds of M. tuberculosis. Jurriaan et al. found that
[75] RIF-R mutation frequencies in Beijing genotype strains were high
compared with those in EAI strains. However, the effect of genetic
background still needs additional research, since other studies did not
draw similar conclusions [73,76]. In addition, the drug concentration is
playing a leading role in the mutation rate, since drug pressure is the
major selection for drug resistance. Higher drug concentration means
more intensive pressure, and consequently, fewer mutations with
high-level resistance could be selected, and lead to reduced mutation
rate. On the contrary, drugs at sub-inhibitory or sub-lethal concentra­
tions can act as mutagens to elevate the mutation rate [77–80]. The
half-life of RIF varies from 1.5 to 5 h, and is progressively shortened by
about 40% during the first 2 weeks of treatment [81], which is more
likely to be lower concentration in the patients. Moreover, the RIF
concentration varies among different tissues within the patient.
Recently, an in-human study using dynamic [11C]rifampin PET-CT,
found that the RIF concentrations in cavity walls and noncavitary le­
sions were less than one-fifth of those in plasma [82]. The target peak
concentration ranges from 8 to 24 μg/ml in serum by 2–4 h after oral
administration. However, this concentration varies among individuals.
G. Xu et al. Tuberculosis 128 (2021) 102083

A pharmacokinetic research in Sweden showed that the peak RIF con­
centration could be more than 45 times difference among patients, and
lower than recommended RIF concentrations were detected in 42% of
the patients after 2 weeks of treatment [83]. Pharmacogenomic factors
of patients may be an explanation for this phenomenon. RIF is a sub­
strate of P-glycoprotein and organic anion transporting polypeptides
(OATPs). Both in vivo and in vitro studies found that genetic poly­
morphisms of ABCB1 (encoding for P-glycoprotein), SLCO1B1 (encoding
for OATP-1B1) and SLCO1B3 (encoding for OATP-1B3) were associated
with reduced rifampin concentrations [84–86]. Therefore, factors from
host, pathogen and drug all may be associated with RIF-R mutation rate
and resistant risk.
in cost of instruments and reagents, hence are not commonly used in
primary medical institutions of high TB burden countries [89]. In
addition, there are other methods for M. tuberculosis DST, such as
microplates [90,91], and phage methods which judge drug resistance by
using M. tuberculosis specific phage to form plaques [92,93]. In general,
due to the extremely long doubling time of M. tuberculosis (about 20 h
per generation [94]), it always takes at least one or three weeks for
liquid or solid culture based DST methods, hindering the early diagnosis
of drug-resistant TB.
4.2. Genotypic RIF DST

Currently, the WHO-recommended commercial methods for geno­

4. Methods of RIF-R detection
There are mainly two types of methods to detect M. tuberculosis drug
resistance, one is phenotypic DST based on culture, and the other is
genotypic DST based on the detection of drug resistance-related muta­
tions. Conventional phenotypic DST is still accepted as the gold standard
for RIF-R detection, while the RIF genotypic DST is also widely used in
clinical settings due to its relatively high specificity and sensitivity.
4.1. Phenotypic RIF DST
The main culture mediums used for M. tuberculosis phenotypic DST
are Löwenstein-Jensen (L-J) solid medium, Middlebrook 7H9 liquid
medium and Middlebrook 7H10 solid medium. There are two DST
methods using solid culture medium: the absolute concentration method
and the proportional method. Compared with the absolute concentra­
tion method, the proportional method has a higher accuracy since it
reduces the influence caused by personal operation. The solid medium-
based DST is lower in cost and contamination rate, while the liquid
medium is mostly chosen in the automated DST instruments. Although,
the automatic method can not only significantly shorten the detection
time but also ensure the high sensitivity and specificity [87,88], it is high
typic RIF DST are GeneXpert MTB/RIF (Xpert) and its optimized Gen­
eXpert MTB/RIF Ultra (Xpert Ultra), and Genotype MTBDRplus
(MTBDRplus, updated to version 2 in 2011). Meta-analysis showed that
the sensitivity and specificity of these methods for RIF-R detection were
all above 90% [95–97]. The biggest advantage of these methods is
time-saving, which can simultaneously detect M. tuberculosis pathogens
and RIF susceptibility within hours. However, RIF-R mutations outside
RRDR cannot be detected by these methods. In South Africa, because the
I572F RIF-R mutation in rpoB is not detected by Xpert, there have been
serious consequences of the transmission of MDR strains [39]. Similar
phenomena of the undetected RIF-R isolates have also been reported in
other countries [98]. In addition, other technologies and methods can be
applied to detect RIF-R susceptibility, such as the rpoB gene sequencing,
melting curve method based real-time PCR [99], DNA chips [100],
whole-genome sequencing [101–104]. The characteristics of these RIF
DST methods are presented in Table 1.
5. Conclusion
However, with the extensive and long-term use of these antibiotics,
the emergence and increasing number of drug-resistant TB is inevitable.
Although new drugs have been developed and used in clinical treatment

Table 1
Comparison of RIF DST methods.
Features Phenotypic RIF DST Genotypic RIF DST

Representative Proportional MGIT 9601 Xpert2 MTBDRplus3 rpoB gene Melting DNA chips Whole-genome
method method sequencing curve sequencing
method
Specificity Very high Very high Very high Determined by method design
Sensitivity Very high High High Determined by method design
Detection Approximately Approximately Less than 2 h 1 day 1–5 days4 3–4 h 1–2 days 3–30 days4
duration 2–6 weeks 1–2 weeks
Cost for Low High High Medium Low Medium High High
detection5
Technique High Medium Low Medium Medium Medium High High
required for
operation
Main Incubator (low) Bactec MGIT GeneXpert PCR machine PCR instrument Real-time PCR system Next-generation
instruments 960 System molecular (medium) (medium) PCR (medium) sequencer (high,
(cost5) (high) diagnostic TwinCubator sequencer (high, system hybridization but this service
system hybridization but this service (high) furnace (medium) can be provided
(high) furnace (medium) can be provided Laser scanner by sequencing
by sequencing (high) company)
company)
Biosafety Low (operation of live bacteria) High (operation of inactivated bacteria)
Other features Traditional Automated Only detect Only detect Can identify the Depend on Informative, and The most
manual DST monitor and mutations in mutations in the specific target the information of informative,
report system the RRDR RRDR for RIF, and mutation sequence multiple genes can genome level
can detect INH location and selection be achieved data, but high
resistance-related nucleotide simultaneously threshold for
mutations change data analysis
simultaneously

Note: 1 MGIT 960 indicates Bactec MGIT 960 system; 2 Xpert indicates GeneXpert MTB/RIF and GeneXpert MTB/RIF Ultra; 3 MTBDRplus indicates Genotype
MTBDRplus; 4 Time depends on whether it is sequenced by the operator self or sent to a sequencing company; 5 Costs may vary across instrument models, sale regions
and channels.

6
G. Xu et al.
such as Bedaquiline and Delamanid [105], in the long run, the speed of
antibiotics development is far behind that of antibiotics resistance.
Therefore, how to use available drugs to treat and control drug-resistant
TB has become one of the most important and tough issues. The study of
drug resistance mechanisms of M. tuberculosis, and establishment of
rapid and effective DST methods would be very helpful to this issue.
Compared with the mechanisms of other drugs resistance, the
mechanism of RIF-R is relatively simpler and clearer. On the one hand, it
provides a convenient condition for the development of rapid detection
methods based on mutations. On the other hand, it is also an ideal object
for studying the general drug resistance mechanisms of M. tuberculosis,
which would be useful to comprehensively understand the phenomenon
of M. tuberculosis drug resistance. Based on the results of current re­
searches, in addition to the main RIF-R mechanism (mutations in rpoB
gene), a large number of genes participate in influencing the suscepti­
bility to RIF, which indicates the phenomenon of M. tuberculosis drug
resistance is very complex. There are two general strategies that
M. tuberculosis cells resist drug pressure. One is decreasing the amount of
drugs entering the cells, and another is enhancing the drugs efflux. Be­
sides, heterogeneous drug resistance, which is conducted under various
mechanisms, also provides a deeper understanding of this drug resis­
tance phenomenon. Those mechanisms which are at transcriptional and
proteic level, or just for subpopulation, seem more general in
M. tuberculosis to resist drug environment. Moreover, the factors of host,
pathogen and drug environment which are interacting within patients,
also may have contributions to RIF-R. The more mechanisms are found,
the more important the combination of multiple effective drugs is in the
TB treatment. Because, even for susceptible M. tuberculosis, it is difficult
to guarantee that a single antibiotic can kill all bacteria.
For clinical purpose, treatment and assessment of susceptibility of TB
are more important. However, not only are a lot of genes involved in
influencing the susceptibility to RIF, but it is also regulated at both gene
level and transcriptional level, which makes it more difficult to fully
assess the RIF-R level of M. tuberculosis. It is not economical to develop
DST methods for the less than 10% RIF-R TB cases without resistance-
related mutations in rpoB gene. But it might be more valuable to study
how to use these mechanisms to treat or control drug resistant TB.
Recently, a study showed that upregulated FhuE transposon could
transfer more rifabutin into Acinetobacter baumannii and cause rifabutin
hyperactivity of killing bacteria [106]. Increasing RIF entrance or
inhibiting RIF efflux may be a better solution to reduce the RIF-R TB
cases without mutations in rpoB gene, or a feasible way to convert RIF-R
cases of low-level resistance to RIF susceptible.
Author contribution
Conceptualization, P. Xu and X. Wang; Investigation, G. Xu and H.
Liu; Writing - original draft, G. Xu; Writing - review & editing, P. Xu and
X. Wang; Visualization, X. Jia; Supervision, P. Xu and X. Wang. All au­
thors have read and agreed to the published version of the manuscript.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgement
This article was funded by national nature science foundation of
China [grant numbers 31700130, 31860706], Guizhou provincial edu­
cation department for young scientists and engineers [grant numbers
Qianjiaohe KY[2017]190], Guizhou Provincial Science and Technology
Foundation [grant numbers Qiankehe jichu [2019]1465], Zunyi Medi­
cal University Foundation for Doctor [grant numbers F-860]. The fun­
ders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
7
Tuberculosis 128 (2021) 102083
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