Tuberculosis is one of the major causes of death from many infectious diseases (3). Out of 9 million people who are infected with mycobacteria, about 2 million deaths occur from tuberculosis every year (3). Unfortunately, the prevalence of tuberculosis is in a continuous increase due to increased number of Human immunodeificnecy virus (HIV) patients, bacterial resistance to anti-tuberculous drugs, and growing number of recreational drug users (3). The pathogen responsible for bacterial infection, potentially causing tuberculosis, is mycobacterium tuberculosis (MTB) (2). Persons with adequate immune system can control the bacterial infection so mycobacteria remain dormant for a long time (11). In a typical tuberculous granuloma, mature macrophages accumulate around a caseous lesion to prevent mycobacteria from leaking into the extracellular matrix (11). Therefore, only those with less effective immune systems will progress to primary progressive tuberculosis (2). When a patient progresses to active tuberculosis, early signs and symptoms include extreme fatigue, weight loss, and fever (7). The inflammatory and immune responses cause wasting that involves a period of rapid fat loss and muscle loss (7). Patients eventually develop cough, blood-streaked sputum from lung abscess, as well as pleuritic chest pain from the inflamed parenchyma (7). To fight against this grave and fatal disease, number of anti-tuberculous drugs was invented in hope of finding a cure. Although some first-line drugs (Isoniazid and Rifampicin) first appeared to be effective in treating tuberculosis, these anti-tuberculous drugs have failed to eradicate all cases of tuberculosis. Instead, anti-tuberculous drugs introduced more challenges in tuberculosis treatment. The current major challenge in treating tuberculosis is the increasing resistance to anti-tuberculous drugs, especially, multidrug-resistant tuberculosis (MDR-TB) in isoniazid (INH) and rifampicin (RIF). This paper will illustrate the transmission dynamics, life cycle, and mechanisms of drug resistance in Mycobacterium tuberculosis.
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Mycobacterium tuberculosis is a pathogen, which its physiology is directly linked to features of tuberculosis that it causes. The crucial feature for a mycobacteria's survival is its unique cell wall structure. The insoluble cell wall core of MTB is formed by a large variety of lipid-containing molecules, such as mycolic acid, that are covalently attached (6). This hydrophobic cell wall provides a physical protection from the host immune response and serves as a barrier against many toxic insults (2). Further, the complex MTB cell wall is impermeable to both hydrophobic and hydrophilic molecules, resulting in inherent resistance of MTB to most common antibiotics (8). Lipoarabinomannan is an antigen on the outside of the organism. This antigen is another important component of the cell wall because it inhibit the fusion of Mycobacterium-containing phagosomes with lysosomal compartments (4). Lipoarabinomannan hinders the fusion of phagosome with lysosome by impairing Ca2+/calmodulin pathway and inactivates macrophages (8). Therefore, this cell-surface component of MTB is able to facilitate the survival of mycrobacteria within macrophages (8). Also, MTB is able to survive the harsh environment of the host tissues by utilizing any available energy sources using alternate electron transport chains (12). This allows them to adapt to conditions of varying availability of oxygen and other terminal electron acceptors (12). Therefore, the structure of cell envelope and metabolic flexibility allows MTB to withstand the harsh environment inside the phagosome. As a result, MTB is able to grow and multiply inside the macrophage for potential latent tuberculosis infection.
Tuberculosis infection is initiated via ingesting, inoculating, or most commonly inhaling the droplet nuclei containing MTB (11). When airborne droplets from an infected person enter the susceptible person's lungs, mycobacteria in the droplets travel along the respiratory tract and reaches alveolar surface (8). In alveolar spaces, alveolar macrophages are readily available and provide an opportunity for the body to destroy invading mycobacteria and prevent infection (4). The macrophage receptor recognizes mycobacterial arabinomannan and quickly engulfs the mycobacteria (7). Engulfed mycobacteria become enclosed inside the phagosome that has a low pH (7). Normally, many bacterial species cannot withstand the harsh environment of the phagocyte; therefore, mature phagosomes are routed to lysosomal compartments for degradation (7). However, mycobacteria avoid this fate in several ways by regulating phagosomal maturation. The mycobacteria are able to inhibit macrophage activation and stimulate the production of cytokines that inhibit macrophage functions (4). The fusion of phagocytic vacuoles with vesicles containing the proton-ATPase is inhibited by MTB; thus, normal maturation of a phagosome into an acidified compartment for digesting engulfed pathogens is prevented (7). Mycobacteria also interfere with phagosome maturation to resist digestion by inhibiting the fusion of phagosome with lysosomes (7). As a result, significantly lowered immune response from the inactivation status of the host macrophage allows bacterial survival and growth (4). However, a person with intact cell-mediate immunity, macrophages and T-lymphocytes are recruited to form a granuloma around MTB to limit the spread of infection (7). The environment inside this granuloma is characterized by low pH, low oxygen level, and limited nutrition (7). Small lesions may form from destroyed macrophages; but an adequate immune system is able to keep the mycobacteria contained in the dormant lesion (14). In case of a tubercle bacilli release, T-cell-activated macrophages ingest and kill the released bacteria (5, 14). Also, infected macrophages presenting mycobacterial antigens are killed by CD4+ cytolytic T lymphocytes (CTL) (5, 14). However, the dormant bacteria can become reactivated in people with HIV infection, history of alcohol abuse, malnutrition, etc that disrupts specific immunity against the bacteria (12). For such less immunocompetent people, the necrotic tissue undergoes liquefaction (7). The liquefied caseous lesion discharges its contents into vicinity, or MTB can enter the bronchus or blood vessel. The bacteria then can disemminate into distant tissues, which leads to the progression of an active disease (7). Many complications arise as different organs in the body are affected with MTB, as well as increase in mortality in tuberculosis patients (7). Fortunately, the development of anti-tuberculous drugs has increased survival rate of tuberculosis patients by interfering with the mycobacteria's life cycle (13). Although proper use of anti-tuberculous drugs treat tuberculosis, misuse of the drugs results in the development of antibiotic resistance bacteria (13).
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Mutlidrug-resistant tuberculosis is mainly a result of chromosomal mutations, which affect the drug target or bacterial enzymes that active prodrugs. Shortly after the first drugs (Streptomycin and INH) were invented, the sudden and seemingly simultaneous appearance of multidrug-resistant tuberculosis (MDR-TB) made the treatment of tuberculosis extremely challenging (7). The MDR mycobacterium is resistant to multiple anti-tuberculous drugs (at least two drugs of isoniazid (INH) and rifampin (RIF)) (5). There are several ways through which a mutation can cause drug resistance. The classic mechanism is a mutation in the gene encoding the drug target, generally an enzyme, whose inhibition accounts for the toxic effects of the drug (5). INH is a prodrug that is only activated and become effective when it is oxidized by KatG, which is a catalase-peroxidase (1). Therefore, any mutation that compromises the ability of KatG to activate INH will result in INH-resistant MTB (13). Not surprisingly, about half of all INH-resistant MTB have KatG gene mutations or deletions (10). A variety of amino acid substitutions can also reduce the enzyme activity or can lead to rapid proteolysis (13). Another way that the INH interferes with tuberculosis infection is that it inhibits the synthesis of mycolic acids in MTB (10). Mycolic acids are fatty acids that form the main component of mycobacteria's cell wall. It is important to inhibit the synthesis of mycolic acids, because it lends the organism increased resistance to chemicals, preventing the activity of hydrophobic antibiotics (10). Mycolic acids also provide protection from host's immune system (1). When INH is oxidized by KatG in the presence of NAD+, INH-NAD adducts are formed. INH-NAD adduct is a tight-binding competitive inhibitor of InhA, an enzyme essential in mycolic acid synthesis. INH-NAD adduct subsequently inhibit InhA binding, and hence mycolic acid biosynthesis (1, 16). However, there are only limited number of INH-NAD adducts available inside the cell to interfere with INH. Thus, overexpression of the InhA leads to a decrease in the effective concentration of the INH-NAD adducts inside the MTB cell, which develops to become resistant to the drug (13). Moreover, alterations of the InhA mediate resistance to INH by reducing the binding INH-NAD adduct to the InhA enzyme. Amino acid substitution within InhA leads to a disruption of a binding of water molecule that is involved in the hydrogen bonding of the INH-NAD adduct (16). RIF is another first-line tuberculosis drug for the treatment of tuberculosis. RIF binds to the upstream of the catalytic center of RNA polymerase β subunit (rpoB gene), which physically blocks transcription (15). Many studies have found numerous mutations and deletions in the rpoB gene of RIF resistant mycobacteria (6, 9, 15). A total of 69 single nucleotide changes, including insertions, deletions, and multiple nucleotide changes, have been reported (9). Most of missense mutations are located in the 51 bp core region of the rpoB gene, which is thus referred to as Rifampicin resistance determining region (9). In 93-98% of RIF-resistant MTB isolates, there are mutations in an 81 bp region of rpoB gene that encodes for the β subunit of RNA polymerase (15). Since the mutation occurs at the binding site of RIF, it can no longer bind to the RNA polymerase. The disappearance of the physical barrier would allow RNA polymerase to transcribe DNA and synthesize RNA chain. In general, drug resistance in tuberculosis is mediated exclusively by naturally occurring genetic mutations in mycobacteria.
Although drug resistance occurs in nature, it is aggravated by human error. In fact, a community with a high rate of drug resistance has a poorly managed tuberculosis control program (5). For instance, the drug resistance can be caused by patients not taking medications properly or health-care providers prescribing treatment incorrectly (5). Medical errors, such as prescription of inadequate therapy, can also lead to the selection of resistant bacilli (5). Inadequate therapy is when a new patient is prescribed of only 2 or 3 drugs during initial phase of treatment. At least 3 drugs must be provided during the initial phase, so mutants resistant to one drug are killed by another drug (9). For instance, prescription of both Isoniazid and Rifampicin will allow mutants resistant to Isoniazid to be killed by Rifampicin, and vice versa (5). Since mutations occur at a low frequency at initial phase, it is very unlikely that a single organism will evolve spontaneous resistance to both drugs (9). Thus, without enough drugs prescribed, resistant mycobactria will be able to survive and eventually develop drug resistant tuberculosis. Also, a failure to fully inform patients to take all the prescribed drugs is also a common medical error (5). Some patients omit one or more drugs, which allow drug resistance development (5). Adding extra drugs in the case of treatment failure can also cause bacterial multi drug resistance (11). If the treatment failure was due to drug resistance, the bacterial population will develop more resistance to the same drug; furthermore, the strains will become sequentially resistant to other agents as well (5). In addition to medical errors, patient co-operation is very important in effective treatment. The problem occurs when the patient cannot adhere to drugs because he/she is homeless, has an alcohol or drug problem, is unemployed, looking for a job, etc (5). Due to their harsh situation, patients cannot manage to complete the full course of prescribed medication (11). Therefore, drug resistance is the result of selection of resistant mutants in the bacterial population after all susceptible bacilli are killed by tuberculosis drugs. The mycobacteria that are vulnerable to antibiotics are killed and resistant mutants will then be able to multiply to cause multidrug-resistant tuberculosis. The problem of drug resistance worsens due to medical errors and failure in patient adherence to prescribed drugs, in addition to naturally occurring drug resistance in the mycobacteria.
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As resistance became more prevalent and developed insights into the molecular basis of drug resistance have led to development of techniques for identifying the mutations associated with drug resistance. The first way to detect INH or rifampicin resistance is probe-based hybridization method (13). A portion of the mutated KatG and rpoB genes are separately amplified by PCR. The resulting PCR products are hybridized with oligonucleotide probes corresponding to sequences present in the wild-type and INH/rifampicin resistance mutations (13). After completing hybridization and washing steps, streptavidin labeled with alkaline phosphatase are added (13). Bound hybrids can be visualized by autoradiography, where the intensity of color will indicate the number of hybrids present. If the probe of a particular gene carries a mutation, it will not have a complementary pair to bind to. Thus, PCR product that is not bound to wild-type sequence can infer the presence of mutation in a gene of interest (either KatG or rpoB gene) (13). When observed under UV light, absence of hybrids will be denoted by dark or low intensity of color. PCR-single strand conformation polymorphism (PCR-SSCP) is another technique used to detect MTB drug resistance. The KatG and rpoB genes are amplified by PCR and then reverse transcribed into a cDNA. The cDNA strands are denatured by heat, and then cooled so single DNA strands will fold into their own conformation (9, 13). Mutated DNA strands will have a different conformation than the wild-type DNA. Therefore, MTB drug resistance can be determined by measuring different electrophoresis mobility on a gel (9). Further research on drug resistance mechanisms and associated mutations can be used for diagnosis and clinical care of individual patients with tuberculosis, as well as potentially combating resistant tuberculosis.
Over many years of biomedical research, there were great advances in understanding of the epidemiology and pathogenesis of tuberculosis. However, the prevalence of tuberculosis is still remains high, mainly due to acquired or developed drug resistance in Mycobacterium tuberculosis. The increase in incidences of multidrug-resistant tuberculosis (MDR-TB) urgently suggests the development of more effective drugs, which specifically targets biochemical pathway and a stage of pathogen's life cycle.