by Tuberculosis (TB) is an infectious disease caused by various species within the Mycobacterium tuberculosis complex. It stands as one of the most pervasive and lethal infectious diseases, claiming over 1.5 million lives annually. According to the World Health Organization (WHO), it is estimated that around a third of the world’s population carries the latent form of the disease. Out of these latent cases, approximately 10% are anticipated to progress to active clinical TB. The predominant form of TB worldwide is pulmonary tuberculosis, which is caused by the bacterium M. tuberculosis.
TB treatment involves a specific regimen comprising drugs like Rifampicin, Isoniazid, Pyrazinamide, Ethambutol, and Streptomycin, either individually or in various combinations. If a strain is resistant to Rifampicin and Isoniazid, or all first-line antibiotics, it’s classified as MDR-TB.
For MDR-TB, a second-line treatment regimen is employed. Key drugs in this category include fluoroquinolones (preferably moxifloxacin, and others like ofloxacin and levofloxacin) and injectable aminoglycosides like amikacin, kanamycin, and capreomycin. Additional drugs like ethionamide, cycloserine, para-aminosalicylic acid, clofazimine, linezolid, and delamanid are also used.
If the MDR-TB strain is resistant to at least one fluoroquinolone and an injectable aminoglycoside, it’s termed as XDR-TB. A combination of pretomanid, bedaquiline, and linezolid is recommended for treating XDR-TB.
However, resistance to available treatment options is emerging in many clinical isolates of the M. tuberculosis complex. The primary mechanisms of resistance involve alterations in target sites, increased efflux, and enzymatic modification of drugs, rendering them ineffective.
Numerous genes are responsible for these drug resistance mechanisms. This note highlights some of the crucial genes associated with drug resistance in tuberculosis-causing bacteria.
Mutated atpE Gene
The mutated atpE gene refers to a genetic alteration in the atpE gene, which is a specific gene that encodes a subunit of the ATP synthase enzyme complex. ATP synthase is a crucial enzyme involved in the production of adenosine triphosphate (ATP), the main energy currency in cells.
When the atpE gene undergoes a mutation, it can lead to changes in the structure or function of the ATP synthase complex. This can potentially affect the efficiency of ATP production and cellular energy metabolism.
Mutations in the atpE gene can occur naturally or as a result of various factors such as exposure to environmental stressors or genetic predispositions. Depending on the nature and location of the mutation, it can have different effects on cellular processes.
Understanding mutated genes like atpE is important in the context of research on cellular biochemistry, energy metabolism, and potentially in the development of targeted therapies for certain conditions associated with mitochondrial dysfunction.
Mechanism of Conferring Resistance of Mutated atpE Gene
The mutated atpE gene can confer resistance to specific antibiotics, particularly those that target bacterial ATP synthase.
- Normal Action of ATP Synthase:
ATP synthase is an enzyme complex found in bacterial membranes that plays a crucial role in energy production. It synthesizes adenosine triphosphate (ATP), the primary energy currency of cells.
- Role of Mutated atpE Gene:
When the atpE gene undergoes a mutation, it leads to changes in the structure or function of the ATP synthase complex.
- Alteration in ATP Synthase Structure:
The mutation in the atpE gene can result in structural changes in the ATP synthase complex. This alteration may affect the binding sites of specific antibiotics that target ATP synthase.
- Reduced Antibiotic Binding:
Due to the structural changes caused by the mutation, the binding affinity of certain antibiotics to the mutated ATP synthase may be reduced. As a result, these antibiotics may be less effective in inhibiting the enzyme.
- Resistance to ATP Synthase-Targeting Antibiotics:
Bacteria carrying the mutated atpE gene and exhibiting altered ATP synthase may show resistance to antibiotics that rely on ATP synthase as their target. This includes specific classes of antibiotics designed to interfere with bacterial energy production.
It’s important to note that resistance conferred by a mutated atpE gene is specific to antibiotics targeting ATP synthase. Other classes of antibiotics with different mechanisms of action may not be affected by this mutation. Understanding these mechanisms of resistance is crucial for the development of effective antibiotic therapies.
Detection Method of Mutated atpE Gene
- DNA Sequencing
- Polymerase Chain Reaction (PCR)
- Restriction Fragment Length Polymorphism (RFLP)
- Allele-Specific PCR
- Fluorescence In Situ Hybridization (FISH)
- Real-Time PCR
- DNA Microarray
- Next-Generation Sequencing (NGS)
- Sanger Sequencing
- High-Resolution Melting Analysis (HRMA)
- Ligase Chain Reaction (LCR)
- Denaturing Gradient Gel Electrophoresis (DGGE)
- Amplification Refractory Mutation System (ARMS)
- Pyrosequencing
- Multiplex Ligation-Dependent Probe Amplification (MLPA)
Mutated Rv0678 Gene
The Rv0678 gene, also known as dosR, is a regulatory gene in Mycobacterium tuberculosis (M. tuberculosis) that plays a crucial role in the bacterium’s response to low oxygen conditions, which are encountered during infection. Mutations in the Rv0678 gene can lead to alterations in the regulatory pathways, potentially affecting the bacterium’s ability to adapt to the host environment.
The dosR regulon, controlled by the Rv0678 gene, is responsible for coordinating the response of M. tuberculosis to various stresses, including hypoxia (low oxygen levels), nitric oxide exposure, and other conditions encountered within the host. Mutations in the Rv0678 gene may disrupt this regulatory pathway, potentially impacting the bacterium’s ability to survive and thrive within the host.
Understanding mutations in the Rv0678 gene is significant in tuberculosis research, as it provides insights into how the bacterium adapts to different environmental conditions and how it may develop resistance mechanisms. Additionally, studying mutated Rv0678 genes can aid in the development of targeted therapies for tuberculosis.
Mechanism of Conferring Resistance of Mutated Rv0678 Gene
The Rv0678 gene is primarily associated with regulating the response of M. tuberculosis to low oxygen conditions and other stresses, but it is not known to directly confer antibiotic resistance.
Detection Method of Mutated Rv0678 Gene
| Detection Method | Description |
| DNA Sequencing | Determines the exact order of nucleotides in the DNA molecule. Can reveal mutations in the gene sequence. |
| Polymerase Chain Reaction (PCR) | Selectively replicates a specific DNA sequence, allowing for the detection of mutations through comparison with a reference sequence. |
| Sanger Sequencing | Widely used DNA sequencing technique that can be employed to identify mutations in the Rv0678 gene. |
| Next-Generation Sequencing (NGS) | High-throughput sequencing technology used to identify mutations in the entire genome, including the Rv0678 gene. |
| Mutagenesis Studies | Involves creating specific mutations in the gene of interest and studying resulting phenotypic changes to confirm functional impact. |
| Allele-Specific PCR | Detects specific mutations by designing primers that specifically anneal to either the wild-type or mutant allele. |
| DNA Microarray | Microarrays can be designed to detect known mutations or sequence variations in specific genes. |
| High-Resolution Melting Analysis (HRMA) | Post-PCR technique used for identifying genetic variations such as single nucleotide polymorphisms (SNPs) or mutations. |
Mutated rpoB Gene
The rpoB gene, also known as the beta subunit of RNA polymerase, is a crucial gene in bacteria responsible for encoding a component of the RNA polymerase enzyme. This enzyme plays a fundamental role in the process of transcription, where genetic information from DNA is transcribed into RNA.
Mutations in the rpoB gene, particularly in specific regions known as the “Rifampicin Resistance Determining Region” (RRDR), are associated with resistance to the antibiotic rifampicin. Rifampicin is a key component of the standard treatment regimen for tuberculosis.
When mutations occur in the RRDR of the rpoB gene, they can alter the structure of the RNA polymerase enzyme, reducing the affinity of rifampicin to its binding site. This leads to reduced effectiveness of the antibiotic in inhibiting bacterial RNA synthesis.
Detection of mutated rpoB genes is essential for identifying rifampicin-resistant strains of bacteria, particularly in cases of tuberculosis. This information guides clinicians in selecting appropriate treatment regimens, as well as in efforts to control the spread of drug-resistant strains.
Mechanism of Conferring Resistance of Mutated rpoB Gene
- Normal Action of RNA Polymerase:
The RNA polymerase enzyme is responsible for transcribing genetic information from DNA to RNA. It is crucial for the synthesis of RNA molecules in bacteria.
- Role of rpoB Gene:
The rpoB gene encodes the beta subunit of RNA polymerase, which is a key component of the enzyme complex.
- Rifampicin Resistance Determining Region (RRDR):
Specific regions of the rpoB gene, known as the RRDR, are particularly important for rifampicin binding. Mutations in this region can alter the structure of the RNA polymerase enzyme.
- Reduced Affinity for Rifampicin:
Mutations in the RRDR can lead to conformational changes in the RNA polymerase enzyme, reducing the affinity of rifampicin to its binding site. This means rifampicin is less effective at inhibiting bacterial RNA synthesis.
- Resistance to Rifampicin:
Bacteria with mutated rpoB genes and altered RNA polymerase are resistant to the inhibitory effects of rifampicin. This can result in the survival and proliferation of rifampicin-resistant strains.
- Clinical Significance:
Detection of mutations in the rpoB gene is crucial for identifying rifampicin-resistant strains, particularly in the context of tuberculosis. This information guides treatment decisions, as alternative antibiotics may be required.
Detection Method of Mutated rpoB Gene
| Detection Method | Description |
| DNA Sequencing | Determines the exact order of nucleotides in the DNA molecule. Can reveal mutations in the gene sequence, including RRDR. |
| Polymerase Chain Reaction (PCR) | Selectively replicates a specific DNA sequence, allowing for the detection of mutations through comparison with a reference sequence. |
| Allele-Specific PCR | Detects specific mutations by designing primers that specifically anneal to either the wild-type or mutant allele, potentially useful for RRDR mutations. |
| Real-Time PCR | Quantitative PCR technique that can be used to monitor the amplification of specific DNA sequences, including mutated sequences. |
| High-Resolution Melting Analysis (HRMA) | Post-PCR technique used for identifying genetic variations, including mutations, by analyzing the melting behavior of DNA fragments. |
| Line Probe Assays (LPA) | Commercial assays designed to identify mutations associated with drug resistance, including RRDR mutations in rpoB. |
Mutated katG Gene
The katG gene, also known as catalase-peroxidase, is a crucial gene in bacteria, including Mycobacterium tuberculosis (M. tuberculosis). It codes for an enzyme that plays a key role in protecting the bacterium from oxidative stress by converting harmful reactive oxygen species (ROS) into less damaging molecules.
Mutations in the katG gene are associated with resistance to the antibiotic isoniazid (INH), which is a first-line drug used in the treatment of tuberculosis. Isoniazid works by inhibiting the activity of the enzyme produced by the katG gene.
When mutations occur in the katG gene, particularly in the region associated with the activation of isoniazid, it can lead to reduced or altered enzyme activity. This results in decreased effectiveness of isoniazid in inhibiting bacterial growth.
Detection of mutated katG genes is crucial for identifying isoniazid-resistant strains of M. tuberculosis. This information is essential for tailoring treatment regimens to target drug-resistant strains and prevent the further spread of resistant tuberculosis.
Mechanism of Conferring Resistance of Mutated katG Gene
Mutations in the katG gene confer resistance to the antibiotic isoniazid (INH) in Mycobacterium tuberculosis. Here’s how it works:
- Normal Action of KatG:
The KatG enzyme is responsible for activating isoniazid, a key component of the standard treatment regimen for tuberculosis.
- Role of Isoniazid:
Isoniazid is a prodrug, meaning it needs to be activated by bacterial enzymes like KatG to become effective.
- Activation of Isoniazid:
KatG activates isoniazid by converting it into its active form, which then inhibits an enzyme involved in cell wall synthesis in Mycobacterium tuberculosis.
- Mutations in katG:
Mutations in the katG gene, particularly in the region associated with the activation of isoniazid, can lead to alterations in the enzyme’s structure or activity.
- Reduced Activation of Isoniazid:
Mutated KatG enzymes may have reduced or altered activity in converting isoniazid into its active form. This leads to decreased effectiveness of isoniazid in inhibiting bacterial growth.
- Resistance to Isoniazid:
Bacteria with mutated katG genes and altered KatG enzyme activity are resistant to the inhibitory effects of isoniazid.
- Clinical Significance:
Detection of mutations in the katG gene is crucial for identifying isoniazid-resistant strains of Mycobacterium tuberculosis. This information guides treatment decisions, as alternative antibiotics may be required.
Detection Method of Mutated katG Gene
| Detection Method | Description |
| DNA Sequencing | Determines the exact order of nucleotides in the DNA molecule. Can reveal mutations in the gene sequence, including those in katG. |
| Polymerase Chain Reaction (PCR) | Selectively replicates a specific DNA sequence, allowing for the detection of mutations through comparison with a reference sequence. |
| Allele-Specific PCR | Detects specific mutations by designing primers that specifically anneal to either the wild-type or mutant allele, potentially useful for katG mutations. |
| Real-Time PCR | Quantitative PCR technique that can be used to monitor the amplification of specific DNA sequences, including mutated sequences in katG. |
| High-Resolution Melting Analysis (HRMA) | Post-PCR technique used for identifying genetic variations, including mutations, by analyzing the melting behavior of DNA fragments. |
| Line Probe Assays (LPA) | Commercial assays designed to identify mutations associated with drug resistance, including those in katG. |
Mutated ethA Gene
The ethA gene in Mycobacterium tuberculosis encodes an enzyme called enoyl-CoA hydratase, which plays a crucial role in fatty acid metabolism. It is involved in the biosynthesis of mycolic acids, which are important components of the bacterial cell wall.
Mutations in the ethA gene can confer resistance to the antibiotic ethionamide. Ethionamide is a second-line drug used in the treatment of tuberculosis. It works by inhibiting the synthesis of mycolic acids, ultimately disrupting the integrity of the bacterial cell wall.
When mutations occur in the ethA gene, they can lead to alterations in the structure or activity of the enzyme, reducing its affinity for ethionamide. This results in decreased effectiveness of the drug in inhibiting bacterial growth.
Detection of mutated ethA genes is crucial for identifying ethionamide-resistant strains of Mycobacterium tuberculosis. This information guides clinicians in selecting appropriate treatment regimens and preventing the further spread of drug-resistant tuberculosis.
Mechanism of Conferring Resistance of Mutated ethA Gene
- Normal Action of EthA:
The EthA enzyme is responsible for activating ethionamide, a key component of the treatment regimen for tuberculosis.
- Role of Ethionamide:
Ethionamide is a prodrug, meaning it needs to be activated by bacterial enzymes like EthA to become effective.
- Activation of Ethionamide:
EthA activates ethionamide by converting it into its active form, which then inhibits the synthesis of mycolic acids in the bacterial cell wall.
- Mutations in ethA:
Mutations in the ethA gene, particularly in regions associated with the activation of ethionamide, can lead to alterations in the enzyme’s structure or activity.
- Reduced Activation of Ethionamide:
Mutated EthA enzymes may have reduced or altered activity in converting ethionamide into its active form. This leads to decreased effectiveness of ethionamide in inhibiting bacterial growth.
- Resistance to Ethionamide:
Bacteria with mutated ethA genes and altered EthA enzyme activity are resistant to the inhibitory effects of ethionamide.
- Clinical Significance:
Detection of mutations in the ethA gene is crucial for identifying ethionamide-resistant strains of Mycobacterium tuberculosis. This information guides treatment decisions, as alternative antibiotics may be required.
Detection Method of Mutated ethA Gene
| Detection Method | Description | Primers |
| DNA Sequencing | Determines the exact order of nucleotides in the DNA molecule. Can reveal mutations in the gene sequence, including those in ethA. | Specific primers designed based on ethA gene sequence. |
| Polymerase Chain Reaction (PCR) | Selectively replicates a specific DNA sequence, allowing for the detection of mutations through comparison with a reference sequence. | Forward and reverse primers flanking the region of interest, including potential mutation sites. |
| Allele-Specific PCR | Detects specific mutations by designing primers that specifically anneal to either the wild-type or mutant allele, potentially useful for ethA mutations. | Wild-type-specific and mutant-specific primers designed to specifically amplify respective alleles. |
| Real-Time PCR | Quantitative PCR technique that can be used to monitor the amplification of specific DNA sequences, including mutated sequences in ethA. | Primers designed to target the specific region of ethA gene, along with a fluorescent probe for real-time detection. |
| High-Resolution Melting Analysis (HRMA) | Post-PCR technique used for identifying genetic variations, including mutations, by analyzing the melting behavior of DNA fragments. | Primers designed to flank the region of interest for ethA, no specific primers for HRMA. |
| Line Probe Assays (LPA) | Commercial assays designed to identify mutations associated with drug resistance, including those in ethA. | Pre-designed primers and probes specific for detecting ethA mutations, provided in the LPA kit. |
Other Genes Responsible for MDR/XDR-TB
| Types of Mutated Genes | Resistant Antibiotics | PCR Primers for Detection |
| rpoB | Rifampicin | Forward: 5′-ACCCGCTGCGGCCCAGGTC-3′ |
| Reverse: 5′-CGTCGACCTGCACGCGCC-3′ | ||
| katG | Isoniazid | Forward: 5′-GTCGGACAGGCCGCTTGCGT-3′ |
| Reverse: 5′-GGTCGCCGGGTTCGATCGTC-3′ | ||
| inhA promoter region | Isoniazid | Forward: 5′-TGGCACCGGCCTTCACCGG-3′ |
| Reverse: 5′-CCCAGCGGTTTCGGACCTG-3′ | ||
| ethA | Ethionamide | Forward: 5′-TTGGTCGTCGGGGTTGTTGTT-3′ |
| Reverse: 5′-CGCGGTGCGTGGGTTGACGTT-3′ | ||
| embB | Ethambutol | Forward: 5′-GGAGTTCGGCGTTACGCGGG-3′ |
| Reverse: 5′-GACGCCGCCACCCGTACCGG-3′ | ||
| pncA | Pyrazinamide | Forward: 5′-GACACCGACGGCTACCTACG-3′ |
| Reverse: 5′-CGTCTCGCCGACTTGTGACG-3′ | ||
| gyrA | Fluoroquinolones | Forward: 5′-TGGCGATGTCGCCCTCGGTC-3′ |
| Reverse: 5′-CGGCCAGCGTCGTCATCGTC-3′ | ||
| rrs | Aminoglycosides | Forward: 5′-AGAGGGAGGTCGAGGCTTGGGACC-3′ |
| Reverse: 5′-GGCAGGTTTCACGTGCGGGCTC-3′ | ||
| embCAB promoter region | Ethambutol | Forward: 5′-GGACGTGGTGGTCCGAGAAGG-3′ |
| Reverse: 5′-CGGTGACCGGCTACCGTGAGC-3′ | ||
| rrs 1401/1402 | Kanamycin/Amikacin | Forward: 5′-TGAGCCAGCAGCCGCTTCGG-3′ |
| Reverse: 5′-CTCGTCCAGGTGCTCACGGT-3′ |
