In this article we will discuss about Atypical Mycobacteria which causes Lung Diseases in Humans:- 1. Classification of Atypical Mycobacteria 2. Culture of Atypical Mycobacteria 3. Biochemical Test 4. Saprophytic Mycobacteria 5. Ecology and Epidemiology 6. Pathogenicity 7. Clinical Features 8. Pulmonary Disease 9. Skin lesions 10. Laboratory Diagnosis and Other Details.
Contents:
- Classification of Atypical Mycobacteria
- Culture of Atypical Mycobacteria
- Biochemical Test for Mycobacteria
- Saprophytic Mycobacteria
- Ecology and Epidemiology of Mycobacteria
- Pathogenicity
- Clinical Features of Mycobacteria
- Pulmonary Disease
- Skin lesions
- Laboratory Diagnosis
- Bacteriophage
- Antigenic Structure of Atypical Mycobacteria
- Mycobacterium Leprae
- Laboratory Diagnosis
1. Classification of Atypical Mycobacteria:
Atypical mycobacteria were first classified into four groups by Runyon (1959) on the basis of production of pigment and rate of growth with include group I—Photochromogens; group II—Scotochromogens, group III—Non-photo-chromogens and group IV—Rapid growers. Runyon’s classification is widely accepted.
2. Culture of Atypical Mycobacteria:
Atypical mycobacteria grow in Lowenstein-Jensen (LJ) medium, sometimes at lower (25°C) or higher (41 °C) temperatures than normal. Colonies appear in 2 to 3 weeks except rapid growers which produce colonies in 4 to 5 days. Photo-chromogens produce pigment when colonies are exposed for one hour in under light and re-incubated for 24 to 48 hours. The pigment is chemically beta-carotene and yellow orange in colour.
The scoto-chromogens produce pigmented growth in the dark and non-photo-chromogens (or non-chromogens) produce pigmentation not related to exposure to light. The rapid growers produce colonies within 4 to 5 days after incubation M. Kansasii and M. intracellular are the common atypical mycobacteria which are the common atypical mycobacteria which are responsible for the production of the disease (Table 40.1).
3. Biochemical Test of Atypical Mycobacteria:
Different species of atypical mycobacteria can be distinguished by their growth at 20°, 25°, 35°, 42° or 45° Celsius; aryl sulphatase test; pigment production, catalase test; nitrate test; Tween hydrolysis; urease test; iron up take; growth rate and agglutination test.
Group I—Photochromogens M. kansaii:
In patients with preexisting lung disease and with impaired immune response, M. kansasii causes pulmonary and systemic disease. The lesions are similar to tuberculosis. Transmission from man to man is not yet known; however, the bacilli are distributed in soil, water and milk. Their pigment is yellow-orange in colour in air under light. M. kansasii are sensitive to rifampicin and other anti-tubercular drugs.
M. simiae:
They are ordinally isolated from pulmonary lesions of monkeys, grow well at 37°C, synthesise niacin like M. tuberculosis, thus they may be falsely identified as M. tuberculosis.
M. marinum (M. balnei):
They are the causative agents of a superficial granulomatous skin disease of man known as swimming pool granuloma or fish tank granuloma which is acquired through contaminated water. They grow at 33°C but not at 37°C, they are nitrate negative, hydrolyse pyrazinamide and show a-L-fucosidase activity.
Group II—Scotochromogens:
M. scrofulaceum
It is a common cause of chronic cervical lymphadenitis (scrofula) in children. In the dark, it produces yellow, orange, red pigmented colonies. It also occurs in the environment as saprophyte.
M. Szulgai:
In 1972, Szulga of Poland discovered M. szulgai causing pulmonary disease and bursitis. At 37°C incubation, it is a scotochromogen; but at 25-27°C, it is photo-chromogenic. Hence it is an unusual scotochromogen.
M.gordonae:
Though this bacterium was known formerly as M. aquae Paa it was named in honour of Dr. Ruth Gordon, a pioneer of mycobacterial taxonomy, as M. gordonae. It was also called tap water scotochromogen as it is very frequently found in water and is a common contaminant of the clinical material. It rarely causes pulmonary disease in man.
Group III—Non-photo-chromogens:
The medically important non-photo-chromogens are M. intracellular, M. avium, and M. xenopi.
M. intracellular:
This organism was first detected in the Battey State Hospital for tuberculosis, USA. Hence it was called as Battey bacillus. It causes chronic pulmonary disease in man, indistinguishable from tuberculosis, which is common in soil and water and about 50% population show positive skin reaction to PPD.
M. avium:
Mostly fowls and sometimes pigs suffer from tuberculosis due to M. avium. In man, the infection due to M. avium is not common; however, in immuno-compromised patients and in children, this organism may cause overt pulmonary disease and cervical lymphadenitis, respectively.
The infection occurs commonly in farmers and their children and also in man with silicosis. These bacteria are thermopiles and grow best at 41°C.
M. avium-intracellular complex:
M. intracellular is closely similar to M. avium and these two are considered to be variants of a single group named M. avium-intracellular (MAI) complex. They are commonly found in soil. Though they are resistant to anti-tuberculosis drugs, they respond to the combined therapy of rifampicin and streptomycin.
M. xenopi:
Though they are originally recovered from xeno pus toads, they produce chronic lung disease in man. They have been also isolated in hot water taps of hospital and they have a limited geographical distribution. In London, South-East England and France, cases of pulmonary lesions due to M. xenopi have been reported. These organisms are thermophiles and grow best at 45°C.
M. ulcerans:
These organisms were isolated from skin lesions of man in Australia and Uganda and are identical to M. buruli. Hence the name of the disease is buruli ulcer. They are saprophytes in the environment and grow in vitro at a temperature of 31-34°C.
M. malmoense:
In 1977, they were isolated in Malmo (Sweden) from patients suffering from pulmonary disease and lymphadenitis. On primary isolation their colonies appear only 10-11 weeks after inoculation.
Group IV—Rapid Growers:
M. fortuitum and M. cheloneii were originally identified as the frog tubercle bacilli and the turtle tubercle bacilli, respectively. Both of them were found in water and soil, grow rapidly at 37°C or 45°C and their colonies appear in 4-6 days and are medically important as they are principally responsible for post-injection abscesses and wound infections.
Pulmonary lesions are sometimes produced by M. fortuitum and are indistinguishable from tuberculosis. M. smegmatis and M. phlei are saprophytes and chromogenic rapid grower.
4. Saprophytic Mycobacteria:
M. phlei and M. gordonae are saprophytes found in soil, water and plants. Whereas M. smegmatis is found in smegma and sebaceous secretion. These organisms are chromogenic rapid growers and may be confused with pathogenic acid fast bacilli.
5. Ecology and Epidemiology:
(A) Distribution:
The atypical mycobacteria are commonly distributed in the environment (soil, marshland, streams, rivers and estuaries). Hence they are also called environmental mycobacteria. False positive sputum findings may be due Ziehl-Neelsen staining reagents prepared by water contaminated with M. terrae, M. marinum, M. gordonae, M. kansasii and M. xenopi.
(B) Environment and Man:
Human beings are often exposed to environmental mycobacteria by drinking contaminated water, washing, showering and inhalation of aerosols. Repeated exposure to these atypical mycobacteria may cause subclinical infection in man that may
(1) Induce the sensitisation to tuberculin and other mycobacterial skin testing reagents;
(2) Affect the subsequent ability of BCG vaccine to induce protective immunity.
6. Pathogenicity:
Though the transmission of atypical mycobacteria from man to man is very rare, its transmission from environment is common. Due to their low virulence, the incidence of overt disease is very low in countries where there is growing number of immuno-compromised patients with AIDS infection. In several European countries, M. malmoense infection is very high.
7. Clinical Features:
Though atypical mycobacteria are saprophytes from the environment, they sometimes cause disease in man and animals.
The diseases are:
Localised lymphadenitis, tuberculosis like pulmonary lesions and skin lesions (Buruli ulcer, swimming pool granuloma (Table). Localised cervical lymphadenitis
It commonly occurs from the tonsillar infection (tonsillitis) in children under 5 years of age. It is a self-limiting disease and can be diagnosed by histological examination of lymph node biopsy. M. scrofulaceum is causal agent in USA; whereas M. avium-intracellular and M. scrofulaceum are responsible for cervical lymphadenitis in Great Britain.
8. Pulmonary Disease:
Cystic fibrosis, malignant disease, damage acquired and congenital immune deficiencies are the predisposing factors for pulmonary disease. Sometimes, persons without underlying disorder may also be affected. This infection, which is indistinguishable from tuberculosis, is caused by M. avium-intracellular and M. kansasii.
These causative organisms must be isolated repeatedly from the sputum over a period of at least one week for the confirmative diagnosis in the laboratory and they should also be differentiated from M. tuberculosis.
9. Skin lesions:
Post Injection Abscesses:
M. cheloneii and M. fortuitum, rapid growers, cause abscesses following injection of drugs contaminated by these bacteria, abscesses appear within a week. They are 8-10 cm in diameter, painful, and last for many months. Farmers may get infected due to M. terrae when their wounds get contaminated with soil while farming.
Buruli Ulcer:
M. ulcerans is the causal agent of Buruli ulcer which was first described in Buruli district of Uganda where there was a large outbreak. Hence, its name was derived. This infection is common in marshy land with surface water of pH 6.1-6.9 and thorns of prickly grass. These contaminated thorns may introduce these organisms into the skin of the body. The leg is vulnerable to this infection.
A painful hard subcutaneous itchy nodule is first formed. This nodule enlarges, becomes soft at its centre, breaks down due to necrosis of the underlying tissue, discharges caseous material and, ultimately, forms a deep ulcer containing many acid fast bacilli.
M. ulcerans elaborates a toxin which cause necrosis of the skin of the experimental guinea pigs. The role of the toxin in Buruli ulcer formation is not yet clear. During the healing stage, acid fast bacilli disappear; cellular infiltration and granuloma formation ensue and the patient becomes immuno-reactive to burulin—a specific skin testing reagent.
Swimming Pool Granuloma:
M. marinum, previously known as M. balnei (balneum meaning bath) is a natural pathogen of cold blooded animals (fish) causing tuberculosis in fish and may also cause swimming pool granuloma in man due to the use of contaminated swimming pools or fish tanks.
The abrasions on the skin of the elbow, knees, ankles, fingers, toe or nose may get infected. The lesions are single or multiple and appear as wart. Though the disease is self- limiting, its resolution is hastened by the use of minocycline, rifampicin or cotrimoxazole.
10. Laboratory Diagnosis of Atypical Mycobacteria:
Microscopy:
It can be done by repeated microscopic examination of Ziehl Neelsen staining of sputum, pus or exudate. Positive smear will show acid fast bacilli.
Culture:
In Lowenstein-Jensen (LJ) and Dubo’s media, atypical mycobacteria grow well; but in ordinary laboratory media, some rapid growers will grow. For distinguishing species, several LJ media should be inoculated with the specimen and incubated in dark and light. When heavy growth of the atypical mycobacteria is repeatedly isolated, then only it should be separated as positive culture.
Allergy and Immunity:
Hypersensitivity (allergy) and immunity (resistance to infection) are two different manifestations of the same mechanism in tuberculosis as both are mediated by T-cells sensitized to bacterial antigen. Humoral antibodies have no influence in the course of the disease.
(1) In the non-immune host, M. tuberculosis are readily phagocytosed, but inside the mononuclear cells, they multiply and resist digestion. The intracellular parasitism is associated with the development of delayed hypersensitivity and of activated macrophages (within 10 days after infection) with an increased ability to destroy the ingested bacilli.
(2) After first infection, the host acquires some resistance against reinfection as evident from quick healing of local ulcer following reinfection of the guinea pig (Koch’s phenomenon). This is due to cell mediated acquired immunity that has led to localisation of tubercle bacilli, inhibition of their multiplication and prevention of their dissemination.
In the immune host the sensitized T-cells (developed during primary infection) proliferate and release lymphokines that make the macrophages bactericidal.
Delayed hypersensitivity can be induced by live, attenuated and killed M. tuberculosis, their products and tubercular-protein with a purified wax extract-protein (tuberculin) injection can detect the hypersensitivity status mycosides. Agglutination test can identify mycobacteria that form stable smooth suspensions.
M. avium-intracellular group has been extensively studied by agglutination test. The original claim that cord factor was a major determinant of virulence is not accepted. The cord factor consists of two mycolic acids linked to trehalose.
Cytoplasmic or Soluble Antigens:
These are proteins used to type the mycobacteria by precipitation test and are divided into four groups:
(a) Group I antigens present in all mycobacteria
(b) Group II antigens occur in slow growing mycobacteria
(c) Group III antigens in rapidly growing mycobacteria
(d) Group IV antigens in individual species of mycobacteria
Group I antigens can also be detected in Nocardia, Listeria and Corynebacterium. All these antigens are obtained from ruptured bacterial cells. When the protein antigen binds with a wax fraction, it elicits the tuberculosis reaction.
11. Bacteriophage:
Some tubercle bacilli are infected with temperate phage. Unlike true lysogenic bacteria, the phage genome appears like a plasmid in many mycobacteria. There are four main phage types of M. tuberculosis—types I, A, B and C; type I is intermediate between A and B.
In India and in neighbouring countries, the infection due to type I is very frequent. But the infection due to phage type A is distributed worldwide and is very common. Phage 33 D recovered from a lysogenic bacterium can kill strains of mycobacteria except Bacilli Calmette Guerin (BCG) mycobacterium.
12. Antigenic Structure of Atypical Mycobacteria:
Antigens of mycobacteria are:
(1) Cell wall (insoluble), and
(2) Cytoplasmic (soluble) antigens
(1) Cell Wall:
Its structure is complex and is made of lipids, proteins and polysaccharides. A particular characteristic cell wall is the lipid content (about 60% of cell wall weight). Lipids of cell wall—particularly the mycolic acid fraction—are responsible for acid-fastness of mycobacteria and also for cellular tissue reactions of the body. The phosphatide fraction is responsible for tubercle-like cellular responses and caseation neferosis.
Cell wall has four layers:
(a) Peptidoglycan (murcin) layer is the innermost layer that maintains the shape and rigidity of the mycobacterial cell wall.
(b) Arabinogalactan layer is external to murin.
(c) Mycolic acid layer is a dense band of characteristic long chain of α-alkyl and β- hydroxy fatty acids attached by ester bonds to the terminal arabinose units of arabinogalacton. Mycolic acid is important constituent of cell wall.
(d) Mycosides (peptidoglycolipids or phenolic glycolipids) constitute the outermost layer of cell wall. In some mycobacteria, the mycoside layer is thin; whereas in intracellular bacteria (M. avium, M. leprae, M. leprae murium) this layer is thick like a capsule.
(2) Insoluble Agglutination Antigens:
These antigens are located as sugar moieties.
(3) Neutral Red:
The virulent strains of tubercle bacilli can bind neutral red in alkaline buffer solution whereas avirulent strains fail to do so. M. tuberculosis, M. bovis, M. avium and M. ulcerans give positive neutral red test.
(4) Amidase Test:
Acetamide, benzamine, carbamide, nicotinamide and pyrazinamide are commonly used amides in this amides test. A typical mycobacteria can be differentiated by their ability to split the amides M. tuberculosis splits the amides by its nicotinamidase and pyrazinamidase production.
(5) Nitrate Reduction Test:
M. tuberculosis can reduce nitrate but M. bovis cannot. The test organism is suspended in a buffer solution containing nitrate and incubated at 37°C for 2 hours. Then sulphanilamide and N-naphthyl-ethylene diamine dihydro- chloride solution is added. Positive reaction is indicated by development of pink or red colour.
(6) Susceptibility to Pyrazinamide:
M. tuberculosis is sensitive to 50 µg/ml pyrazinamide whereas other mycobacteria including M. bovis are resistant.
(7) Susceptibility to Thiopen-2-Carboxylic Acid Hydrazide (TCH):
The addition of 10 µg / ml TCH will not inhibit the growth of M. tuberculosis, but the growth of South Indian strains of M. tuberculosis and M. bovis are inhibited by this TCH.
Treatment:
Though the atypical mycobacteria are resistant to various anti-tubercular drugs in vitro there is a good clinical response to the combination of these drugs.
(1) A triple therapy of rifampicin, isoniazid and ethambutol can cure pulmonary disease caused by M. avium intracellular and M. kansasii.
(2) M. malmoense and M. xenopi are sensitive to the standard triple drug regimen for a period of 18 months.
(3) A simple excision of the ulcerative lesions due to M. ulcerans is beneficial along with clofazimine therapy.
(4) M. cheloneii and M. fortuitum are sensitive to the combination of erythromycin and trimethoprim therapy.
(5) M. kansasii, M. xenopi and M. fortuitum infections respond very well to ciprofloxacin and ofloxacin.
(6) The combination of rifabutin (ensamycin) and clofazimine (antileprosy drug) is effective in M. avium-intracellular infection in immuno-compromised hosts’ including AIDS.
13. Mycobacterium Leprae:
Causes Leprosy in man. It was discovered in 1873—9 years before the discovery of tubercle bacilli in 1882. These bacilli occur singly, in parallel bundles. They are stained by Z-N method by substituting 5% sulphuric acid for 20%. They are found in skin, mucous membrane, endothelial cells of blood vessels and mononuclear cells.
They have limited growth in living animals (footpads of mice or armadillos). M. leprae from armadillo or human tissue contains a unique O-diphenoloxidase, enzyme. The lesions are confined to cooler tissue of the human body. Leprosy is divided into 2 types, lepromatous (skin nodule) tuberculoid (skin nodule, nerve involvement).
14. Laboratory Diagnosis:
Skin or nasal mucosal scrapings or biopsy of ear lobe, skin are smeared on a slide and stained by Z-N technique to demonstrate M. leprae. No serological tests are of value and are in experimental stage.
Treatment:
Dapsone, DDS, and rifampin suppress the growth, clofazimine is an oral drug used in sulfone resistant leprosy. Recent vaccine (Leprovac) stimulates immune system to kill M. leprae and accelerates Bacterial clearance which enables the patient to get cured.