Genes associated with resistance to various anti‐TB drugs.
Tuberculosis (TB) remains to be a serious health problem worldwide. There is an increased transmission of Mycobacterium tuberculosis strains with drug resistance, hence complicating TB control. The deciphering of the M. tuberculosis genome, together with the implementation of new molecular biology tools, has allowed the identification of changes in nucleic acid sequences with a functional impact. These mutations have become important in the design of early‐diagnostic kits to identify the resistance profile of M. tuberculosis. Since the conventional methods to determine the identity of M. tuberculosis strains based in cultures are laborious, time‐consuming and performed by specialized technicians, the result is generated until 4 months after receiving the samples. During this time, patients with TB are not adequately treated, and resistant strains may be transmitted to the rest of the population. In this chapter, we describe the most relevant mutations in genes associated with drug resistance in M. tuberculosis, the analysis of gene expression to identify new markers of drug resistance strains, and the development of new antituberculosis drugs against drug‐resistant strains.
- Mycobacterium tuberculosis
- drug resistance
- gene expression
- antituberculosis drugs
Tuberculosis (TB) has remained a serious health problem since Mycobacterium tuberculosis, the main agent of this disease, infects about one third population. In 2015, 10.4 million cases of TB were estimated and, although only a small percentage (5–10%) develops the illness, its control has complicated due to the emergence of drug resistance strains . Tuberculosis regiment treatment includes the first line drugs rifampicine, isoniazide, ethambutol and pyrazinamide and strains that develop resistance to the two more effective antituberculosis drugs, isoniazide and rifampicine, named multidrug resistance strains [2, 3]. The resistance phenomenon in M. tuberculosis has been highly related to mutations in specific genes , and this association has been the base for the implementation of rapid diagnostics kits  but unfortunately mutations do not explain completely the resistance in all cases [6, 7], suggesting that other mechanisms could be involved. New approaches to search new markers and mechanisms of resistance have been performed. One of these is the evaluation of changes in gene expression . Together with the diagnostics of TB, the implementation of new schemes of treatments is necessary to restrict the transmission. The development of new drugs against drug resistance M. tuberculosis has resulted promissory to control TB .
2. Mutations that confer resistance in Mycobacterium tuberculosis
Although the rate of resistance to first and second line drugs in M. tuberculosis varies among countries, the resistance phenomenon has complicated the tuberculosis control worldwide. There two types of observed resistance in M. tuberculosis, one is the genetic resistance where mutations in genomic regions, on target genes, confer the capacity to avoid the drug effect; the second is the phenotypic resistance, where epigenomic modifications, including alteration of protein structures, generate resistance to drugs without mutation on DNA. Although the current knowledge of the molecular genetic basis of resistance to antituberculosis drugs has advanced rapidly the last years , there are unknown mechanisms in how bacilli is able to resist to drugs. Identification of clinical isolates with resistance to antituberculosis drugs would facilitate the timely and accurate diagnosis to initiate an appropriate treatment.
Many works have revealed, using microbiological and clinical data, mutational events in clinical isolates from patients with tuberculosis. Multidrug resistance appears to result from the sequential acquisition of mutations. Possible reasons for the acquisition of mutations include inadequate prescription and delivery of chemotherapy, poor compliance, or an insufficient number of active drugs in the treatment regimen . Mutations or combinations of mutations have been found in strains displaying single or multidrug resistance. Here, we summarized the most common mutation found in clinical isolates that confer resistance to the first and second line antituberculosis drugs (Table 1).
|Drugs||MIC (µg/mL)||Drug mode of action||Gene||Target enzyme||Frequency of mutations (%) associated with resistance|
|Isoniazide||0.02–0.2||Inhibits mycolic acid synthesis and other multiple effects||katG||Catalase peroxidase||30–60|
|InhA||Fatty acid enoyl acyl carrier protein reductase A||70–80|
|ahpC||Alkyl hydroperoxidase reductase||Not known|
|kasA||β‐ketoacyl‐ACP synthase||Not known|
|Rifampicin||0.05–1||Inhibits RNA synthesis||rpoB||β subunit of RNA polymerase||95|
|Streptomycin||2–8||Inhibits protein synthesis||rpsL||Ribosomal protein S12||65–67|
|Ethambutol||1–5||Inhibits arabinogalactan synthesis||embCAB||Arabinosyl transferase||70–90|
|Pyrazinamide||16–100||Disrupts plasmamembrane and energy metabolism (inhibits pantothenate and CoA synthesis)||pncA||Pyrazinamidase||>70|
|IS6110 insertion||Not known|
|Fluoroquinolones||0.5–2.5||Introduces negative supercoils in DNA molecules||gyrA||DNA gyrase||42‐85|
|Kanamycin/Amikacin||2–4||Inhibits protein synthesis||rrs||16S rRNA||>60|
|Capreomycin/Viomycin||2–4||Inhibits protein synthesis||rrs||16S rRNA||40‐100|
|Ethionamide||2.5–10||Disrupts cell wall biosynthesis by inhibition of mycolic acid synthesis||InhA||Fatty acid enoyl acyl carrier protein reductase A||>60|
|ethR||Transcriptional repressor||Not known|
Due to its properties as a bactericidal drug, isoniazid has been widely used as the first line drug in the treatment against M. tuberculosis complex members. Mutations on katG and mabA‐InhA genes have repeatedly been associated with isoniazid resistance  (Table 1). In the case of katG, the most common of mutation is S315, which is present in 50–95% of isoniazid resistant clinical isolates . The occurrence of mutations is also observed in the promoter region of mabA/inhA. Mutations in the inhA promoter can also confer cross‐resistance to ethionamide . There are other genes as ahpC, kasA, and ndh, encoding for alkyl hydroperoxidase reductase, β‐ketoacyl‐ACP synthase, and NADH dehydrogenase, respectively, which have also been associated with isoniazid resistance.
Rifampicin is highly bactericidal for M.tuberculosis, where this drug is able to bind to the β subunit of RNA polymerase, and this event induces hydroxyl radical formation in susceptible mycobacteria . Resistance to rifampicin is acquired by mutations in a region of the 81‐bp region of the rpoB gene, encoding the β subunit of RNA polymerase, and these mutations have been found in ∼96% of rifampicin‐resistant clinical isolates. The most frequent mutations are located in positions 516, 526, and 531. Also, there is evidence that mutations in the rpoB gene generate cross‐resistance to rifamycins . It is important to mention that not all mutations in rpoB are associated with rifampicin resistance .
It has been considered as a second line antituberculosis drug, which binds to the 30S subunit of the ribosome and blocks protein synthesis. The resistance is provoked by mutations in the rpsL gene, which encoded the S12 protein, and the rrs gene, which encoded the 16S rRNA. The mutations in both genes are the main mechanism of streptomycin resistance, and it has been found in 65–67% of resistant clinical isolates . There is another gene, gidB, involved in streptomycin resistance. This gene encoded a 7‐methylguanosine (m(7)G) methyltransferase, and mutations have been found in 33% of clinical isolates resistant to streptomycin.
Ethambutol has a target, the inhibition of the enzyme arabinosyl transferase, which is involved in the biosynthesis of cell wall arabinogalactan. The enzyme is encoded by the embB gene, harboured in the embCAB operon, and mutation in this gene is related to ethambutol resistance. The most frequent mutation found in the embB gene is located in codon 306. More than 68% of resistant clinical isolates have mutations in the embB gene .
This pro‐drug is converted to its active form, pyrazinoic acid, and it only kills non‐growing persistent bacteria. The mutations of the pncA gene are scattered along this genomic region, and it is the main mechanism of pyrazinamide resistance. The majority of pyrazinamide‐resistant clinical isolates (72–99%) have showed mutations on the pncA gene sequence. Due to a high correlation between mutations and pyrazinamide resistance, it has been suggested that the use of mutations to predict the resistance profile to pyrazinamide; however, there are silent mutations that do not confer resistance .
Fluoroquinolones are able to inhibit the activity of DNA gyrase. When the activity of DNA gyrase is affected, the chromosomal DNA acquires a supercoiled conformation . Then, mutations on gyrA, encoding DNA gyrase, are strongly associated with fluoroquinolone resistance. There are many reports where mutations located in the gyrA region are present in 42–85% of clinical isolates resistant to fluoroquinolones (Louw et al., 2009). Also, mutations on the gyrB gene have also been associated with fluoroquinolone resistance, where 3% of clinical isolates harbor the mutation in this gene. The most common mutations of the gyrA gene are located in codons 90, 91 and 94 .
Amikacin and kanamycin are considered as second‐line antituberculosis drugs. It has been identified that the rrs gene as the target of action of these drugs; however, the molecular mechanisms that confer resistance are focused to inhibition of protein synthesis . About 60% of the clinical isolates resistant to amikacin/kanamycin have mutations on the rrs gene . The most common mutations are located at the position 1400 of the rrs gene, which causes high‐level resistance these drugs.
This prodrug requires to be activated by the mono‐oxygenase EtaA/EthA. It has been described as the only bactericidal agent against M. tuberculosis. Ethionamide inhibits mycolic acid synthesis. Mutations in inhA also confer resistance to ethionamide. Frequency of mutations on etaA/ethA, ethR, and inhA genes in clinical isolates resistant to ethionamide reaches 60% .
Mutations described in M. tuberculosis have led to the implementation of rapid molecular diagnostic kits with the aim to diagnose TB and detect drug resistance in a shorter period compared to drug susceptibility testing based on the culture of the microorganism .
Within the rapid methods approved by the WHO, there are real‐time PCR‐based assays, as Xpert MTB/RIF, the line probe assays Genotype MTBDRplus and Genotype MTBDRsl. The XpertMTB/RIF tests allow M. tuberculosis detection as well as resistance to rifampicine. A multicenter study in which 6648 patients were evaluated, the Xpert MTB/RIF test allowed the detection of 90.3% of the TB confirmed cases based on culture, as well as 67.1% of the TB cases diagnosed by microscopy. For detection of rifampicine resistance, a sensitivity of 94.4% and specificity of 98.3% were reported, and an indeterminate rate of 2.4%, which was lower than that of culture diagnose with 4.6% . On the other hand, the Genotype MTBDRplus allows detection of resistance for the first line drugs, while Genotype MTBDRsl detects resistance to the second line drugs. A meta‐analysis of Genotype MTBDRplus reported a pooled sensitivity of 0.91, 0.96, and 0.91 and a pooled specificity of 0.99, 0.98 and 0.99 for the detection of isoniazide‐, rifampicine‐, and multidrug‐resistance, respectively. Both, sensitivity and specificity settings have 95% confidence intervals . Finally, in a multicenter study realized in 2012, the accuracy of the Genotype MTBDRsl was evaluated in 200 M. Tuberculosis isolates; in this study, the sensitivity reported was between 77.3 and 92.3% for the detection of resistance to fluoroquinolones, ethambutol, amikacin, and capreomycin while for kanamycin was 42.7 and 22.6% for XDR detection; the specificity was 82% for all drugs .
3. Searching for new markers to identify drug resistance of Mycobacterium tuberculosis
In the understanding and linking‐up of genetic associations with the drug resistance phenotype in M. tuberculosis, mutations in specific genes have been the most common association as previously described; nevertheless, not all resistant M. tuberculosis strains have the related mutations previously reported suggesting that other mechanisms could be involved in this phenomenon [6, 23, 24]. For this purpose, the expression level of some genes has been studied. One of them is efflux pump genes, these are important elements that play a role in the extrusion of drugs out of the cells conferring M. tuberculosis resistance to drugs . The efflux pumps have been classified in super families: ATP‐binding cassette (ABC), major facilitator super‐family (MFS), resistance nodulation division (RND), small multidrug resistance (SMR), and multidrug and toxic‐compound extrusion (MATE) . In M. tuberculosis, the efflux pumps consist of (a) 12 mycobacterial large membrane proteins (MmpL) belonging to RND‐type transporters , (b) 37 ABC transporters (26 complete and 11 incomplete) from which 21 are putative exporters which include antibiotic transporters that represent the 2.5% of the genome [27, 28] and (c) 16 putative MFS drug efflux pumps . Some findings have reported efflux pump genes to be overexpressed in drug resistance M. tuberculosis strains (Table 2) [25, 30, 31, 32]. The importance of efflux pumps involved in drug resistance has led to suggest the analysis of the implementations of a combined therapy of antituberculosis drugs together with efflux pump inhibitors .
|Locus||Symbola||Gene namea||Drug‐resistant phenotype||References|
|Rv2688c||Antibiotic‐transport ATP‐binding protein ABC transporter||XDR|||
|Rv1634||Drug efflux membrane protein||XDR|||
|Rv2936||drrA||Daunorubicin‐dim‐transport ATP‐binding protein ABC transporter drrA||XDR|||
|Rv2937||drrB||Daunorubicin‐dim‐transport membrane protein ABC transporter drrB||XDR|||
|Rv0820||phoT||Phosphate‐transport ATP‐binding protein ABC transporter phoT||XDR|||
|Rv2136c||uppP||Conserved membrane protein||MDR|||
|Rv2846c||efpA||Membrane efflux protein efpA||MDR|||
|Rv0849||Conserved membrane transport protein||MDR|||
|Rv1410||Aminoglycosides/tetracycline‐transport membrane protein||MDR|||
|Rv1634||Drug efflux membrane protein||MDR|||
|Rv2994||Conserved membrane protein||MDR|||
|Rv2333c||stp||Involved in transport of drug across the membrane (export)||MDR|||
|Rv2459||Conserved membrane transport protein||MDR|||
Furthermore, studies in drug resistance strains have reported other genes as differentially expressed between sensitive and drug‐resistant strains. Functional categories of these genes are among others, stress response and translation (Table 3). On the other hand, expression of intergenic regions (IGs) has also been associated with a drug resistance phenomenon in M. tuberculosis [8, 25, 31], suggesting that an additional analysis is necessary to evaluate and confirm the contribution of these regions in drug resistance.
|Locus||Symbola||Gene namea||Expression level modification||Drug‐resistant phenotype||References|
|Rv1181||pks4||Polyketide beta‐ketoacyl synthase pks4||I||XDR|||
|Rv1182||ppA3||Polyketide synthase associated protein papA3||I||XDR|||
|Rv1184c||Hypothetical exported protein||I||XDR|||
|Rv0826||Conserved hypothetical protein||I||XDR|||
|Rv1483||fabG1||3‐oxoacyl‐[acyl‐carrier protein] reductase fabG1||I||XDR|||
|Rv1592c||Conserved hypothetical protein||I||XDR|||
|Rv1623c||cydA||Membrane cytochrome D ubiquinol oxidase subunit I cydA||I||XDR|||
|Rv3269||Conserved hypothetical protein||I||XDR|||
|Rv0287||esxG||Esat‐6 like protein esxG||I||MDR|||
|Rv0288||esxH||Low molecular weight protein antigen 7 esxH||I||MDR|||
|Rv1037c||esxI||Esat‐6 like protein esxI||I||MDR|||
|Rv1642||rpmI||50S ribosomal protein L35 rpmI||I||MDR|||
|Rv1630||rpsA||30S ribosomal protein S1 rpsA||I||MDR|||
|Rv3418c||groES||10 kda chaperonin groES||R||MDR|||
|Rv1161||narG||Respiratory nitrate reductase alpha chain narG||R||MDR|||
|Rv1819c||Drugs‐transport transmembrane ATP‐binding protein ABC transporter||R||MDR|||
With the aim to demonstrate the resistance association between the level of expression of some genes and drug resistance, assays using recombinant strains of M. tuberculosis as well as other Mycobacterium strains treated with different drugs and/or overexpressing genes of interest have been analyzed [25, 33, 34, 35]. The new findings related to differences of gene basal expression between susceptible and resistant M. tuberculosis strains can contribute to identify newly genetic drug‐resistant markers that could contribute in the early diagnosis of drug‐resistant tuberculosis, which could be applied in the establishment of a more efficient drug therapy [8, 30].
4. Development of new drugs against Mycobacterium tuberculosis drug resistance strains
Even though tuberculosis antibiotic treatment therapy is described, drug resistance in M. tuberculosis complicates the TB control. In 2015, 480,000 MDR tuberculosis cases were estimated and, in addition, 100,000 more cases were added which had resistance to rifampicin , these cases are more likely to develop multi‐drug resistance. Drug‐resistant TB has led to the implementation of new therapeutic regimens involving second line drugs, once drug susceptibility testing results are available .
Drug therapy for a patient infected with a susceptible M. tuberculosis strain lasts 6 months with diverse combinations of the first‐line drugs rifampicine, isoniazid, ethambuthol, and pyrazinamide, while treatment therapy for a patient with DR tuberculosis can last up to 20 months and include a fluoroquinolone, an injectable aminoglycoside plus an oral bacteriostatic second line drug and a first line drug (for details consult D’Ambrosio et al. ).
Because the problem of resistant tuberculosis is increasing, searching for new drugs continues with the aim of improving the therapeutic regimens currently used, shorten treatment duration in addition to find more effective drugs for latent TB and drug‐resistant strains. The development of new antituberculosis drugs implicates the following stages: basic research, discovery of new antituberculosis compounds or drugs, preclinical and clinical studies conformed by phases I, II, and III to finally get to the technology transfer; all these processes entail long periods of time . In this continuous search for better antituberculosis drugs, many natural, semi‐synthetic, and synthetic compounds have been evaluated in vitro and in vivo. We will mention some new drugs that are based on the structure of first line drugs, among which some analogues have been described with activity against sensitive and drug resistant M. tuberculosis strains. Thereby based on ethambutol, some of the novel described compounds comprise SQ109 and analogues based on carbamate prodrugs , S2824 and analogues with a homopiperazine ring , 1,2 diamines , ferrocenyl compounds  and dihydrosphingosine‐ethambutol analogues . Within pyrazinamide analogues, it has been described POEs (pyrazinoic acid esters) and 5‐Cl‐substituted pyrazinoic acid derivatives . However, it is necessary to consider the adverse effects of these compounds. For isoniazide‐based compounds, there has been reported aromatic and heterocyclic aldehydes containing electron‐withdrawing or donating groups , and rifampicin has been described within the rifamycins as well as among others as rifabutin, rifapnetine, rifalazil, and rifametane .
As general conclusion, although mutations are commonly associated with drug resistance in M. tuberculosis, other studies are necessary to discover genetic markers that support the early diagnostic of drug resistance in strains that enable the establishment of optimized therapeutic schemes limiting their transmission.
This work was partially supported by Instituto Mexicano del Seguro Social (FIS/IMSS/PROT/G15/1457).