Mycobacterium

            Mycobacteria are slender rods, sometimes show branching filamentous forms. In liquid cultures they form a mold-like pellicle, hence the name mycobacteria, meaning fungus-like bacteria. They do not stain readily, but once stained resist decolurisation with dilute mineral acids, therfore they are called “Acid Fast Bacilli” or AFB. They are aerobic, nonmotile, noncapsulated, nonsporing. Generally slow growth, obligate parasites, opportunistic pathogens and saprophytes.

Important pathogenic species are Mycobacterium tuberculosis, Mycobacterium leprae

M. tuberculosis – A straight or slightly curved rod, occuring singly, in pairs or small clumps. Tubercle bacilli after staining with basic dyes they resist decolourisation by alcohol. When stained with carbol fuchsin by the Ziehl-Neelson method, they resist decolourisation by sulphuric acid and alcohol. Acid fastness is due to the presence of mycolic acid. Electron micrographs show that the thick wall is composed of three layers enclosing a trilaminar plasma membrane.

            It grow slowly, the generation time is 14-15 hours. Colonies appear in about 2 weeks, sometimes 8 weeks. Optimum temperature is 37⁰C, no growth below 25⁰C or above 40⁰C, optimum pH is 6.4-7.0. It is an obligate aerobe. The addition of 0.5% glycerol improves the growth of the tubercle bacilli, but not to other species. They are sensitive to traces of toxic substances like fatty acids in culture media. The toxicity is neutralised by serum albumin. The solid media containing egg, Lowenstein-Jensen(LJ) medium without starch is the recommended media. It consists of coagulated hen’s egg, mineral salt solution, asparagine, malachite green(a selective agent inhibiting other bacteria). Liquid media generally not employed for routine cultivation, but are used for sensitive testing, chemical analysis, preparation of antigens, vaccines.

            On solid media, M. tuberculosis  forms dry, rough, raised, irregular colonies with a wrinkled surface. They are creamy white, becoming yellowish or buff coloured on further incubation. They are not easily emulsified. In liquid media the growth begins  at the bottom, creeps up the sides and forms a prominent surface  pellicle which may extend along the sides  above the medium.

            Mycobacteria are killed at 60⁰C in 15-20 minutes. Survival is influenced by the material in which the bacteria are present. Cultures may be killed by exposure to direct sunlight for 2 hours, but bacilli in sputum may remain alive for 20-30hours. They are resistant to chemical disinfectants, survive 5% phenol, 15% H₂SO₄, 3% nitric acid, 5% oxalic acid, 4% NaOH. They are destroyed by tincture of iodine in 5 minutes and by 80% ethanol in 2-10 minutes. Ethanol is a suitable disinfectant  for skin, gloves and clinical thermometers.

Biochemical Reactions

Niacin test- Human tubercle bacilli form niacin when grown on an egg medium. When 10% cyanogen bromide  and 4% aniline in 96% ethanol are added to a suspension of the culture, a canary yellow colour indicates a positive reaction. The test is positive with human type, so useful in identifying human strains.

Aryl Sulphatase Test- Positive with atypical bacteria only. The bacilli are grown in a medium containing 0.001 tripotassium phenolphthalein disulphate, then 2N NaOH is added drop by drop to the culture, a pink colour indicates a positive reaction.

Neutral Red Test – Virulent strains of tubercle bacilli are able to find neutral red in alkaline buffer solution, while avirulent strains are unable to do so.

Catalase- Peroxidase Test- to differentiate tubercle bacilli from atypical mycobacteria, also provides an indication of the sensitivity of the strain to isoniazid. Atypical mycobacteria are strongly catalase positive, but peroxidase negative, while tubercle bacilli are weakly catalase positive and peroxidase positive. Both these activities are lost when tubercle bacilli become INH resistant. A mixture of equal volumes of 30vol.H₂O₂ and 0.2% catechol in distilled water is added to 5ml. Of the test culture and allowed to stand for few minutes. Effervescence indicates catalase production and browning indicates peroxidase activity.

Amidase Test- The ability to split amides has been used to differentiate mycobacteria. Five amides such as acetamide, benzamide ,carbamide, nicotinamide and pyrazinamide are tested.

A 0.00165M solution of the amide is incubated with the bacillary suspension at 37⁰C and 0.1ml of MnSO₄.4H₂O, 1ml of phenol solution and 0.5ml hypochlorite solution added. The tubes are placed in boiling water for 20 minutes. A blue colour indicates a positive test.

Nitrate Reduction test- Here nitrate is reduced to nitrite by the enzyme reductase, M. tuberculosis is positive with the formation of red colour.

Antigenic Properties

            Following infection of tubercle bacilli delayed hypersensitivity is developed to the  bacillary protein, tuberculin. Also polysaccharide, protein antigens present. They also produce bacteriocins.

Pathogenicity

             Tubercle bacilli causes natural infection in humans, other primates, dogs. M. bovis produces tuberculosis in cattles, dogs and cats, also in humans. The disease is acquired by the inhalation of droplet nuclei of respiratory secretions or particles of dry sputum containing tubercle bacilli. After inhalation they multiply very slowly inside WBC that have phagocytized them. They stimulates a host response that includes neutrophil infiltration and fluid accumulation within the alveoli of the lungs. The organisms eventually rupture and destroy the neutrophils. Later macrophages and lymphocytes move into the area. Alveolar macrophages also phagocytize the living tubercle bacilli, which are again able to multiply within and destroy their new hosts. Rupture of phagocytes releases infective organisms. No toxins, but an acute inflammatory response occurs as additional cells are infected. A large quantity of fluid is released, especially in lung tissue, where it produces pneumonia-like symptoms. Sometimes lesions heal, if not massive tissue necrosis or solidify to become chronic granulomas or tubercles. Tubercles consist of central accumulations of macrophages, multinucleate Langerhans giant cells containing tubercle bacilli, peripheral lymphocytes, macrophages and newly formed connective tissue. The central portion of the tubercle undergoes destruction, giving it a cheesy appearance. Some organisms gain access to the lymphatic and circulatory systems. About 3-4 weeks after exposure, delayed hypersensitivity and cell mediated immunity develop. On occasion host fails to respond immunologically. Then uncontrollable multiplication of tubercle bacilli takes place in the lungs, therefore numerous tubercles. Organisms spread by the circulatory system to other body tissues, this condition is miliary tuberculosis(as it resembles millet seeds). Lesions near blood vessels perforate the vessels and cause hemorrhage, leads to bloody sputum, a major symptom.

            Granulomas keep viable organisms walled off by encapsulation for decades. When the immune system becomes impaired by age or other infections , granulomas open and the disease can be  reactivated,eg., people with AIDS have TB. TB on bones can cause extensive erosion, especially in the spine. The urogenital tract , meninges, lymphatic system, peritoneum are prone to develop extrapulmonary TB. In disseminated TB (frequent in AIDS patients) infected cells' organelles and membranes destroyed by  multiplying bacilli and a clump of bacilli in the shape or cast of the cell remains. When this process occurs in the intestine, a replica of the intestine consisting of live Mycobacterium can be seen. Today digestive tract TB occurs as a secondary infection site, when pathogen-laded sputum coughed up from a primary lung infection is swallowed.

Laboratory Diagnosis

            Laboratory diagnosis of TB may be established by demonstrating the bacillus in the lesion, sputum  culturing etc..

            For pulmonary TB, the specimen tested is sputum, which is collected in the morning before any meal. If scanty 24 hrs sample may be tested. Sputum sampling on 3 days increases chances of detection. If sputum is not available, laryngeal swabs, bronchial washings may be collected, and in small children gastric lavage collected.

Microscopy : Direct or concentrated smears of sputum examined. New slides should be used for smears, and should not be reused, as AFB may not removed from slides by cleaning. Smears are dried, heat fixed, stained by Ziehl- Neelson technique,ie., the smear is covered with carbol fuchsin, gently heated to steaming for 5-7 minutes(stain shouldn’t boil or dry), slide is then washed with water and decolourised with 20% H₂SO₄, then with 95% ethanol for 2 mts. After washing the smear is counterstained with Loeffler’s Methylene Blue, 1% picric acid or 0.2% malachite green for 1 minute. Under the oil immersion, AFB seen as bright red rods, while the background is blue, yellow or green. At least 100 fields examined and report expressed as 1+ if 3-9 bacilli, 2+ if 10 or more and 3+ if 10 or more in oil-immersion.

Concentration methods : Petroff’s method- sputum incubated with an equal volume of 4% NaOH solution at 37⁰C with frequent shaking till it becomes clear. It is then centrifugedat 3000rpm for 20 mts, sediment neutralised with N/10 HCl and used for smear, culture, animal inoculation.

Culture : The concentrated material is inoculated onto LJ medium. Cultures examined for growth after incubation at  37⁰C for 4 days. Any growth is smeared, tested by ZN staining, confirmation by biochemical studies. Culturing can be detected 10-100 bacilli/ml.

Sensitivity tests : are of 3 types-(a) Absolue concentration method in which a number of media containing serial concentrations of the drugs are inoculated, minimum inhibitory concentrations calculated. (b) Resistance ratio method in which 2 sets of media containing graded concentrations of the drugs are inoculated, one set with test strain and other with a standard strain. (c) Proportion method which indicates the average sensitivity of the strain.

Animal Inoculation : The concentrated material is inoculated intramuscularly into the thigh of two healthy guinea pigs. Animals weighed before and after inoculation. Progressive loss of weight is an indication of infection. Infected shows positive tuberculin skin reaction. One killed after 4 weeks and autopsied, other after 8 weeks.

Nucleic Acid Technology : PCR and LCR used as diagnostic techniques. Demonstration of mutation in drug sensitivity genes is a useful indicator of drug resistance.

Immunodiagnosis : Antibody-Antigen reaction studied.

For extrapulmonary TB also microscopy, culture are used for diagnosis. For the allergic tests tuberculin test is done.

Treatment & Prevention

            In the prevention of TB, measures such as adequate nutrition, good housing and health education are important.

Immunoprophylaxis is by intradermal injection of the live attenuated vaccine developed by Calmette and Guerin in 1921, at Pasteur institute in Paris,the Bacilli Calmette Guerin or BCG. This is a strain of M. bovis attenuated.

Chemoprophylaxis is the administration of  antituberculous drugs such as isoniazid and rifampin for at least one year. But atypical forms in AIDS patients are now resistant to isoniazid. Antituberculous drugs are of two types, bactericidal and bacteriostatic

Bactericidal Drugs – Rifampicin, Pyrazinamide(sterilizing drugs- effectively kill bacteria in the lesions). Isoniazid- effective only against replicating bacilli. Streptomycin- only against extracellular bacilli.

Bacteristatic Drugs – Ethambutol

If both these are combined and given, it is first line drugs in the therapy. The long course treatment has been replaced by short course, ie., 7 months, which are effective and convenient. A very serious consequence of unchecked drug resistance to the spread of Multidrug Resistant TB(MDR-TB)

M. leprae – It causes leprosy or Hansen’s disease, recognised from vedic times and Biblical times. Leprosy has always been held in superstitious, and the person suffering from leprosy considered ‘unclean’ and a social outcaste. The leprae bacilli was observed first by Hansen.

            The bacilli is a straight or slightly curved rod. Polar bodies and other intracellular elements may be present. Clubbing, lateral buds,branching may be observed. It is Gram positive and Acid Fast, but not as tubercle bacillus, So 5% H₂SO₄  employed for decolourisation. It has not so far been possible to cultivate leprae bacilli either in bacteriological media or in tissue culture. But recently they are found growing in nine-banded armadillos, chimpanzees, mangabey monkeys, footpad of mice, made the bacterium available for research. It reproduces very slowly,ie.,12 day division cycle. The genome has been mapped and the genes for its major protein antigens cloned and sequenced. It may now be possible to develop a vaccine. They remain viable in a warm humid environment for 9-16 days and in moist soil for 46 days. They survive exposure to direct sunlight for 2 hours and UV light for 30 minutes..

Pathogenicity

            Leprosy is a chronic granulomatous disease of humans primarily involving the skin, peripheral nerves, nasal mucosa, but capable of affecting any tissue or organ. The disease may be classified into 4 types, which is a reflection of the immune status of the host.

1. Lepromatous : It is seen where the host resistance is low. Superficial nodular lesions develop which consist of granulation tissue containing a dense collection of vacuolated cells. The nodules ulcerate, becomes secondarily infected and cause distortion and mutilation. Incubation period is 9-12 years. Bacilli invade the mucosa of the nose, mouth and upper respiratory tract and are shed in large numbers in nasal and oral secretions. The bacilli are seen in large numbers inside lepra cells or extracellularly.

2. Tuberculoid : Seen in patients with a high degree of resistance, also known as anesthetic form, here areas of skin lose pigment and sensation. Bacilli are scanty in lesions. Incubation time averages 2-5 years.

3. Dimorphous : Lesions possessing characteristics of both tuberculoid and lepromatous types. Depending on alterations in host resistance, they may shift to tuberculoid or lepromatous type.

4. Indeterminate : Not characteristic of tuberculoid or lepromatous type. This type of lesions undergo healing spontaneously. In some, the lesions progress to tuberculoid or lepromatous types.

     Indian classification includes pure neuritic type, shows neural involvement.

            M. leprae destroys skin and mucus membranes. The organism seen in ears, nose and fingers, except central nervous system. Large numbers of bacilli are shed in respiratory secretions and in pus discharged from lesions. The shedding of organisms transmits the disease to those with in close contact with patients, such as children of infected parents. As disease progresses, it deforms hand and feet. Severe lepromatous disease erodes bone, fingers and toes become needle-like, pits develop in the skull, nasal bones are destroyed, teeth fall from the jaw as bone surrounding them is lost. Sometimes surgery can restore the use of extremely crippled hands and feet.

Immunity- Infection with lepra bacilli induces both humoral and cellular immune responses. Humoral antibodies do not have deleterious effect on the bacilli, while cellular immune mechanisms are capable of destroying them.

Lepromin test : This is a skin test for delayed type of hypersensitivity. The original antigen, lepromin was boiled, emulsified and given as intradermal injection. The test is employed to classify the lesions of leprosy patients. The test is positive in tuberculoid, negative in lepromatous and variable in other types.

Laboratory Diagnosis

            Hansen’s disease is diagnosed by PCR, or by finding the organism in acid-fast stained smears and in scrapings from lesions or biopsies. For routine examination, specimens are coleected from the nasal mucosa, skin lesions and ear lobules. A blunt, narrow scalpel is introduced into the nose and the internal septum scrapped sufficiently to remove a piece of mucus membrane, which is transferred to a slide and made into a uniform smear. Samples from the skin should be obtained from the edges of the lesion. In Ziehl-Neelson technique smears are graded,

1-10 bacilli in 100 fields = 1+

1-10 bacilli in 10 fields   = 2+

1-10 bacilli per field       = 3+

10-100 per field             = 4+

100-1000 per field         = 5+

1000                               = 6+

            The bacteriological index or BI calculated by totalling the number of pulses scored in all smears, divided the number of smears.

Treatment – Dapsone, Clofazimine, Rifampin are the chemotherapeutic agents used in therapy, but dapsone resistant forms had appeared. Treatment reduces nodules of lepromatous tissue, but cannot restore lost tissue. Vaccines not available. Leprosy is not highly communicable. Case finding and adequate therapy are essential for control.