MDR/XDR TB Antibiotic Resistance Genes

Antibiotic Resistance Genes in MDR/XDR Tuberculosis (TB)

Tuberculosis (TB) is caused by various species within the Mycobacterium tuberculosis complex, representing a group of infectious agents. As one of the most fatal and widespread infectious diseases, TB is responsible for over 1.5 million deaths annually, with approximately one-third of the world's population estimated to be latently infected according to the WHO. Of those latently infected, around 10% are expected to develop active clinical disease. Globally, pulmonary tuberculosis caused by M. tuberculosis remains the most prevalent form of TB.

Antibiotic Resistance Genes in Tuberculosis (TB)

Tuberculosis (TB) is treated with a specific regimen that includes first-line drugs such as Rifampicin, Isoniazid, Pyrazinamide, Ethambutol, and Streptomycin, either in combination or as monotherapy. MDR-TB (Multidrug-resistant TB) is diagnosed when the pathogen is resistant to at least Rifampicin and Isoniazid, or all first-line antibiotics. In such cases, a second-line treatment is used, consisting of drugs like fluoroquinolones (preferably moxifloxacin, with alternatives like ofloxacin and levofloxacin) and injectable aminoglycosides such as amikacin, kanamycin, and capreomycin. Additional second-line anti-TB drugs include ethionamide, cycloserine, para-aminosalicylic acid, clofazimine, linezolid, and delamanid.

When the MDR-TB pathogen shows resistance to at least one fluoroquinolone and one injectable aminoglycoside, it is termed XDR-TB (Extensively drug-resistant TB). For treating XDR-TB, a combination of pretomanid, bedaquiline, and linezolid is recommended.

However, the effectiveness of XDR-TB treatment options is being challenged as many clinical isolates of the Mycobacterium tuberculosis complex have developed resistance to them. The main mechanisms of resistance observed in TB pathogens include modification of drug target sites, overexpression of efflux pumps, and enzymatic modification of the drugs, rendering them ineffective.

Various genes are responsible for the development of these mechanisms of antimicrobial resistance (AMR). This note highlights some important genes involved in drug resistance in tubercle bacilli.

Table of Contents

• Mutated atpE Gene
• Mechanism of Conferring Resistance ofMutated atpE Gene
• Detection Method of Mutated atpE Gene
• Mutated Rv0678 Gene
• Mechanism of Conferring Resistance ofMutated Rv0678 Gene
• Detection Method of Mutated Rv0678 Gene
• Mutated rpoB Gene
• Mechanism of Conferring Resistance ofMutated rpoB Gene
• Detection Method of Mutated rpoB Gene
• Mutated katG Gene
• Mechanism of Conferring Resistance ofMutated katG Gene
• Detection Method of Mutated katG Gene
• Mutated ethA Gene
• Mechanism of Conferring Resistance ofMutated ethA Gene
• Detection Method of Mutated ethA Gene
• Other Genes Responsible for MDR/XDR-TB

 

Mutated atpE Gene

atpE gene is a Mycobacterium gene that encodes for the synthesis of subunit ‘C’ of the ATP synthase protein. It is present in the H37Rv genome of Mycobacterium spp. and is found in all the members of the Mycobacterium tuberculosis complex.

The atpE gene product, subunit C of ATP synthase, is a target for a novel antimycobacterial antibiotic: bedaquiline. Bedaquiline binds to the c-subunit and inhibits the action of the ATP synthase enzyme resulting in the deficiency of energy molecules, the ATP molecules. Scarcity of the ATP molecules will result in energy deficiency for biochemical processes and eventually cause the death of the bacterium.  

Mechanism of Conferring Resistance of Mutated atpE Gene

Mutation in the atpE gene results in the synthesis of modified subunit C of the ATP synthase enzyme. Bedaquiline has a very low affinity with the modified form of subunit C and hence can’t bind with them and can’t disturb the process of ATP synthesis. This results in an increase in the MIC of bedaquiline and higher survival of the infecting Mycobacterium.

  

Detection Method of Mutated atpE Gene

Complete genome analysis and PCR are the available molecular techniques that can be used to detect the mutated atpE gene. The atpE gene is first detected and amplified using PCR. After amplification, it is subjected to gene sequencing to draw its complete nucleotide sequence and the nucleotide sequence is cross-referred with the reference M. tuberculosis H37Rv genome sequence (NCBI Reference Sequence: NC_000962.3) to detect the mutation.

Primers that can be used for the amplification of the atpE gene are:

Primers	References
F: 5’- TGT ACT TCA GCC AAG CGA TGG -3’ R: 5’- CCG TTG GGA ATG AGG AAG TTG -3’	Singh BK, Soneja M, Sharma R, Chaubey J, Kodan P, Jorwal P, Nischa N, Chandra S, Ramachandran R, Wig N. Mutation in atpE and Rv0678 genes associated with bedaquiline resistance among drug-resistant tuberculosis patients: A pilot study from a high-burden setting in Northern India. Int J Mycobacteriol [serial online] 2020 [[cited 2023 Jan 3];9:212-5.

 

Mutated Rv0678 Gene

Rv0678 gene is a Mycobacterial gene responsible for the expression of the MmpS5 – MmpL5 efflux pumps in the Mycobacterium tuberculosis complex.

These genes are naturally found in different bacteria and archaea including the Mycobacterium species; however, a mutation in the Rv0678 gene results in overexpression of the efflux pumps like Mycobacterial membrane protein Large (mmpL) and Mycobacterial membrane protein Small (mmpS).

Mechanism of Conferring Resistance of Mutated Rv0678 Gene

Mutations in the Rv0678 gene result in the overexpression of the mmpL and mmpS type efflux pumps, particularly MmpL5 and MmpS5 are the efflux pump of concern. These efflux pumps provide efflux-associated resistance to Bedaquiline. Overexpression of the MmpL5/MmpS5 efflux pumps is associated with the increase of the MIC value of the Bedaquiline antibiotic by 2- to 16- folds. 

Detection Method of Mutated Rv0678 Gene

There is no unique sequence to read and identify the mutation in the Rv0678 gene. The only available and used method to know about the mutation is to do the complete nucleotide sequencing of the Rv0678 gene from the suspected isolate and map it with the nucleotide sequence of the Rv0678 gene from the reference M. tuberculosis H37Rv genome sequence.

The primer that can be used to amplify the Rv0678 gene is tabulated below. 

Primers	References
F: 5’-  AGC CGG AAA CTT CGT ACT CCAC -3’ R: 5’-  GCT GGA CAA CAC GGT CAC CT -3’	Singh BK, Soneja M, Sharma R, Chaubey J, Kodan P, Jorwal P, Nischa N, Chandra S, Ramachandran R, Wig N. Mutation in atpE and Rv0678 genes associated with bedaquiline resistance among drug-resistant tuberculosis patients: A pilot study from a high-burden setting in Northern India. Int J Mycobaceriol [serial online] 2020 [[cited 2023 Jan 3];9:212-5.
F: 5’-  CTT CGG AAC CAA AGA AAG TG -3’ R: 5’-  CCA ACC GAG TCA AAC TCC TG -3’	Crystal Structure of the Transcriptional Regulator Rv0678 of Mycobacterium tuberculosis. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 23, pp. 16526 –16540, June 6, 2014. DOI:https://doi.org/10.1074/jbc.M113.538959

 

Mutated rpoB Gene

The rpoB gene is a bacterial gene encoding for the -subunit of bacterial RNA polymerase enzyme. It has been extensively used in the identification and phylogenetic analysis of bacteria.  In Mycobacterium tuberculosis also, the rpoB gene encodes for the -subunit of DNA-dependent RNA polymerase. This -subunit is the target site of rifampicin antibiotic i.e. rifampicin binds at the -subunit of RNA polymerase and physically blocks amino-acid elongation step and prevents bacteria from synthesizing the required proteins.

In about 70 – 90% of rifampicin-resistant M. tuberculosis, mutations are reported in the rpoB gene, mainly in three codons: 531, 526, and 516 codons.  

Mechanism of Conferring Resistance of Mutated rpoB Gene

The presence of mutations in the rpoB gene leads to the production of a modified β-subunit of DNA-dependent RNA polymerase (β-subunit). This modified β-subunit prevents rifampicin from binding effectively, rendering the antibiotic unable to halt the protein elongation step in the bacterium. As a result, the minimum inhibitory concentration (MIC) value of rifampicin increases, and the likelihood of treatment failure rises, as the bacteria become resistant to its effects.

Detection Method of Mutated rpoB Gene

The initial step in this process involves the utilization of PCR to detect and amplify the rpoB gene from the isolates. After amplification, gene sequencing is conducted to determine the complete nucleotide sequence of the rpoB gene. This obtained sequence is subsequently compared with the reference nucleotide sequence of the rpoB gene from M. tuberculosis H37Rv.

The following primers can be employed for the PCR amplification:

By using these primers in PCR, the specific target region (rpoB gene) is amplified from the isolates, enabling further gene sequencing and comparison with the reference sequence.

Primers	References
F: 5′- GAG GCG ATC ACA CCG  CAG ACGT-3′  R: 5′- GAT GTT GGG CCC CTC AGG GGTT-3′	(2014). Mutation pattern in rifampicin resistance determining region of rpoB gene in multidrug-resistant Mycobacterium tuberculosis isolates from Pakistan. International Journal of Mycobacteriology, 3(3), 173-177. https://doi.org/10.1016/j.ijmyco.2014.06.004
F: 5′- GTC GCC GCG ATC AAG GA -3′  R: 5′- TGA CCC GCG CGT ACA C -3′	Gupta, Anamika & Prakash, Pradyot & Singh, Surya & Anupurba, Shampa. (2013). Rapid Genotypic Detection of rpoB and katG Gene Mutations in Mycobacterium tuberculosis Clinical Isolates from Northern India as Determined by MAS-PCR. Journal of clinical laboratory analysis. 27. 31-7. 10.1002/jcla.21558.
F: 5′- CGA ATA TCT GGT CCG CTT G -3′  R: 5′- GGT CAG GTA CAC GAT CTC -3′	Motavaf, B., Keshavarz, N., Ghorbanian, F., Firuzabadi, S., Hosseini, F., & Bostanabad, S. Z. (2021). Detection of genomic mutations in katG and rpoB genes among multidrug-resistant Mycobacterium tuberculosis isolates from Tehran, Iran. New Microbes and New Infections, 41. https://doi.org/10.1016/j.nmni.2021.100879

 

Mutated katG Gene

The katG gene, also known as the Rv1908c gene, is responsible for encoding the catalase-peroxidase enzyme (KatG) in mycobacteria. KatG functions as a catalase enzyme, facilitating the breakdown of hydrogen peroxide into harmless oxygen and water molecules, protecting the bacteria from oxidative damage.

In addition to this essential cellular role, the KatG enzyme also plays a crucial role in activating a pro-drug used against tuberculosis, called isoniazid. The katG gene encodes for a specific type of catalase enzyme, which catalyzes the conversion of isoniazid into an isonicotinic acyl radical. This radical combines spontaneously with NADH, forming an Isonicotinic acyl-NADH complex. The complex then binds to InhA, an enzyme known as enoyl-acyl carrier protein reductase. This interaction disrupts the formation of mycolic acid, which is essential for the bacterial cell wall, leading to cell lysis and the bacterium's destruction.

Mutations in the katG gene have been strongly associated with isoniazid-resistant Mycobacterium tuberculosis strains. The S315T variant of the KatG catalase-peroxidase enzyme has been particularly observed in about 94% of isoniazid-resistant clinical isolates of mycobacteria.

Mechanism of Conferring Resistance of Mutated katG Gene

Mutations in the katG gene result in the production of modified KatG enzymes, particularly the S315T variant. These altered KatG enzymes lose their ability to catalyze the formation of the Isonicotinic acyl-NADH complex. Consequently, the prodrug isoniazid remains in its inactive prodrug stage.

With the modified KatG enzymes unable to activate isoniazid, the drug becomes ineffective in preventing the formation of mycolic acid and fails to induce the degradation of the mycobacterial cell wall. As a result, the bacterium's cell wall remains intact, and cell lysis associated with its degradation does not occur.

Detection Method of Mutated katG Gene

To identify mutations in the katG gene, the recommended approach is to obtain the complete nucleotide sequence of the gene and then compare it with the katG gene sequence of M. tuberculosis H37Rv. This method allows for the detection of any variations or mutations in the gene.

For PCR amplification of the katG gene, the following primers can be utilized:

Using these primers in PCR enables the specific amplification of the katG gene, facilitating subsequent gene sequencing and mutation analysis.

Primers	References
F: 5′- GCG ACG CGT GAT CCG CTC ATA G-3′ R: 3′-TCG GCG GTC ACA CTT T-5′	Samad, Gusai H.Abdel, Solima M. A. Sabeel, Walaa A. Abuelgassim, Abeer E. Abdelltif, Wisam M. Osman, Mona A. Haroun, Somaya M. Soliman, Sami. A. B. Salam, Hamid. A. Hamdan, and Mohamed A. Hassan. “Molecular Characteristic and Insilico Analysis of KatG Gene in Isoniazid Resistance Mycobacterium Tuberculosis Isolate from Sudan.”  American Journal of Microbiological Research 2, no. 6 (2014): 227-233.
F: 5′- GCA GAT GGG GCT GAT CTA CG -3′ R: 5′- AAC GGG TCC GGG ATG GTG -3′	Gupta, Anamika & Prakash, Pradyot & Singh, Surya & Anupurba, Shampa. (2013). Rapid Genotypic Detection of rpoB and katG Gene Mutations in Mycobacterium tuberculosis Clinical Isolates from Northern India as Determined by MAS-PCR. Journal of clinical laboratory analysis. 27. 31-7. 10.1002/jcla.21558.
F: 5′- CTC GGC GAT GAG CGT TAC -3′ R: 5′- TCC TTG GCG GTG TAT TGC -3′	Kardan Yamchi J., Haeili M., Gizaw Feyisa S., Kazemian H., Hashemi Shahraki A., Zahednamazi F. Evaluation of efflux pump gene expression among drug susceptible and drug resistant strains of Mycobacterium tuberculosis from Iran.  Inf Genetic Evol. 2015;36:23–26

 

Mutated ethA Gene

The ethA gene in mycobacteria encodes the Baeyer Villiger monooxygenase EthA enzyme. This enzyme plays a critical role in activating the pro-drug ethionamide into an active radical. Once activated, the radical forms a toxic complex with NADH. This complex then inhibits the enoyl-acyl carrier protein reductase InhA, leading to the prevention of mycolic acid formation in the mycobacterial cell wall. Consequently, disruption of the mycolic acid occurs, inducing cell lysis.

In the majority of ethionamide-resistant clinical isolates of Mycobacterium species, the ethA gene shows specific mutations, particularly loss-of-function frameshift mutations and nonsense mutations.

Ethionamide is a crucial second-line drug widely used to treat MDR-TB. However, the mutations in the ethA gene have rendered this drug ineffective against a significant number of MDR-TB pathogens. As a result, its effectiveness as a treatment option for MDR-TB has been compromised in these cases.

Mechanism of Conferring Resistance of Mutated ethA Gene

The mutated ethA genes encode for modified EthA enzymes which can’t activate the prodrug ethionamide. Thus unactivated ethionamide can’t disrupt the mycolic acid and the bacterium can survive.

Detection Method of Mutated ethA Gene

To identify mutations in the ethA gene, the recommended method is to sequence the ethA gene and then compare it with the reference nucleotide sequence of the ethA gene from the M. tuberculosis H37Rv reference strain.

For the PCR amplification of the ethA gene, the following primers can be employed:

Using these primers in PCR facilitates the specific amplification of the ethA gene, enabling subsequent gene sequencing and mutation analysis.

Primers	References
F: 5′- ATC ATC GTC GTC TGA CTA TGG -3′ R: 5′- ACT ACA ACC CCT GGG ACC -3′	Moazemi, S., Arjomandzadegan, M., Ahmadi, A., Tayebun, M., & Shojapor, M. (2015). Study of ethA gene sequence in Ethionamide resistant clinical isolates of Mycobacterium tuberculosis.

 

Other Genes Responsible for MDR/XDR-TB

Types of Mutated Genes	Resistant Antibiotics	PCR Primers for Detection	References
Mutated rrs Genes	Aminoglycosides like streptomycin, amikacin, kanamycin, capreomycin, viomycin	F: 5’- TTA AAA GCC GGT CTC AGT TC-3’  R: 5’- TAC GCC CCA CCA GTT GGG GC-3’	Suzuki, Y., Katsukawa, C., Tamaru, A., Abe, C., Makino, M., Mizuguchi, Y., & Taniguchi, H. (1998). Detection of Kanamycin-Resistant Mycobacterium tuberculosis by Identifying Mutations in the 16S rRNA Gene. Journal of Clinical Microbiology, 36(5), 1220-1225. https://doi.org/10.1128/jcm.36.5.1220-1225.1998
Mutated rspL Gene	Aminoglycosides like streptomycin, amikacin, kanamycin, capreomycin, viomycin	F: 5’- GAA TTC GGT AGA TGC CAA CCA TCC -3’ R: 5’- TGA AGC TTG ACC AAC GGA CGC TTG GG-3’	Katsukawa C, Tamaru A, Miyata Y, Abe C, Makino M, Suzuki Y. Characterization of the rpsL and rrs genes of streptomycin-resistant clinical isolates of Mycobacterium tuberculosis in Japan. J Appl Microbiol. 1997 Nov;83(5):634-40. doi: 10.1046/j.1365-2672.1997.00279.x. PMID: 9418025
Mutated inhA Gene	Isoniazid	F: 5′-ACA TAC CTG CTG CGC AAT -3′ R: 5′-TCA CAT TCG ACG CCA AAC -3′	Hosny AM, Shady HMA, Essawy AKE (2020) rpoB, katG and inhA Genes: The Mutations Associated with Resistance to Rifampicin and Isoniazid in Egyptian Mycobacterium tuberculosis Clinical Isolates. J Microb Biochem Technol.12:3. doi: 10.35248/1948-5948.20.12.428
Mutated aphC Gene	Isoniazid	—	—
Mutated gyrA and gyrB Gene	Fluoroquinolones like moxifloxacin, levofloxacin, ofloxacin	—	—
Mutated pncA Gene	Pyrazinamide	F: 5’- AAG GCC GCG ATG ACA CCT CT -3’ R: 5’- GTG TCG TAG AAG CGG CCG AT -3’	Juréen Pontus et al. “Pyrazinamide Resistance and PncA Gene Mutations in Mycobacterium Tuberculosis.” Antimicrobial Agents and Chemotherapy 52.5 (2008): 1852–1854. Web.
Mutated rpoB Gene	Rifampicin	—	—
Mutated embB Gene	Ethambutol	—	Lee, S. G., Khadijah Othman, S. N., Ho, Y. M., & Wong, S. Y. (2004). Novel Mutations within the embB Gene in Ethambutol-Susceptible Clinical Isolates of Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy, 48(11), 4447-4449. https://doi.org/10.1128/AAC.48.11.4447-4449.2004
Mutated gidB Gene	Kanamycin, amikacin	—	—
Mutated tlyA Gene	Capreomycin	—	—