1,3-Diarylpyrazolyl-acylsulfonamides Target HadAB/BC Complex in Mycobacterium tuberculosis.

Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.

NSC19723, a Thiacetazone-Like Benzaldehyde Thiosemicarbazone Improves the Efficacy of TB Drugs In Vitro and In Vivo.

The complexity and duration of tuberculosis (TB) treatment contributes to the emergence of drug resistant tuberculosis (DR-TB) and drug-associated side effects. Alternate chemotherapeutic agents are needed to shorten the time and improve efficacy of current treatment. In this study, we have assessed the antitubercular activity of NSC19723, a benzaldehyde thiosemicarbazone molecule. NSC19723 is structurally similar to thiacetazone (TAC), a second-line anti-TB drug used to treat individuals with DR-TB. NSC19723 displayed better MIC values than TAC against Mycobacterium tuberculosis and Mycobacterium bovis BCG. In our checkerboard experiments, NSC19723 displayed better profiles than TAC in combination with known first-line and recently approved drugs. Mechanistic studies revealed that NSC19723 inhibits mycolic acid biosynthesis by targeting the HadABC complex. Computational studies revealed that the binding pocket of HadAB is similarly occupied by NSC19723 and TAC. NSC19723 also improved the efficacy of isoniazid in macrophages and mouse models of infection. Cumulatively, we have identified a benzaldehyde thiosemicarbazone scaffold that improved the activity of TB drugs in liquid cultures, macrophages, and mice. IMPORTANCE Mycobacterium tuberculosis, the causative agent of TB is among the leading causes of death among infectious diseases in humans. This situation has worsened due to the failure of BCG vaccines and the increased number of cases with HIV-TB coinfections and drug-resistant strains. Another challenge in the field is the lengthy duration of therapy for drug-sensitive and -resistant TB. Here, we have deciphered the mechanism of action of NSC19723, benzaldehyde thiosemicarbazone. We show that NSC19723 targets HadABC complex and inhibits mycolic acid biosynthesis. We also show that NSC19723 enhances the activity of known drugs in liquid cultures, macrophages, and mice. We have also performed molecular docking studies to identify the interacting residues of HadAB with NSC19723. Taken together, we demonstrate that NSC19723, a benzaldehyde thiosemicarbazone, has better antitubercular activity than thiacetazone.

Fluorescently labelled thioacetazone for detecting the interaction with Mycobacterium dehydratases HadAB and HadBC.

Thioacetazone (TAC) used to be a highly affordable, bacteriostatic anti-TB drug but its use has now been restricted, owing to severe side-effects and the frequent appearance of the TAC resistant M. tuberculosis strains. In order to develop new TAC analogues with fewer side-effects, its target enzymes need to be firmly established. It is now hypothesized that TAC, after being activated by a monooxygenase EthA, binds to the dehydratase complex HadAB that finally leads to a covalent modification of HadA, the main partner involved in dehydration. Another dehydratase enzyme, namely HadC in the HadBC complex, is also thought to be a possible target for TAC, for which definitive evidence is lacking. Herein, using a recently exploited azido naphthalimide template attached to thioacetazone and adopting a photo-affinity based labelling technique, coupled with electrophoresis and in-gel visualization, we have successfully demonstrated the involvement of these enzymes including HadBC along with a possible participation of an alternate mycobacterial monooxygenase MymA. In silico studies also revealed strong interactions between the TAC-probe and the concerned enzymes.

Mycobacterial Epoxide Hydrolase EphD Is Inhibited by Urea and Thiourea Derivatives.

The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell.

Resistance to Thiacetazone Derivatives Active against Mycobacterium abscessus Involves Mutations in the MmpL5 Transcriptional Repressor MAB_4384.

Available chemotherapeutic options are very limited against Mycobacterium abscessus, which imparts a particular challenge in the treatment of cystic fibrosis (CF) patients infected with this rapidly growing mycobacterium. New drugs are urgently needed against this emerging pathogen, but the discovery of active chemotypes has not been performed intensively. Interestingly, however, the repurposing of thiacetazone (TAC), a drug once used to treat tuberculosis, has increased following the deciphering of its mechanism of action and the detection of significantly more potent analogues. We therefore report studies performed on a library of 38 TAC-related derivatives previously evaluated for their antitubercular activity. Several compounds, including D6, D15, and D17, were found to exhibit potent activity in vitro against M. abscessus, Mycobacterium massiliense, and Mycobacterium bolletii clinical isolates from CF and non-CF patients. Similar to TAC in Mycobacterium tuberculosis, the three analogues act as prodrugs in M. abscessus, requiring bioactivation by the EthA enzyme, MAB_0985. Importantly, mutations in the transcriptional TetR repressor MAB_4384, with concomitant upregulation of the divergently oriented adjacent genes encoding an MmpS5/MmpL5 efflux pump system, accounted for high cross-resistance levels among all three compounds. Overall, this study uncovered a new mechanism of drug resistance in M. abscessus and demonstrated that simple structural optimization of the TAC scaffold can lead to the development of new drug candidates against M. abscessus infections.

A new dehydratase conferring innate resistance to thiacetazone and intra-amoebal survival of Mycobacterium smegmatis.

Nontuberculous mycobacteria are innately resistant to most antibiotics, although the mechanisms responsible for their drug resistance remain poorly understood. They are particularly refractory to thiacetazone (TAC), a second-line antitubercular drug. Herein, we identified MSMEG_6754 as essential for the innate resistance of Mycobacterium smegmatis to TAC. Transposon-mediated and targeted disruption of MSMEG_6754 resulted in hypersusceptibility to TAC. Conversely, introduction of MSMEG_6754 into Mycobacterium tuberculosis increased resistance 100-fold. Resolution of the crystal structure of MSMEG_6754 revealed a homodimer in which each monomer comprises two hot-dog domains characteristic of dehydratase-like proteins and very similar to the HadAB complex involved in mycolic acid biosynthesis. Gene inactivation of the essential hadB dehydratase could be achieved in M. smegmatis and M. tuberculosis only when the strains carried an integrated copy of MSMEG_6754, supporting the idea that MSMEG_6754 and HadB share redundant dehydratase activity. Using M. smegmatis-Acanthamoeba co-cultures, we found that intra-amoebal growth of the MSMEG_6754 deleted strain was significantly reduced compared with the parental strain. This in vivo growth defect was fully restored upon complementation with catalytically active MSMEG_6754 or HadABC, indicating that MSMEG_6754 plays a critical role in the survival of M. smegmatis within the environmental host.

The new tuberculosis drug Perchlozone® shows cross-resistance with thiacetazone.

Perchlozone(®) (PCZ), a new thiosemicarbazone developed by JSC Pharmasyntez (Moscow, Russia) for the treatment of tuberculosis (TB), was approved for use against multidrug-resistant disease in Russia in 2012. The mechanism of action of the drug is unknown. A well-studied thiosemicarbazone is the old TB drug thiacetazone (TAC). It has a narrow spectrum and inhibits the FASII dehydratase complex HadABC, which is involved in cell wall biosynthesis in Mycobacterium tuberculosis. TAC is a prodrug, requiring activation by the monooxygenase EthA. In this study, a comparative in vitro analysis of both drugs was performed. The two compounds had an identical spectrum of activity, spontaneous resistant mutants showed cross-resistance, and resistance was mapped to HadABC and EthA. These results suggest that PCZ, like TAC, is a prodrug and that both drugs share EthA as an activating enzyme and HadABC as their principal target.

Recycling and refurbishing old antitubercular drugs: the encouraging case of inhibitors of mycolic acid biosynthesis.

One of the first approaches undertaken in the quest for antitubercular compounds was that of understanding the mechanism of action of old drugs and proposing chemical modifications or other strategies to improve their activity, generally lost to the mechanisms of resistance developed by Mycobacterium tuberculosis. A leading case was the work carried out on a set of compounds with proven activity on the essential pathway of the synthesis of mycolic acids. As a result, different solutions were presented, improving the activity of those inhibitors or producing novel compounds acting on the same molecular target(s), but avoiding the most common resistance strategies developed by the tubercle bacilli. This review focuses on the activity of those compounds, developed following the completion of the studies on several of the classic antitubercular drugs.

Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues.

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the ß-hydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors.

A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone.

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.

Mutations in the essential FAS II ß-hydroxyacyl ACP dehydratase complex confer resistance to thiacetazone in Mycobacterium tuberculosis and Mycobacterium kansasii.

It has recently been shown that the anti-mycobacterial pro-drug thiacetazone (TAC) inhibits the conversion of double bonds of mycolic acid precursors into cyclopropyl rings in Mycobacterium bovis var BCG, M. marimum and M. chelonae by affecting the cyclopropyl mycolic acid synthases (CMASs) as judged by the build-up of unsaturated mycolate precursors. In our hands, TAC inhibits mycolic acid biosynthesis in Mycobacterium tuberculosis and M. kansasii with almost negligible accumulation of those precursors. Our observations that 'de novo' biosynthesis of all the mycolic acid families decreased upon TAC treatment prompted us to analyse the role of each one of the Type II Fatty Acid Synthase (FASII) enzymes. Overexpression of the hadABC operon, encoding the essential FASII dehydratase complex, but not of any of the remaining FASII genes acting on the elongation of fatty acyl chains leading to the synthesis of meromycolic acids, resulted in high level of resistance to TAC in M. tuberculosis. Spontaneous M. tuberculosis and M. kansasii TAC-resistant mutants isolated during this work revealed mutations in the hadABC genes strongly supporting our proposal that these enzymes are new players in the resistance to this anti-mycobacterial compound.

Exposure of mycobacteria to cell wall-inhibitory drugs decreases production of arabinoglycerolipid related to Mycolyl-arabinogalactan-peptidoglycan metabolism.

The "cell wall core" consisting of a mycolyl-arabinogalactan-peptidoglycan (mAGP) complex represents the hallmark of the mycobacterial cell envelope. It has been the focus of intense research at both structural and biosynthetic levels during the past few decades. Because it is essential, mAGP is also regarded as a target for several antitubercular drugs. Herein, we demonstrate that exposure of Mycobacterium bovis Bacille Calmette-Guérin or Mycobacterium marinum to thiacetazone, a second line antitubercular drug, is associated with a severe decrease in the level of a major apolar glycolipid. This inhibition requires MmaA4, a methyltransferase reported to participate in the activation process of thiacetazone. Following purification, this glycolipid was subjected to detailed structural analyses, combining gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance. This allowed to identify it as a 5-O-mycolyl-ß-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1)-Gro, designated dimycolyl diarabinoglycerol (DMAG). The presence of DMAG was subsequently confirmed in other slow growing pathogenic species, including Mycobacterium tuberculosis. DMAG production was stimulated in the presence of exogenous glycerol. Interestingly, DMAG appears structurally identical to the terminal portion of the mycolylated arabinosyl motif of mAGP, and the metabolic relationship between these two components was provided using antitubercular drugs such as ethambutol or isoniazid known to inhibit the biosynthesis of arabinogalactan or mycolic acid, respectively. Finally, DMAG was identified in the cell wall of M. tuberculosis. This opens the possibility of a potent biological function for DMAG that may be important to mycobacterial pathogenesis.

Phenylethyl butyrate enhances the potency of second-line drugs against clinical isolates of Mycobacterium tuberculosis.

Ethionamide (ETH) is a second-line drug for the treatment of tuberculosis. As a prodrug, ETH has to be activated by EthA. ethA is controlled by its repressor EthR. 2-Phenylethyl-butyrate (2-PEB) inhibits EthR binding, enhances expression of EthA, and thereby enhances the growth-inhibitory effects of ethionamide, isoxyl, and thiacetazone in Mycobacterium tuberculosis strains with resistance to ETH due to inhA promoter mutations but not ethA mutations.

Mycolic acid methyltransferase, MmaA4, is necessary for thiacetazone susceptibility in Mycobacterium tuberculosis.

Susceptibility of Mycobacterium tuberculosis to the second-line antitubercular drug thiacetazone (TAC) requires activation by the monoxygenase, EthA. Here, we report isolation of spontaneous mutants in Mycobacterium bovis BCG that are highly resistant to TAC, but carry a functional EthA. Unexpectedly, a majority of the TAC-resistant mutants lacked keto-mycolic acids, which are long-chain fatty acids associated with the cell wall and which contribute significantly to the physiopathology of tuberculosis. Predictably, causative mutations in the above mutants were in the gene encoding methyltransferase MmaA4, which is required for synthesis of keto- and methoxy-mycolic acids. Drug-resistant phenotype of the BCG mutants was reproduced in a mmaA4, but not in a mmaA3 null mutant of M. tuberculosis CDC1551. Susceptibility to TAC could be restored by complementation with a functional mmaA4 gene. Interestingly, overexpression of MmaA4 in M. bovis BCG made it more susceptible to TAC. We provide novel mechanistic insights into antitubercular drug activation by co-ordinated actions of EthA and MmaA4. This study is the first demonstration of the participation of an enzyme linked to the synthesis of oxygenated mycolates in a drug activation process in M. tuberculosis, and highlights the interplay between mycolic acid synthesis, drug activation and mycobacterial virulence.

In silico fasciolicide activity of three experimental compounds in sheep.

The aim of the present study was to evaluate the fasciolicide activity of three experimental drugs, selected by an in silico system called TOMOCOMD-CARDD, in sheep. Drugs were identified by the computer system, and, after statistical selection, 24 Pelibuey sheep were infected on days 0 and 30, each with 200 metacercariae of Fasciola hepatica. When the infection reached 8 and 4 weeks of age, respectively, four groups of six animals each were formed. Group 1 received thiacetazone 150 mg/animal/p.o. Group 2 was treated with 3,5,5, trimethyloxazolidine 2,4-dione at 450 mg/animal/p.o. G3 received guanabenz acetate at a dose of 1.5 mg/animal/p.o. G4 served as an untreated control. Monitoring of the animals was followed by individual coprological examinations and slaughter of the animals 15 days after treatment to collect and count flukes from the liver. Efficacy was measured as the reduction in the percentage of flukes of treated animals relative to untreated controls. Results indicated an efficacy of 80.0, 43.8, and 100% for 8-week-old flukes and 62.1, 57.9, and 100% for 4-week-old flukes in the three experimental groups, respectively. Even though guanabenz acetate showed a high efficacy, it was highly toxic since two animals died approximately 24 h after being treated. We conclude that further investigations should be conducted to perform computer-aided prediction of drugs aimed to detect fasciolicide activity.

Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria.

BACKGROUND: Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. METHODOLOGY/PRINCIPLE FINDINGS: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the alpha- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. CONCLUSIONS/SIGNIFICANCE: This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

The lipophilicity estimation of 5-arylidene derivatives of (2-thio)hydantoin with antimycobacterial activity.

The lipophilicity of antituberculotic 5-arylidene derivatives of (thio)hydantoin, thioacetazone and isoniazid has been determined by reversed-phase thin-layer chromatography (RP-TLC). Mixtures of acetone and water (with acetone content 60-85%) were used as the mobile phase. The R(M) (relative lipophilicity) of each compound (except isoniazid) decreased linearly with the increasing concentration of acetone. The partition coefficients (log P) of the compounds were calculated by the use of eight computer programs (ClogP, KowWin, XlogP, AlogPs, CAChe, Pallas, Interactive analysis and Slipper) and compared with the experimental lipophilicity (R(M0)). According to the observations the best tools for in silico predicting log P of (2-thio)hydantoins are programs KowWin and AlogPs.

EthA, a common activator of thiocarbamide-containing drugs acting on different mycobacterial targets.

Many of the current antimycobacterial agents require some form of cellular activation unmasking reactive groups, which in turn will bind to their specific targets. Therefore, understanding the mechanisms of activation of current antimycobacterials not only helps to decipher mechanisms of drug resistance but may also facilitate the development of alternative activation strategies or of analogues that do not require such processes. Herein, through the use of genetically defined strains of Mycobacterium bovis BCG we provide evidence that EthA, previously shown to activate ethionamide, also converts isoxyl (ISO) and thiacetazone (TAC) into reactive species. These results were further supported by the development of an in vitro assay using purified recombinant EthA, which allowed direct assessment of the metabolism of ISO. Interestingly, biochemical analysis of [(14)C]acetate-labeled cultures suggested that all of these EthA-activated drugs inhibit mycolic acid biosynthesis via different mechanisms through binding to specific targets. This report is also the first description of the molecular mechanism of action of TAC, a thiosemicarbazone antimicrobial agent that is still used in the treatment of tuberculosis as a second-line drug in many developing countries. Altogether, the results suggest that EthA is a common activator of thiocarbamide-containing drugs. The broad specificity of EthA can now be used to improve the activation process of these drugs, which may help overcome the toxicity problems associated with clinical thiocarbamide use.

[The rates of resistance to second-line drugs in multidrug resistant Mycobacterium tuberculosis strains]. / Cok ilaca dirençli Mycobacterium tuberculosis suslarinin minör antitüberküloz ilaçlara duyarliliginin saptanmasi.

The treatment of multidrug resistant tuberculosis (MDR-TB) requires the use of minor antituberculosis drugs, thus susceptibilities against these minor drugs should be performed. The aim of this study was to establish and standardize the susceptibility testing methods for minor antituberculosis drugs in this laboratory and to obtain data about the resistance rates. Drug susceptibility tests were performed by proportion method in Lowenstein-Jensen media for 50 MDR Mycobacterium tuberculosis isolates that have been collected from different regions of our country. Resistance rates to ethionamide, cycloserin, kanamycin, p-aminosalicylic acid, ofloxacin, thiacetasone, and capreomycin were found as 22%, 8%, 6%, 6%, 2%, 2%, and 0%, respectively. Multidrug resistance was observed in 8% of the isolates. As a result, the resistance rates of multidrug resistant M. tuberculosis strains against the tested minor antituberculosis drugs seem to be low in this study.