Mycobacterium tuberculosis

Mycobacterium tuberculosis

Mycobacterium tuberculosis is the etiologic agent of tuberculosis. This bacterium, belonging to the Mycobacteriaceae family, was discovered in 1882 by Robert Koch and, as such, it is also called Koch’s bacillus.

In 1920, Albert Calmette and Camille Guérin developed a vaccine against tuberculosis: BCG or Bacillus Calmette-Guérin, a strain of Mycobacterium bovis that lost the virulence by successive subcultures. In 1944, Selman Waksman discovered the first antibiotic active against Mycobacterium tuberculosis. Then, other drugs, active against this Bacillus, like isoniazid or rifampicin, were discovered.

Characterization

Mycobacterium tuberculosis are immobile bacilli whose dimensions range from 0.2 to 0.3 µm in diameter and 3 to 5 µm in length, slightly curved and with rounded edges. They are aerobic bacteria which have neither capsule nor spores and they do not produce toxins.

Koch’s Bacillus is sensitive to heat, to UV radiation and x-rays. The 70º alcohol destroys it in 5 min. However, it is resistant to cold and drying, to dilute acids, to antiseptics and to detergents.

Despite having the structure of a Gram-positive bacterium, it colours hardly through this technique (it’s the case of all Mycobacteria). This is due to the fact that the cell wall of Mycobacteria is very rich in structural lipids that provide a great hydrophobicity. Thus, during the process of Gram stain, the action of the mordants and the aqueous differentiators is hampered.

The large amount of lipids gives to these bacilli a property called acid-alcohol resistance. They are visualized by Ziehl-Neelsen method, during which the primary dye, carbol fuchsin, fixes tightly to the lipids of the cell wall and is not removed by mixing alcohol and hydrochloric acid. The acid-alcohol resistant bacilli then appear stained in red while the non acid-alcohol resistant bacteria appear blue, the colour of the methylene blue counterstain.

Culture

The culture time of Mycobacterium tuberculosis is very slow; its doubling time is about 20 hours. Its culture requires an enriched medium, for example Löwenstein-Jensen medium (used in the isolation of Mycobacteria), a solid medium which incorporate whole egg, mineral salts, glycerin, asparagine, potato starch and malachite green.

After about 21 days of culture, in aerobic conditions, Mycobacterium tuberculosis colonies can be observed. These colonies are beige in colour, dry, with a rough surface and with a cauliflower form characteristic.

Identification techniques

The standard identification methods of Mycobacteria are culture and morphological analysis of colonies on solid media associated with the use of biochemical tests. In the identification of Mycobacterium tuberculosis, three tests are used: a positive niacin test, a positive nitrate reductase test and also a weak catalase activity at 22ºC which disappears at 68ºC. However, these tests are time consuming and laborious.

Molecular probe technique is currently the most used to identify and distinguish Mycobacteria. These probes, produced commercially, are quick use (about 2 hours), sensitive and specific.

Habitat

The natural reservoir of Mycobacterium tuberculosis is humans and its transmission is done directly by air, through saliva droplets, called Flügge droplets, expelled by the patient when coughing, sneezing or speaking. About 2 million of Koch bacilli are expelled by a patient suffering from tuberculosis when he sneezes. Occasionally, domestic animals, dogs and cats, can be contaminated.

Mechanism of infection

A patient suffering from tuberculosis with pulmonary infection expels, speaking, sneezing or coughing, Flügge droplets which carry 1 to 3 Koch bacilli (by droplet). These droplets, inhaled by healthy individuals, reach the pulmonary alveoli.

The bacilli are phagocytised by circulating macrophages, within which they multiply; they are released after the bursting of the cells and, phagocytised again.

Meantime, the macrophages release cytokines that recruit other macrophages and other immune system mononuclear cells that clump together around the primary infectious outbreak to form a tuber. This tuber can remain intact for several months or even years; it can decrease in size or calcify. In 90% of cases, the infection is asymptomatic and is called latent primary infection.

In all other cases, if the immune system is weakened, the disease can develop. The bacilli can cross the barrier of the alveoli and be expelled by the patient (patent primary infection).

After days, months or years, an individual who pass by an immunocompromised state can suffer a secondary infection, whether by reactivation of a latent focus (endogenous) or by reinfection (exogenous), and develop the disease.

In rare cases, the bacilli migrate and infect other organs (extrapulmonary infection) as the lymph nodes, bones, kidneys or, in a more serious form, the meninges.

References:

• Alexander, D.C. and Liu, J.. (2006). Mycobacterial genomes. In: Chan, R.V.L. et al. Bacterial Genomes and Infectious Diseases. New Jersey: Humana Press Inc. p151-174.
• Cole, S.T. et al. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 393, p537-544.
• Gengenbacher, M. and Kaufmann, S. (2012). Mycobacterium tuberculosis: success through dormancy. FEMS Microbiology Reviews. 36 (3), p514-532.
• Murray, P. et al. (2013). Mycobacterium. In: Medical microbiology. 7th ed. Philadelphia: Elsevier Inc. p235-347.