**2.4. Ethambutol**

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.

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 rifampicin-

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

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.

resistant *M. tuberculosis* isolates [22].

166 Tuberculosis - Current Issues in Diagnosis and Management

**2.3. Pyrazinamide**

administration.

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

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 new mechanisms of EMB resistance.

#### **2.5. Streptomycin**

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 kanamycin or capreomycin. Hearing and renal function should be monitored in patients getting SM.

less frequently, in *gyrB* are the primary mechanism of FQ resistance in *M. tuberculosis* [66, 67]. Analysis of QRDR alone by genotypic tests has been suggested as sufficient for rapid identification of vast majority of FQ-resistant *M. tuberculosis* strains as additional targeting

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Mutations within the QRDR of *gyr*A have been identified in clinical and laboratory-selected isolates of *M. tuberculosis*, largely clustered at codons 90, 91 and 94 [69-73], with Asp94 being relatively frequent [71, 74]. Codon 95 (Ser95Thr) contains a naturally occurring polymor‐ phism that is not related to fluoroquinolone resistance, as it occurs in both fluoroquinolonesusceptible and fluoroquinolone-resistant strains [75]. A less common involvement is codon 88 [76]. For clinical isolates, *gyr*B mutations appear to be of much rarer occurrence [72, 73]. Generally, two mutations in *gyr*A or concomitant mutations in *gyr*A plus *gyr*B are required

The aminoglycosides amikacin (AMK)/kanamycin (KAN) and the cyclic polypeptide cap‐ reomycin (CAP) are important injectable drugs in the treatment of multidrug-resistant tu‐ berculosis. Although belonging to two different antibiotic families, all exert their activity at the level of protein translation. Renal toxicity occurs from these drugs. Regular monitoring

AMK and KAN are aminoglycosides that have a high level of cross-resistance between them [78-80]. The cyclic polypeptide CAP is structurally unrelated to the aminoglycosides and thus is a potential candidate to replace AMK or KAN if resistance to either of them is sus‐ pected [81, 82]. It has been demonstrated that the risk of treatment failure and mortality in‐ crease when CAP resistance emerges among MDR-TB cases [83]. However, cross-resistance in *M. tuberculosis* between AMK/KAN and CAP has been observed in both clinical isolates

AMK/KAN and CAP primarily affect protein synthesis in *M. tuberculosis* and resistance to these drugs is associated with changes in the 16S rRNA (*rrs*) [78, 80, 81, 85, 86]. The *rrs* muta‐ tion A1401G causes high-level AMK/KAN and low-level CAP resistance. C1402T is associat‐ ed with CAP resistance and low-level KAN resistance. G1484T is linked to high-level AMK/KAN and CAP resistance [79, 80, 84, 86]. Low-level resistance to kanamycin has been correlated to mutations in the promoter region of the *eis* gene encoding aminoglycoside ace‐

Resistance to the cyclic peptide capreomycin has also been associated with mutations in *tlyA* [86]. The gene *tlyA* encodes a putative 2'-O-methyltransferase (TlyA) that has been suggested to methylate nucleotide C1402 in helix 44 of 16S rRNA and nucleotide C2158 in helix 69 of 23S rRNA in *M. tuberculosis* [81, 88]. Capreomycin binds to the 70S ribosome and inhibits mRNA–tRNA translocation [89]. It is believed that TlyA methylation enhan‐ ces the antimicrobial activity of capreomycin [81] and that disruption of *tlyA* leads to cap‐

of *gyrB* did not enhance the sensitivity significantly [67, 68].

for the development of higher levels of resistance [69, 77].

of hearing and renal function is recommended.

and laboratory-generated mutants [79, 80, 84, 85].

tyltransferase, the enhanced intracellular survival protein, Eis [87].

**3.2. Aminoglycosides (kanamycin, amikacin and capreomycin)**

Mutations associated with streptomycin resistance have been identified in the genes encoding 16S rRNA (*rrs*) [53] and ribosomal protein S12 (*rpsL*) [54-57]. Ribosomal protein S12 stabilizes the highly conserved pseudoknot structure formed by 16S rRNA [58]. Amino acid substitu‐ tions in RpsL affect the higher-order structure of 16S rRNA [51] and confer streptomycin resistance. Alterations in the 16S rRNA structure disrupt interactions between 16S rRNA and streptomycin, a process that results in resistance [59]. Mutations in *rps*L and *rrs* are the major mechanism of SM resistance [54, 56, 57], accounting for respectively about 50% and 20% of SM-resistant strains [54, 56, 57]. The most common mutation in *rps*L is a substitution in codon 43 from lysine to arginine [54, 56, 57], causing high-level resistance to SM. Mutation in codon 88 is also common [54, 56, 57]. Mutations of the *rrs* gene occur in the loops of the 16S rRNA and are clustered in two regions around nucleotides 530 and 915 [39, 54, 56, 57]. However, about 20–30% of SM-resistant strains with a low level of resistance (MIC < 32 μg/ml) do not have mutations in *rps*L or *rrs* [60], which indicates other mechanism(s) of resistance. A mutation in *gid*B, encoding a conserved 7-methylguanosine (m(7)G) methyltransferase specific for 16S rRNA, has been found to cause low-level SM resistance in 33% of resistant *M. tubercu‐ losis* isolates [61]. A subsequent study showed that while Leu16Arg change is a polymorphism not involved in SM resistance, other mutations in *gid*B appear to be involved in low-level SM resistance [62]. In addition, some low-level SM resistance seems to be caused by increased efflux as efflux pump inhibitors caused increased sensitivity to SM, although the exact mechanism remains to be identified [62].
