Isoniazid is one of the main drugs

Isoniazid is one of the main drugs for the treatment of TB. It has a simple structure containing a pyridine ring and a hydrazide group, with both components being essential for the high activity against M. tuberculosis. Despite this simple structure, the mode of action of isoniazid is more complex and isoniazid-resistant strains had already been isolated as soon as its anti-TB activity was recognized. Very early on, it was also shown that Mycobacterium bovis and M. tuberculosis isoniazid-resistant strains lacking catalase activity were highly attenuated in guinea pigs.24,25

Resistance to isoniazid is a complex process. Mutations in several genes, including katG, ahpC, inhA, kasA and ndh, have all been associated with isoniazid resistance. Isoniazid is a pro-drug requiring activation by the catalase/peroxidase enzyme encoded by katG.26 Activated isoniazid interferes with the synthesis of essential mycolic acids by inhibiting NADH-dependent enoyl-ACP reductase, which is encoded by inhA.27 Two molecular mechanisms have been shown to be the main cause for isoniazid resistance: mutations in katG and mutations in inhA, or more frequently in its promoter region.28,29

A study by Hazbón et al.30 analysed 240 alleles previously described in association with isoniazid resistance and found that mutations in katG, inhA and ahpC were most strongly associated with isoniazid resistance, while mutations in kasA were not associated with resistance. Similar results were reported in another previous study by Larsen et al.31

A decrease in or total loss of catalase/peroxidase activity as a result of katG mutations are the most common genetic alterations associated with isoniazid resistance.26 So far, more than a hundred mutations in katG have been reported, with MICs ranging from 0.2 to 256 mg/L. Missense and nonsense mutations, insertions, deletions, truncation and, more rarely, full gene deletion have been observed. The most common mutation is S315T, which results in an isoniazid product that is highly deficient in forming the isoniazid-NAD adduct related to isoniazid antimicrobial activity.32

Interestingly, it has been shown that the mutation S315T in katG occurs more frequently in MDR than in isoniazid mono-resistant strains, and it has been postulated that this alteration does not produce a fitness cost, while inhA overexpression would produce a fitness cost.30,33 Also, isoniazid-resistant strains with the katG mutation S315T were found to be in clusters almost as frequently as the isoniazid-susceptible strains showing no transmissibility difference with the latter.34 It has been proposed that the combination of isoniazid resistance with retention of virulence presumably offers the basis for persistence and the possibility to gain additional resistance to other drugs. This could be partially explained by the hypothesis that katG S315T mutants are the result of a second-step mutation, occurring after a long period of inappropriate chemotherapy.30

Mutations in inhA cause not only resistance to isoniazid, but also resistance to the structurally related second-line drug ethionamide.35 The most common inhA mutation occurs in its promoter region (−15C→T) and it has been found more frequently associated with mono-resistant strains.36

In M. tuberculosis, 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.37 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.38

Described for the first time in M. smegmatis, mutations in ndh reduce the activity of NADH dehydrogenase and produce resistance to isoniazid and ethionamide.39 In M. tuberculosis, mutations in ndh have been associated with isoniazid resistance alone or in combination with other gene mutations such as inhA and katG. Although the mutations A13C and V18A in ndh were reported in two isoniazid-resistant M. tuberculosis strains, both also had mutations in katG. Moreover, mutation V18A was previously described in an isoniazid-susceptible strain.29,30,40

Arylamine N-acetyltransferases are cytosolic conjugating enzymes that transfer an acetyl group from acetyl coenzyme A to an acceptor substrate. Mammalian arylamine N-acetyltransferases are involved in drug detoxification, showing an important pharmacogenetic role.41 In humans, there are two isoenzymes encoded by polymorphic genes that lead to different rates of inactivation of drugs, including isoniazid.42 Genomic and experimental studies have identified N-acetyltransferase homologues in bacteria, including M. tuberculosis, that inactivate isoniazid.43 It would be expected that mutations resulting in the loss of N-acetyltransferase activity would improve susceptibility to isoniazid; however, no clear correlation between nat mutations and isoniazid susceptibility has been found in studies with clinical strains, although this does not permit the conclusion that arylamine N-acetyltransferase is directly related to isoniazid resistance.44 Interestingly, the arylamine N-acetyltransferase protein appears to play an important role in the synthesis of the mycobacterial cell wall in slow-growing mycobacteria and has been suggested to be a target for anti-mycobacterial therapy.45

Down-regulation of katG expression has also been recently shown to be associated with resistance to isoniazid.46 Three novel mutations in the furA-katG intergenic region were identified in 4% of 108 isoniazid-resistant strains studied; none of these was present in 51 isoniazid-susceptible strains. Reconstructing these mutations in the furA-katG intergenic region of isogenic strains decreased the expression of katG and conferred resistance to isoniazid.

Similarly, mutations in the intergenic region oxyR-ahpC can reduce the level of expression of inhA and have been associated with resistance to isoniazid. A study by Dalla Costa et al.47 found mutations in the intergenic region oxyR-ahpC in 8.9% of 224 isoniazid-resistant strains studied, confirming its less frequent involvement as a cause of resistance to isoniazid. The role of some of these genes in isoniazid resistance, however, has not been completely elucidated.

Isoniazid and tolerance
Antibiotic tolerance can be defined as the ability of bacteria to stop growing in the presence of an antibiotic, while still surviving to resume growth once the antibiotic has been removed. In M. tuberculosis, tolerance or phenotypic resistance occurs when changes in the metabolism or physiological status of the bacteria make them temporarily resistant to a certain drug. It has been shown that isoniazid induces alterations in the expression of several genes in both drug-resistant and drug-tolerant M. tuberculosis strains. Some of these genes fall into the functional class of lipid metabolism, while others belong to the class of cell wall and cell processes or transporters.48

It has also been described that M. tuberculosis iniA gene (Rv0342), part of the three-gene operon (Rv0341, Rv0342, Rv0343) induced in the presence of isoniazid, participates in the development of tolerance to both isoniazid and ethambutol. The same study also suggested that iniA functions through an MDR-pump-like mechanism, although IniA does not appear to directly transport isoniazid from the cell.49

Isoniazid also induces several other genes, including an operon cluster of five genes that code type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase. Other genes also induced are efpA, fadE23, fadE24 and ahpC, which mediate processes linked to the toxic activity of the drug and efflux mechanisms.50

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