**2. Genetic mechanisms of drug resistance in Mtb**

one-third of the world's population has latent TB, providing a source of infection. Efforts to curb TB have resulted in 2% annual decline in the global incidence of TB, except in sub-Saharan Africa [1]. However, the emergence of antibiotic-resistant strains of Mtb is a threat to TB control. If antibiotic-resistant TB is not rapidly and appropriately diagnosed, it may lead to an increase in mortality rates and the spread of resistant strains in the population.

80 Basic Biology and Applications of Actinobacteria

The first line antibiotics rifampicin (RIF) and isoniazid (INH), were developed against Mtb in the 1950s and 1960s, and are still the most effective treatments for TB. An estimated 20% of all Mtb isolates are resistant to at least one of the major antibiotics [2]. Multidrug-resistant tuberculosis (MDR-TB) is defined as TB that does not respond to at least rifampicin (RIF) and isoniazid (INH), while extensively drug-resistant TB (XDR-TB) is defined as TB resistant to INH and RIF in addition to resistance to any of the fluoroquinolones (FLQ) and to at least one of the three second-line injectable drugs: amikacin (AMK), capreomycin (CAP) or kanamycin (KAN). Antibiotic resistance arises when bacteria acquire mutations in drug target genes in an infected patient receiving antibiotics, usually as a result of mismanagement of treatment. Primary resistance arises when resistant strains are transmitted from one patient to another.

Efforts to control drug-resistant TB have relied on two beliefs: that most drug resistance is acquired *de novo* during Mtb treatment regiments, i.e., secondary resistance, and that drug-resistance mutations would have an associated fitness cost reducing the transmissibility and virulence of resistant strains [2]. Therefore, TB control has focused on increasing the effectiveness of the first line treatment and of drug-susceptibility tests only in patients who have received anti-TB medication previously. An added challenge is that diagnosing MDR- and XDR-TB requires drug-susceptibility testing with six different drugs, which can take several weeks to months [3].

Results from improved molecular diagnostic methods have challenged these two beliefs. First, an increase in the prevalence of MDR- and XDR-TB appeared to be driving the spread of TB in some areas. For example, primary transmission of MDR- and XDR-TB is the main driving force of drug-resistant TB spread in sub-Saharan Africa [2]. Second, drug-resistance mutations have variable effects on fitness and transmissibility. Mutations associated with resistance to INH, RIF, and streptomycin (SM) have even been associated with low or no fitness costs [4]. Secondary mutations that compensate for drug resistance mutations appear rapidly after the emergence of drug resistance, in the same gene or in genes involved in linked metabolic pathways, and act to restore virulence and may even increase transmissibility [2].

The WHO recommends the Xpert MTB/RIF assay for the diagnosis of rifampicin resistance, and molecular line probe assays for the detection of resistance to first and second line drugs. Many countries with a high TB burden now implement the Xpert MTB/RIF assay, which can be used as a marker for MDR-TB, as INH resistance generally precedes RIF resistance [5]. Microbiological culture is still the reference standard for diagnosis of TB and of drug-resistance. TB remains very difficult to manage in resource-poor areas. Whole-genome sequencing (WGS) and detection of variants holds great promise for characterizing all of the resistance markers (as opposed to a limited range of mutations) as well as genotyping the strain of Mtb, but relies on a more complete understanding of the relationship between genotype, specific drug resistance mutations, activity states of multiple genes and encoded proteins, and the The major antibiotics for the treatment of TB have four different mechanisms of action: (i) inhibition of RNA synthesis; (ii) inhibition of protein synthesis; (iii) inhibition of cell wall biosynthesis; and (iv) by interfering with the synthesis of cell membranes [8].

Since the early 1990s, numerous studies have described the genetic mechanisms of drug resistance in Mtb, and there is a quantity of data on the polymorphisms found in isolates resistant to specific antibiotics. Mtb is highly clonal, and as such there is little or no horizontal gene transfer, implying that antibiotic resistance is due to point mutations or deletions. Drugresistance mutations occur in genes coding for the antibiotic target itself (e.g., *gyrA*, *gyrB*, *rrs*), in genes that code for enzymes needed for activating the antibiotic (e.g., *katG*, *inhA*, *rpoB*, *pncA*, *embB*), or in promoter regions of these genes [2, 9]. To date, there are 1031 mutations in the Mtb genome believed to be associated with resistance to nine major groups of antibiotics, with different combinations of mutations causing MDR-TB [10]. Many of the mutations identified are thought to play roles other than causing resistance directly, e.g., compensatory or adaptive roles, to increase fitness, which is being reduced by the drug-resistance mutations [8].

Researchers have not fully elucidated the mechanisms by which drug resistance emerges and is preserved in Mtb populations [11]. Early mathematical models of MDR-TB suggested that DR mutations would impose fitness costs that would tend to select against the mutation in the population and thus limit the spread of TB [12]. However, current research has shown that DR mutations have a variable effect on fitness and transmissibility. INH, RIF and SM resistance have even been associated with low or no fitness costs [2, 4].

**Table 1** summarizes the literature data [7, 13–15] on the roles of the major antibiotics used to treat TB and known genes involved in drug-resistance, as well as the mechanisms thought to be responsible for drug-resistance. Drug resistant phenotype in Mtb is associated exclusively with mutations at specific positions in bacterial genomes. No events of a horizontal acquisition of drug resistance genetic determinants were reported for Mtb. Mutations in protein coding genes either alter drug target molecules or reduce activity of enzymes converting prodrug molecules into active antibiotics, e.g., *katG* gene, which encodes a catalase converting isoniazid to an active isonicotinoyl-NAD adduct [16]. Mutations affecting activities of bacterial enzymes usually reduce viability of bacteria. This phenomenon is known as the fitness cost of drug resistance. Overcoming of the fitness cost requires from bacteria an acquisition of secondary mutations to compensate the side effects of DR mutations. We hypothesized in this paper that the necessity for bacteria to compensate the side effects of DR mutations potentially opens new ways to identify molecular targets for new drugs to induce the reversion of antibiotic resistance in bacterial populations.


**Antibiotic name Mechanism of action Some polymorphisms in** 

Binds to the small 16S rRNA of the 30S subunit of bacterial ribosome, interfering with the binding of tRNA to the 30S

synthesis through modification of ribosomal structures at the 16S rRNA

by monooxygenase EthA, inhibits mycolic acid synthesis by binding the ACP reductase InhA

Trapping gyrase on DNA as ternary complexes, thereby blocking the movement of replication forks and transcription complexes

PAS is a prod-drug and thymidylate synthase A is required for conversion to active

PAS inhibits folic acid biosynthesis and uptake of iron

peptidoglycan synthesis (for cell wall) by inhibiting the enzymes d-alanine racemase (AlrA) and d-alanine:d-alanine ligase (Ddl)

**Table 1.** The main anti-TB drugs, mechanisms of actions and resistance-conferring polymorphisms.

form

subunit

Capreomycin, CAP Inhibits protein

Ethionamide, ETH ETH requires activation

Second line drugs

Aminoglycosides: kanamycin KAN, amikacin AMK

Fluoroquinolones (FLQ), e.g., ofloxacin (OFX), moxifloxacin (MOX)

Para-aminosalicylic acid,

Cycloserine, CS Interrupts

PAS

**Mtb causing resistance**

Mutation of the ribosome target binding sites genes *rrs*, but not cross-resistant with streptomycin

Clade-Specific Distribution of Antibiotic Resistance Mutations in the Population…

Mutations in the *rrs* gene encoding 16S rRNA mutations in the gene *tlyA* encoding a 2'-O-methyltransferase of 16S rRNA and 23S rRNA

70% due to mutations in

Usually multiple mutations in conserved quinolone resistance-determining region (QRDR) of *gyrA* and *gyrB*, most often at positions Ala-90 and Asp-

[Mutations at position 80 of *gyrA* cause hypersusceptibility to fluoroquinolones]

Mutations in the *thyA* gene encoding the enzyme thymidylate synthesis of the folate biosynthesis pathway, mostly Thr202Ala Also: mutations in *folC*,

*ethA* or *inhA*

94 in *gyrA*

*ribD*, *dfrA*

To be determined *alr, ddl, cycA*

**Mechanism of drug resistance**

83

Alteration of drug

Alteration of drug

Similar to INH: *inhA* promoter: overexpression of the

FLQ traps the DNAgyrase complex in which the DNA is broken. Resistant GyrA prevents chromosome

Pro-drug cannot be converted to active

enzyme

breakage.

drug

Unknown

target

http://dx.doi.org/10.5772/intechopen.75181

target


**Antibiotic name Mechanism of action Some polymorphisms in** 

polymerase by binding it. When RIF binds to the RpoB target, hydroxyl radicals are formed and this has a cytotoxic effect.

pathways, mostly arabinogalactan biosynthesis through inhibition of cell wall arabinan polymerization; RNA metabolism, transfer of mycolic acid into cell wall, phospholipid synthesis, spermidine

synthesis

acid.

Pyrazinamide, PZA Activated by enzyme

Aminoglycosides: streptomycin, SM

activated by the catalase-peroxidase enzyme KatG and then binds to InhA. Disrupts multiple pathways, mainly interferes with synthesis of mycolic

pyrazinamidase (PZase). Mechanism poorly understood. Disruption of the proton motive force required for essential membrane transport functions by POA at

acidic pH.

subunit

Binds to the small 16S rRNA of the 30S subunit of bacterial ribosome, interfering with the binding of tRNA to the 30S

Rifampicin, RIF Inhibits bacterial RNA

82 Basic Biology and Applications of Actinobacteria

Ethambutol, EMB Affects several cellular

Isoniazid, INH INH is a pro-drug,

First line drugs

**Mtb causing resistance**

Most mutations occur in cluster I of *rpoB*(β subunit of RNA pol), in the 81 bp rifampicin resistance determining region (RRDR)

Point mutations in the *embCAB* operon or the *emb* genes, affecting expression of the *embA*, *embB*, and

Mutations to *katG* gene (50–80%): Mostly S315 T. Mutations to *inhA*, or the promoter region Mutations in *ndh* gene (NADH dehydogenase), *kasA* and *ahpC* genes Mutations in *kasA* gene

Mutations in the *pncA* gene encoding PZase, most are in 561-bp region of the open reading frame or in an 82-bp region of its

Mutation of the ribosome target binding sites: 50% in the *rpsL* gene, which encodes the ribosomal protein S12, usually K43R 20% mutations to the *rrs*

Also mutations in *gidB*, which encodes 16S rRNA methyltransferase

promoter.

gene.

*embC* genes

**Mechanism of drug resistance**

Drug target is altered. In resistant bacteria, hydroxyl radicals are not formed when RIF binds to RpoB, so cells

Alteration of the drug

*katG*: mutations decrease catalase and peroxidase activity, so reduce activation

*inhA* promoter: overexpression of the

Pro-drug cannot be converted to its active

Alteration of the drug

of INH

enzyme

form

target

do not die.

target

**Table 1.** The main anti-TB drugs, mechanisms of actions and resistance-conferring polymorphisms.
