**2.1. Isoniazid**

First- and second-line drugs, minimum inhibitory concentrations (MICs) and mechanisms of drug resistance are presented in Table 1 [4]. Antituberculosis drugs are mainly divided into

**1.** First-line antituberculosis drugs- Isoniazid (INH), rifampicin (RIF), ethambutol (EMB),

**i.** Fluoroquinolones- Ofloxacin (OFX), levofloxacin (LEV), moxifloxacin (MOX)

**ii.** Injectable antituberculosis drugs- Kanamycin (KAN), amikacin (AMK) and

**iii.** Less-effective second-line antituberculosis drugs- Ethionamide (ETH)/Prothio‐ namide (PTH), Cycloserine (CS)/Terizidone, P-aminosalicylic acid (PAS).

*inhA* enoyl reductase

*rrs* 16S rRNA

*ahpC* alkyl hydroperoxide reductase

*gidB* 7-methylguanosine methyltransferase

*eis* aminoglycoside acetyltransferase

*tylA* rRNA methyltransferase

**Drug MIC (mg/L) Gene Role of gene product** Isoniazid 0.02–0.2 (7H9/7H10) *katG* catalase/peroxidase

Rifampicin 0.05–0.1 (7H9/7H10) *rpoB* β-subunit of RNA polymerase

Streptomycin 2–8 (7H9/7H10) *rpsL* S12 ribosomal protein

Ethambutol 1–5 (7H9/7H10) *embB* arabinosyl transferase

Fluoroquinolones 0.5–2.0 (7H9/7H10) *gyrA/gyrB* DNA gyrase Kanamycin 2–4 (7H9/7H10) *rrs* 16S rRNA

Amikacin 2–4 (7H9/7H10) *rrs* 16S rRNA Capreomycin 2-4 (7H9/7H10) *rrs* 16S rRNA

**Table 1.** First- and second-line drugs, MICs and mechanisms of drug resistance

Ethionamide 2.5–10 (7H11) *inhA* enoyl reductase

*p*-aminosalicylic acid 0.5 (LJ) *thyA* thymidylate synthase A

Pyrazinimide 16–50 (LJ) *pncA* PZase

pyrazinamide (PZA) and streptomycin (SM).

164 Tuberculosis - Current Issues in Diagnosis and Management

and ciprofloxacin (CIP).

capreomycin (CAP).

**2.** Second-line antituberculosis drugs- Sub divided into two

two parts.

Isoniazid (INH) is one of the most effective and specific antituberculosis drugs, which has been a key to treatment since its introduction in 1952 [5]. *M. tuberculosis* is highly susceptible to INH (MIC 0.02–0.2 μg/ml). INH is only active against growing tubercle bacilli, and is not active against non-replicating bacilli or under anaerobic conditions. INH enters the mycobacterial cell by passive diffusion [6]. The most significant adverse reactions associated with isoniazid administration are hepatotoxicity and neurotoxicity.

Resistance to isoniazid is a complex process. Mutations in several genes, including *katG*, *ahpC*, and *inhA*, have all been associated with isoniazid resistance. INH is a prodrug that is activated by the mycobacterial enzyme KatG [7]. INH-resistant clinical isolates of *M. tubercu‐ losis* often lose catalase and peroxidase enzyme encoded by *kat*G [8], especially in high-level resistant strains (MIC > 5 μg/ml) [9]. Low-level resistant strains (MIC < 1 μg/ml) often still possess catalase activity [9]. Although mutations in *katG* have been shown to be responsible for INH resistance [10], it is not clear whether the regulation of *katG* expression plays a role in INH resistance. The *katG* gene encodes a bifunctional catalase-peroxidase that converts INH to its active form [7]. Activated INH inhibits the synthesis of essential mycolic acids by inactivating the NADH-dependent enoyl-acyl carrier protein reductase encoded by *inhA* [11].

A study by Hazbo´n et al. [12] analysed 240 alleles and found that mutations in *katG*, *inhA* and *ahpC* were most strongly associated with isoniazid resistance. 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 [7]. Ser315Thr is the most widespread *katG* mutation in clinical isolates, but there are many mutations that result in inactivation of catalaseperoxidase, with MICs ranging from 0.2 to 256 mg/L.

Mutations in *inh*A or its promoter region are usually associated with low-level resistance (MICs = 0.2 −1 μg/ml) and are less frequent than *kat*G mutations [10, 12]. INH-resistant *M. tuberculo‐ sis* harboring *inh*A mutations could have additional mutations in *kat*G, conferring higher levels of INH resistance [13]. The most common *inhA* mutation occurs in its promoter region (-15C → T) and it has been found more frequently associated with mono-resistant strains [14].

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 [15]. 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 [4, 16].

#### **2.2. Rifampicin**

Rifampicin (RIF) was introduced in 1972 as an antituberculosis drug and has excellent steriliz‐ ing activity. Rifampicin acts by binding to the β-subunit of RNA polymerase (*rpoB*) [17], the en‐ zyme responsible for transcription and expression of mycobacterial genes, resulting in inhibition of the bacterial transcription activity and thereby killing the organism. An important characteristic of rifampicin is that it is active against actively growing and slowly metabolizing (non-growing) bacilli [18]. RIF produces relatively few adverse reactions. It may cause gastro‐ intestinal upset. Hepatotoxicity occurs less frequently than with isoniazid administration.

**2.4. Ethambutol**

new mechanisms of EMB resistance.

**2.5. Streptomycin**

Ethambutol (EMB) [dextro-2,2'-(ethylenediimino)di-1-butanol], which is an essential first-line drug in tuberculosis treatment, plays an important role in the chemotherapy of drug-resistant TB [36]. EMB is also an important antimycobacterial drug as it enhances the effect of other companion drugs including aminoglycosides, rifamycins and quinolones. The most common side effects observed with ethambutol are dizziness, blurred vision, color blindness, nausea, vomiting, stomach pain, loss of appetite, headache, rash, itching, breathlessness, swelling of the face, lips or eyes, numbness or tingling in the fingers or toes. Patients taking ethambutol

First– and Second–Line Drugs and Drug Resistance

http://dx.doi.org/10.5772/54960

167

The MICs of EMB for *M. tuberculosis* are in the range of 0.5–2 μg/ml. EMB is a bacteriostatic agent that is active for growing bacilli and has no effect on non-replicating bacilli. EMB interferes with the biosynthesis of cell wall arabinogalactan [37]. It inhibits the polymerization of cell-wall arabinan of arabinogalactan and of lipoarabinomannan, and induces the accumu‐ lation of D-arabinofuranosyl-P-decaprenol, an intermediate in arabinan biosynthesis [38, 39].

Arabinosyl transferase, encoded *by embB*, an enzyme involved in the synthesis of arabinoga‐ lactan, has been proposed as the target of EMB in *M. tuberculosis* [40] and *M. avium* [41]. In *M. tuberculosis*, *embB* is organized into an operon with *embC* and *embA* in the order *embCAB*. *embC, embB* and *embA* share over 65% amino acid identity with each other and are predicted to encode transmembrane proteins [40]. Mutations in the *embCAB* operon, in particular *embB*, and occasionally *embC*, are responsible for resistance to EMB [40]. Point mutations of the *em‐ bABC* gene commonly occurred in *embB* codon 306 [40, 42, 43], and mutations in the *embB306* codon have been proposed as a marker for EMB resistance in diagnostic tests [44]. However, point mutations in the *embB306* locus occur in only 50 to 60% of all EMB-resistant clinical isolates [42, 45-47], and *embB306* mutations can also occur in EMB-susceptible clinical isolates [46, 47]. Five different mutations were uncovered in this codon (ATG→ GTG/CTG/ATA, ATC and ATT), resulting in three different amino acid shifts (Met→ Val, Leu, or Ile) [43]. Although the association between *embB306* mutation and ethambutol resistance or broad drug resistance has been observed in several groups' studies with either clinical or laboratorial isolates [48, 49], the exact role of *embB306* mutations play in the development of ethambutol resistance and multidrug resistance in *M. tuberculosis* is not fully understood. About 35% of EMB-resistant strains (MIC <10 μg/ml) do not have *embB* mutations [39, 45], suggesting that there may be other mechanisms of EMB resistance. Further studies are necessary to identify the potential

Streptomycin (SM), an aminocyclitol glycoside antibiotic, was the first drug to be used in the treatment of TB, in 1948 [50]. SM kills actively growing tubercle bacilli with MICs of 2–4 μg/ml, but it is inactive against non-growing or intracellular bacilli [23]. The drug binds to the 16S rRNA, interferes with translation proofreading, and thereby inhibits protein synthesis [51, 52]. Ototoxicity and nephotoxicity are associated with SM administration. Vestibular dysfunc‐ tion is more common than auditory damage. Renal toxicity occurs less frequently than with

should have their visual acuity and color vision checked at least monthly.

Rifampicin MICs ranging from 0.05 to 1 μg/ml on solid or liquid media, but the MIC is higher in egg media (MIC = 2.5–10 μg/ml). Strains with MICs < 1 μg/ml in liquid or agar medium or MICs < 40 μg/ml in Lowenstein-Jensen (LJ) medium are considered RIF-susceptible. The great majority of *M. tuberculosis* clinical isolates resistant to rifampicin show mutations in the gene *rpoB* that encodes the β-subunit of RNA polymerase. This results in conformational changes that determine a low affinity for the drug and consequently the development of resistance [19]. Mutations in a 'hot-spot' region of 81 bp of *rpoB* have been found in about 96% of rifampicinresistant *M. tuberculosis*isolates. This region, spanning codons 507–533 (numbering according to the *Escherichia coli rpoB* sequence), is also known as the rifampicin resistance-determining region (RRDR) [17]. Mutations in codons 531, 526 and 516 (Ser531Leu, His526Tyr, and Asp516Val) are the most frequently reported mutations in most of the studies [20, 21]. Some studies have also reported mutations outside of the hot-spot region of *rpoB* in rifampicinresistant *M. tuberculosis* isolates [22].

### **2.3. Pyrazinamide**

Pyrazinamide (PZA) is an important first-line antituberculosis (anti-TB) drug that is used in short-course chemotherapy and is one of the cornerstone drugs in the treatment of MDR-TB [23]. One key characteristic of pyrazinamide is its ability to inhibit semidormant bacilli residing in acidic environments [23]. Pyrazinamide is a structural analogue of nicotinamide and is a pro-drug that needs to be converted into its active form, pyrazinoic acid, by the enzyme pyrazinamidase/nicotinamidase (PZase) [24]. PZA is only active against *M. tuberculosis* at acid pH (e.g., 5.5) [25]. Even at acid pH (5.5), PZA activity is quite poor, with MICs in the range of 6.25–50 μg/ml [26]. Hypersensitivity reactions and gastrointestinal upset may occur with PZA administration.

PZase is encoded in *M. tuberculosis* by the gene *pncA* [27]. Mutations in the *pncA* gene may cause a reduction in PZase activity which may be the major mechanism of PZA resistance in MTB [28, 29]. The mutations of the *pncA* gene in PZA-resistant MTB isolates has been well characterized, however the correlation varies between different geographical areas including missense mutations, one or more base insertions or deletions, and complete deletion [28-32]. Despite the highly diverse and scattered distribution of *pnc*A mutations, there is some degree of clustering of mutations within different regions of the *pncA* gene such as at amino acid residues 3–17, 61–85 and 132–142 has been reported [33, 34]. The highly diverse mutation profile in the *pncA* gene observed in PZA-resistant strains is unique among drug-resistance genes in *M. tuberculosis* [28]. While the reason behind this diversity is still unclear, it is thought that this could be due to adaptive mutagenesis or due to deficiency in DNA mismatch repair mechanisms [23]. Most PZA-resistant *M. tuberculosis* strains (72–97%) have mutations in *pnc*A; [28, 29, 34, 35] however; some resistant strains do not have *pnc*A mutations.
