Minor contribution of mutations at iniA codon 501 and embC-embA intergenic region in ethambutol-resistant clinical Mycobacterium tuberculosis isolates in Kuwait
Annals of Clinical Microbiology and Antimicrobials
0 Address: Department of Microbiology, Faculty of Medicine, Kuwait University , Kuwait City , Kuwait
Background: Ethambutol (EMB) is a first-line drug for the treatment of tuberculosis (TB). Resistance to EMB in Mycobacterium tuberculosis isolates is mediated by mutations in several genes involved in arabinan synthesis notably three emb (arabinosyl transferase) and iniA (isoniazidinducible) genes. Most epidemiologically unrelated EMB-resistant M. tuberculosis strains contain mutations at embB codons 306, 406 and 497, embC-embA intergenic region (IGR) and iniA codon 501 (iniA501). Objective: To develop a more comprehensive molecular screen for EMB-resistance detectioamong epidemiologically unrelated EMB-resistant M. tuberculosis strains previously analyzed for embB codon 306, 406 and 497 mutations by including analysis of mutations at iniA501 and in embC-embA IGR. Methods: Fifty consecutive and phenotypically documented EMB-resistant and 25 pansusceptible M. tuberculosis strains isolated from 75 different TB patients over a four-year period in Kuwait were analyzed. Mutations at iniA501 were detected by PCR amplification followed by restriction fragment length polymorphism (RFLP) patterns generated with Hpy 99 I. Direct DNA sequencing was used to confirm RFLP results and for detecting mutations in embC-embA IGR. Results: Nearly same number of EMB-resistant M. tuberculosis strains were resistant to EMB alone and EMB together with additional resistance to rifampicin and isoniazid (9 of 50, 18% and 11 of 50, 22%, respectively). All the 25 pansusceptible strains contained wild-type sequences at iniA501 and in embC-embA IGR. The analysis of 50 EMB-resistant M. tuberculosis isolates showed that only one strain contained a mutated iniA501 while no mutation was detected in embC-embA IGR in any of the isolate. Conclusion: Analysis of iniA501 and embC-embA IGR in epidemiologically unrelated EMB-resistant M. tuberculosis isolates in Kuwait indicate that mutations at these locations occur very infrequently and their inclusion for the development of a comprehensive molecular screen will make only minor contribution towards rapid EMB resistance detection.
The tuberculosis (TB) epidemic continues unabated.
Despite intense efforts made over the past two decades,
the morbidity and mortality associated with TB remain
high, with 8 million active disease cases and 2 million
deaths occurring worldwide every year [1,2]. Two factors,
co-infection with human immunodeficiency virus (HIV)
and increasing incidence of infections with drug-resistant
strains of Mycobacterium tuberculosis are steadily worsening
the problem of TB [3,4]. The latest World Health
Organization (WHO)-sponsored study showed that
drug-resistant TB among new cases is prevalent in 74 of 77 (96%)
settings or countries with resistance to at least one anti-TB
drug varying from 0% in some rich, developed countries
to >30% in several developing countries . Infections
with drug-resistant, particularly multidrug-resistant
(MDR) (resistant to at least rifampicin and isoniazid)
strains of M. tuberculosis (MDR-TB) are associated with
higher mortality [4,5]. Molecular methods for rapidly
identifying drug-resistant strains of M. tuberculosis are
urgently needed to avoid inadequate treatment since
phenotypic drug susceptibility testing by radiometric method
requires 414 days after the primary culture has been
isolated . A simple and rapid line probe assay has been
developed recently for detection of resistance of M.
tuberculosis to rifampicin and isoniazid (MDR-TB) in cultured
isolates and clinical samples 
Ethambutol (EMB), an arabinose analogue, is a
bactericidal, first-line drug for the treatment of TB. The EMB is
often used, along with isoniazid, rifampicin and
pyrazinamide, as an alternative to streptomycin, in the four-drug
regimens advocated by World Health Organization under
the directly observed chemotherapy short-course. Global
data on drug resistance patterns have shown that
resistance of M. tuberculosis to EMB in newly diagnosed as well
as previously treated cases is much less compared to
streptomycin [4,8]. Similar pattern is also noted in the rate of
resistance of M. tuberculosis isolates to EMB (1.98%) and
streptomycin (4.31%) in Kuwait . Since resistance of
M. tuberculosis to EMB is also generally associated with
resistance to other anti-TB drugs [8,10], early detection of
resistance will not only abolish drug-associated adverse
reactions, particularly optic neuritis, it will also suggest
modifications in therapy regimens.
The mechanism of action and the molecular genetic basis
of resistance to EMB are complex and are not completely
defined. The enzymes participating in synthesis and
polymerization of cell wall arabinan are implicated as the
main target for EMB. These include three homologous
and membrane associated arabinosyltransferases encoded
by three contiguous genes, embC-embA-embB,
isoniazidinducible genes particularly iniA, acyl carrier proteins and
regulatory proteins modulating their expression [11-13].
Mutations in embB particularly involving codon 306
(embB306) and less frequently, codons 406 (embB406)
and 497 (embB497) have been identified as most
common genetic alterations conferring resistance to EMB in
clinical M. tuberculosis isolates [11-14]. The frequency of
these mutations in EMB-resistant strains in some studies
has been reported to be ~70% [14-17]. However, in
epidemiologically unrelated strains, these mutations accounted
for EMB resistance in only around 30% EMB-resistant
isolates [13,18]. Only one study has so far detected
mutations in other emb (embA and embC) or other genes
conferring resistance to EMB in epidemiologically
unrelated EMB-resistant strains . The data showed that,
mutations in embC-embA intergenic region (IGR) (four
nucleotide positions), embA (several codons) and iniA
(mainly codon 501, iniA501) occurred in ~30%
EMBresistant strains while mutations in eight other genes were
either absent or occurred infrequently . However, the
role of mutations in these loci in conferring resistance to
EMB remain unclear as these mutations, particularly in
embC-embA IGR, were found in isolates with other
wellcharacterized EMB resistance conferring mutations .
The TCG to TGG mutation at iniA501 most likely confers
resistance of M. tuberculosis to EMB as it alters the
structural property of the encoded protein . However, the
resistance conferring mechanism explaining the role of
mutations in embC-embA IGR is not well characterized
and the paucity of information on the frequency of
mutations in embC-embA IGR in EMB-resistant M. tuberculosis
strains from different regions of the world makes it
difficult to ascertain their exact role in conferring resistance to
EMB. The present study was carried out to detect the
frequency of mutations at iniA501 and in embC-embA IGR in
50 consecutive and epidemiologically unrelated
EMBresistant M. tuberculosis strains that were analyzed
previously for embB306, embB406 and embB497 mutations 
in an effort to develop a more comprehensive molecular
screen for EMB-resistance detection. The EMB-susceptible
isolates were also analyzed simultaneously to ensure that
such mutations are not present in EMB-susceptible
Materials and methods
EMB-resistant and -susceptible M. tuberculosis strains
A single TB control unit and the Kuwait National
Tuberculosis Reference Laboratory (KNTRL) under the Ministry of
Health are responsible for the diagnosis and treatment of
all TB patients in Kuwait. Appropriate specimens from
suspected TB patients are sent to KNTRL for culture. All
active TB cases are diagnosed by culture and susceptibility
testing to first line anti-TB drugs is performed on all the
isolates identified as M. tuberculosis . All the 50
EMBresistant M. tuberculosis strains (KE1 to KE50) isolated
throughout Kuwait from 50 different TB patients during
2000 to 2003 were included in this study. The clinical
background and demographic data of patients yielding
EMB-resistant M. tuberculosis strains are shown in Table 1.
Repeat isolates were recovered from eight TB patients
within one to two months of isolation of the first isolate.
A total of 25 M. tuberculosis strains susceptible to all
firstline drugs (pansusceptible strains) were also included.
The isolation and identification of M. tuberculosis strains
from clinical specimens was performed using the
mycobacterial growth indicator tube (MGIT) 960 system as
described previously . The drug susceptibility testing
of M. tuberculosis isolates was performed using the
BACTEC 460 TB system as reported earlier [6,21]. The
isolates were considered EMB resistant when bacterial
growth occurred at a concentration of 2.5 g EMB per ml.
Resistance of all the isolates to isoniazid (0.1 g/ml),
rifampicin (2 g/ml) and streptomycin (2 g/ml) was also
Sample preparation for PCR
The M. tuberculosis reference strain H37Rv was used as a
control in PCR-RFLP and DNA sequencing. The reference
strain and clinical EMB-resistant and pansusceptible M.
tuberculosis isolates were obtained as heat killed BACTEC
liquid cultures. One ml of BACTEC culture was heated
with 40 mg Chelex-100 (Sigma) at 95C for 20 min
followed by centrifugation at 12,000 g for 15 min and the
supernatant obtained was used as the source of genomic
DNA  for the amplification of various gene regions.
Primer design and PCR amplification
Two primer pairs were synthesized. One primer pair
(INIAF and INIAR) was used for the amplification of the
iniA gene (Genbank accession no. Z95324) region around
codon 501. Another primer pair (IGRF and IGRR) was
synthesized for the amplification of embC-embA IGR
(Genbank accession no. AL123456) including nucleotide
positions that are mutated in EMB-resistant strains .
The DNA sequences of the primers, their positions on the
target genes and the expected size of the PCR products are
shown in Table 2.
The PCR amplification of the target DNA was carried out
in a final volume of 50 l and contained 5 l of 10
Perkin-Elmer PCR buffer, 10 pmol of the appropriate forward
(F) and reverse (R) primers (INIAF and INIAR or IGRF and
IGRR, Table 2), 2 l of template DNA, 0.1 mM dNTPs, 2
units of AmpliTaq DNA polymerase (Perkin-Elmer) and
sterile distilled water. The cycling parameters were same as
described previously . Following amplification, a
portion of the product (10 l) was run on 2% agarose gels
 to confirm the amplification of a DNA fragment of
expected size. The remaining PCR product was purified by
using PCR purification columns (Qiagen) that were used
as instructed by the manufacturer. The purified amplicons
were used for further studies.
Detection of mutations at iniA501 by PCR-RFLP
The mutations at iniA501 were detected by restriction
digestion of amplicons obtained with primers INIAF and
INIAR with Hpy 99I as described previously . Briefly,
the reaction mixture in a final volume of 25 l contained
5 l of the purified amplicon, 10.25 l of sterile water,
6.25 l of NE Buffer 4, 2.5 l of 10 BSA and 1 l (2 units)
of Hpy99 I (New England Biolabs). After incubation at
37C for 1 h, the digested products were separated on
2.5% agarose gels. The amplicon from M. tuberculosis
H37Rv as well as M. tuberculosis strains containing TCG at
iniA501 (iniA501TCG) yield two fragments of 104 bp and
137 bp while the amplicon from isolates containing TGG
at iniA501 (iniA501TGG) yield the original undigested
fragment of 241 bp . The results of RFLP for some of
the isolates were confirmed by DNA sequencing. The
DNA sequencing was performed by using the cycle DNA
sequencing kit (DTCS CEQ2000, Beckman Coulter) as
described in detail previously . Briefly, the reaction
mixtures in a final volume of 20 l contained, 8.0 l of
purified amplicon, 1.0 l (3.2 pmol) of primer INIAF or
INIAR (Table 2) or an internal primer (INIAS,
5'-CGCTGGGCCGGATGGAATCGAA-3'), 8.0 l of the pre-mix
reaction components supplied in the kit and 3.0 l sterile
water. The cycling parameters were same as described
previously . Reactions products were precipitated and
loaded on the DNA sequencer as directed by the
manufacturer (Beckman Coulter Model CEQ8000).
Detection of mutations in embC-embA IGR by DNA
The mutations at -11, -12, -16 and -43 positions (relative
to the translational start point of embA) in embC-embA IGR
 were detected by direct DNA sequencing of purified
amplicons, obtained by using IGRF and IGRR primers.
The DNA sequencing was carried out, as described above
except that primer IGRF or IGRR (Table 2) or an internal
primer (IGRS, 5'-CCGCTGATCTGAACCTAGGAAC-3')
was used as sequencing primer.
Molecular fingerprinting of EMB-resistant M. tuberculosis
The molecular fingerprinting of EMB-resistant M.
tuberculosis isolates was performed by genetic group analysis 
based on polymorphisms at katG codon 463 (katG463)
and gyrA codon 95 (gyrA95) and by touchdown
doublerepetitive-element (DRE)-PCR. The presence of Arg463/
Leu463 at katG463 and Ser95/Thr95 at gyrA95 were
determined by PCR-RFLP as described previously [24,25].
Briefly, the amplification of katG463 and gyrA95 DNA
regions was carried out as described above except that
(5'-CCCGAGGAATTGGCCGACGAGTTC-3') + KatG1R
(5'-GGTGCGAATGACCTTGCGCAGATC-3')  and GYRA95F (5'-CGCAGCTACATCGA
CTATGCGATG-3') + GYRA95R
(5'-GGGCTTCGGTGTACTable 1: Clinical background, resistance patterns and demographic information of patients yielding EMB-resistant M. tuberculosis
isolates in Kuwait
Isolate no. Year of isolation Clinical specimena Resistance patternb
Table 1: Clinical background, resistance patterns and demographic information of patients yielding EMB-resistant M. tuberculosis
isolates in Kuwait (Continued)
After incubation at 37C for 4 h, the digested products
were separated on 2.5% agarose gels. The amplicon from
M. tuberculosis H37Rv as well as M. tuberculosis strains
containing Arg at katG463 (katG463CGG) yield two fragments
of 187 bp and 173 bp while the amplified fragment from
isolates containing Leu at katG463 (katG463CTG) yield
Primer pair Primer name
Target genea Directionb/position Target region Amplicon size (bp) Mutations detected
ACGAAGCGCCGC ACATTGGCCTT-3' 5'
CGACGGCTGCTA3' R, 16431621 R, 2932129298
Hpy 99 I RFLP
aThe accession numbers for target genes are iniA, Z95324; embC-embA IGR, AL123456
bF, forward; R, reverse
cIGR, he embC-embA intergenic region
d-11 to -43 corresponds to -11, -12, -16 and -43 positions (relative to the start codon of embA) in embC-embA IGR
the original undigested fragment of 360 bp . The
purified gyrA95 region amplicons were digested with
restriction enzyme Ale I to obtain RFLP patterns. The reaction
mixture in a final volume of 25 l contained, 2.5 l of NE
Buffer 4, 1.25 l of 10 BSA (New England Biolabs), 6.0
l of the amplified DNA, 1 l (5 units) of Ale I and 14.25
l of sterile water. After incubation at 37C for 3 h, the
digested products were separated on 2.5% agarose gels.
The amplicon from M. tuberculosis H37Rv as well as M.
tuberculosis strains containing Ser at gyrA95 (gyrA95AGC)
yield the original undigested fragment of 322 bp while the
amplified fragment from isolates containing Thr at gyrA95
(gyrA95ACC) yield two fragments of 212 bp and 110 bp
The typing of EMB-resistant M. tuberculosis isolates was
performed by touchdown DRE-PCR as described
previously . Briefly, the reaction mixtures in a final volume
of 50 l contained 1 Perkin-Elmer PCR buffer II, 2.5 mM
MgCl2, 10 pmol each of the four primers (IS6110-5'R,
5'TCCCCGCCGTTGCCGTACAG-3' and PGRS-3'F,
5'-CTTGGGAAACCCGGCCAGCTG-3') , 2 l of template
DNA, 0.2 mM dNTPs and 2 units of AmpliTaq DNA
polymerase. The cycling parameters were same as
described previously . The amplified products (20 to
25 l) were resolved on 2% agarose gels. The M.
tuberculosis isolates belonging to different genetic groups and/or
yielding unique patterns of DNA amplified fragments in
DRE-PCR were considered as genotypically distinct
Nucleotide sequence accession numbers
The DNA sequencing data for the mutant strain reported
in this study have been deposited in EMBL under the
accession number AJ973188.
Results and Discussion
A total of 50 EMB-resistant strains of M. tuberculosis were
isolated from 50 different TB patients throughout Kuwait
during the year 2000 to 2003 and all the isolates were
included for analysis. Twenty of these isolates were from
patients of Middle-Eastern origin (including seven
Kuwaiti nationals), 27 from patients of South-Asian
origin and three from patients of Southeast Asian countries
(Table 1). All the isolates were recovered from
HIV-negative adult TB patients (18 to 65 years) and included 15
female and 35 male patients. The clinical specimens
yielding these strains included sputum (n = 29), pus (n = 6),
fine needle aspirate (n = 7), lymph node (n = 3), pleural
fluid (n = 2), abscess (n = 1), bronchoalveolar lavage
(BAL) (n = 1), and gastric secretion (n = 1) (Table 1). In
addition, 25 pansusceptible clinical M. tuberculosis isolates
were also included in the study. Nine, 12 and four of these
isolates were from patients of Middle-Eastern (including
three Kuwaiti nationals), South Asian, and Southeast
Asian origin, respectively. The clinical specimens yielding
pansusceptible strains included sputum samples (n = 15),
pus (n = 6), fine needle aspirate (n = 3), and tissue biopsy
(n = 1). The data are consistent with previously reported
observations that majority (~80%) of M. tuberculosis
infections in Kuwait occur in foreign-born expatriate workers
mostly within the first few years of their migration even
though all expatriates entering Kuwait are screened for TB
(chest radiograph) as well as for HIV infection [9,27].
Only nine of 50 (18%) EMB-resistant strains were
monoresistant to EMB while the remaining (41 of 50,
82%) isolates were additionally resistant to at least one
more first-line drug. Most (39 of 41) of the latter isolates
were resistant to isoniazid with or without additional
resistance to other drugs. Twenty-four (48%) M.
tuberculosis strains were resistant to two drugs while eight (16%)
isolates were resistant to three drugs (including three
MDR-TB strains). Only eight (16%) EMB-resistant strains
were also resistant to the other three first-line drugs. All
the isolates were resistant to EMB on first isolation
indicating that the patients were already infected with
EMBresistant strains. Furthermore, nearly same number (9 of
50, 18% and 11 of 50, 22%) of EMB-resistant M.
tuberculosis strains were resistant to EMB alone or EMB together
with additional resistance to rifampicin and isoniazid
(MDR-TB strains). Worldwide, monoresistance to EMB is
rare and majority of EMB-resistant strains particularly
those originating from TB endemic countries are MDR-TB
The PCR amplification of iniA501 DNA region from 25
pansusceptible and 50 EMB-resistant M. tuberculosis
strains yielded an amplicon of 241 bp, as expected. The
Hpy 99 I digestion patterns of purified amplicons showed
that all the 25 pansusceptible and 49 EMB-resistant strains
contained TCG at iniA501 (iniA501TCG) while only one
EMB-resistant strain contained a mutated iniA501. The
results of PCR-RFLP for three pansusceptible strains
containing iniA501TCG, three EMB-resistant strains also
containing iniA501TCG and one EMB-resistant strain
containing a mutated iniA501 were confirmed by DNA
sequencing. The solitary isolate with a mutation at
iniA501 contained iniA501TGG (Table 3). Likewise, the
PCR amplification of DNA region encompassing
embCembA IGR from 25 pansusceptible and 50 EMB-resistant
M. tuberculosis strains also yielded an amplicon of 261 bp,
as expected. Direct DNA sequencing of the purified
amplicons showed that all the 25 pansusceptible and 50
EMBresistant strains contained wild-type sequences including
nucleotide positions -11, -12, -16 and -43  relative to
the translational start point for embA gene (Table 3). The
repeat isolates recovered from eight TB patients yielded
the same resistance pattern and the same results for
iniA501 and embC-embA IGR analyses as the parent isolate
(data not shown).
Consistent with previously reported data showing that
majority of rifampicin-resistant and isoniazid-resistant M.
tuberculosis infections in Kuwait occur in foreign-born TB
patients as a result of reactivation of previously acquired
infection [21,29,30], genetic group analysis and
fingerprinting patterns obtained in DRE-PCR showed that
majority of EMB-resistant M. tuberculosis strains were also
unique strains (data not shown).
Majority (~70%) of EMB-resistant M. tuberculosis strains
from several geographical locations around the world
have been shown to contain mutations within embB gene
particularly at embB306, embB406 and embB497 [14-17].
However, in most of these studies, all or consecutive
EMBsusceptible and EMB-resistant M. tuberculosis strains from
a single location and/or isolated over a specified period of
time were not included and the proportion of MDR-TB
strains or strains resistant to several anti-TB drugs was
unusually high [15-17]. When the 50 phenotypically
documented EMB-resistant M. tuberculosis strains used in this
study were tested for genotypic resistance detection
targeting mutations at embB306, embB406 and embB497, only
38% (19 of 50) isolates contained a resistance conferring
mutation . These findings suggested that mutations at
other codon positions within embB gene or in other loci
are responsible for conferring resistance to EMB in
majority of EMB-resistant M. tuberculosis strains in Kuwait and
their identification will be crucial for the development of
a molecular screen for rapid detection of majority of
The results presented in this study showed that mutations
at iniA501 and in embC-embA IGR in EMB-resistant M.
tuberculosis strains in Kuwait also occur rather infrequently
(1 of 50, 2%). Thus, their inclusion in the molecular
screen will have only minor contribution towards
detection of EMB resistance in Kuwait. Only one previous study
has detected the occurrence of mutations at iniA501 and
in embC-embA IGR in EMB-resistant M. tuberculosis strains
. Although the authors reported that mutations at
iniA501 and in embC-embA IGR occurred in nearly 20%
Table 3: Occurrence of mutations at iniA501 and embC-embA IGR in 25 pansusceptible and 50 EMB-resistant clinical M. tuberculosis
strains isolated in Kuwait
Susceptibility to ethambutol No. of isolates
No. of isolates with iniA501 as
No. of isolates with wild-type nucleotides at -11, -12, -16 and
43 in embC-embA IGR
Wild-type (TCG) Mutant (TGG)
(15 of 75) of EMB-resistant strains, majority of isolates
with mutations at iniA501 and all the isolates with a
mutation in embC-embA IGR also contained other well
characterized EMB resistance-conferring mutations,
particularly at embB306, embB406 and embB497 .
Previous studies have shown that drug-resistant M. tuberculosis
strains isolated from TB patients with limited previous
exposure to first-line anti-TB drugs usually contain single
point mutations in resistance conferring genes while
strains from previously treated patients usually contain
multiple mutations in target genes [15,18,20,31-33]. The
susceptibility of EMB-resistant strains to other first-line
drugs and the HIV status of the TB patients in the study
that analyzed mutations at iniA501 and in embC-embA
IGR was not indicated . However, the presence of
mutations in two or more genes involved in EMB
resistance in 25% (19 of 75) EMB-resistant strains and the
geographical origin of the tested isolates indicates that the
isolates were either MDR-TB strains or strains additionally
resistant to multiple first-line drugs . These
observations are also consistent with the role of mutations at
iniA501 and in embC-embA IGR in conferring resistance to
The TCG to TGG mutation at iniA501 most likely confers
resistance of M. tuberculosis to EMB by altering the
structural property of the encoded protein . However, the
resistance conferring mechanism explaining the role of
mutations in embC-embA IGR in EMB resistance is not well
characterized. The suggestion that C to T mutations at -12
and -16 positions lead to creation of TATA box-like
sequences  seems less likely since these elements are
found in promoter elements while the hot-spot DNA
segment is immediately upstream of the translational start
point for EmbA and putatively involves ribosomal
binding site. Furthermore, other characteristic features of
promoter elements (such as -35 element sequences) are also
absent in the surrounding DNA region. An alternative
(titration) mechanism to explain the role of mutations in
embC-embA IGR is summarized below and most likely
involves premature termination of some transcripts
originating from embCAB promoter before embA and embB
genes are transcribed. The embC-embA IGR of 85
nucleotides is rather long for a polycistronic bacterial mRNA. A
closer look at embC-embA IGR and N-terminal region of
embA indicates that it contains an I-shaped transcriptional
terminator typically found in mycobacteria including M.
tuberculosis . The RNA transcript involving -19 to +23
nucleotides (relative to translational start site of embA)
can form a hairpin structure that is stabilized by nine G.C
pairs including the two 'C' residues (at -12 and -16
positions) that are mostly mutated in some EMB-resistant
strains . Thus, mutations of -12C or -16C residues will
decrease the stability of the hair-pin structure resulting in
decreased termination of transcripts and consequently,
increased expression of embA and embB encoded gene
products. These assumptions are further supported by
occurrence of nearly all the mutations in embC-embA IGR
in isolates with a mutated embB gene. The above also
explains the absence of these mutations in EMB-resistant
M. tuberculosis isolates in Kuwait as such mutations are
more likely to occur in TB patients with previous history
of prolonged therapy with anti-TB drugs
In conclusion, the results presented in this study showed
that mutations at iniA501 and in embC-embA IGR in
EMBresistant M. tuberculosis strains isolated from TB patients in
Kuwait occur infrequently. Consequently, inclusion of
mutation detection at these loci in the molecular screen
for detecting majority of EMB-resistant M. tuberculosis
strains will contribute minimally towards this goal in
Kuwait as well as, perhaps, in other countries. Further
studies from other geographical regions are clearly
warranted to validate our observations.
The authors declare that they have no competing interests.
SA and EM participated in the design of the study,
analyzed the data and drafted the manuscript. They also
arranged financial support for the study. A-AJ carried out
the experimental work. All authors have read and
approved the final manuscript.
This study was supported by Research Administration grant MI 06/02 and
the college of Graduate Studies, Kuwait University.
1. Harries AD , Dye C : Tuberculosis. Ann Trop Med Parasitol 2006 , 100 : 415 - 31 .
2. World Health Organization: Global tuberculosis control: surveillance, planning, financing . WHO report 2007 . Geneva, Switzerland, World Health Organization . WHO/HTM/TB/ 2007 376: 1 - 277 .
3. Wells CD , Peter Cegielski J , Nelson LJ , Laserson KF , Holtz TH , Finlay A , Castro KG , Weyer K : HIV infection and multidrug-resistant tuberculosis-the perfect storm . J Infect Dis 2007 , 196 (Suppl 1): S86 - 107 .
4. Aziz MA , Wright A , Laszlo A , De Muynck A , Portaels F , Van Deun A , Wells C , Nunn P , Blanc L , Raviglione M , for the WHO/International Union Against Tuberculosis and Lung Disease global project on antituberculosis drug resistance surveillance: Epidemiology of antituberculosis drug resistance (the Global Project on Anti-tuberculosis Drug Resistance Surveillance): an updated analysis . Lancet 2006 , 368 : 2142 - 54 .
5. Zignol M , Hosseini MS , Wright A , Lambregts-van Weezenbeek C , Nunn P , Watt CJ , Williams BG , Dye C : Global incidence of multidrug-resistant tuberculosis . J Infect Dis 2006 , 194 : 479 - 85 .
6. Siddiqi SH , Hawkins J , Laszlo A : Interlaboratory drug susceptibility testing of Mycobacterium tuberculosis by radiometric procedure and two conventional methods . J Clin Microbiol 1985 , 22 : 919 - 23 .
7. Lacoma A , Garcia-Sierra N , Prat C , Ruiz-Manzano J , Haba L , Ross S , Maldonado J , Domnguez J : GenoType MTBDRplus assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical samples . J Clin Microbiol 2008 , 46 : 3660 - 7 .
8. Espinal MA , Laszlo A , Simonsen L , Boulahbal F , Kim SJ , Reniero A , Hoffner S , Rieder HL , Binkin N , Dye C , Williams R , Raviglione MC , for the World Health Organization-International Union Against Tuberculosis and Lung Disease working group on anti-tuberculosis drug resistance surveillance .: Global trends in resistance to antituberculosis drugs . N Engl J Med 2001 , 344 : 1294 - 303 .
9. Mokaddas E , Ahmad S , Samir I : Secular trends in susceptibility patterns of Mycobacterium tuberculosis isolates in Kuwait , 1996 - 2005 . Int J Tuberc Lung Dis 2008 , 12 : 319 - 25 .
10. Cavusoglu C , Hilmioglu S , Guneri S , Bilgic A : Characterization of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates from Turkey by DNA sequencing and line probe assay . J Clin Microbiol 2006 , 44 : 2338 - 42 .
11. Alcaide F , Pfyffer GE , Telenti A : Role of embB in natural and acquired resistance to ethambutol in mycobacteria . Antimicrob Agents Chemother 1997 , 41 : 2270 - 3 .
12. Telenti A , Phillipp W , Sreevatsan S , Bernasconi C , Stockbauer KE , Wieles B , Musser JM , Jacobs WR Jr: The emb operon, a unique gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol . Nat Med 1997 , 3 : 567 - 70 .
13. Ramaswamy S , Amin AG , Koksel S , Stager CE , Dou S-J , El Sahly H, Moghazeh SL , Kreiswirth BN , Musser JM : Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis . Antimicrob Agents Chemother 2000 , 44 : 326 - 36 .
14. Sreevatsan S , Stockbauer KE , Pan X , Kreiswirth BN , Moghazeh SL , Jacobs WR Jr, Telenti A , Musser JM : Ethambutol resistance in Mycobacterium tuberculosis : critical role of embB mutations . Antimicrob Agents Chemother 1997 , 41 : 1677 - 81 .
15. Ramaswamy SV , Dou S-J , Rendon A , Yang Z , Cave MD , Graviss EA : Genotypic analysis of multidrug-resistant isolates from Monterrey , Mexico. J Med Microbiol 2004 , 53 : 107 - 13 .
16. Parsons LM , Salfinger M , Clobridge A , Dormandy J , Mirabello L , Polletta VL , Sanic A , Sinyavskiy O , Larsen SC , Driscoll J , Zickas G , Taber HW : Phenotypic and molecular characterization of Mycobacterium tuberculosis isolates resistant to both isoniazid and ethambutol . Antimicrob Agents Chemother 2005 , 49 : 2218 - 25 .
17. Pinke C , Rusch-Gerdes S , Nieman S : Significance of mutations in embB codon 306 for prediction of ethambutol resistance in clinical Mycobacterium tuberculosis isolates . Antimicrob Agents Chemother 2006 , 50 : 1900 - 2 .
18. Ahmad S , Jaber A-A , Mokaddas E : Frequency of embB codon 306 mutations in ethambutol-susceptible and -resistant clinical Mycobacterium tuberculosis isolates in Kuwait . Tuberculosis 2007 , 87 : 123 - 9 .
19. Mokaddas E , Ahmad S : Development and evaluation of a multiplex PCR for rapid detection and differentiation of Mycobacterium tuberculosis complex members from non-tuberculous mycobacteria . Jpn J Infect Dis 2007 , 60 ( 2-3 ): 140 - 144 .
20. Ahmad S , Mokaddas E : The occurrence of rare rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolates from Kuwait . Int J Antimicrob Agents 2005 , 26 : 205 - 12 .
21. Ahmad S , Mokaddas E , Fares E : Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai . Diagn Microbiol Infect Dis 2002 , 44 : 245 - 52 .
22. Ahmad S , Mokaddas E , Jaber A-A : Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCRRFLP targeting embB codons 306 and 497 and iniA codon 501 mutations . Mol Cell Probes 2004 , 18 : 299 - 306 .
23. Sreevatsan S , Pan X , Stockbauer KE , Connell ND , Kreiswirth BN , Whittam TS , Musser JM : Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination . Proc Natl Acad Sci USA 1997 , 94 : 9869 - 74 .
24. Ahmad S , Mokaddas E , Abal AT , Araj GF , Fares E , Mustafa AS : Genetic polymorphism at codon 463 in the katG gene in isoniazid-sensitive and -resistant Mycobacterium tuberculosis isolates from the Middle East . Med Princ Pract 2001 , 10 : 129 - 34 .
25. Mokaddas E , Ahmad S , Abal AT , Al-Shami AS : Molecular fingerprinting reveals familial transmission of rifampin-resistant tuberculosis in Kuwait . Ann Saudi Med 2005 , 25 : 150 - 3 .
26. Mokaddas E , Ahmad S , Abal AT : Molecular fingerprinting of isoniazid-resistant Mycobacterium tuberculosis isolates from Chest Disease Hospital in Kuwait . Microbiol Immunol 2002 , 46 : 767 - 71 .
27. Mokaddas E , Ahmad S : Species spectrum of nontuberculous mycobacteria isolated from clinical specimens in Kuwait . Curr Microbiol 2008 , 56 : 413 - 7 .
28. Hazbon MH , Bobadilla del Valle M , Guerrero MI , Varma-Basil M , Filliol I , Cavatore M , Colangeli R , Safi H , Billman-Jacobe H , Lavender C , Fyfe J , Garcia-Garcia L , Davidow A , Brimacombe M , Leon CI , Porras T , Bose M , Chaves F , Eisenach KD , Sifuentes-Osornio J , Ponce de Leon A , Cave MD , Alland D : Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance . Antimicrob Agents Chemother 2005 , 49 : 3794 - 802 .
29. Abal AT , Ahmad S , Mokaddas E : Variations in the occurrence of the S315T mutation within the katG gene in isoniazid-resistant clinical Mycobacterium tuberculosis isolates from Kuwait . Microb Drug Resist 2002 , 8 : 99 - 105 .
30. Ahmad S , Mokaddas E : Contribution of AGC to ACC and other mutations at codon 315 of the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolates from the Middle East . Int J Antimicrob Agents 2004 , 23 : 473 - 9 .
31. Cardoso RF , Cooksey RC , Morlock GP , Barco P , Cecon L , Forestiero F , Leite CQF , Sato DN , Shikama MdeL , Mamizuka EM , Hirata RDC , Hirata MH : Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil . Antimicrob Agents Chemother 2004 , 48 : 3373 - 81 .
32. Siddiqi N , Shamim M , Hussain S , Choudhary RK , Ahmed N , Prachee , Banerjee S , Savithri GR , Alam M , Pathak N , Amin A , Hanief M , Katoch VM , Sharma SK , Hasnain SE : Molecular characterization of multidrug-resistant isolates of Mycobacterium tuberculosis from patients in North India . Antimicrob Agents Chemother 2002 , 46 : 443 - 50 .
33. Caws M , Duy PM , Tho DQ , Lan NT , Hoa DV , Farrar J : utations prevalent among rifampin- and isoniazid-resistant Mycobacterium tuberculosis Misolates from a hospital in Vietnam . J Clin Microbiol 2006 , 44 : 2333 - 7 .
34. Mitra A , Angamuthu K , Nagaraja V : Genome-wide analysis of the intrinsic terminators of transcription across the genus Mycobacterium . Tuberculosis 2008 , 88 : 566 - 75 .