MOLECULAR BASIS & MECHANISMS OF DRUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS

MOLECULAR BASIS & MECHANISMS OF DRUG RESISTANCE INMYCOBACTERIUM TUBERCULOSIS

Presenter: Dr.KalaiyarasanModerator: Dr.V.P.Myneedu

04-Jan-2015

Introduction

• Drug-resistant TB poses a major threat to control of TB worldwide.

• Among notified pulmonary TB patients in 2014, an estimated 300 000 (range: 220 000–370 000) had MDR-TB.

• Globally in 2014, 123 000 patients with MDR -TB or rifampicin resistant tuberculosis (RR-TB) were notified.

MDR TB

• Globally, 3.3% of new cases (95% CI: 2.2–4.4%) and 20% of previously treated cases (95%CI: 14–27%) have MDR-TB.

• In India (2014), an estimated 2.2% of new cases (95% CI: 1.9–2.6%) and 15% of previously treated cases (95%CI: 11–19%) have MDR-TB.

WHO Global tuberculosis report 2015

XDR TB

• Extensively drug-resistant TB (XDR-TB) has been reported by 105 countries.1

• On average, an estimated 9.7% of people with MDR-TB have XDR-TB (95% CI: 7.4–12%).1

• Sub-national drug resistance surveys (2005-09) showed 4-7% XDR & 21-24% Ofx resistance in MDR isolates.

1. WHO Global tuberculosis report 2015

ANTIBIOTIC RESISTANCE

“ Resistance is defined as a decrease in sensitivity of sufficient degree to be reasonably certain that the strain concerned is different from a sample of wild strains of human type that have never come into contact with the drug. ” 1

Drug resistance comes at a biological cost, e.g. reduced growth in the absence of antibiotics.

1. Mitchison, 1962

TYPES OF DRUG RESISTANCE

Intrinsic drug resistance Occurs naturally in all or most strains of that species Chromosomally encoded Eg: Gram -ves are resistant to Vancomycin

Acquired drug resistance Results from a mutation in the existing DNA of an organism or

acquisition of new genes.

Mono resistance TB - Resistant in vitro to one first line anti-TB drug.

Poly drug resistance TB - Resistance to INH or RIF (not both) with resistance to one or more other 1st line drugs.

MDR TB - Resistance to INH & Rif with or without resistance to other anti-TB drugs.

XDR TB - Resistance to INH & Rif in addition to any fluoroquinolone and at least one of the three 2nd line injectable drugs.

Primary drug resistance - No previous TB treatment implying transmitted resistance.

Acquired drug resistance - Development of drug resistance in a previously treated patient due to inappropriate treatment.

New drug resistance - Resistance in cultures from patients who have not previously received TB treatment or who have received TB treatment for <1month.

Previously treated drug resistance - Resistance in cultures from patients previously treated for ≥1 month with only first line drugs.

Transmitted drug resistance - Due to infection with already drug resistant strain, with or without anti-TB treatment.

Tuberculosis: Simon Schaaf & Alimuddin Zumla, 2009

MECHANISMS OF RESISTANCE

S.NO.

FACTORS DESCRIPTION REFERENCES

1 GENETIC FACTORS • Accumulation of changes in genomic content• Gene acquisition and loss• Spontaneous mutation

Kato-Maeda et al 2001Sharma and Mohan 2004Ramaswamy et al 1998

2 INCOMPLETE & INADEQUATE TREATMENT

• History of treatment of TB• Shortage of drugs• Increased cost of drugs• Physician error (drugs, dosing interval & duration)• Use of single drug to treat TB• Addition of a single drug to a failing regimen• Failure to identify preexisting resistance• Initiation of an inadequate primary regimen• Variations in bioavailability of anti-TB drugs

Mwinga 2001Chan and Iseman 2002Iseman 1993Sharma and Mohan 2003

3 INADEQUATE TREATMENT ADHERENCE

• Poor compliance• Psychiatric illness• Alcoholism• Drug addiction• Homelessness• Travel to different places• Symptom relief• Adverse drug reactions• Inability to afford treatment• Length of treatment• Adverse drug reactions

Jacaban 1994Malian and Adarm 1995Goble et al 1993Sharma and Mohan 2004Johnson et al 2003

4 OTHER FACTORS • Poor infrastructure of NTCP• Poor administrative control on purchase anddistribution of the drugs• No proper mechanism on quality control• No proper mechanism of bioavailability tests• No appropriate laboratory support leading toover diagnosis of TB• Unnecessary treatment• Side effects without benefit• Service inefficiencies• No specific therapy for MDR-TB DOTS Strategy

Prasad 2005Floyd et al 2006Sharma and Mohan 2006

The mutation rate, rather than the mutation frequency, is the most reliable measure, as it records the risk of mutation per cell division rather than the proportion of mutant cells.

The rate at which resistance emerges, being highest for ethambutol and lowest for rifampicin & quinolones.

McGrath M, Gey van Pittius NC, van Helden PD, Warren RM, Warner DF, J Antimicrob Chemother. 2014 Feb;69(2):292-302.

Drug Avg. mutation rateIsoniazid 2.56 x 10-8

Rifampicin 2.25 x 10-10

Ethambutol 1 x 10-7 to 6.4 × 10−7

Streptomycin 2.95 x 10-8

MUTATION RATE

For example the risk of resistance for a combination of RIF, STM, & INH is 10-25/bacterium/generation.

Risk of resistance may be more accurately calculated using the formula P =1-(1-r)n

Where,P is the probability of drug resistance emergingr is the mutation raten is the number of bacilli in a lesion, usually calculated to be 108 per lesion

If single-drug therapy with a risk of mutation of 10-6 is used, the risk of resistance emerging is 100%.

If two drugs with a combined mutation rate of 10-12 are used, then the risk is 0.01%

Shimao, T. 1987. Drug resistance in tuberculosis control. Tubercle 68:5–18.

INTRINSIC DRUG RESISTANCE IN TB

Unusual structure of its mycolic acid-containing cell wall.

low permeability for many antibiotics.1

Intrinsic resistance is important .

Limits the number of drugs available for treatment & favors the emergence of strains with a high level of drug resistance.

1 Jarlier V, Nikaido H. Mycobacterial cell wall: structure and role in natural resistance to antibiotics. FEMS Microbiol Lett 1994; 123: 11–8.2 De Rossi E, Aıńsa JA, Riccardi G. Role of mycobacterial efflux transporters in drug resistance: an unresolved question. FEMS Microbiol Rev 2006; 30: 36–52.

Recently, the role of efflux mechanisms has also been recognized as an important factor in the natural resistance against antibiotics such as Tetracycline, fluoroquinolones and aminoglycosides, among others.2

In M. smegmatis, mutants lacking the major porin MspA and MspC had an increased MIC of the b-lactam antibiotics ampicillin and cefaloridine.

Deletion of the mspA gene also increased the MIC of vancomycin, erythromycin and rifampicin by 2-10 fold.1

1 Stephan J, Mailaender C, Etienne G et al. Multidrug resistance of a porin deletion mutant of Mycobacterium smegmatis. Antimicrob Agents Chemother 2004; 48: 4163–70.

ACQUIRED DRUG RESISTANCE

In other bacteria generally mediated through horizontal transfer by mobile genetic elements, such as plasmids, transposons or integrons.

In M. tuberculosis, caused mainly by spontaneous mutations in chromosomal genes, producing the selection of resistant strains during sub-optimal drug therapy.1

1 Kochi A, Vareldzis B, Styblo K. Multidrug-resistant tuberculosis and its control. Res Microbiol 1993; 144: 104–10.

In prokaryotes, spontaneous mutations occur at a low rate of 0.0033 per replication.

The mutation rate per division is inversely proportional to the genome size.

Rate of mutation depends on the nature of the drug selection, but for most of the main anti-TB drugs, this occurs at a rate of 10-9 mutations per cell division.

This is the main reason why anti-TB drugs are given as a combination, as the risk of a mutant containing two resistance mutations is <10-18 .1

1 Gillespie SH. Tuberculosis: evolution in millennia and minutes. Biochem Soc Trans 2007; 35: 1317–20.

Anti-TubercularDrugs

ISONIAZID

First synthesized in the early 20th century

One of the main drugs for TB since recognition of its clinical activity in 1952.1

Simple structure - pyridine ring & a hydrazide group – both essential for the high activity against M.TB.

INH is a nicotinamide analog

structurally related to ethionamide and pyrazinamide.

Active against growing TB bacilli but is bacteriostatic if the mycobacteria are slow-growing.2

1 Robitzek and Selikoff 19522 Ahmad, Z.; Klinkenberg, P. C.(2009). "Biphasic Kill Curve of Isoniazid Reveals the Presence of Drug Tolerant, Not Drug Resistant,Mycobacterium ‐ ‐tuberculosis in the Guinea Pig". The Journal of Infectious Diseases 200 (7): 1136–1143.

MECHANISM OF ACTION

Pro-drug, requiring oxidative activation by the M. tuberculosis catalase-peroxidase enzyme encoded by KatG.1

Appears to penetrate host cells readily and diffuses across the cell membrane.2

Reported to inhibit multiple essential cellular pathways including synthesis of nucleic acids3, phospholipids4 & NAD metabolism5

Primary pathway responsible for the killing activity is inhibition of mycolic acid synthesis6 by inhibiting NADH dependent enoyl-ACP reductase, which is encoded by inhA.7,8,9

1 Zhang Y, Heym B, Allen B et al. The catalase/peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 1992; 358:591–3 2 Mackaness and Smith 1952; Suter 1952; Bardou, Raynaud et al. 1998; 3 Gangadharam, Harold et al. 1963; 4 Brennan, Rooney et al. 1970; 5 Zatman, Kaplan et al. 1954; Bekierkunst 1966; 6 Winder and Collins 1970; Takayama, Wang et al. 1972; Takayama, Schnoes et al. 19757 Rawat R, Whitty A, Tonge PJ. The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance. Proc Natl Acad Sci USA 2003; 100: 13881–6.; 8 Banerjee, Dubnau et al. 1994; 9 Quemard, Sacchettini et al. 1995

MECHANISM OF RESISTANCE

Most commonly used anti-TB drug, so resistance occurs more frequently among clinical isolates than resistance to any other agent.1

Mutations in several genes, including katG, ahpC, inhA, kasA & ndh have all been associated with isoniazid resistance.

Two molecular mechanisms have been shown to be the main cause for isoniazid resistance:

i. Mutations in katG ii. Mutations in inhA, or more frequently in its promoter region.2,3

1 Karakousis 20092 Rawat R, Whitty A, Tonge PJ. The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance. Proc Natl Acad Sci USA 2003; 100: 13881–6.3 Silva MS, Senna SG, Ribeiro MO et al. Mutations in katG, inhA, and ahpC genes of Brazilian isoniazid-resistant isolates of Mycobacterium tuberculosis. J Clin Microbiol 2003; 41: 4471–4.

katG

Located in a highly variable and unstable region of the M. tuberculosis genome.

Decrease or total loss of catalase/peroxidase activity are the most common genetic alterations.1

Occurs in 50–80% of cases

So far, more than a hundred mutations in katG have been reported, with MICs ranging from 0.2 to 256 mg/L.

Down-regulation of katG expression has also been recently shown to be associated with resistance to isoniazid.2

1 Zhang Y, Heym B, Allen B et al. The catalase/peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 1992; 358:591–3.2 Ando H, Kitao T, Miyoshi-Akiyama T et al. Downregulation of katG expression is associated with isoniazid resistance in Mycobacterium tuberculosis. Mol Microbiol 2011; 9: 1615–28.

Point mutations in katG are more commonly observed.

Substitution of threonine for serine at residue 315 (S315T) accounts for the majority of INH resistance among clinical isolates.1

Results in a significant reduction in catalase & peroxidase activity 2 & causes high-level INH resistance (MIC = 5–10 μg/mL).3

Interestingly, the mutation S315T in katG occurs more frequently in MDR than in isoniazid mono resistant strains.4,5

1 Marttila, Soini et al. 1998; Abate, Hoffner et al. 20012 Vilche`ze C, Jacobs WR Jr. The mechanism of isoniazid killing: clarity through the scope of genetics. Annu Rev Microbiol 2007; 61: 35–50.3 Rouse, DeVito et al. 1996; Saint-Joanis, Souchon et al. 19994 Hazboń MH, Brimacombe M, Bobadilla del Valle M et al. Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 2006; 50: 2640–9. 5 van Doorn HR, de Haas PE, Kremer K et al. Public health impact of isoniazid-resistant Mycobacterium tuberculosis strains with a mutationat amino-acid position 315 of katG: a decade of experience in The Netherlands. Clin Microbiol Infect 2006; 12: 769–75.

inhA

Mutations result in reduced affinity of the enzyme for NADH without affecting its enoyl reductase activity1

Most common mutation occurs in promoter region & has been found more frequently associated with mono-resistant strains.2

Usually confer low-level resistance (MIC = 0.2–1 mg/L).3

Mutations in inhA also cause resistance to the structurally related second-line drug ethionamide.4

1 Basso, Zheng et al. 19982 Leung ET, Ho PL, Yuen KY et al. Molecular characterization of isoniazid resistance in Mycobacterium tuberculosis: identification of a novel mutation in inhA. Antimicrob Agents Chemother 2006; 50: 1075–8.3 Wade and Zhang 20044 Banerjee A, Dubnau E, Quemard A et al. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis.Science 1994; 263: 227–30.

ndh

Described for the first time in M. smegmatis

Mutations reduce the activity of NADH dehydrogenase and produce resistance to isoniazid and ethionamide.1

Defective NADH dehydrogenase leads to an increased ratio of NADH/NAD, interfering with KatG-mediated peroxidation of INH, or by displacing the INH/NAD adduct from the InhA active site.1

In M. tuberculosis, mutations in ndh have been associated with isoniazid resistance alone2 or in combination with other gene mutations such as inhA and katG.

1 Miesel L, Weisbrod TR, Marcinkeviciene JA et al. NADH dehydrogenase defects confer isoniazid resistance and conditional lethality in Mycobacterium smegmatis. J Bacteriol 1998; 180: 2459–67.2 Lee, Teo et al. 2001

ahpC

Codes for an alkyl hydroperoxidase reductase that is implicated in resistance to reactive oxygen and reactive nitrogen intermediates.

It was initially proposed that mutations in the promoter of ahpC could be used as surrogate markers for the detection of isoniazid resistance.1

However, several other studies have found that an increase in the expression of ahpC seems to be more a compensatory mutation for the loss of catalase/peroxidase activity rather than the basis for isoniazid resistance.2

1 Rinder H, Thomschke A, Ru¨sch-Gerdes S et al. Significance of ahpC promoter mutations for the prediction of isoniazid resistance in Mycobacterium tuberculosis. Eur J Clin Microbiol Infect Dis 1998; 17:508–11.2 Sherman DR, Mdluli K, Hickey MJ et al. Compensatory ahpC gene expression in isoniazid-resistant Mycobacterium tuberculosis. Science 1996; 272: 1641–3.

As many as a quarter of all clinical INH-resistant isolates do not have mutations in any of the above genes, suggesting alternative mechanisms of INH resistance.1

A study by Hazboń et al.2 analysed 240 alleles previously described in association with INH resistance and found that mutations in katG, inhA and ahpC were most strongly associated with INH resistance.

Similar results were reported in another previous study by Larsen et al.3

1 Karakousis 20092 Hazboń MH, Brimacombe M, Bobadilla del Valle M et al. Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 2006; 50: 2640–9.3 Larsen MH, Vilche`ze C, Kremer L et al. Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis. Mol Microbiol 2002; 46:453–66.

ISONIAZID & TOLERANCE

INH induces alterations in the expression of several genes in both drug-resistant and drug-tolerant M. tuberculosis strains.

M.TB iniA gene induced in the presence of isoniazid, participates in the development of tolerance to both isoniazid and ethambutol.

1 Fu LM, Shinnick TM. Understanding the action of INH on a highly INH-resistant Mycobacterium tuberculosis strain using Genechips. Tuberculosis (Edinb) 2007; 87: 63–70.

RIFAMPICIN

Lipophylic ansamycin introduced in 1972.

Together with INH considered to be the basis of the short-course treatment regimen for TB.1

It is effective against Gram +ve & some Gram –ve bacteria, but also against the highly resistant Mycobacteria.

e.g. M. leprae, and M. avium intracellulare.

1 Rattan A, Kalia A, Ahmad N. Multidrug-resistant Mycobacterium tuberculosis: molecular perspectives. Emerg Infect Dis 1998; 4: 195–209.

MECHANISM OF ACTION

The target of rifampicin in M. tuberculosis is the b-subunit of RNA polymerase, where it binds and inhibits the elongation of mRNA.1

Active against actively growing and slowly metabolizing (non-growing) bacilli.2

1 Blanchard JS. Molecular mechanisms of drug resistance in Mycobacterium tuberculosis. Annu Rev Biochem 1996; 65: 215.2 Mitchison DA. Basic mechanisms of chemotherapy. Chest 1979; 76 Suppl 6: 771–81.


The great majority of clinical isolates shows mutations in the gene rpoB that encodes the b-subunit of RNA polymerase.

This results in conformational changes that determine a low affinity for the drug and consequently the development of resistance.1

Mutations in a ‘hot-spot’ region of 81 bp of rpoB have been found in about 96% of rifampicin-resistant M. tuberculosis isolates.

1 Telenti A, Imboden P, Marchesi F et al. Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis. Antimicrob Agents Chemother 1993; 37: 2054–8.

This region, spanning codons 507–533, is also known as the rifampicin resistance-determining region (RRDR).1

Mutations in codons 531 and 526 are the most frequently reported mutations in most of the studies.2,3

Some studies have also reported mutations outside of the hot-spot region of rpoB in rifampicin-resistant M. tuberculosis isolates.4

1 Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis 1998; 79: 3–29.2 Somoskovi A, Parsons LM, Salfinger M. The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respir Res 20013 Caws M, Duy PM, Tho DQ et al. Mutations prevalent among rifampinand isoniazid-resistant Mycobacterium tuberculosis isolates from a hospital in Vietnam. J Clin Microbiol 2006; 44: 2333–7.4 Heep M, Rieger U, Beck D et al. Mutations in the beginning of the rpoB gene can induce resistance to rifamycins in both Helicobacter pylori and Mycobacterium tuberculosis. Antimicrob Agents Chemother 2000; 44:1075–7.

Cross-resistance between rifampicin and other rifamycins do exist.

some mutations in codons 516, 518, 522, 529 and 533 have been associated with low-level resistance to rifampicin but susceptibility to rifabutin and rifalazil.1,2

An important finding is that almost all rifampicin-resistant strains also show resistance to other drugs, particularly to isoniazid.

For this reason, rifampicin resistance detection has been proposed as a surrogate molecular marker for MDR.3

1 Yang B, Koga H, Ohno H et al. Relationship between antimycobacterial activities of rifampicin, rifabutin and KRM-1648 and rpoB mutations of Mycobacterium tuberculosis. J Antimicrob Chemother 1998; 42: 621–8.2 Cavusoglu C, Karaca-Derici Y, Bilgic A. In-vitro activity of rifabutin against rifampicin-resistant Mycobacterium tuberculosis isolates with known rpoB mutations. Clin Microbiol Infect 2004; 10: 662–5.3 Traore H, Fissette K, Bastian I et al. Detection of rifampicin resistance in Mycobacterium tuberculosis isolates from diverse countries by a commercial line probe assay as an initial indicator of multidrug resistance. Int J Tuberc Lung Dis 2000; 4: 481–4.

Cross Resistance

PYRAZINAMIDE

Discovered in 1952 and introduced into TB chemotherapy in the early 1950s.

Structural analogue of nicotinamide.

It’s use allowed the length of treatment to be reduced from 9 to 6 months.

Its largely bacteriostatic, but can be bacteriocidal on actively replicating tuberculosis bacteria.

Key characteristic - ability to inhibit semid dormant bacilli residing in acidic environments.1

1 Mitchison DA. The action of antituberculosis drugs in short-course chemotherapy. Tubercle 1985; 66: 219–25.

MECHANISM OF ACTION

Pro-drug

Converted into its active form, pyrazinoic acid, by the enzyme pyrazinamidase/nicotinamidase (PZase).1

Disrupts bacterial membrane energetics & inhibits membrane transport.

PZase is encoded in M. tuberculosis by the gene pncA.2

1 Konno K, Feldmann FM, McDermott W. Pyrazinamide susceptibilityand amidase activity of tubercle bacilli. Am Rev Respir Dis 1967; 95:461–9.2 Scorpio A, Zhang Y. Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nat Med 1996;2: 662–7.

Studies have shown that pyrazinamide enters M. tuberculosis by passive diffusion, is converted into pyrazinoic acid by PZase, and is excreted by a weak efflux pump.

Under acid conditions, the protonated pyrazinoic acid is reabsorbed and accumulates inside the cell due to an inefficient efflux pump, resulting in cellular damage.1

Another proposed theory states that pyrazinoic acid and its n-propyl ester inhibit the fatty acid synthase type I in replicating bacilli.2

1 Zhang Y, Mitchison D. The curious characteristics of pyrazinamide: a review. Int J Tuberc Lung Dis 2003; 7: 6–21.2 Zimhony O, Vilche`ze C, Arai M et al. Pyrazinoic acid and its n-propyl ester inhibit fatty acid synthase type I in replicating tubercle bacilli. Antimicrob Agents Chemother 2007; 51: 752–4.


Mutations in pncA is the main mechanism.

Most alterations occur in a 561 bp region of the open reading frame or in an 82 bp region of its putative promoter.1,2

Some PZA-resistant strains do not show mutations in pncA or its promoter region.

In this case, it has been postulated that resistance could be due to mutations in an unknown pncA regulatory gene.3

A small proportion have low-level resistance and retain PZase activity are considered to have another alternative mechanism of resistance.4

1 Scorpio A, Lindholm-Levy P, Heifets L et al. Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 1997; 41: 540–3.2 Jureén P, Werngren J, Toro JC et al. Pyrazinamide resistance and pncA gene mutations in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2008; 52: 1852–4.3 Cheng SJ, Thibert L, Sanchez T et al. pncA mutations as a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis:spread of a monoresistant strain in Quebec, Canada. Antimicrob Agents Chemother 2000; 44: 528–32.4 Sreevatsan S, Pan X, Zhang Y et al. Mutations associated with pyrazinamide resistance in pncA of Mycobacterium tuberculosis complex organisms. Antimicrob Agents Chemother 1997; 41:636–40.

STREPTOMYCIN

An aminoglycoside antibiotic.

First antibiotic used in the treatment of TB.

Isolated from the soil microorganism Streptomyces griseus.

Bactericidal antibiotic.

MECHANISM OF ACTION

Inhibition of initiation of translation by binding to the 16S rRNA.1

1 Moazed D, Noller HF. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature 1987; 327: 389–94.2 Crofton J, Mitchison DA. Streptomycin resistance in pulmonary tuberculosis. Br Med J 1948; 2: 1009–15.

As it was used as mono-therapy, resistance to emerged quite rapidly.2

Due to mutations in rrs or rpsL, which produce alterations in the STM binding site.

The majority of point mutations resulting in STM resistance occur in rpsL, with the most common mutation being K43R.


However, such mutations in rpsL or rrs genes are identified in slightly more than 50% of the strains studied to date suggesting alternative mechanism for streptomycin resistance.1,2,5

Recently it has been shown that mutations in gidB, which encodes a conserved 7-methylguanosine methyltransferase specific for the 16S rRNA, can confer a low level of streptomycin resistance.3,4

1 Zhang Y, Telenti A. Genetics of drug resistance in Mycobacterium TB. In: Hatfull GF, Jacobs WR, eds. Molecular Genetics Mycobacteria. Washington,DC: American Society for Microbiology Press,2000; 235–56.2 Silva PE, Bigi F, Santangelo MP et al. Characterization of P55, a multidrug efflux pump in Mycobacterium bovis and Mycobacterium tuberculosis. Antimicrob Agents Chemother 2001; 45: 800–4.3 Okamoto S, Tamaru A, Nakajima C et al. Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria. Mol Microbiol 2007; 63: 1096–106.4 Spies FS, da Silva PE, Ribeiro MO et al. Identification of mutations related to streptomycin resistance in clinical isolates of Mycobacterium tuberculosis and possible involvement of efflux mechanism. Antimicrob Agents Chemother 2008; 52: 2947–9.5 Gillespie SH. Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob Agents Chemother 2002; 46: 267–74.

ETHAMBUTOL

Ethambutol was first used in 1966 against TB.

Bacteriostatic anti-mycobacterial drug

Also used to treat Mycobacterium avium complex and Mycobacterium kansasii.

MECHANISM OF ACTION

It is active against multiplying bacilli.

Inhibits the enzyme arabinosyl transferase & interferes in the biosynthesis of cell wall arabinogalactan.1

Increases permeability of the cell wall.

1 Takayama K, Armstrong EL, Kunugi KA et al. Inhibition by ethambutol of mycolic acid transfer into the cell wall of Mycobacterium smegmatis. Antimicrob Agents Chemother 1979; 16: 240–2.

Close to 50% strains had mutations in codon 306 of embB.1

Sreevatsan et al.,2 showed that in 50% of ethambutol-resistant isolates there were mutations in embB.

Other studies have found that there were mutations present in embB codon 306 in up to 20% of ethambutol-susceptible isolates evaluated.3,4

1 Telenti A, Philipp WJ, Sreevatsan S et al. The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat Med 1997; 3: 567–70.2 Sreevatsan S, Stockbauer KE, Pan X et al. Ethambutol resistance in Mycobacterium tuberculosis: critical role of embB mutations. Antimicrob Agents Chemother 1997.3 Lee AS, Othman SN, Ho YM et al. Novel mutations within the embB gene in ethambutol-susceptible clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2004; 48: 4447–9.4 Ahmad S, Jaber AA, Mokaddas E. Frequency of embB codon 306 mutations in ethambutol-susceptible and -resistant clinical Mycobacterium tuberculosis isolates in Kuwait. Tuberculosis (Edinb) 2007; 87: 123–9.


Studies with MDR strains susceptible to ethambutol by phenotypic methods found an association between mutations in embB306 and broad drug resistance rather than with resistance to ethambutol.1

It has been postulated that mutations in embB306 may be related to variable degrees of ethambutol resistance but not sufficient for high-level ethambutol resistance.

There is an important percentage that don’t have mutations in embB, suggesting undiscovered mechanism for ethambutol resistance.2

1 Hazboń MH, Bobadilla del Valle M, Guerrero MI et al. 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; 49:3794–802.2 Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2009; 13: 1320–30.

FLUOROQUINOLONES

Both ciprofloxacin and ofloxacin are synthetic derivatives of nalidixic acid, the parent compound discovered in 1965 as a by-product in the purification of the anti-malarial drug chloroquine.1

New generation of fluoroquinolones, such as moxifloxacin and gatifloxacin are being proposed as first-line antibiotics with the goal of shortening the duration of TB treatment.2–4

1 Goss WA, Deitz WH, Cook TM. Mechanism of action of nalidixic acid on Escherichia coli. II. Inhibition of deoxyribonucleic acid synthesis. J Bacteriol 1965; 89: 1068–74.2 Alvirez-Freites EJ, Carter JL, Cynamon MH. In vitro and in vivo activities of gatifloxacin against Mycobacterium tuberculosis. Antimicrob Agents Chemother 2002; 46: 1022–5.3 Nuermberger EL, Yoshimatsu T, Tyagi S et al. Moxifloxacin-containing regimens of reduced duration produce a stable cure in murine tuberculosis. Am J Respir Crit Care Med 2004; 170: 1131–4.4 Rustomjee R, Diacon AH, Allen J et al. Early bactericidal activity and pharmacokinetics of the diarylquinoline TMC207 in treatment of pulmonary tuberculosis. Antimicrob Agents Chemother 2008; 52:2831–5.

MECHANISM OF ACTION

In M. tuberculosis, only type II topoisomerase (DNA gyrase) is present and thus is the only target for fluoroquinolone activity.1

Type II topoisomerase is a tetramer composed of two A and B subunits encoded by the genes gyrA and gyrB, respectively, that catalyses the supercoiling of DNA.2–4

1 Aubry A, Pan XS, Fisher LM et al. Mycobacterium tuberculosis DNA gyrase: interaction with quinolones and correlation with antimycobacterial drug activity. Antimicrob Agents Chemother 2004; 48: 1281–8.2 Wang JC. DNA topoisomerases. Annu Rev Biochem 1996; 65: 635–92.3 Drlica K. Mechanism of fluoroquinolone action. Curr Opin Microbiol 1999; 2: 504–8.4 Takiff HE, Salazar L, Guerrero C et al. Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations. Antimicrob Agents Chemother 1994;38: 773–80.


Initial studies showed amino acid substitutions in the putative fluoroquinolone binding region in gyrA or gyrB.1

Quinolone resistance-determining region (QRDR) of gyrA and gyrB and resistance to fluoroquinolones has been confirmed now in multiple studies.

Resistant strains show mutations in a conserved region of gyrA, with Ala-90 and Asp-94 as the most frequently mutated positions;

Mutations at Ala-74, Gly-88 and Ser-91 have also been reported.2,3

1Takiff HE, Salazar L, Guerrero C et al. Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations. Antimicrob Agents Chemother 1994; 38: 773–80.2 Cheng AF, Yew WW, Chan EW et al. Multiplex PCR amplimer conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates. Antimicrob Agents Chemother 2004; 48: 596–601.3 Sun Z, Zhang J, Zhang X et al. Comparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates. Int J Antimicrob Agents 2008;31: 115–21.

Studies showed that for the same mutation in gyrA, the MIC value of Ofloxacin was higher in the laboratory-selected strains than clinical isolates.1

These findings indicated that other factors are also involved in resistance, especially in laboratory-selected strains, such as alterations in membrane permeability or increased expression of efflux mechanisms.

Several studies have already shown the presumptive participation of efflux mechanisms in resistance to fluoroquinolones.2

1 Sun Z, Zhang J, Zhang X et al. Comparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates. Int J Antimicrob Agents 2008;31: 115–21.2 Escribano I, Rodrı´guez JC, Llorca B et al. Importance of the efflux pump systems in the resistance of Mycobacterium tuberculosis to fluoroquinolones and linezolid. Chemotherapy 2007; 53: 397–401.

Hegde et al.,1 described a fluoroquinolone-resistance protein from M.TB that mimics DNA.

MfpA protein, in M.TB caused resistance to ciprofloxacin & sparfloxacin.

By binding to DNA gyrase, MfpA inhibits its activity.

Determination of its three-dimensional structure revealed a resemblance in terms of size, shape and electrostatic similarity to B-form DNA.

This DNA mimicry would explain the inhibitory effect on gyrase and fluoroquinolone resistance.

Significance of the MfpA protein in fluoroquinolone resistance is yet to be evaluated.

1 Hegde SS, Vetting MW, Roderick S et al. A fluoroquinolone resistance protein from Mycobacterium tuberculosis that mimics DNA. Science 2005; 308: 1480–3.

Kanamycin, Amikacin, Capreomycin & Viomycin

Kanamycin and amikacin are aminoglycoside antibiotics.

Capreomycin and viomycin are cyclic peptide antibiotics.

Used in the treatment of DR-TB.

MECHANISM OF ACTION

Although belonging to two different antibiotic families, all exert their activity at the level of protein translation.


The most common molecular mechanism of drug resistance has been associated with an A1401G mutation in the rrs gene coding for 16S rRNA.

This mutation occurs more frequently in strains with high-level resistance to kanamycin and amikacin.1

1 Jugheli L, Bzekalava N, de Rijk P et al. High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene. Antimicrob Agents Chemother 2009; 53: 5064–8.

Cross-resistance between kanamycin and amikacin or kanamycin, capreomycin and viomycin to variable degrees have been reported.1,2,3

Capreomycin and viomycin are structurally similar antibiotics with full cross-resistance shown in previous studies.4

Mutations in the gene tlyA have been implicated in resistance to capreomycin and viomycin.

This gene codes an rRNA methyltransferase specific for 2 -O-methylation ′of ribose in rRNA.

1 Tsukamura M, Mizuno S. Cross-resistant relationships among the aminoglucoside antibiotics in Mycobacterium tuberculosis. J Gen Microbiol 1975; 88: 269.2 Alangaden GJ, Kreiswirth BN, Aouad A et al. Mechanism of resistance to amikacin and kanamycin in Mycobacterium tuberculosis. Antimicrob Agents Chemother 1998; 42: 1295–7.3 Maus CE, Plikaytis BB, Shinnick TM. Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2005; 49:3192–7.4 McClatchy JK, Kanes W, Davidson PT et al. Cross-resistance in M.tuberculosis to kanamycin, capreomycin and viomycin. Tubercle 1977;58: 29–34.

CROSS RESISTANCE

Kanamycin & amikacin cross-resistance is not absolute with highly variable patterns and levels of resistance.1

Studies found that mutations in the promoter region of the eis gene in M. tuberculosis conferred low-level resistance to kanamycin but not amikacin.2,3,4

1 Kruü¨ner A, Jureén P, Levina K et al. Discordant resistance to kanamycin and amikacin in drug-resistant Mycobacterium tuberculosis.Antimicrob Agents Chemother 2003; 2 Zaunbrecher MA, Sikes RD Jr, Metchock B et al. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2009; 106: 20004–9.3 Engstro¨m A, Perskvist N, Werngren J et al. Comparison of clinical isolates and in vitro selected mutants reveals that tlyA is not a sensitive genetic marker for capreomycin resistance in Mycobacterium tuberculosis. J Antimicrob Chemother 2011; 66: 1247–54.4 Campbell PJ, Morlock GP, Sikes RD et al. Molecular detection of mutations associated with first and second-line drug resistance compared with conventional drug susceptibility testing in M. tuberculosis. Antimicrob Agents Chemother 2011; Epub ahead of print 7 February 2011.

CROSS RESISTANCE

ETHIONAMIDE

Synthetic compound discovered in 1956.

Structurally related to INH.

Also structurally similar to methimazole & was reported to cause hypothyroidism in several TB patients.1

1 New England journal of med 352;26 www.nejm.org june 30, 2005 page 2757

MECHANISM OF ACTION

Pro-drug, requiring activation by the mono-oxygenase EthA .1

Similar to INH, ethionamide inhibits mycolic acid synthesis by binding the ACP reductase InhA.

More recently, mshA, a gene encoding a glycosyltransferase involved in mycothiol biosynthesis, has also been suggested as a possible target for ethionamide.2

1 Baulard, Betts et al. 2000; DeBarber, Mdluli et al. 2000; Vannelli, Dykman et al. 20022 Vilche`ze C, Av-Gay Y, Attarian R et al. Mycothiol biosynthesis is essential for ethionamide susceptibility in Mycobacterium tuberculosis. Mol Microbiol 2008; 69: 1316–29.


Mutations in ethA and inhA confer resistance to ethionamide.1

Other potential mechanisms of resistance have been identified, as mshA deletion mutants were found to be defective in mycothiol biosynthesis and resistant to ethionamide, likely due to defective activation of the drug.2

Co-resistance to INH and ethionamide can be mediated by mutations that alter the InhA target or cause their over expression or by mutations in ndh that increase the intracellular concentration of NADH.3,4

1 Hazboń MH, Brimacombe M, Bobadilla del Valle M et al. Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 2006; 50: 2640–9.2 Vilcheze, Av-Gay et al. 20083 Wang F, Langley R, Gulten G et al. Mechanism of thioamide drug action against tuberculosis and leprosy. J Exp Med 2007; 204:73–8.4 Vilche`ze C, Wang F, Arai M et al. Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid. Nat Med 2006; 12: 1027–9.

p-Amino salicylic acid

Discovered by the Swedish chemist Jörgen Lehmann.

Used as an oral anti-TB therapy late in 1944.

Was used in the treatment of the disease in combination with isoniazid and streptomycin.1

Also used for the treatment of inflammatory bowel diseases (IBDs).

1 Lehmann J. para-Aminosalicylic acid in the treatment of tuberculosis. Lancet 1946; i: 15–6.

MECHANISM OF ACTION

Mechanism of action was never clearly elucidated.

Suggested that PAS may compete with para-amino benzoic acid (PABA) for dihydropteroate synthase, an enzyme needed in folate biosynthesis.1

1 Wade and Zhang 2004


Mutations in the thyA gene encoding the enzyme thimidylatesynthesis of the folate biosynthesis pathway have been identified.1

Mutations in thyA results in decreased enzyme activity.

Other studies assessing the role of enzymes in the folate pathway determined that PAS was a pro-drug whose activation required a viable ThyA.2

1 Rengarajan J, Sassetti CM, Naroditskaya V et al. The folate pathway is a target for resistance to the drug para-aminosalicylic acid (PAS) in mycobacteria. Mol Microbiol 2004; 53: 275–822 Mathys V, Wintjens R, Lefevre P et al. Molecular genetics of para-aminosalicylic acid resistance in clinical isolates and spontaneous mutants of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2009; 53: 2100–9.

However, only 37% of the evaluated clinical isolates of M.TB & spontaneous mutants had mutations in thyA, suggesting the existence of additional mechanisms for PAS resistance.

Some studies reported Thr202Ala as the most common mutation associated with PAS resistance, although a few susceptible isolates have been found to contain this same mutation.1

1 Leung KL, Yip CW, Yeung YL et al. Usefulness of resistant gene markers for predicting treatment outcome on second-line anti-tuberculosis drugs. J Appl Microbiol 2010; 109: 2087–94.

MACROLIDES

Clarithromycin

Broad-spectrum antibiotics.

Commonly used to treat infections caused by the MAC & other NTM.

Little or no effect on mycobacteria belonging to the M. tuberculosis complex.1,2

1 Nguyen L, Thompson CJ. Foundations of antibiotic resistance in bacterial physiology: the mycobacterial paradigm. Trends Microbiol 2006; 14: 304–12.2 Bermudez and Yamazaki 2004

MECHANISM OF ACTION

Binds to the bacterial 50S ribosomal subunit and inhibits RNA-dependent protein synthesis.1

However, these drugs have limited activity against wild-type M. tuberculosis.2

1 Piscitelli, Danziger et al. 19922 Bermudez and Yamazaki 2004

Intrinsic resistance to macrolides such as clarithromycin has been associated with low cell wall permeability

Expression of the erm37 gene, which encodes an enzyme that methylates a specific site in the 23S rRNA, preventing antibiotic binding.

Studies found that intrinsic resistance was inducible with sub inhibitory concentrations of clarithromycin.

The same study also found an increase in the levels of erm37 mRNA.1


1 Andini N, Nash KA. Intrinsic macrolide resistance of the Mycobacterium tuberculosis complex is inducible. Antimicrob Agents Chemother 2006; 50: 2560–2.

Sub inhibitory concentrations of ethambutol reversed resistance to clarithromycin in clinical isolates of M. tuberculosis.

Supporting the concept that macrolide intrinsic resistance could be related to a permeability barrier.1

Studies have found the occurrence of synergy when combined with sub inhibitory concentrations of other antibiotics or anti-TB drugs.2,3

1 Bosne-David S, Barros V, Verde SC et al. Intrinsic resistance of Mycobacterium tuberculosis to clarithromycin is effectively reversed by subinhibitory concentrations of cell wall inhibitors. J Antimicrob Chemother 2000; 46: 391–5.2 Bhusal Y, Shiohira CM, Yamane N. Determination of in vitro synergy when three antimicrobial agents are combined against Mycobacterium tuberculosis. Int J Antimicrob Agents 2005; 26: 292–7.3 Stoffels K, Traore H, Vanderbist F et al. The effect of combined tobramycin-clarithromycin on Mycobacterium tuberculosis isolates. Int J Tuberc Lung Dis 2009; 13: 1041–4.

LINEZOLID

First compound belonging to the oxazolidinones approved for clinical use.

Discovered in the 1990s

FDA approved linezolid on April 18, 2000

Commonly used to treat drug resistant TB

Use has been limited by toxicity concerns, particularly leukopenia & thrombocytopenia, as well as peripheral neuropathy, which may be irreversible.

MECHANISM OF ACTION

Due to its ability to penetrate macrophages, linezolid is active against intracellular bacilli.

Binds to the ribosomal 50S subunit and thus inhibits an early step in protein synthesis.1

Linezolid has shown significant in vitro and in vivo activity in animal models against M. tuberculosis.2,3

1 Zhang Y. The magic bullets and tuberculosis drug targets. Annu Rev Pharmacol Toxicol 2005; 45: 529–64.2 Alcala L, Ruiz-Serrano M, Turegano C et al. In vitro activities of linezolid against clinical isolates of M. tuberculosis that are susceptible or resistant to first-line antituberculous drugs. Antimicrob Agents Chemother 2003; 47: 416–7.3 Cynamon M, Klemens S, Sharpe C et al. Activities of several oxazolidinones against Mycobacterium tuberculosis in a murine model. Antimicrob Agents Chemother 1999; 43: 1189–91.

Resistance in M. tuberculosis has been reported as rare.

Study by Richter et al (2007) found 1.9% resistance among 210 MDR strains.

In vitro-selected mutants with high-level resistance to linezolid (MIC = 16–32 mg/L) have been found to contain mutations at G2061T and G2576T in the 23S rRNA gene.1

On the other hand, mutants with lower level linezolid resistance (MIC = 4–8 mg/L) lacked these mutations, implicating other possible mechanisms such as involvement of efflux pumps2 or other non-ribosomal alterations, as has been shown in M. smegmatis mutants.3

1 Hillemann D, Rusch-Gerdes S, Richter E. In vitro-selected linezolid-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 2008; 52: 800–1.2 Escribano I, Rodrı´guez JC, Llorca B et al. Importance of the efflux pump systems in the resistance of Mycobacterium tuberculosis to fluoroquinolones and linezolid. Chemotherapy 2007; 53: 397–401.3 Sander P, Belova L, Kidan YG et al. Ribosomal and non-ribosomal resistance to oxazolidinones: species-specific idiosyncrasy of ribosomal alterations. Mol Microbiol 2002; 46: 1295–304.


D-CYCLOSERINE

4-amino-3-isoxazolidinone.

An analogue of D-alanine.

The compound was first isolated from Streptomyces orchidaceus and also synthetically.

MECHANISM OF ACTION

Inhibits peptidoglycan synthesis by blocking the action of D-alanine:D-alanine ligase (Ddl).1

It also inhibits D-alanine racemase (Alr) involved in the interconversion of L-alanine and D-alanine, which then serves as a substrate for Ddl.2

1 Caceres, Harris et al. 19972 Zhang Y. The magic bullets and tuberculosis drug targets. Annu Rev Pharmacol Toxicol 2005; 45: 529–64.


Overexpression of M. tuberculosis AlrA and Ddl on a multicopy vector results in resistance to D-cycloserine in M. smegmatis and M. bovis BCG.1,2

1 Caćeres NE, Harris NB, Wellehan JF et al. Overexpression of the d-alanine racemase gene confers resistance to d-cycloserine in Mycobacterium smegmatis. J Bacteriol 1997; 179:5046–55.2 Feng and Barletta 2003

NEW DRUGSNEW TARGETS

&NEW RESISTANCE

MECHANISMS

NITROIMIDAZOLES

Pretomanid (PA-824), a nitroimidazo-oxazine.

Delamanid (OPC-67683) a nitroimidazo-oxazole.

Shows activity against M. tuberculosis strains susceptible and resistant to classical anti-TB drugs.1,2

1 Stover CK, Warrener P, VanDevanter DR et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 2000; 405: 962–6.2 Rivers EC, Mancera RL. New anti-tuberculosis drugs in clinical trials with novel mechanisms of action. Drug Discov Today 2008; 13:1090–8.

Pretomanid (PA-824)

MIC values reported for Pretomanid are in the range of 0.015–0.25 mg/L for drug-susceptible strains and 0.03–0.53 mg/L for drug-resistant strains.1

Has activity against both replicating, hypoxic non-replicating bacilli.2

1 Ginsberg AM, Laurenzi MW, Rouse DJ et al. Safety, tolerability, and pharmacokinetics of PA-824 in healthy subjects. Antimicrob Agents Chemother 2009; 53: 3720–5.2 Singh R, Manjunatha U, Boshoff HI et al. PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release. Science 2008;322: 1392–5.

MECHANISM OF ACTION

Pro-drug needs to be metabolized by M.TB in order to be activated.

The mechanism of action has been found to be inhibition of cell wall lipid and protein synthesis1

PA-824 also acts directly as an Nitric Oxide donor.2

This respiratory poisoning through NO release seems to be a crucial element of anaerobic activity by Pretomanid.2

1 Stover CK, Warrener P, VanDevanter DR et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis.Nature 2000; 405: 962–6.2 Singh R, Manjunatha U, Boshoff HI, Ha YH, Niyomrattanakit P, Ledwidge R, et al. PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release. Science 2008; 322:1392-5.


Mediated by the loss of a specific glucose-6-phosphate dehydrogenase or its deazaflavin cofactor F420, which could provide electrons for reduction.1

Rv3547, a protein with high structural specificity for binding to Pretomanid, is also involved in the activation process.2

1 Stover CK, Warrener P, VanDevanter DR et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis.Nature 2000; 405: 962–6.2 Manjunatha UH, Boshoff H, Dowd CS et al. Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2006; 103:431–6.

DELAMANID (OPC-67683)

Pro-drug that needs to be activated by M. tuberculosis.

Has shown MIC values of 0.006–0.024 mg/L with no cross-resistance with first-line drugs.

In phase II clinical trials, the drug was used in combination with standard TB drugs.1,2

Healing rates (measured as sputum culture conversion) were significantly better in patients who additionally took delamanid. 1,2

1 H. Spreitzer (18 February 2013). "Neue Wirkstoffe – Bedaquilin und Delamanid". sterreichische Apothekerzeitung (in German) (4/2013): 222 "Delamanid for Multidrug-Resistant Pulmonary Tuberculosis". New England Journal of Medicine 366 (23): 2151–2160.

MECHANISM OF ACTION

Inhibition of the synthesis of methoxy- and keto-mycolic acids.


Drug-resistant strains do not metabolize the drug and contain a mutation in the Rv3547 gene, suggesting its involvement in drug activation.1

1 Matsumoto M, Hashizume H, Tomishige T et al. OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice. PLoS Med 2006; 3: e466.

SQ109 1,2-diamine analogue of ethambutol obtained after analysis of a library of 63,238 compounds.

Shows good activity, with MIC values ranging from 0.16 to 0.63 mg/L.1

1 Protopopova M, Hanrahan C, Nikonenko B et al. Identification of a new antitubercular drug candidate, SQ109, from a combinatorial library of 1,2-ethylenediamines. J Antimicrob Chemother 2005; 56: 968–74.

On October 16th, 2007, declared as Orphan drug by the U.S. FDA for use against drug-susceptible and drug-resistant TB bacteria.

Shows activity against both drug susceptible and MDR TB, including XDR strains.

In preclinical studies SQ109 enhanced the activity of anti-TB drugs INH & rifampin

Reduced >30% the time required to cure mice of experimental TB.

MECHANISM OF ACTION

Not well known. Believed that it affects mycobacterial cell wall synthesis in a different manner to that exerted by ethambutol.


In strains resistant to isoniazid, ethambutol & SQ109, there is up-regulation of ahpC, suggesting a possible role in the development of resistance to this drug.1

acolE1 Jia L, Coward L, Gorman GS et al. Pharmacoproteomic effects of isoniazid, ethambutol, and N-geranyl-N -(2-adamantyl)ethane-1,2-diamine (SQ109) on ′Mycobacterium tuberculosis H37Rv. J Pharmxp Ther 2005; 315: 905–11.

BEDAQUILINE (TMC207/R207910)

Diarylquinoline with inhibitory activity against both drug-susceptible, drug-resistant M. tuberculosis and other mycobacteria.1

Approved by the FDA on the 28th December 2012, the first new medicine to fight TB in more than 40 years.

Specifically approved to treat MDR TB.

when added to standard therapy for MDR-TB, reduces the time & increases sputum conversion.2

1 Andries K, Verhasselt P, Guillemont J et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 2005; 307: 223–7.2 Diacon AH, Pym A, Grobusch M et al. The diarylquinoline TMC207 for multidrug-resistant tuberculosis. N Engl J Med 2009; 360: 2397–405.

MECHANSIM OF ACTION

Specific inhibition of mycobacterial ATP synthase.1,2

1 Andries K, Verhasselt P, Guillemont J et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 2005; 307: 223–7.2 Koul A, Dendouga N, Vergauwen K et al. Diarylquinolines target subunit c of mycobacterial ATP synthase. Nat Chem Biol 2007; 3: 323–4.3 Huitric E, Verhasselt P, Koul A et al. Rates & mechanisms of resistance development in Mycobacterium tuberculosis to a novel diarylquinoline ATP synthase inhibitor. Antimicrob Agents Chemother 2010; 54: 1022–8.

MECHANSIM OF RESISTANCE In vitro generated mutants showed an A63P mutation in the atpE gene, suggesting that the drug acts on the proton pump of ATP synthase.

Petrella et al.3 found the same mutation, A63P, and a new mutation, I66M, in atpE in vitro-generated mutants.

More studies with in vitro-generated mutants suggest that alternative mechanisms for drug resistance might exist, since in 38 out of 53 mutants evaluated no mutation in atpE could be observed.

NAS-21 and NAS-91

The anti-malarial agents.

Inhibits mycolic acid biosynthesis and profoundly altering the production of oleic acids.1

In studies with M. bovis BCG, it has been suggested that the main target could be the FAS-II dehydratase coded by Rv0636.

MECHANISM OF RESISTANCEIt has been shown that strains resistant to these compounds over express Rv0636 gene analogues.2

1 Gratraud P, Surolia N, Besra GS et al. Antimycobacterial activity and mechanism of action of NAS-91. Antimicrob Agents Chemother 2008; 52:1162–6.2 Bhowruth V, Brown AK, Besra GS. Synthesis and biological evaluation of NAS-21 and NAS-91 analogues as potential inhibitors of the mycobacterial FAS-II dehydratase enzyme Rv0636. Microbiology 2008;154: 1866–75.

PHENOTHIAZINES

Thioridazine and chlorpromazine can be considered as calmodulin antagonists

Anti-TB activity has been related to the presence of a calmodulin-like protein in the bacilli.1

Thioridazine has shown in vitro activity against drug-susceptible and drug-resistant strains of M. tuberculosis.

More importantly, they have shown activity against bacilli inside macrophages.2

1 Zhang Y. The magic bullets and tuberculosis drug targets. Annu Rev Pharmacol Toxicol 2005; 45: 529–64.2 Ordway D, Viveiros M, Leandro C et al. Clinical concentrations of thioridazine kill intracellular multidrug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 2003; 47: 917–22.

MECHANSIM OF ACTION

Has not been completely elucidated.

May be related to its inhibition of calcium transport and enzymes dependent on calcium thus inhibit the generation of cellular energy and ATP hydrolysis.

Among these are efflux pumps, and for this reason phenothiazines are considered as putative efflux pump inhibitors.1

More recently thioridazine has been shown to have an effect on the sigma factor network in M. tuberculosis that would play a role in the bacteria’s defense against cell damage.2

1 Martins M, Dastidar SG, Fanning S et al. Potential role of non-antibiotics (helper compounds) in the treatment of multidrug-resistant Gram-negative infections: mechanisms for their direct and indirect activities. Int J Antimicrob Agents 2008; 31: 198–208.2 Dutta NK, Mehra S, Kaushal D. A Mycobacterium tuberculosis sigma factor network responds to cell-envelope damage by the promising anti-mycobacterial thioridazine. PLoS One 2010; 5: e10069.

BENZOTHIAZINONES

A new class of antimycobacterial agents, recently been synthesized and characterized.

Kills M. tuberculosis in vitro, ex vivo and in a mouse model of TB infection.1

1 Makarov V, Manina G, Mikusova K et al. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science 2009; 324: 801–4.

MECHANSIM OF ACTION

DprE1 subunit of the enzyme decaprenylphosphoryl-β-D-ribose 2 -′epimerase has been identified as the major target.

When this enzyme activity is inhibited, it aborts the formation of decaprenylphosphoryl arabinose, a precursor in the synthesis of cell wall arabinan.

When spontaneous resistant laboratory mutants were generated, codon Cys387 of dprE1 was replaced by an Ser or Gly.

On the other hand, in M. avium, which is naturally resistant to 1,3-benzothiazin-4-ones, codon Cys387 is replaced by an Ala codon.

MECHANSIM OF RESISTANCE

No resistance has been found yet in clinical isolates of M.TB.

Recent evaluation performed in 4 hospitals in Europe found uniform susceptibility among the 240 isolates evaluated, with susceptibility in the range of 0.75–30 ng/mL.

None of them showed any mutation in dprE1.1

Overexpression of nitroreductase NfnB inactivated the drug by reducing a critical nitro group to an amino group in M. smegmatis. 2

M. TB seems to lack nitroreductases able to inactivate these drugs.

1 Pasca MR, Degiacomi G, Ribeiro AL et al. Clinical isolates of Mycobacterium tuberculosis in four European hospitals are uniformly susceptible to benzothiazinones. Antimicrob Agents Chemother 2010;54: 1616–8.2 Manina G, Bellinzoni M, Pasca MR et al. Biological and structural characterization of the Mycobacterium smegmatis nitroreductase NfnB, and its role in benzothiazinone resistance. Mol Microbiol 2010; 77:1172–85.

Conclusion

• Drug resistance in TB remains largely a man-made phenomenon which renders the bacteria resistant to the most commonly used anti-TB drugs.

• Among the reasons for this, the non-compliance with the treatment regimens is signaled as the first cause.

• There are still many cases where resistant strains do not harbor any known mutation.

• More clarification is needed on the role of specific gene mutations and the development of MDR- or XDR-TB.

Conclusion

• A recent study using whole-genome sequencing identified new genes and intergenic regions that were associated with drug resistance, showing that TB drug resistance is a phenomenon more complex than previously assumed.1

• A better knowledge is also required on the intrinsic resistance to certain antibiotics, efflux pump mechanisms and the development of clinical drug resistance.

• It is, thus, quite important to further our knowledge of additional mechanisms of drug resistance to the available anti-TB drugs.

• This could have a major impact on the dynamics of TB transmission and for the discovery and development of new anti-TB drugs.

1 Zhang, H.; Li, D.; Zhao, L.; Fleming, J.; Lin, N.; Wang, T.; Liu, Z.; Li, C.; Galwey, N.; Deng, J.; et al. Genome sequencing of 161 Mycobacterium tuberculosis isolates from China identifies genes and intergenic regions associated with drug resistance. Nat. Genet. 2013, 45, 1255–1260

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Health & Medicine

MOLECULAR BASIS & MECHANISMS OF DRUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS