Journal of Pharmacy and Pharmacology 2 (2014) 170-176

D DAVID PUBLISHING

Molecular Analysis of the Resistance of Mycobacterium

tuberculosis to Ethionamide Using PCR-SSCP Methods
and the Implementation of Its Nuclear Technique

Mukh Syaifudin and Zubaidah Alatas

Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jakarta 12070, Indonesia

Received: February 13, 2014 / Accepted: March 12, 2014 / Published: March 31, 2014.

Abstract: With a population of more than 231 million, Indonesia carries the fifth highest TB (tuberculosis) burden globally.
Therefore, many efforts to support TB control program are being undertaken. One of them is strengthening the capabilities in
molecular analysis of the resistance of TB bacteria to the drugs such as ETH (ethionamide). ETH is prodrug that needs to be
activated by mycobacterial enzymes in order to exert their antimicrobial activity. The aim of this study was to understand the
molecular basis for Mycobacterium tuberculosis resistance to ETH. DNA (Deoxyribonucleic acid) extraction of M. tuberculosis was
amplified with PCR (polymerase chain reaction) on ethA gene and analyzed their drug resistance by using SSCP (single strand
conformation polymorphism) technique. The DNA of M. tuberculosis (H37Rv) strain was used as positive control. Of 53 extracts of
DNA amplified, 11 (20.75%) were positive for the target gene and none of samples was resistent to ETH based on the alteration of
DNA band mobility shift on acrylamide gel. This low number of positive PCR amplicons may be related to annealing temperature
and initial DNA content in specimen. Study on the implementation of hot-SSCP by labeling the DNA with 32P radioisotope shows
that this nuclear based technique has the higher quality in term of the visualization.

Key words: Tuberculosis, resistance, ethionamide, PCR-SSCP, 32P labeling.

1. Introduction national laboratory network including the capabilities

in molecular analysis of the resistance of TB bacteria
TB (tuberculosis) is a communicable disease with
to the drugs. Preliminary data obtained in Java island
significant morbidity and mortality, where it accounts
suggest an MDR rate of 1.8% among the new cases and
for 26% of all preventable adult deaths globally [1].
16.7% among retreatment cases [4]. Though this rate is
Medications are the cornerstone of TB treatment.
still relatively low, the total number of MDR-TB cases
Around 2% of newly diagnosed TB cases are
is considerable due to the large numbers of TB
estimated to have developed MDR (multi-drug
patients. The development of MDR in classical
resistance) and will be very difficult to cure by
treatment options for M. tuberculosis has led to the
standard treatment. MDR-TB defined as tuberculosis
consideration of alternative antimicrobial agents
that is resistant to at least isoniazid and rifampicin [2].
including ETH (ethionamide).
According to the World Health Organization’s Global
ETH is the second-line anti-TB drug and structurally
Tuberculosis Control data, Indonesia ranks fifth
analogue to INH (isoniazid) and the cornerstone of
worldwide for total number of TB cases, with 429,730 [3].
front-line TB treatment. ETH is considered to be the
There are many efforts to develop and strengthen the
most active anti-TB drug after aminoglycosides and
Coresponding author: Mukh Syaifudin, Ph.D., researcher, fluoroquinolones and is a component of most of the
research field: radiation biology. E-mail: drug regimens used for treating MDR-TB or suspected
mukh_syaifudin@batan.go.id.
 Molecular Analysis of the Resistance of Mycobacterium tuberculosis to Ethionamide Using PCR-SSCP 171
Methods and the Implementation of Its Nuclear Technique
MDR-TB [5, 6]. ETH, which activated by the enzyme
EthA, appears to disrupt cell wall biosynthesis and
have at least one common cellular target, the
enoyl-acyl carrier protein reductase InhA [7]. To date,
DST (drug susceptibility testing) for ETH relies mainly
on phenotypic tests because the molecular mechanisms
of ETH resistance are not fully understood. Therefore,
a genotypic or molecular approach would be of great
value to improve and hasten DST and the management
of MDR-TB [8].
Several alternative approaches have been attempted
for the rapid and specific diagnosis of TB. Among
these the PCR is the most sensitive of the existing
rapid methods to detect this microbial pathogens in
clinical specimens. It requires only 1-2 days and has a
specificity and sensitivity and particularly useful for
slow-growing or nonculturable microorganisms and
for the detection of point mutations or certain
genotypes [9, 10], while traditional phenotypic
determination of resistance may take up to 10 weeks
after referral of a sample to the laboratory.
The genotypic methods of PCR-SSCP analysis has
been an important tool for detecting point mutations
underlying the drug resistance of diseases causing
microorganisms [11, 12]. It is commonly used for the
detection of drug resistant strains of bacteria such as
M. tuberculosis [13]. Depending on the quantity
produced and the size of the amplified fragment, the
reaction products can be visualized directly by
staining with EtBr (ethidium bromide) or a tube and shaked in rotary shaker for 20 min. Samples
silver-staining protocol, or by means of radioisotopes
and followed by autoradiography.
This PCR-SSCP assay has been used in some
studies for the rapid detection of drug-resistant M.
tuberculosis in our center by introducing nuclear
technique based molecular methods to detect the
mutations in rifampin and isoniazid encoding genes
[14-16]. This paper describes the results of the
analysis of ETH resistance using PCR-SSCP on
sputum of TB suspected patients, and provides an
example on the implementation of nuclear technique
in that drug resistance detection method.
2. Materials and Methods
2.1 Clinical Samples
Fifty three sputum specimens were obtained from
out patients (32 male and 21 female) with age ranged
from 19-60 years old (mean: 33.53 ± 9.44 years) in
Health Center for Lung Diseases or Indonesian PPTI
(Tuberculosis Eradication Association), Pasar Senen,
Jakarta with permission. These patients were
suspected of having tuberculosis, with positive
response to AFB (acid fast bacilli) by Ziehl-Neelsen
staining. Two to five milliliters of expectorated
early-morning sputa was transferred in sterile cups to
the laboratory, where a direct smear was prepared. If
any of the samples contained more than three bacilli, it
was categorized as positive AFB. After
decontamination and homogenization, sputum samples
were washed with saline, and pellet was used for DNA
extraction with procedure as described previously [15].
2.2 Extraction of Mycobacterial DNA from Sputum
Samples
DNA isolation from clinical specimens was
performed as previously described [15, 17]. Briefly,
500 μL of sputum specimens were digested and
decontaminated with the same volume of N-acetyl
L-cysteine NaOH-Na citrate solution. Decontaminated
specimen was transferred to a sterile microcentrifuge
were then centrifuged, and the pellet was washed
twice with sterile distilled water. After
recentrifugation the pellet was resuspended in 500 μL
of deionized water containing 5 μL of proteinase-K
(0.25 mg/mL) and incubated at 56 °C for 1 h followed
by boiling for 10 min. Samples were then centrifuged,
and the supernatant was used as DNA template for
PCR amplification.
2.3 Amplification of DNA with PCR and Detection
One tube PCR process was done with primers of
172 Molecular Analysis of the Resistance of Mycobacterium tuberculosis to Ethionamide Using PCR-SSCP
Methods and the Implementation of Its Nuclear Technique
ethA-1 (5’-ATC ATC GTC GTC TGA CTA TGG-3’)
and ethA-5 (5’-ACT ACA ACC CCT GGG ACC-3')
produced a 667-bp product designated for ETH1 [18].
PCR was performed in a tube (AccuPower PCR
PreMix; Bioneer) containing 2 U of Taq polymerase,
10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2 and 20
pmol of each ethA-1 and ethA-5 primers and then the
volume was adjusted to 20 μL. The solution was
subjected to preheating at 95 °C for 5 min and then
followed by 35 cycles (30 s at 95 °C, 1.5 min at 65 °C,
1.5 min at 72 °C) followed by a 7-min extension at
72 °C. The Rifr (rifampicin resistant) reference strain
(H37Rv ATCC 35838) and deionized water was used
as positive and negative control, respectively. The
amplifications were carried out using an automated
thermal cycler (Perkin-Elmer, GeneAmp PCR System
9600). The detection of amplified products was done
on 2% agarose gel (Invitrogene) with EtBr staining.
2.4 Detection of Mutation with SSCP
Six microlitter of the PCR product was mixed with
6 μL of SSCP loading dye (95% formamide (Biorad),
0.5 M EDTA and 0.05% bromophenol blue (Biorad)
denaturants). The samples were denatured at 95 °C for
4 min and snap cooled on ice and loaded on the 0.5 X
MDE (Mutation Detection Enhancement) gel (BMA,
Rockland, ME, USA). Electrophoreis was run at
constant voltage (110 V) at room temperature (±
25 °C) for 1.5 h. MDE gel was stained with EtBr for
20 min.
For hot-SSCP (radioactive SSCP), DNA was
labeled by adding one microlitter (0.1 μCi) of
[alpha-32P] dCTP (Specific activity of 3,000 Ci/mmol,
Fig. 2 Visualization of amplified DNA on agarose gel stained with EtBr.
Amersham International) to the reaction mixture of
PCR after template DNA addition. PCR process was
done as above and SSCP was done by
electrophoration of esis for 1.5 h, the MDE gels were
exposed to X-ray film (AGFA) in a X-ray cassette at
-80 °C for 24-72 h, and developed with Fuji Medical
Processor 1200 Japan [19].
3. Results and Discussion
In this research AFB test that was done with
Ziehl-Neelsen staining showed that mostly the
specimens (31 samples) were +1 categorization of
IUAT-LD (International Union Against Tuberculosis
and Lung Diseases), 7 samples were +2 and 15
speciments were +3. An example of microscopic view
of AFB observation at 1,000 magnification is
presented in Fig. 1.
In the molecular analysis of the resistance of
bacteria to ETH, 53 sputum extracts of DNA were
analyzed, 11 (20.75%) were PCR positive for the
target gene for ETH1 with the size of 667-bp product.
Some of these PCR amplification results are presented
in Fig. 2. The figure shows agarose gel electrophoresis
Fig. 1 Result of AFB test on sputa specimen smear
showing the existence of bacteria (red color).
 Molecular Analysis of the Resistance of Mycobacterium tuberculosis to Ethionamide Using PCR-SSCP 173
Methods and the Implementation of Its Nuclear Technique

of the PCR amplification for ETH1 where only four
samples were successfully amplified its target DNA
used the designed oligonucleotide primers from eleven
samples.
In this research, only 20.75% samples were positive
PCR amplification, even though some other PCR
conditions with different annealing temperatures were
done so that there was a lack in the determination of
appropriate annealing temperature. Annealing
temperature is one of the most important parameters
that need adjustment in the PCR reaction. It can be
solved by decreasing or increasing annealing
temperature gradually to the lowest or highest possible.
Moreover, the flexibility of this parameter allows
optimization of the reaction in the presence of variable
amounts of other ingredients (especially template
DNA). Other reasons are insufficient number of PCR
cycles, degeneration of DNA-template, the presence
of inhibitors which slow down the PCR, unsuitable
reaction conditions, badly defined primers, incorrect
primer specificity, no optimal reaction conditions, and
no target present in DNA template [20, 21]. The
inhibitor such as hemoglobin can copurify with DNA
and cause loss of detection of the targets [22]. On the
other hand, the low number of positive result in PCR
amplicons was also caused by the low contents of
DNA originally present in the sample or only a few
bacteria are expected to be in specimens. The solution
is using high quality and purified DNA templates [23]
or nested PCR for detection of only a few bacteria in
clinical specimens [24].
SSCP analysis revealed that there was no any
sample of positive PCR with the alteration of DNA
band mobility shift on acrylamide gel indicating no
samples were resistant to ETH. Therefore, this
analysis of Eth mutation with SSCP technique did not
identify the alteration of mobility shift of DNA band
on acrylamide gel conferring mutations in the 667-bp
amplified product Eth gene in 11 (20.75%) clinical
isolates of M. tuberculosis. The results of SSCP
analysis were presented in Fig. 3. It can be seen from
the figure that there is no alteration in DNA band
mobility shift that related to the resistance pattern of
bacteria.
Study on the implementation of hot-SSCP by
labeling the DNA with 32P radioisotope shows that
this nuclear based technique has the relatively higher
quality in term of the visualization (Fig. 4). This
informs us that the ability to detect point mutations
and polymorphisms can be improved by using
radioisotope-labeled SSCP (hot-SSCP), instead of
conventional EtBr staining. Excellent results have

Fig. 3 Visualization of SSCP gel of acrylamide stained with EtBr.

Fig. 4 Visualization of hot-SSCP gel of acrylamide with autoradiography on samples examined for rifampin resistance
without mobility shift of DNA.
174 Molecular Analysis of the Resistance of Mycobacterium tuberculosis to Ethionamide Using PCR-SSCP
Methods and the Implementation of Its Nuclear Technique
been obtained in resolving mutant PCR fragments
from M. tuberculosis resistance to drug. However,
compared to standard hot-SSCP, the conventional
(non-isotopic labeling) method has additional
advantages of dramatically increased speed, precise
temperature control, reproducibility, easily and
inexpensively obtainable reagents and equipment.
This new method also lacks the safety and hazardous
waste management concerns associated with
radioactive materials.
Many radioisotope-based methods can be described
as important tools for human diseases research and
diagnosis. It allows detection of as little as 0.1 pg of
target DNA due to its exquisite sensitivity, probes
labeled with isotopes such as 32P has an important
place in DNA hybridization, which is a widely used
molecular biology technique. Even though molecular
imaging of hot-SSCP followed by autoradiography
technique is relatively laborious and requires long
time for exposure, this isotope-labeled products can
provide up to approximately 125-fold increase in
sensitivity over EtBr staining, with a maximum of
625-fold greater sensitivity with a 3-day exposure [25].
Another finding showed that the sensitivity of
radioisotope labeled probes was only 10 fold than
EtBr staining [26].
Some other investigators used molecular technique
to detect the resistance of M. tuberculosis to ETH.
Brossier et al. [26] searched for mutations in 87 clinical
isolates and found that 47 isolates were ETH-resistant
and 40 were ETH-susceptible. These mutations were
found in ethA, ethR, or inhA or its promoter. Whereas
the analysis by Boonaiam et al. [27] on 160
drug-resistant M. tuberculosis clinical isolates from
Thailand showed that 13 of them were
ethionamide-resistant isolates and had mutations in
the ethA gene. Mutation analysis related to ETH
resistance among M. tuberculosis isolates from Korea
was done by Lee et al. [28] and found that 12 out of 14
isolates were resistant.
Diagnosis of TB in a resource-poor country is
problematic. During the past decade, efforts to
develop new molecular detection and analysis for TB
resistance have been intensified and considerable
progress has been made. A notable advantage of
molecular tests is their rapid turnaround time, which
may have implications for patient management and
transmission of drug-resistant M. tuberculosis.
Molecular tests such as PCR-SSCP, whose prices are
typically higher than those of conventional tests
(culture) may be popular in resource-rich settings.
However, the most resource-constrained countries
tend to have the highest burden of MDR-TB cases and
are the least likely to benefit from expensive
technologies because of high costs and a lack of
appropriate laboratory capacity [29].
Screening for mutations prior to sequencing can
reduce the time and costs of identifying mutations.
When the DNA sequence is known, the technique of
detecting mutations as SSCP is a convenient method
of screening for possible mutations due to its technical
simplicity and relatively high sensitivity for the
detection of mutations. It has the ability of detecting a
single base change, and has been applied to a big
number of genes. It must be considered that detection
and amplification of mutations in genes in a cheap and
100% effective manner is a major objective in modern
molecular genetics. SSCP is a reproducible, rapid and
quite simple method for the detection of deletions,
insertions and rearrangements in PCR amplified DNA [17].
The results of study demonstrate the feasibility of
using a PCR-based assay directly on sputum
specimens for analysis/detection of M. tuberculosis
resistance, and suggest that patients with
smear-positive, untreated tuberculosis and those
presenting with suspected drug-resistant tuberculosis
are the most appropriate groups for testing by PCR.
4. Conclusions
Fifty-three extracts of DNA was analyzed that 11
(20.75%) were positive for the target gene of ETH and
none of the samples show the alteration of DNA band
 Molecular Analysis of the Resistance of Mycobacterium tuberculosis to Ethionamide Using PCR-SSCP 175
Methods and the Implementation of Its Nuclear Technique
mobility shift on acrylamide gel that may cause the
resistance of bacteria to ETH. This low positive result
in PCR may be caused by annealing temperature and
DNA content problems. Study on the implementation
of hot-SSCP by labeling the DNA with 32P
radioisotope shows that this nuclear based technique
has the higher quality in terms of the visualization.
Acknowledgments
We deeply appreciate to Ms. Sakinah Alamanda for
technical assistance during experiment. Work of our
laboratory presented herein has been supported by
DIPA (Annual Research Projects) of the Center for
2006-2010 fiscal years.
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