1. Mycobacteriaceae

2. Classification – Family Mycobacteriaceae 1 genus of medical importance= Mycobacteria All are slow growing All are acid-fast and contain large amounts of lipids in their cell walls Tubercle bacilli= M. tuberculosis, M. africanum, and M. bovis

3. Mycobacteriaceae Mycobacteria other than tubercle bacilli (MOTT) or the atypicals= all other Mycobacteria species The Mycobacteria are sometimes divided into 4 groups (Runyon groups) based on growth rate and pigmentation: Photochromagens – are non-pigmented when grown in the dark, but produce photoactivated pigments upon exposure to light Scotochromogens – produce deep yellow to orange pigments when grown in light or dark, but the color will deepen upon two weeks exposure to light Nonphotochromagens – may produce pigment ranging from white to yellow, but pigment does not intensify upon exposure to light. M. tuberculosis belongs in this group. Rapid growers – organisms that form colonies within seven days.

4. Mycobacteriaceae Mycobacterium leprae does not fall into any of these groups and it can’t be grown on ordinary lab media. It is grown in the 9 banded armadillo or in the footpads of mice (takes 3-4 weeks to replicate!) Morphology and cultural characteristics Always work under a biosafety cabinet! Slender G+ rods that stain poorly because of the large amount of lipids in the cell wall. Are all acid-fast – the acid-fast stain is an important first step for presumptive diagnosis of mycobacterial disease. A concentrated specimen is often used.

5. Mycobacteriaceae Heating is required for stain penetration because of the high lipid content of the cell wall (mycolic acid and waxD). Once the stain penetrates and binds to the mycolic acid in the cell wall, it cannot be removed even with acid alcohol treatment.

6. Mycobacteriaceae There are several different acid fast stains that may be used: Ziehl-Neelsen – uses heat to get the primary stain of carbol fuchsin to penetrate the cell wall; acid alcohol destaining; and methylene blue as the counterstain. Kinyon – uses a higher content of phenol (organic solvent) in the carbol fuchsin primary stain to allow penetration of the stain without the need to apply heat. Also uses acid alcohol to destain and methylene blue as the counterstain.

7. Acid-fast bacilli

8. Mycobacteriaceae Auramine-rhodamine fluorochrome (a fluorescent stain) requires a fluorescent microscope, but allows one to scan the slide a high dry so that slides may be read much faster Stain with auramine-rhodamine for 10 minutes (phenol in the solution allows for penetration) Destain with acid alcohol Counterstain with acridine orange A positive result is a bright yellow fluorescence. Reporting results: 1-2 AFB/300 fields +/-; 1-9 AFB/100 fields 1+

9. Mycobacteriaceae If the specimen is highly contaminated with organic debris and normal flora, before plating the specimen should be digested and decontaminated with NaOH Addition of N-acetyl-L-cystine helps to liquify sputum. Most will grow on simple media, but for primary isolation complex media should also be used – use of a nonselective, a selective and possibly a liquid media is recommended. Nonselective – may be egg or agar based and may include malachite green to suppress growth of contaminating bacteria. Lowenstein-Jensen – egg based; colonies grow in 18-24 days)

10. Mycobacteriaceae Middlebrook 7H10 and 7H11 – agar based; colonies grow in 12-14 days. Selective media – consists of one of the nonselective media plus added antimicrobial agents (malachite green, cyclohexamide, and nalidixic acid are often used) The colonies of M. tuberculosis on the solid media are rough, dry, granular, nonpigmented to buff or tan colored colonies.

11. M. tuberculosis culture

12. Mycobacteriaceae Liquid media - the media usually contains tween 80 and albumin and the organisms will grow faster than on solid media Most Mycobacteria grow best in 5-10% CO 2 and at 35-37 0 C. Organisms from skin lesions grow best at 30-33 0 C. Cultures should be kept 6-8 weeks before being discarded as - Skin lesion cultures should be kept for 12 weeks. CDC wants results in 1-2 weeks and this is only possible with the new PCR based methods of identification. Identification Rate of growth and growth in relation to temperature Pigmentation and photoreactivity Further biochemical testing includes:

13. Mycobacteriaceae Niacin – a niacin + slow growing organism producing rough, dry, granular colonies that resemble Tb is strongly presumptive of Tb, but it must be differentiated from M. simiae M. tb. Is nitrate reduction+ and – for catalase at 68 0 C M. simiae is – for nitrate reduction and + for catalase at 68 0 C. M simiae is also a photochromagen, but its photoreactivity may be delayed. Tween hydrolysis, arylsulfatase production, tellurite reduction, salt tolerance, and pyrazinamidase production may also be used in differentiating the different species of Mycobacteria.

14. Mycobacteriaceae Virulence factors (M. tb.) Cord factor – is a glycolipid, trehalose 6,6’ dimycolate that is responsible for the serpentine growth (filaments or cords) of M. tb. in which the bacilli grow in close parallel arrangement. It is toxic to leukocytes, activates macrophages and dendritic cells, interferes with mitochondrial function in mice and plays a role in the development of granulomatous lesions Iron capturing ability – required for survival inside phagocytes Sulfolipids prevent phagosome-lysosome fusion so that the organisms are not exposed to lysosomal enzymes (important in intracellular survival)

15. Mycobacteriaceae Clinical significance (M.tb.) Both M. tuberculosis and M. bovis can cause Tb, but M. bovis is rare in the U.S. The organism can gain entrance by inhalation (most common), ingestion (usually from contaminated milk), or through mucous membranes of the genital-urinary tract or conjunctiva, or through skin abrasions. Man is very susceptible to infection, but remarkably resistant to tuberculous disease.

16. Mycobacteriaceae Progression of the infection after inhalation: Tubercle bacilli that reach the alveoli of the lung are ingested by macrophages The organisms multiply and cause a chemotactic response that recruits other macrophages and T cells to the area. Enzymes and cytokines are released to start an inflammatory response to wall off the organism (tubercle formation), but the inflammatory response also causes lung damage. At his point the patient becomes skin test +.

17. Mycobacteriaceae After a few weeks many of the macrophages die, releasing tubercle bacilli and forming a caseous center in the tubercle. In healthy individuals, the disease is usually arrested at this time and the lesions may may become calcified (Ghon complexes). Tubercle bacilli may remain dormant in the lesion and serve as a basis for later reactivation of the disease. When the defenses fail, a mature tubercle may form whereby the caseous center will enlarge and liquify to form a tuberculous cavity where the bacilli multiply outside the macrophages.

18. Mycobacteriaceae The tubercle eventually ruptures, releasing tubercle bacilli that can disseminate throughout the lungs and then to the circulatory and lymphatic systems (Miliary Tb). This is the progressive form of the disease and it is characterized by weight loss, coughing with blood, and loss of vigor (The old name for this disease was consumption because it is as if the body is being consumed by the bacteria)

19. Progression of tuberculosis

22. Mycobacteriaceae Clinical significance – the atypicals Are usually transmitted form soil or water to man Some produce pulmonary diseases that resemble Tb and others cause skin lesions The M. avium-intracellulare group has become a major problem in AIDs patients where the organisms may be cultured from the blood and stools. Clinical significance – M. leprae Causes leprosy which is also called Hansen’s disease

23. Mycobacteriaceae The mycobacteria are obligate intracellular parasites found in histiocytes, Schwann cells, and in epitheloid structures called Lepra cells The mode of transmission is unknown, but in lepromatous leprosy there are large numbers of bacilli in the sputum and nasal secretions The disease is contagious, but requires prolonged, intimate contact for transmission The incubation is 2-4 years The clinical manifestations depend upon the adequacy of the host cell mediated response against the organism. Like Tb, many are infected, but few develop clinical symptoms.

24. Mycobacteriaceae Subclinical – the infection is contained by the cell mediated immune response Tuberculoid – this form of the disease occurs when the cell mediated immune response is partially effective: Lesions are confined to the skin and peripheral nerves Lesions are solitary or few in number Lesions have a raised, erythematous margin and a flat center Nerve damage due to the cell mediated immune response causes a loss of sensation to touch, temperature, and pain within the lesion A skin biopsy reveals many lymphocytic and epithelial cells, but no AFB. The lepromin test is +

25. Tuberculous form of leprosy

26. Mycobacteriaceae Lepromatous – this form of the disease occurs when there is defective cell mediated immunity against the organism May involve all areas of the skin and lesions have a waxy, nodular appearance There is destruction of cutaneous nerves The skin may thicken and fold There is a loss of eyebrows and eyelashes There may be destruction of the nasal septum A skin biopsy reveals no lymphocytes and many Lepra cells packed with AFB. The lepromin test is -

27. Lepromatous form of leprosy

31. Mycobacteriaceae Indeterminate There is a hypopigmented macule There may or may not be sensory loss within the macule The macule may completely resolve Reactionary state Erythema nodosum leprosum (ENL) This is an antibody mediated reaction in which immune complexes form Painful, erythematous nodules form May occur in individuals with the lepromatous form of the disease following drug therapy

32. Mycobacteriaceae Lucio phenomena – necrotizing skin lesions which blister and ulcerate may occur. Treatment -Tb Use a combination of drugs to help prevent the emergence of resistant strains Isoniazid Rifampin Para-aminosalicylic acid Ethambutol Streptomycin Antibiotics must be given over an extended period of time – at least 9 months

33. Mycobacteriaceae BCG is a preventative vaccine (not used in the U.S.). It was made by passaging the organism in culture over a long period of time to attenuate it. Tuberculin skin test – the Mantoux test uses PPD (purified protein derivative) which is injected just under the skin. A delayed-type hypersensitivity reaction indicates a previous or current infection, but does not mean that you have the disease. If results are +, X-ray for Ghon complexes

34. Tuberculin skin test

35. Ghon complexes

36. Mycobacteriaceae Treatment – atypicals Must use multiple drugs because resistance is common Teatment – leprosy Dapsone – For those with the lepromatous form of the disease, treatment is for life For those with the tuberculoid form of the disease, treatment is for 5-10 years (Note that treatment may cause the development of ENL) Rifampin Clofazimine Thalodimide – treats ENL symptoms Lepromin skin test is similar to the Tb skin test