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Advances in Environmental Biology, 8(22) November 2014, Pages: 893-897
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Molecular Mechanisms of Resistance to Injectable Agents in Mycobacterium
Tuberculosis
1Khodadadi
5Shiva
Ali, 2Roshdi Maleki Mehdi, 3Abbasi Masoumeh, 4Habibollahi Mohammad Hossein,
Ahmadishoar
1
Department of Veterinary, Malekan Branch, Islamic Azad University, Malekan, Iran.
Department of Microbiology, College of Sciences , Karaj Branch, Islamic Azad University, Alborz, Iran.
Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
5
Department of Microbiology, Malekan Branch, Islamic Azad University, Malekan, Iran.
2,4
3
ARTICLE INFO
Article history:
Received 25 October 2014
Received in revised form 26 November
2014
Accepted 29 December 2014
Available online 15 January 2015
Keywords:
Molecular mechanisms,
injectable agents, TB
resistance,
ABSTRACT
Tuberculosis (TB) has remained one of the most difficult infections to treat. Most
current TB regimens consist of six to nine months of daily doses of four drugs that are
highly toxic to patients. The purpose of these lengthy treatments is to completely
eradicate Mycobacterium tuberculosis, notorious for its ability to resist most
antibacterial agents, thereby preventing the formation of drug resistant mutants. On the
contrary, the prolonged therapies have led to poor patient adherence. This, together with
a severe limit of drug choices, has resulted in the emergence of strains that are
increasingly resistant to the few available antibiotics. Here we review our current
understanding of molecular mechanisms the profound drug resistance of M.
tuberculosis. This knowledge is essential for the development of more effective
antibiotics that not only are potent against drug resistant M. tuberculosis strains but also
help shorten the current treatment courses required for drug susceptible TB.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Khodadadi Ali, Roshdi Maleki Mehdi, Abbasi Masoumeh, Habibollahi Mohammad Hossein, Shiva Ahmadishoar.,
Molecular Mechanisms of Resistance to Injectable Agents in Mycobacterium tuberculosis. Adv. Environ. Biol., 8(22), 893-897, 2014
INTRODUCTION
The emergence of drug-resistant TB in many countries has become a major public health problem and an
obstacle to effective tuberculosis control. In 2011, the World Health Organization (WHO) reported nearly
60,000 new cases of multidrug-resistant tuberculosis (MDR-TB) [1] and estimates of the annual global
incidence are much higher. The emergence of drug-resistant strains has made the treatment of TB complex,
costly, toxic, time-intensive, and less efficacious. Design of a treatment regimen for drug-resistant TB includes
the administration of first-line drugs to which the strains remain susceptible together with second-line drugs.
These second-line agents are more expensive, more difficult to administer (several require intravenous
administration), and are often associated with severe toxicities, including hepatic and renal dysfunction. In
comparison to the 6 months required to treat drug-susceptible TB, drug-resistant TB requires a prolonged
treatment duration of 18 to 24 months. These logistics constitute considerable hardships for patients as well as
for overburdened public health services. Too frequently, premature discontinuation of therapy occurs, leading to
treatment failure and the emergence of Mycobacterium tuberculosis strains with additional drug resistance.
Definitions of drug resistant tuberculosis (TB):
MDR-TB is defined as M. tuberculosis with in vitro resistance to two first-line medications: ionized
(isonicotinic acid hydrazide, INH) and rifampin (RIF). Extensively drug-resistant TB (XDR-TB) is defined as
M. tuberculosis that is resistant to not only INH and RIF, but also to other medication classes that comprise the
backbone of drug-resistant TB therapy, namely a quinolone and one of the second-line inject able drugs
(kanamycin [KAN], amikacin [AMK], or capreomycin [CAP]) [2]. Totally drug-resistant TB (TDR-TB) refers
to strains that are resistant to all available TB drugs, although the number and degree of resistance to each drug
Corresponding Author: Khodadadi Ali, Department of Veterinary, Malekan Branch, Islamic Azad University, Malekan,
Iran.
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Khodadadi Ali et al, 2014
Advances in Environmental Biology, 8(22) November 2014, Pages: 893-897
has not yet been precisely defined. The emergence of these strains further underscores the medical and public
health urgency to control drug-resistant TB [3,4].
Mechanisms of drug resistance:
The traditional mechanisms by which bacteria achieve antimicrobial resistance are (i) barrier mechanisms
(decreased permeability/efflux), (ii) degrading/inactivating enzymes, (iii) modification of pathways involved in
drug activation/metabolism, and (iv) drug target modification or target amplification. As will be discussed
below, M. tuberculosis uses all of these mechanisms to achieve resistance. For the aim of this article we define
drug resistance as genetic changes that alter the phenotypic resistance levels. Antibiotic tolerance (not discussed
herein) addresses mechanisms that do not require genetic changes in order to achieve an altered phenotypic
resistance level and is usually reversible after removal of the drug or a change in the growth conditions.
Amino glycosides have been used to treat TB since the introduction of the first antituberculous agent,
streptomycin (STR). Currently, STR remains a first-line agent, although its use is argely restricted to retreatment
cases because like other amino glycosides it must be given parent rally. AMK and KAN are the other major
amino glycosides used for TB. These may be given intravenously or intramuscularly and are second-line agents
used to treat MDR- or XDR-TB. Amino glycosides are considered bactericidal drugs and achieve rapid bacterial
killing during the initiation phase of treatment. They have poor sterilizing activity and must be combined with
agents such as rifamycins and pyrazinamide (PZA) in order to achieve durable cure.
Mechanism of action:
Amino glycosides inhibit protein synthesis by binding to the 30S subunit of the mycobacterium ribosome.
The majority of mutations that confer amino glycoside resistance lead directly or indirectly to alterations in the
amino glycoside binding pockets of the ribosome, which prevent drug binding but preserve ribosome function as
occurs with mutations in the rpsL, rrs, and gidB genes. A minor mechanism of amino glycoside resistance is
drug modification like that which occurs with the Eis protein, an amino glycoside acetyl transferase.
STR is a streptidine amino glycoside. On binding to the 30S ribosomal subunit, STR inhibits translational
initiation and may also cause misreading of mRNA (6). AMK and KAN are deoxystreptamine amino
glycosides, which bind to a different locus on the 30S ribosome. Because of these differences in ribosome
binding site between STR and AMK/KAN, different drug-conferring mutations are associated with these two
drug groups. In contrast, owing to the structural and functional similarity between AMK and KAN, there is
extensive cross-resistance between them.
Mechanism of resistance:
Target modification mutations:
rpsL gene:
The rpsL gene encodes the 12S protein, which is a structural component of the ribosome. The RpsL protein
serves to stabilize the pseudo knot that is formed by the 16S rRNA component of the 30S ribosome. While rpsL
is an essential gene, no synonymous mutations are tolerated for ribosomal function but result in reduced amino
glycoside binding. Common mutations in amino glycoside resistance that confer resistance to STR are RpsL
K43R and K88R [7,8].
Rrs gene:
The rrs gene encodes the 16S rRNA itself. The 16S rRNA contains key structures such as the 530 stem loop
and the 915 turn; both of these rRNA structures form contacts for the binding of amino glycosides. Like rpsL,
rrs is an essential gene. No synonymous SNPs in the rrs gene result in reduced amino glycoside binding while
allowing for preserved ribosome function. Many bacteria harbor multiple copies of the rrs gene, and
consequently single-allele rrs mutations confer low-level amino glycoside resistance. In contrast, M.
tuberculosis has only one rrs gene, and mutations in the M. tuberculosis rrs gene are usually associated with
high-level amino glycoside resistance. While more than 20 rrs mutations have been associated with amino
glycoside resistance, the common polymorphisms are A1401G, A514C, and C517T. Some rrs mutations also
confer resistance to the cyclic peptide antibiotic CAP . The most common mutation for second-line amino
glycoside resistance is A1401G, which was found in 78%, 56%, and 76% of AMK, KAN, and CAP resistant
strains, respectively, in one large systematic review [9].
gidB gene:
The gidB gene encodes a 7- methylguanosine (m7G) methyltransferase that specifically modifies residues
on the 16S rRNA (rrs). gidB is a nonessential gene, and loss-of-function mutations in gidB result in failure to
methyl ate G527 within the 530 loop of the 16S rRNA molecule [10]. Reduced ribosomal methylation confers
low-level amino glycoside resistance by reducing the affinity of the drugs for the 16S rRNA binding site. Many
different gidB mutations including deletions are associated with amino glycoside resistance, suggesting that loss
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Advances in Environmental Biology, 8(22) November 2014, Pages: 893-897
of function confers resistance. Polymorphisms in gidB have also been identified in drug-susceptible strains,
indicating that as for other resistance-conferring loci, the presence of a mutation in a gene is not necessarily
indicative of resistance.
Mechanism of resistance:
Inactivating mutations: eis:
The eis gene encodes an aminoglycoside acetyltransferase, which has an affinity for KAN. KAN acetylation
inactivates the drug by preventing it from binding to the 30S ribosome. Promoter upregulating mutations in the
5´ untranslated region of eis are associated with clinically relevant M. tuberculosis resistance to KAN. While the
Eis protein is capable of acetylating AMK, its affinity for AMK is low, and studies of clinical isolates with eis
promoter-up mutations reveal selective KAN resistance with relative preservation of AMK susceptibility [11].
Nevertheless, clinical isolates with eis mutations that are KAN and AMK-resistant have been described [7], so
the specificity of eis mutations for KAN resistance remains uncertain.
Mechanism of resistance:
up regulation of drug in activator and drug efflux: whiB7 Recently, mutations in the promoter region of the
transcriptional activator whiB7 have been identified in clinical isolates. These promoter mutations result in up
regulation of the whiB7 regulon, leading to an increase in eis expression and resulting KAN resistance [12]. The
whiB7 regulon also includes Rv1258c, which encodes an efflux pump, tap, that is upregulated in tandem with
eis, resulting in STR resistance. whiB7 promoter mutations therefore can result in both a target modification and
efflux mechanism of resistance, leading to cross-resistance to two amino glycoside drug groups [12].
STR resistance-conferring mutations:
rpsL, rrs, gidB:
High-level STR resistance is achieved predominantly by mutations in rpsL (∼50% of STR-resistant strains)
and rrs (∼15% of STR-resistant strains). Important rrs mutations associated with STR resistance are rrs A514C
and A908C. While the rrs A514C mutation is associated with high-level resistance to STR, the most common
rrs mutation conferring AMK and KAN resistance (rrs A1401G) does not confer STR cross-resistance. gidB
mutations are less frequent among STR-resistant strains (∼20% of strains) and are associated with low-level
STR resistance [7]. Mechanisms of low-level resistance have not been well characterized but probably involve
drug efflux.
Amikacin and kanamycin resistance conferring mutations: rrs, eis:
The rrs A1401G is the most frequent mutation conferring AMK and KAN resistance, occurring in ~85% or
more of strains that are AMK or KAN resistant. The presence of A1401G appears to be 100% specific for co
resistance to AMK and KAN. Of AMK and KAN resistant strains, 10 to 15% harbor eis mutations, indicating
that eis is a minor determinant of AMK and KAN resistance [5,7].
Capreomycin:
CAP and viomycin (VIO) are cyclic peptide antibiotics with structural similarity. The drugs have uniform
cross-resistance in M. tuberculosis and appear to have similar mechanisms of action. While VIO is rarely used
due to high toxicity, CAP is an inject able drug commonly used as a second-line agent in the management of
MDR and XDR-TB that is resistant to amino glycosides.
Like the structurally unrelated amino glycosides, CAP and VIO are bactericidal drugs that inhibit protein
synthesis. VIO has been shown to bind both the 30S and 50S ribosome subunits and to inhibit ribosomal
translocation by interference with the peptidyl tRNA acceptor site [13,14]. Due to overlap in the binding region
of CAP and the amino glycosides, certain mutations confer cross-resistance to CAP and AMK/KAN. In
contrast, cross-resistance between CAP and STR is rare. The major mechanisms of CAP resistance are
mutations that result in ribosome modification, particularly rrs and tlyA. Interestingly, tlyA mutations uniquely
affect CAP resistance and do not appear to play a role in resistance to aminoglycosides.
Mechanism of resistance:
Target modification mutations
Rrs:
The rrs A1401G mutation is found in ∼85% of CAP resistant XDR-TB strains. Other rrs mutations
including C1402T and G1484T are also associated with CAP resistance [7].
tlyA:
The tlyA gene is a nonessential gene found in many bacteria. Transposon mutants of tlyA as well as
spontaneous point mutants display significant CAP and VIO resistance. Biochemical, genetic, and comparative
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genomics suggest that the tlyA gene is an rRNA methyltransferase and that loss of methyltransferase activity
yields an unmethylated ribosome that is resistant to CAP inhibition [15]. This mechanism is similar to that of
the eis gene that confers resistance to KAN. Numerous tlyA mutations have been reported including L180R,
S265T, S64W, frame shift at 218L, N236K, and L150P [7,15,16].
Conclusion:
It has only been less than 100 years since antibiotics were first used to treat bacterial infections. This time
period is very short considering the pre-antibiotic era dated back thousands of years during which infectious
diseases might have acted as selective forces of human evolution. For a long time, humans and bacteria had coevolved in duels. However, everything has changed since antibiotics were discovered and applied in mass
amounts to modern medicine: bacteria now must evolve under the additional selective pressure of these killing
molecules. Evidence thus far indicates that pathogenic bacteria such as M. tuberculosis are well able to cope
with this pressure and that they have evolved to become progressively resistant to antibiotics. Acquired
resistances due to mutations in genes encoding target proteins or genes required for drug activities have allowed
rapid evolution of mutants that become newly resistant to the antibiotics used. Accumulation of these resistance
mutations has led to the emergence of M. tuberculosis strains that are more and more resistant to the available
antibiotics. More alarmingly, recent studies indicate that these drug resistant mutants are able to evolve to regain
fitness via compensatory mutations, thus enhancing their transmissibility and/or virulence. In addition to the
acquired resistances caused by chromosomal mutations, M. tuberculosis is naturally resistant to most antibiotics.
The profound intrinsic drug resistance in M. tuberculosis includes both passive and specialized mechanisms, the
latter of which are able to respond to the presence of antibiotics. In fact, structural proteins capable of antibiotic
resistance might have existed long before the clinical applications of these molecules. While these “drug
resistance” proteins might still play roles in the physiology or metabolism of M. tuberculosis and other bacteria,
inducible expression upon antibiotic exposure allows activities of these proteins important for antibiotic
resistance.
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