Mycobacterium tuberculosis: An overview

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The History of Discovery


  • Causative agent of tuberculosis (Mycobacterium tuberculosis) was first discovered and thoroughly investigated in 1882 by R. Koch.
  • Robert Koch and coworkers created the experimental animal models of the disease that made possible to study pathogenesis and immunity in tuberculosis.
  • In 1890 R. Koch obtained complex antigenic substance (tuberculin) from tubercle bacilli and tried to use it for tuberculosis treatment. This attempt appeared to be unsuccessful, but later tuberculin was applied for tuberculosis immunodiagnostics.
  • In 1919 A. Calmette and Ch. Guerin created live attenuated vaccine against tuberculosis. The strain BCG (or bacillus Calmette-Guerin) was introduced into medical practice, and it is used now for specific prophylaxis of the disease.
  • At the middle of XX century the first efficient drugs for tuberculosis treatment were worked out (streptomycin in 1944, para-aminosalicylic acid in 1946, and isoniazid in 1952), thereby making possible the control of this severe disorder.

Classification of Pathogenic Mycobacteria


  • Mycobacteria pertain to the order Actinomycetales, family Mycobacteriaceae, and genus Mycobacteria. To date more than 130 mycobacterial species are known.
  • Mycobacterium tuberculosis is the predominant causative agent of tuberculosis in humans.
  • Mycobacterium bovis causes tuberculosis in cattle and much more seldom in humans (about 2-5% of cases).
  • Several other mycobacteria, e.g. M. africanum can rarely produce human tuberculosis infection.
  • Causative agents of mycobacterioses comprise more than 60 species. These bacteria exert severe opportunistic infections.
  • The predominant pathogens here are Mycobacterium avium-intracellulare, M. kansasii M. ulcerans and others. They usually affect immunocompromised individuals, e.g. AIDS patients.
  • Mycobacterium leprae is the causative agent of leprosy.
  • Many of mycobacteria are acid-resistant saprophytes, e.g. M. smegmatis.

Structure and Properties of Mycobacteria


Morphology

  • Most of mycobacteria are thin straight rods without capsule, spores and flagella.
  • Being highly pleomorphic, the bacteria can appear in granular, coccoid, thread-like, branching and filtering forms. The latter can pass through bacterial filters similar with mycoplasmas and viruses. Various microbial forms can be found intracellularly.
  • Mycobacteria are considered to be gram-positive, albeit they are poorly stained by aniline dyes.
  • Microorganisms reveal striking acid and alkaline resistance (so-called “acid-fastness”). These abilities ensue from particular chemical composition of mycobacteria.
  • They contain a great number of chemically inert lipids, phosphatides and waxes, usually termed as mycolic acids (various high molecular weight hydroxy fatty acids) in a complex with cell wall mucopeptides. Besides lipid fractions, bacteria include various proteins and polysaccharides.
  • Acid-fast mycobacteria stain red by basic phenol fuchsin in Ziehl-Neelsen stain, whereas other bacteria are sensitive to sulfuric acid treatment and counterstain with methylene blue.
  • Also mycobacteria are successfully stained by fluorescent dyes, e.g. auramine.

Cultivation

  • Mycobacteria grow very slowly in aerobic conditions. It depends on long period of microbial replication (about 15 h for doubling). Growth is possible within temperature range from 24°C to 42°C with optimum at 37°C.
  • Various selective and special media are used for their cultivation. Lowenstein-Jensen medium contains agar, egg yolk, glycerol, potato extract, asparagine, milk, salts and malachite green to inhibit concomitant microflora. Composition of Finn medium is almost the same, but asparagine is substituted by additional number of salts.
  • Middlebrook semisynthetic agar is composed of oleic acid, albumin, vitamins and cofactors, various salts, catalase, glycerol, glucose, and malachite green.
  • Primary growth on solid media is observed in 3-6 weeks. Mycobacterium tuberculosis usually grows in warty dry colonies (R forms) with cream-colored “ivory” pigment.
  • Saprophytic mycobacteria proliferate more rapidly and appear in few days. They are able to produce orange or yellow pigment.
  • Different broth media support the cultivation of small amounts of mycobacteria. Liquid media growth reveals thin, brittle, wrinkled film resulted from microbial hydrophobic substances.
  • Pryce’s microculture method on narrow glass slides is available for rapid cultivation of mycobacteria in citrate blood.
  • Rapid advanced cultural methods (e.g., BACTEC radiometric broth system) allow swift identification of M. tuberculosis in minimal amounts.
  • Modern BACTEC radiometric system is composed of liquid medium supplemented with [14С]-labeled palmitic acid. The medium also contains a number of antibiotics with broad spectrum of action to inhibit concomitant bacteria.
  • During cultivation M. tuberculosis metabolizes palmitic acid with formation of radiolabeled carbon dioxide, which is further registered by radioactivity counter. By this method the infection is detected in 7-8 days of culturing.

Biochemical properties

  • Mycobacteria are aerobic microorganisms. M. tuberculosis produces a number of oxidation-reduction enzymes, including thermolabile catalase-peroxidase and superoxide dismutase. Also the bacteria express lecithinase, phosphatase, and urease.
  • M. tuberculosis can utilize various carbohydrates and proteins.

Antigenic structure

  • Lipids and phosphatides, encased in mycobacteria, are generally regarded as moderate antigens or haptens.
  • Nevertheless, in complex with mycobacterial proteins they elicit both cellular and humoral immune responses. M. tuberculosis infection induces cell-mediated reactions of delayed type hypersensitivity with chronic inflammation.
  • Also mycobacterial antigens activate production of specific antibodies usually in low or moderate titers.
  • Tuberculin is a peculiar antigenic complex composed of various tuberculoproteins and wax fractions. It causes hypersensitivity reactions evaluated by tuberculin skin test (TST).

Virulence factors

  • Toxic substances of mycobacteria are closely associated with microbial body and mainly release after microbial decomposition.
  • For instance, mycolic acids render toxic effects against host cells and tissues.
  • Cell wall structures of glycolipid nature (mycosides, mannosides, etc.) participate in microbial adhesion and inhibit phagocytosis.
  • In addition, M. tuberculosis possesses highly specific type VII secretion system (T7SS) that promotes active secretion of micobacterial proteins across lipid-containing cell wall of mycobacteria.
  • T7SS is characteristic for virulent strains of M. tuberculosis and M. bovis being absent in vaccine BCG strain.
  • With the aid of T7SS M. tuberculosis expels some virulence effector proteins into the cytoplasm of infected cells (e.g., macrophages).
  • Among them are CFP-10 and ESAT-6, which inhibit respiratory burst and secretion of proinflammatory cytokines by macrophages. Other proteins prevent the recognition of mycobacteria by Toll-like receptors of immune cells.
  • Microbial enzymes catalase and superoxide dismutase contribute to the inhibition of respiratory burst by macrophages.
  • Also M. tuberculosis carries so-called “cord factor” – potent toxic glycolipid fraction (trehalose dimycolate), which inhibits biological oxidation in the host cells and induces chronic immune inflammatory response with granuloma formation.
  • The production of cord factor is determined in Pryce’s microculture method, where virulent mycobacterial cells are grouped in tightly braced chains (or “serpentine cords”) visible under the microscopy with Ziehl-Neelsen stain.
  • Tuberculin is toxic for guinea pigs in course of experimental infection and elicits hypersensitivity in human infections, followed by tissue inflammation.
  • Mutations in genes, encoding bacterial enzymes catalase and RNA-polymerase confer the resistance of mycobacteria to basic tuberculosis drugs of the first line – isoniazid and rifampicin.

Resistance

  • Mycobacteria show high resistance in the environment. They remain viable in water for 1 year, in soil for 6 months, in the home dust and dried sputum for several months.
  • Due to high lipid contents the bacteria can withstand the action of generally used disinfectants, thus ordinary disinfection regimens should be prolonged to inactivate mycobacteria.
  • The most efficient are chlorine-containing chemical agents.
  • Mycobacteria are resistant to majority of antimicrobial drugs.
  • Nevertheless, heating at a temperature of 100°C readily kills all mycobacteria. Pasteurization inactivates M. bovis in dairy products, thereby preventing alimentary transmission of the disease.
  • Also mycobacteria are susceptible to sunlight and UV irradiation.

Epidemiology, Pathogenesis and Clinical Findings in Tuberculosis


  • Tuberculosis is one of the most important threats for human health at the beginning of XXI century. According to WHO data, annual total number of disease cases is about 9.6 million.
  • It is generally assumed that about one quarter of affected persons dies from tuberculosis and its consequences, and most of patients are young adults.
  • Moreover, mortality rate in untreated or untreatable disease exceeds 50%. Thus, tuberculosis remains the leading infectious cause of human death resulting in approximately 1.4 million lethal cases every year.
  • Moreover, tuberculosis is the major AIDS-indicator disease in HIV-infected persons. It develops at least in 30-40% of HIV-infected individuals being the major cause of death of AIDS patients (25-40% of total AIDS lethality).
  • Finally, the uprising threat that faces public health nowadays is the rapid spread of multidrug resistant (MDR) and extensively drug-resistant (or XDR) tuberculosis. Now about 3.5% of tuberculosis cases worldwide are produced by MDR mycobacteria, and in certain world regions (African countries, several Chinese and Russia provinces, Baltic states, etc.) their incidence greatly exceeds 10-20%. Therefore, the global spread of MDR tuberculosis is a problem of great medical and social importance.
  • Overall, tuberculosis is a ubiquitous disease that affects various living beings including animals, birds, and humans. It is generally ascertained that one-third of the world’s human population (about 2 billion people) is infected with M. tuberculosis.
  • However, humans demonstrate high natural resistance to tubercle bacilli; therefore, tuberculosis remains the social disease that strongly correlates with poverty, adverse living and working conditions and general economic decline.
  • The main sources of infection are persons with active tuberculosis. Sick animals (cattle) can also spread the disease.
  • The infection is transmitted predominantly by the airborne (air droplet) route and more seldom by contact route. Ingestion of contaminated foodstuffs, usually milk, is also possible especially for M. bovis infection. Very seldom the disease can be transmitted by direct inoculation that may occur among health care workers.
  • Overcrowding, malnutrition and starvation, inaccessibility of medical care and other hard socioeconomic conditions, as well as suppression of patient’s immune system (e.g., by HIV infection), are in the direct relationships with tuberculosis susceptibility and pathogen dissemination.
  • After primary penetration mycobacterial infection usually remains latent. Without prophylaxis about 5-10% of infected persons produce tuberculosis disease.
  • Inhaled mycobacteria are ingested by lung macrophages and transported to regional bronchial lymph nodes. M. tuberculosis survives within phagocytes, blocking phagolysosome fusion. Cord-factor inhibits cell migration within the inflammatory focus.
  • Primary lung tuberculosis is characterized by acute exudative lesion affecting lung acinary tissue with subsequent rapid involvement of lymphatic vessels and regional bronchial lymph nodes (primary tubercular complex).
  • Cell-mediated inflammation leads to formation of tubercular granulomas (tubercles) with caseous necrosis in their centers. Multinucleated giant cells, epithelioid cells, lymphocytes, macrophages and fibroblasts surround inflammation focus with mycobacteria.
  • Typical symptoms of progressive pulmonary disease include intoxication, fever, productive cough with hemoptysis, enlargement of lymph nodes, and abnormal results of chest X-ray examination.
  • Cellular immune reactions restrict inflammation area and terminate the propagation of M. tuberculosis. Primary specific process is resolved with connective tissue progression, fibrosis, and calcification. Remaining live mycobacteria come into dormant state and usually persist intracellularly within macrophages or epithelial cells lifelong.
  • In case of decreased immune resistance especially combined with large microbial load further spread of pathogen is possible via bronchi, lymphatic and blood vessels.
  • Disseminated disease affects lungs (miliary tuberculosis) or leads to extrapulmonary tuberculosis (tuberculosis of eyes, intestine, kidneys, tubercular meningitis, etc). These forms are much more severe and may cause patient’s death.
  • Reactivation of viable mycobacteria is possible due to many unfavorable external or internal stimuli, and easily affects immunocompromised persons (e.g., HIV patients).
  • Secondary tuberculosis is characterized by chronic tissue lesions (tubercles, cavities with caseous necrosis, etc.), followed by disseminated fibrosis. Secondary lesions are very difficult in treatment showing no tendency to self-recovery.
  • Immunity in tuberculosis is predominantly cell-mediated and non-sterile, maintained by viable mycobacteria. Macrophages, dendritic cells and Th1 cell subsets produce the vast number of proinflammatory cytokines (e.g., γ-interferon), thereby inhibiting microbial propagation. Antibodies are not proven to possess substantial antimicrobial activity.

Laboratory Diagnosis of Tuberculosis


  • Patient’s sputum, lymph nodes puncture contents, urine, pleural or cerebrospinal fluid, etc. are used for bacteriological examination. Conventional methods comprise the acid-fast stain, culture, and biochemical tests for detecting and identifying M. tuberculosis.
  • However, rising spread of tuberculosis with high incidence of MDR mycobacteria has required new rapid laboratory tests for M. tuberculosis identification.
  • Microscopy of acid-fast bacilli is a valuable primary test for laboratory diagnosis of the disease. Tubercle bacilli stain red by fuchsin in Ziehl-Neelsen method due to the remarkable acid resistance of bacteria.
  • Fluorescent microscopy is a more sensitive method than Ziehl-Neelsen technique. Mycobacteria easily stain with luminescent dyes (auramine or rhodamine).
  • In case of small amounts of pathogen the clinical sample is treated to enrich microbial content. The material is digested with a mucolytic agent (e.g., N-acetyl-L-cysteine) and treated with sodium hydroxide that kills acid-sensitive microorganisms. After centrifugation the smears from the sediment are prepared and stained.
  • Nevertheless, microscopy can reveal only 104-105 microbial cells.
  • For culture isolation both solid and liquid media may be used.
  • After sulfuric acid treatment specimens are usually inoculated into egg-based media (e.g., Lowenstein-Jensen agar, Finn medium, etc.) After long-term cultivation bacteria are identified by cultural, biochemical and virulent properties.
  • M. tuberculosis growth appears in 15-60 days resulting in typical dry colonies with “ivory”-colored pigment (R forms). Bacteria grow only at 37-38°С, being unable to propagate in ordinary media or when treated with salicylates. M. tuberculosis carries thermolabile catalase, produces urease, and reduces nitrates into nitrites. Guinea pigs are very sensitive to M. tuberculosis.
  • Cord factor production is essential for M. tuberculosis. It is estimated by Pryce’s microculture method using several narrow glass slides placed into citrate blood. After 4-5 days of cultivation slides are stained by Ziehl-Neelsen acid-fast stain. Cord factor elicits “serpentine cord”-like aggregations of microbial cells visible on microscopy.
  • Mycobacterium bovis grows within about 40 days. Bacterial growth renders smooth round colonies, or S forms. Microbial cells produce thermolabile catalase, urease, but can’t reduce nitrates into nitrites. M. bovis is highly virulent for rabbits.
  • Atypical mycobacteria (e.g. Mycobacterium avium-intracellulare complex, M. kansasii, M. microti, M. ulcerans, etc.) are virulent in S form, can grow at 22-45oС and in salicylate presence, produce orange pigment, carry thermostable catalase, being lack of cord factor and urease.
  • Acid-fast saprophytic mycobacteria (e.g., M. smegmatis) are non-virulent, microbial growth evolves within 3-4 days; bacteria propagate in ordinary media resulting in S-form colonies with orange pigment.
  • Rapid advanced cultural methods like BACTEC radiometric broth system greatly accelerate identification of M. tuberculosis taken in minimal amounts.
  • By this method the infection is detected in 7-8 days of culturing.
  • For rapid mycobacteria identification in clinical specimens polymerase chain reaction (PCR) with specific primers is used. This method is the most promising technique for express-detection of virulent mycobacteria.
  • Experimental animal infection has only a little worth for diagnosis. Similar, determination of specific antibodies against M. tuberculosis is also of limited value due to low specifity of serological reactions in tuberculosis.
  • Tuberculin skin test (TST) or Mantoux reaction evaluates delayed hypersensitivity in M. tuberculosis infection. Tuberculin is a multi-component antigenic complex of M. tuberculosis, composed of various tuberculoproteins and wax fractions.
  • R. Koch obtained it as glycerol-based filtrated suspension of killed tubercle bacilli. He applied it for tuberculosis treatment but without evident success.
  • Nevertheless, tuberculin was proven to be worthy for diagnostics of tuberculosis. It was further purified to derive protein fractions (tuberculin PPD or purified protein derivative).
  • After intracutaneous injection of a definite amount (usually 2 tuberculin units) of tuberculin PPD to the patient previously exposed to M. tuberculosis, the papule (induration and redness) appear, being maximal in 24-72 hours.
  • A positive TST indicates that the person has been infected with M. tuberculosis. Test explanation may be difficult, because previous BCG vaccination, specific chemotherapy, or host immune status can influence the reaction. However, test conversion from negative to positive implies recent infection and possible current activity of tuberculosis.
  • A positive skin test assists in diagnosis, and it is also helpful for evaluation of tuberculosis treatment.
  • More advanced version of allergic skin test in tuberculosis is recently devised Diaskintest (DST). It uses recombinant proteins of M. tuberculosis CFP-10 and ESAT-6 as infectious mycobacterial allergens for intracutaneous injection.
  • This test has serious advantages against TST as it is not influenced by primary BCG vaccination (BCG bacilli are lack of CFP-10 and ESAT-6 virulence proteins).
  • Blood lymphocyte culture tests like interferon-gamma release assay (IGRA test) are also of rising value in immunological diagnosis of tuberculosis. They determine patient’s lymphocyte sensitization to M. tuberculosis.
  • The test is based on detection of γ-interferon release after the challenge of lymphocyte culture with specific micobacterial antigens.
  • Finally, adequate patient management in tuberculosis is impossible without rapid antibiotic susceptibility testing of isolated M. tuberculosis culture.
  • For these purpose both cultural and genetic methods are used. In the latter case PCR (like in GeneXpert system) and hybridization techniques are employed to detect bacterial genes, conferring the resistance to antimicrobial drugs.

Specific Treatment and Prophylaxis of Tuberculosis


  • Treatment course of tuberculosis patient lasts from 6 to 12 months. Long duration of treatment period ensues from slow metabolism of tubercle bacilli as they tend to intracellular persistency and permanent evasion from host immune system.
  • Very short list of antimicrobial drugs was proven to be effective in tuberculosis treatment.
  • They pertain to the first line drugs. Among them are isoniazid, rifampicin, pyrazinamide, ethambutol, and streptomycin. Pyrazinamide is a sole antimycobacterial drug that can affect intracellular forms of M. tuberculosis.
  • Combined use of the first line drugs for 6 months of so-called “short chemotherapy course”, recommended by WHO, yields cure rates rather than 80-90% and prevents the emergence of drug resistance.
  • The second line preparations (e.g., fluoroquinolones, ethionamide, cycloserine, etc.) demonstrate lower efficacy, but often increased toxicity, being of more seldom use.
  • The treatment of MDR and XDR tuberculosis poses serious difficulties.
  • MDR bacteria are resistant to isoniazid and rifampicin, whereas XDR microbial cells are additionally resistant to fluoroquinolones and one more drug of the second line.
  • In these clinical cases the treatment course may last for 1.5-2 years.
  • The worsened situation with a highly limited list of efficient drugs against tuberculosis stimulated the design of novel antimycobacterial agents.
  • Some of them are already introduced into clinical practice (e.g., bedaquiline and delamanid). They are predominantly administered in MDR and XDR tuberculosis.
  • Non-specific prophylaxis of the disease is achieved by isolation and adequate treatment of tuberculosis patients. Hospital disinfection is made by 5% carbolic acid or chlorine-containing disinfectants.
  • Vaccination with live attenuated BCG vaccine is used for specific prophylaxis of tuberculosis. Vaccine contains avirulent strain of M. bovis, obtained by A. Calmette and Ch. Guerin after 13-year continuous bacterial passage through bile-containing media.
  • Newborn infants undergo primary vaccination at 3-5 day of life. Human immunization with BCG vaccine reduces the risk of tuberculosis in vaccinated persons by about 50%.