How does Multi-Drug Resistance TB occurs?

1.WHAT IS MDR-TB?

Tuberculosis (TB) is a disease caused by bacteria that are spread from person to person through the air. TB usually affects the lungs, but it can also affect other parts of the body, such as the brain, the kidneys, or the spine. In most cases, TB is treatable and curable; however, people with TB can die if they do not get proper treatment. Sometimes drug-resistant TB occurs when bacteria become resistant to the drugs used to treat TB. This means that the drug can no longer kill the TB bacteria.

A better knowledge of the mechanisms of drug resistance of M. tuberculosis and the relevant molecular mechanisms involved will improve the available techniques for rapid drug resistance detection and will help to explore new targets for drug activity and development.

In 2012, there were an estimated 450,000 cases of multidrug resistant (MDR)-TB and 170,000 deaths were due to it. MDR-TB is caused by strains of Mycobacterium tuberculosis that are resistant to at least rifampicin and isoniazid, two key drugs in the treatment of the disease. Since 2006, it has been recognized the presence of even more resistant strains of M. tuberculosis labelled as extensively drug resistant (XDR)-TB.These strains in addition to being MDR are also resistant to any fluoroquinolone and to at least one of the injectable second-line drugs: kanamycin, capreomycin or amikacin. More recently, a more worrying situation has emerged with the description of M. tuberculosis strains that have been found resistant to all antibiotics that were available for testing, a situation labelled as totally drug resistant (TDR)-TB.

Early detection of all forms of drug resistance in TB is a key factor to reduce and contain the spread of these resistant strains. A better knowledge of the mechanisms of action of anti-TB drugs and the development of drug resistance will allow identifying new drug targets and better ways to detect drug resistance. The following sections will review the mode of action and resistance mechanisms of the main anti-TB drugs as well as new drugs recently described with anti-TB activity.

2. WHICH DRUGS ARE USED FOR TREATMENT OF TB AS FIRST LINE OF DEFENCE?

2.1. Rifampicin:

Rifampicin is a rifamycin derivative introduced in 1972 as an anti-tuberculosis agent. It is one of the most effective anti-TB antibiotics and together with isoniazid constitutes the basis of the multidrug treatment regimen for TB. Rifampicin is active against growing and non-growing (slow metabolizing) bacilli. The mode of action of rifampicin in M. tuberculosis is by binding to the β-subunit of the RNA polymerase, inhibiting the elongation of messenger RNA. The majority of rifampicin-resistant clinical isolates of M. tuberculosis harbor mutations in the rpoB gene that codes for the β-subunit of the RNA polymerase. As a result of this, conformational changes occur that decrease the affinity for the drug and results in the development of resistance.

Gene of mutation due to Rifampicin:

In about 96% of M. tuberculosis isolates resistant to rifampicin, there are mutations in the so-called “hot-spot region” of 81-bp spanning codons 507–533 of the rpoB gene. This region is also known as the rifampicin resistance-determining region. Mutations in codons 516, 526 and 531 are the most commonly associated mutations with rifampicin resistance in the majority of studies. Although less frequent, some reports have also noted the occurrence of mutations outside of the hot-spot region of rpoB . Cross-resistance with other rifamycins can occur. Mutations in some codons (e.g., 518 or 529) have been associated with low-level resistance to rifampicin but still susceptible to other rifamycins, such as rifabutin or rifalazil. This is important for TB patients that need to receive antiretroviral therapy since rifabutin is a less effective inducer of the cytochrome P450 CYP3A oxidative enzyme. On the other hand, mono-resistance to rifampicin is quite rare and almost all rifampicin-resistant strains are also resistant to other drugs, especially to isoniazid. This is the reason why rifampicin resistance is considered as a surrogate marker for MDR-TB.

Recent genome sequencing studies have uncovered the acquisition of compensatory mutations in rpoA and rpoC, encoding the α and β' subunits of RNA polymerase, in rifampicin-resistant strains with mutations in rpoB. These compensatory mutations would be responsible for restoring the fitness of these strains in vivo and have also been associated with a higher transmissibility in some settings.

2.2. Isoniazid:

Isoniazid was introduced in 1952 as an anti-TB agent and it remains, together with rifampicin, as the basis for the treatment of the disease. Unlike rifampicin, isoniazid is only active against metabolically-active replicating bacilli. Also known as isonicotinic acid hydrazide, isoniazid is a pro-drug that requires activation by the catalase/peroxidase enzyme KatG, encoded by the katG gene, to exert its effect. Isoniazid acts by inhibiting the synthesis of mycolic acids through the NADH-dependent enoyl-acyl carrier protein (ACP)-reductase, encoded by inhA. Although simple in its structure, resistance to this drug has been associated with mutations in several genes, such as katG, inhA, ahpC, kasA and NDH.

Gene of mutation due to Isoniazid:

The two main molecular mechanisms of isoniazid resistance are associated with gene mutations in katG and inhA or its promoter region. Indeed, numerous studies have found mutations in these two genes as the most commonly associated with isoniazid resistance. Among these, the most prevalent gene mutation has been identified as S315T in katG resulting in an isoniazid product deficient in forming the isoniazid-NAD adduct needed to exert its antimicrobial activity. This mutation has been consistently associated with high-level resistance (MIC > 1 µg/mL) to isoniazid and occurs more frequently in MDR strains. The second most common mutation occurs in the promoter region of inhA causing an overexpression of InhA or less frequently, a mutation in its active site, which decreases its affinity for the isoniazid-NAD adduct. The most prevalent mutation found is at position −15C/T and is more commonly associated with low level resistance to isoniazid (MIC < 1 µg/mL). Mutations in inhA not only cause resistance to isoniazid but also to the structurally related drug ethionamide, which shares the same target. A recent study found that a mutation in the inhA regulatory region together with a mutation in the inhA coding region produced high-level isoniazid resistance and also cross-resistance to ethionamide.

One recent interesting finding showed that the 4R isomer of the isoniazid-NADP adduct causes inhibition of the dihydrofolate reductase (DfrA) in M. tuberculosis, suggesting that mutations in dfrA could possibly play a role in resistance to isoniazid. Moreover, an analysis of the proteome of isoniazid targets in M. tuberculosis identified 16 other proteins, in addition to InhA and DfrA, that were bound by these adducts with high affinity, which could signal other not yet clearly defined actions of isoniazid on the bacteriai. Two recent studies, however, have failed to identify any mutation in dfrA associated with resistance to isoniazid.

In M. tuberculosis, ahpC encodes an alkyl hydroperoxidase reductase that is implicated in resistance to reactive oxygen intermediates and it was initially proposed that mutations in the promoter of ahpC could be used as proxy markers for isoniazid resistance. It is now better understood that mutations in the promoter of ahpC are compensatory mutations for the loss of catalase/peroxidase activity rather than the cause for isoniazid resistance. Moreover, overexpression of AhpC does not confer resistance to isoniazid.

Several studies have found single nucleotide polymorphisms in other genes in isoniazid resistant clinical isolates of M. tuberculosis, including kasA and the oxyR-ahpC and furA-katG intergenic regions. However, their direct role as a cause of isoniazid resistance has not been fully demonstrated. On the other hand, co-resistance to isoniazid and ethionamide has been clearly demonstrated to be caused by mutations in ndh in M. smegmatis and M. bovis BCG, by altering the NADH/NAD ratios inside the cell, leading to a competitive inhibition of the INH-NAD adduct. A recent study has also found that a silent mutation in mabA conferred isoniazid resistance through upregulation of inhA in M. tuberculosis.

2.3. Ethambutol:

Ethambutol was first introduced in the treatment of TB in 1966 and is part of the current first-line regimen to treat the disease. Ethambutol is bacteriostatic against multiplying bacilli interfering with the biosynthesis of arabinogalactan in the cell wall. In M. tuberculosis, the genes embCAB, organized as an operon, code for arabinosyl transferase, which is involved in the synthesis of arabinogalactan, producing the accumulation of the intermediate d-arabinofuranosyl-P-decaprenol.

Gene of mutation due to Ethambutol:

The recognized mechanism of resistance to ethambutol has been linked to mutations in the gene embB with mutations at position embB306 as the most prevalent in most of the studies performed. Some studies, however, have also found mutations in embB306 in ethambutol susceptible isolates. Moreover, a study with a large number of M. tuberculosis isolates found that mutations in embB306 were not necessarily associated with resistance to ethambutol but with a predisposition to develop resistance to increasing number of drugs and to be transmitted. In fact, allelic exchange studies have shown that individual mutations causing certain amino acid substitutions produced ethambutol resistance, while other amino acid substitutions had little or no effect on ethambutol resistance. The same authors have more recently reported that mutations in the decaprenylphosphoryl-B-d-arabinose (DPA) biosynthetic and utilization pathway genes, Rv3806c and Rv3792, together with mutations in embB and embC accumulate, giving rise to a range of MICs of ethambutol depending on mutation type and number. These findings could have influence on the correct detection of ethambutol resistance by current molecular methods. Mutations in embB306 then, cause variable degrees of ethambutol resistance and are required but are not enough to cause high-level resistance to ethambutol. There remain about 30% ethambutol resistant strains that do not present any mutation in embB stressing the need to identify other possible mechanisms of drug resistance to this drug.

2.4. Pyrazinamide:

Pyrazinamide was introduced into TB treatment in the early 1950s and constitutes now part of the standard first-line regimen to treat the disease. Pyrazinamide is an analog of nicotinamide and its introduction allowed reducing the length of treatment to six months. It has the characteristic of inhibiting semi-dormant bacilli residing in acidic environments such as found in the TB lesions. Pyrazinamide is also a pro-drug that needs to be converted to its active form, pyrazinoic acid, by the enzyme pyrazinamidase / nicotinamidase coded by the pncA gene. The proposed mechanism of action of pyrazinamide involves conversion of pyrazinamide to pyrazinoic acid, which disrupts the bacterial membrane energetics inhibiting membrane transport. Pyrazinamide would enter the bacterial cell by passive diffusion and after conversion to pyrazinoic acid it is excreted by a weak efflux pump. Under acid conditions, the protonated pyrazinoic acid would be reabsorbed into the cell and accumulated inside, due to an inefficient efflux pump, resulting in cellular damage. One study has also found that pyrazinoic acid and its n-propyl ester can inhibit the fatty acid synthase type I in replicating M. tuberculosis bacilli.

Gene mutation due to Pyrazinamide:

A recent study, however, has challenged the previous model by proposing that pyrazinoic acid inhibits trans-translation, a process of ribosome-sparing in M. tuberculosis. The study was performed in pyrazinamide-resistant strains lacking mutations in pncA but that had mutations in rpsA identifying the ribosomal protein 1 (RpsA) as the proposed target. Overexpression of RpsA conferred increased resistance to pyrazinamide and pyrazinoic acid was confirmed to be bound to RpsA. While a very intriguing hypothesis as a target for pyrazinamide, the failure to perform allelic transfers in this study makes it difficult to conclude that in fact mutations in rpsA are the target of pyrazinamide.

Mutations in the gene pncA remain as the most common finding in pyrazinamide resistant strains. These mutations, however, are scattered throughout the gene but most occur in a 561-bp region in the open reading frame or in an 82-bp region of its putative promoter. Some few studies have reported the occurrence of pyrazinamide resistant strains without any mutation in pncA stating that the resistance could be due to mutations in another not yet identified regulatory gene. Based on the current evidence, the contribution of mutations in rpsA to pyrazinamide resistance remains limited.

2.5. Streptomycin:

Originally isolated from the soil microorganism Streptomyces griseus, streptomycin was the first antibiotic to be successfully used against TB. Unfortunately, as soon as it was prescribed, resistance to it emerged, a result of being administered as monotherapy. Streptomycin is an aminocyclitol glycoside active against actively growing bacilli and its mode of action is by inhibiting the initiation of the translation in the protein synthesis . More specifically, streptomycin acts at the level of the 30S subunit of the ribosome at the ribosomal protein S12 and the 16S rRNA coded by the genes rpsL and rrs, respectively.

Gene mutation due to Streptomycin:

Consequently, mutations in rpsL and rrs are the major mechanisms of resistance to streptomycin but account for 60%–70% of the resistance found. Among the mutations reported in rpsL, a substitution in codon 43 from lysine to arginine has been the most commonly reported. This mutation produces high-level resistance to streptomycin. In rrs the most common mutations occur around nucleotides 530 and 915. There remain an important percentage of strains resistant to streptomycin that lack mutations in either of these two genes, suggesting additional mechanisms of resistance.

In the last years, it has also been reported that mutations in gidB, a gene encoding a conserved 7-methylguanosine methyltransferase specific for the 16S rRNA, confers low-level resistance to streptomycin.

3. WHICH DRUGS ARE USED FOR TREATMENT OF TB AS SECOND LINE OF DEFENCE?

3.1. Fluoroquinolones:

Fluoroquinolones are currently in use as second-line drugs in the treatment of MDR-TB. Both ciprofloxacin and ofloxacin are synthetic derivatives of the parent compound nalidixic acid, discovered as a by-product of the antimalarial chloroquine. Newer-generation quinolones such as moxifloxacin and gatifloxacin are being evaluated in clinical trials and proposed as first-line antibiotics with the purpose of shortening the length of treatment in TB .

The mode of action of fluoroquinolones is by inhibiting the topoisomerase II (DNA gyrase) and topoisomerase IV, two critical enzymes for bacterial viability. These proteins are encoded by the genes gyrA, gyrB, parC and parE, respectively. In M. tuberculosis, only type II topoisomerase (DNA gyrase) is present and, thus, is the only target of fluoroquinolone activity. Type II topoisomerase is a tetramer formed by two α and β subunits, coded by gyrA and gyrB, respectively, which catalyzes the supercoiling of DNA.

Gene mutation due to fluroquinolones:

The main mechanism of development of fluoroquinolone resistance in M. tuberculosis is by chromosomal mutations in the quinolone resistance-determining region of gyrA or gyrB. The most frequent mutations found are at position 90 and 94 of gyrA but mutations at position 74, 88 and 91 have also been reported. A recent systematic review of fluoroquinolone-resistance-associated gyrase mutations in M. tuberculosis has been published.

One interesting finding in M. tuberculosis is the presence of a natural polymorphism at position 95 in gyrA that is not related to fluoroquinolone resistance since it is also found in fluoroquinolone-susceptible strains. Another interesting finding has been the report that the simultaneous occurrence of mutations T80A and A90G in gyrA led to hypersusceptibility to several quinolones. This finding could point out that the problem of fluoroquinolone resistance in M. tuberculosis might be more complex than was thought initially.

Cross-resistance is assumed to occur between fluoroquinolones although isolated reports have acknowledged the presence of strains resistant to gatifloxacin and moxifloxacin that were still susceptible to ofloxacin. Also, the involvement of efflux mechanisms has been suggested as a possible cause for fluoroquinolone resistance in M. tuberculosis.

3.2. Kanamycin, Capreomycin, Amikacin, Viomycin:

These four antibiotics have the same mechanism of action by inhibiting the protein synthesis but, while kanamycin and amikacin are aminoglycosides, capreomycin and viomycin are cyclic peptide antibiotics. All four are second-line drugs used in the management of MDR-TB.

Gene of mutation due to these drugs:

Kanamycin and amikacin inhibit protein synthesis by alteration at the level of 16S rRNA. The most common mutations found in kanamycin-resistant strains are at position 1400 and 1401 of the rrs gene, conferring high-level resistance to kanamycin and amikacin. However, mutations at position 1483 have also been reported. Full cross-resistance between kanamycin and amikacin is not complete, as previously thought. Some studies have shown variable levels and patterns of resistance suggesting that other mechanisms of resistance might be possible. In concordance with this, a low-level resistance to kanamycin has been associated with mutations in the promoter region of the eis gene, encoding an aminoglycoside acetyltransferase. Mutations at position −10 and −35 of the eis promoter led to an overexpression of the protein and low-level resistance to kanamycin but not to amikacin. These mutations were found in up to 80% of clinical isolates showing low-level resistance to kanamycin.

Capreomycin and viomycin, on the other hand, have a similar structure and bind at the same site in the ribosome, at the interface of the small and large subunits. They show full cross-resistance as reported in previous studies. Mutations in the tlyA gene have also been associated with resistance to capreomycin and viomycin. TlyA is an rRNA methyltransferase specific for 2'-O-methylation of ribose in rRNA. Mutations in tlyA determine the absence of methylation activity. Although some studies did not find this association, a recent meta-analysis, evaluating the association of genetic mutations and resistance to second-line drugs, has confirmed the presence of tlyA mutations in addition to mutations in rrs and eis.

3.3.Ethionoamide:

Ethionamide is a derivative of isonicotinic acid structurally similar to isoniazid. It is also a pro-drug requiring activation by a monooxygenase encoded by the ethA gene. It interferes with the mycolic acid synthesis by forming an adduct with NAD that inhibits the enoyl-ACP reductase enzyme. EthA is regulated by the transcriptional repressor EthR. 

Resistance to ethionamide occurs by mutations in etaA/ethA, ethR and also mutations in inhA, which cause resistance to both isoniazid and ethionamide. Moreover, studies with spontaneous isoniazid- and ethionamide-resistant mutants of M. tuberculosis found that they map to mshA, encoding an enzyme essential for mycothiol biosynthesis.

3.4. Para-amino Salicylic Acid:

Although it was one of the first anti-tuberculosis drugs used in the treatment of the disease, together with isoniazid and streptomycin, para-amino salicylic acid or PAS is now considered as a second-line drug part of the treatment regimen for MDR-TB. Until recently, its mechanism of action was not completely defined. It has been proposed that being an analog of para-amino benzoic acid, it must compete with it for dihydropteroate synthase, interfering in the process of folate synthesis. A study using transposon mutagenesis identified mutations in the thyA gene associated with resistance to PAS that were also present in clinical isolates resistant to PAS. A recent study has also identified various missense mutations in folC encoding dihydrofolate synthase that conferred resistance to PAS in laboratory isolates of M. tuberculosis. In a panel of 85 clinical MDR-TB isolates, mutations in folC were identified in five isolates resistant to PAS. Nevertheless, just less than 40% of PAS-resistant strains had mutations in thyA indicating that still other mechanisms of resistance to the drug might exist.

REFERENCES:
https://en.wikipedia.org/wiki/Multi-drug-resistant_tuberculosis
https://www.who.int/features/qa/79/en/
https://www.ncbi.nlm.nih.gov/books/NBK247431/
https://www.intechopen.com/books/tuberculosis-current-issues-in-diagnosis-and-management/first-and-second-line-drugs-and-drug-resistance