Antibiotic Resistance Genes in MDR/XDR Tuberculosis (TB)

Tuberculosis (TB) is an infectious disease caused by a group of different Mycobacterium species known as the Mycobacterium tuberculosis complex. TB is one of the most deadliest and most common infectious diseases that is held responsible for more than 1.5 million death per year and is supposed to have infected about a third of the world’s population in latent form (according to the WHO). Among these latently infected ones, about 10% are expected to develop clinical disease. Pulmonary tuberculosis caused by M. tuberculosis is the most common form of TB prevalent globally.

A specific treatment regimen is developed to treat TB. Rifampicin, Isoniazid, Pyrazinamide, Ethambutol, and Streptomycin, either in a different form of combinations of each other or in monotherapy form are used as first-line treatment. Resistant to at least rifampicin and isoniazid or all antibiotics of the first-line treatment is reported as MDR-TB.

For MDR-TB treatment, the second line of treatment was designed. The major drugs of the second line include fluoroquinolones (preferably moxifloxacin, and others like ofloxacin and levofloxacin) and injectable aminoglycosides including amikacin, kanamycin, and capreomycin. Besides, other drugs like ethionamide, cycloserine, para-aminosalicylic acid, clofazimine, linezolid, and delamind are other second-line anti-TB drugs.

If the MDR-TB pathogen is resistant to at least one fluoroquinolone and an injectable aminoglycoside, then the MDR-TB is now defined as XDR-TB. A combination of pretomanid, bedaquiline, and linezolid is recommended for treating the XDR-TB.

However, even the treatment options available for XDR-TB are now seen as resistant in many clinical isolates of the M. tuberculosis complex. The major mechanisms of resistance developed by TB pathogens are modification of target sites, overexpression of effluxes, and enzymatic modification of the drugs making them ineffective.

Several genes are responsible for the development of such AMR mechanism; some of the important genes responsible for drug resistance in tubercle bacilli are described in this note.           

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:

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.  

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

• Mutated rpoB gene encodes for the synthesis of modified -subunit of DNA-dependent RNA polymerase (‘-subunit). Rifampicin can’t bind with this modified  ‘-subunit and fails to prevent the protein elongation step in the bacterium ensuring an increase in the MIC value of rifampicin and a higher chance of treatment failure. 

Detection Method of Mutated rpoB Gene

• PCR is first used to identify and amplify the rpoB gene from isolates followed by gene sequencing to identify the complete nucleotide sequence. The obtained nucleotide sequence of the rpoB gene is then compared with the reference nucleotide sequence of the rpoB gene of   M. tuberculosis H₃₇Rv. The primers that can be used in the process are: 

Mutated katG Gene

• katG gene (Rv1908c gene) is a mycobacterial gene that encodes for the synthesis of the catalase-peroxidase enzyme (KatG). KatG is a catalase enzyme that catalyzes the decomposition of hydrogen peroxide into oxygen and water molecules and thus prevents the bacteria from oxidative damage. 
• Besides this cellular function, the KatG enzyme is responsible for activating a pro-anti-TB-drug, isoniazid. 
• The katG gene encodes for the KatG type catalase enzyme that catalyzed the formation of isonicotinic acyl radical from the isoniazid molecule. Thus formed isonicotinic acyl radical spontaneously combines with the NADH molecule forming an Isonicotinic acyl-NADH complex which combines with InhA, the enoyl-acyl carrier protein reductase enzyme. This binding will prevent the formation of mycolic acid; hence disrupting the bacterial cell wall resulting in cell lysis. 
• Mutation in the katG gene has been associated with the majority of isoniazid-resistant Mycobacterium tuberculosis. In about 94% of isoniazid-resistant clinical isolates of mycobacteria, the S315T variant of the KatG catalase-peroxidase enzyme has been observed.   

Mechanism of Conferring Resistance of Mutated katG Gene

• The mutated katG gene encodes for the modified type of KatG enzyme, mainly the S315T variant. These modified KatG enzymes can’t catalyze the formation of the Isonicotinic acyl-NADH complex and the prodrug isoniazid remains in its inactive prodrug stage. The isoniazid can’t prevent the formation of mycolic acid and can’t induce cell wall degradation-associated mycobacterial cell lysis. 

Detection Method of Mutated katG Gene

• Getting the complete nucleotide sequence of the katG gene and comparing the sequence with the katG gene of   M. tuberculosis H₃₇Rv is the ideal method to detect the mutation in the katG gene. Some of the primers that can be used to amplify the katG gene via the PCR method are:

Mutated ethA Gene

• ethA gene is a mycobacterial gene that encodes for the production of the Baeyer Villiger monooxygenase EthA enzyme. This enzyme activates the pro-anti-TB-drug ethionamide into an active radical which then couples with the NADH molecule making it an active toxic complex. Thus activated complex then inhibits the enoyl-acyl carrier protein reductase InhA which ultimately prevents the formation of mycolic acid and induces the disruption of the mycolic acid in the mycobacterial cell wall inducing the cell lysis. 
• In the majority of the ethionamide-resistant clinical isolates of Mycobacterium spp., a mutation in the ethA gene, particularly loss-of-function frameshift mutation and nonsense mutation, has been recorded.  
• Ethionamide is an important anti-TB drug used widely to treat MDR-TB as a second-line drug. And, the mutation in the ethA gene has rendered this drug ineffective against a significant number of MDR-TB pathogens.  

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

• For the detection of mutation in the ethA gene, the ethA gene must be sequenced and compared with the reference nucleotide sequence of the ethA gene of the reference of   M. tuberculosis H₃₇Rv. The PCR primers that can be used for the identification and amplification of the ethA gene are:

Other Genes Responsible for MDR/XDR-TB

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