MYCOBACTERIA

Course: Microbiology Professor Nadir Khan, Ph.D.

Objectives
Students should be aware of the unique morphologic and biochemical characteristics of the mycobacteria. An understanding of the basic pathogenesis of tuberculosis is expected.

Student should be able to:

1. Describe the morphological and cultural characteristics of M. tuberculosis. 2. List the members of the genus Mycobacterium which cause disease in man. 3. Explain the difference between acid-fast bacilli and gram-positive and gram-negative organisms. 4. Outline the methods available for staining mycobacteria. 5. Describe the constituents of tubercle bacilli and their role, if any, in the pathogenesis of the disease process.

Student should be able to:

6. Define the role of M. bovis in human disease and BCG. 7. Define atypical mycobacteria and their role in human disease and their differences from classical mycobacteria. 8. Define Koch phenomenon and how it explains immunity to tuberculosis. 9. Define OT and PPD. 10. Describe characteristics of the organism which causes leprosy. 11. Define Actinomyces, Nocardia and Streptomyces and their disease causing a beneficial potential.

I. PERSPECTIVE
Mycobacteria are ubiquitous microorganisms that can be readily found in animals and humans. They are rod-shaped, non-motile, non-spore forming, non-capsulated organisms which are relatively impermeable to various basic dyes, but once stained, resist decolorization.

I. PERSPECTIVE (cont)
The genus Mycobacterium includes nearly 37 different species and the members of the genus include obligate parasites, saprophytes and intermediate forms. These organisms have several other important characteristics. First, they are obligate aerobes. Second they are slow growing. Third, they have very high cell-wall lipid content. Finally, they are extremely resistant to chemical and physical agents.

I. PERSPECTIVE (cont)
Two disease of medical importance, tuberculosis (TB) and leprosy, are caused by mycobacteria. The causative agent of tuberculosis in humans is M.tuberculosis, the human strain, and M.bovis, the strain found in cattle. Tuberculosis, usually is a respiratory infection but occasionally acquired by ingestion of mycobacteria.

I. PERSPECTIVE (cont)
Leprosy, primarily a disease of the skin and nerves, is caused by M.leprae. More than 95% of all human infections are caused by six species:  M.tuberculosis  M.avium-intracellulare  M.kansasi  M.fortuitum  M.chelonae  M.leprae

Table 22-1 Mycobacteria Associated with Human Disease

Mycobacterium M.tuberculosis M.bovis M.leprae M.kansasu M.mannum M.scofulaceum Environmental Contaminant No No No Rarely Rarely Possibly Reservoir Humans Humans, Cattle, Other mammals Humans, armadillos Water, Cattle, Swine(rarely) Pumates, possibly water Soil, Water, Foodstuffs

Table 22-1 Mycobacteria Associated with Human Disease (cont)

Mycobacterium M.szulgai Environmental Contaminant No Reservoir Unknown Soil, Water, Swine, Cattle, Birds Water Soil, water, animals, manne life Soil, water, animals, manne life Unknown

M.avium-intracellulare Possibly M.xenopi M.forcurcum M.chelonae M.ulcerans Possibly Yes Yes No

II. GENERAL CHARACTERISTICS

A. B. C. D. E. F. G. H. Morphology Staining Lipids Cord Factor Wax D Proteins Polysaccharides Growth

Morphology
The tubercle bacilli are thin, usually straight or slightly curved rods, which occur singly. They are non-motile, do not form spores, do not form capsules, and, when stained, often appear beaded.

Staining
Ziehl-Neelsen Method: The staining characteristics of mycobacteria are defined on the basis of the distinctive staining property that depends on the lipid-rich cell wall. Tubercle bacilli routinely are stained by using the Ziehl-Neelsen technique, in which a smear is covered with the red stain carbol-fuchsia, and the dye-covered smear is heated by steaming for a few minutes to aid the penetration of the dye into the cell.

Staining (cont)
The stained smear is then washed with acid alcohol and counter-stained with methylene blue. The mycobacteria retain the original red stain. Malachite green, methylene blue or picric acid can be used for background staining. A cold-stain modification of this procedure, in which the carbol-fuchsin is first dissolved in a detergent (Turgitol #7), is also used to stain mycobacteria. In this case, no steaming is necessary and staining is accelerated. Because of their resistance to decolorization, the mycobacteria are called ACIDFAST.

Staining (cont)
Mycobacteria possess a peptidoglycan layer but the usual gram-staining reagents cannot penetrate it. This peptidoglycan layer is surrounded by layers of lipids. The most striking chemical feature of the mycobacteria is the abundance of lipids in the cell wall (up to 60% of its dry weight).

Staining (cont)
This property accounts for the:  Hydrophobic character o the organisms  Relative impermeability to stains  Acid-fastness  Unusual resistance to killing by acids and alkali  Resistance to bactericidal action of antibodies and complement

Lipids
Although many different fatty acids are found in mycobacteria, the MYCOLIC ACIDS appear to be unique to the cell walls of Mycobacteria, Nocardia and Corynebacteria. They are very high molecular fatty acids which can occur free or bound to carbohydrates as glycolipids which are called MYCOSIDES.

Cord Factor
This is a mycoside and its presence is responsible for a phenomenon in which individual bacteria grow parallel to each other, forming large serpentine cords. Hence the name, cord factor.

Several lines of evidence relate cord factor to virulence:

      Its extraction renders cells non-virulent It is toxic It inhibits migration of PMNs Subcutaneous injection will kill a mouse It is more abundant in virulent strains Young cultures of tubercle bacilli are more virulent, and have a higher content of cord factor

Wax D
It is another complex mycoside. When emulsified with water and oil, Wax D acts as an ADJUVANT. It also seems to be responsible for the induction of the cellular immune response to tubercle bacilli. By mixing Wax D with mycobacterial proteins, one obtains a cellular immune response to the proteins, whereas this does not occur using proteins alone.

Proteins
M.tuberculosis also possesses a number of protein antigens that, by themselves, do not appear to be toxic or involved in virulence. These are responsible for delayed hypersensitivity. Skin testing with PPD (Purified Protein Derivative) detects hypersensitivity to these proteins. Antibodies against some of these proteins are present in patients sera but do not play a role in protection.

Polysaccharides
A variety of polysaccharides are present but their role in the pathogenesis of the disease process is, at best, uncertain.

Growth
M.tuberculosis is an OBLIGATE AEROBE. Most pathogenic mycobacteria usually grow very slowly. The generation time for M.tuberculosis is 18-24 hours, compared to every 20 minutes for E.coli. This lengthy doubling time is reflected in the time it takes for visible colonies to develop on laboratory media (from two to ten weeks, depending on the species) and in the time for symptoms to develop in the disease process (several weeks to months).

Growth (cont)
The organisms can be grown in simple media consisting of only inorganic salts, asparagine, and glycerol. The medium most commonly used in the microbiology clinical laboratory is a very complex one and is called LOWENSTEINJENSEN medium. The organisms are readily killed by heat, and the conditions of pasteurization practiced in the United States are sufficient to kill tubercle bacilli that might be present in the milk.

Growth (cont)
They are quite resistant to chemical agents are are particularly resistant to drying, making it possible for M.tuberculosis to remain alive for long periods in rooms, bedding, sputum, and other environmental niches.

III. MYCOBACTERIUM BOVIS

The ingested organisms produce lesions primarily in the bones, joints, and cervical lymph nodes. When inhaled (e.g. by dairy farmers), the organisms can also cause pulmonary tuberculosis indistinguishable from that caused by M.tuberculosis. One attenuated bovine strain, carried through several hundred serial cultures on unfavorable media, is known as Bacille Calmette Guerin (BCG). This strain, which is used to immunize humans against tuberculosis, has remained avirulent for more than 60 years. It is a live vaccine.

IV. ATYPICAL MYCOBACTERIA

 (see next slide)

Table 22-2 Classification of Important Mycobacteria

Mycobacteria Unclassified Mycobacteria M.leprae M.tuberculosis M.bovis M.ulcerans Strict Pathogen Strict Pathogen Strict Pathogen Strict Pathogen Positive No No No Positive Slow Slow Slow Clinical Significance Pigmentation* Rate of Growth

Table 22-2 Classification of Important Mycobacteria (cont)

Mycobacteria Clinical Significance Pigmentation* Rate of Growth RUNYON GROUP I M.kansasii M.marinum M.simiae Usually pathogenic Usually pathogenic Usually pathogenic Photo Photo Photo Slow Moderate slow

Table 22-2 Classification of Important Mycobacteria (cont)

Mycobacteria Clinical Significance Pigmentation* Rate of Growth RUNYON GROUP II M.scrofulaceum M.szulgai M.xenopi M.gardonae M.flavescens Rarely pathogenic Strict pathogen Rarely pathogenic Nonpathogenic Nonpathogenic Scoto Scoto Scoto Scoto Scoto Slow Slow Slow Slow moderate

Table 22-2 Classification of Important Mycobacteria (cont)

Mycobacteria Clinical Significance Pigmentation* Rate of Growth RUNYON GROUP III M.aviumintracellulare Usually pathogenic No Slow

RUNYON GROUP IV M.fortuitum M.chelonae Rarely pathogenic Rarely pathogenic No No Rapid Rapid

Table 22-2 Classification of Important Mycobacteria (cont)

Modified from the Manual of clinical microbiology. Ed 4 Washington DC. American Society for Microbiology, 1985.

*Pigmentation, nonpigmented, no photochromogen, photo, scotochromogen. +M.leprae does not grow in vivo.

IV. ATYPICAL MYCOBACTERIA (cont)

In addition to mammalian tubercle bacilli, various other acid-fast bacilli occasionally have been cultured from respiratory secretions of apparent tuberculous individuals. Several of them are normal inhabitants of soil and water.

IV. ATYPICAL MYCOBACTERIA (cont)

As the incidence of tuberculosis decreases, the relative importance of pulmonary disease attributable to non-tuberculosis mycobacteria is increasing. Within the United States, in some parts of the south, between 1/3 to of the population has been infected with them.

IV. ATYPICAL MYCOBACTERIA (cont)

Currently, the most commonly used classification divides these organisms into the following four groups:  Photochromogens  Schotochromogens  Nonphotochromogens  Rapidly-growing Mycobacteria

A. Photochromogens
These organism produce a yellow pigment only if grown in light. There is no color if grown in the dark. M.kansasi is the most prevalent human pathogen in this group. M.marinum, another photochromogen, has been isolated from swimming pools and lakes.

B. Schotochromogens
Organisms belonging to this group produce an orange pigment when grown in the dark. M.scrofulaceum (cervical lymph node lesion is called scrofula) is the most prevalent pathogen of this group. It is a common cause of cervical lymphadenitis in children. The organism is primarily a soil saprophyte.

C. Nonphotochromogens
These organisms have variable pigmentation not related to light exposure. Two major pathogens, M.avium and M.intracellulare, are so closely related that many refer to them as M.aviumintracellulare complex (MAC). The MAC has acquired a new significance in those individuals with AIDS, in whom it is found to be the most common cause of tuberculosis. Members of the MAC can be isolated from sputum, blood, aspirates of bone marrow and even stool.

D. Rapidly-growing Mycobacteria
This group of mycobacteria has a very short generation time and colonies become visible after 2-3 days. This group includes non-pathogens, such as M.smegmatis and M.phlei, as well as several species which cause human infection such as M.fortuitum complex.

Differences from Classic Tuberculosis

Atypical mycobacteria and the diseases they produce differ in several important respects from classic tuberculosis: 1. Evidence of close contact with patients with disease caused by an atypical mycobacteria reveals no evidence of human-to-human transmission. 2. Atypical mycobacteria are more likely to be resistant to the first-line anti-tuberculosis drugs than is M.tuberculosis.

Differences from Classic Tuberculosis (cont)

3. Patients infected with atypical mycobacteria usually give a negative tuberculin test to a 5-TU test dose of tuberculin prepared from M.tuberculosis

V. PATHOGENESIS
Tuberculosis epitomizes the pathogenesis of chronic infectious diseases. Humans become infected with M.tuberculosis most frequently by inhaling droplet nuclei, which contain tubercle bacilli and because of their very small size, remain airborne for long periods of time. The organisms, though readily phagocytized, resist the destructive properties of normal macrophages and are capable of multiplying intracellularly.

VI. IMMUNE RESPONSE TO TUBERCULOSIS INFECTION

A. The Koch Phenomenon: Kochs initial injection, in a previously non-exposed guinea pig, resulted in an ulcer at the injection site that failed to heal and was followed by a spread to the regional lymph nodes. A second injection, several weeks later, into the same animal but at a different site, resulted in a more localized ulcer that eventually healed, and the regional lymph nodes were not infected again. This observation is called the KOCH PHENOMENON.

VI. IMMUNE RESPONSE TO TUBERCULOSIS INFECTION (cont)

A. Koch Phenomenon (cont): The infected animal has thus acquired increased resistance. Serum antibodies do not account for this immunity. This kind of immunity can be passively transferred with viable lymphoid cells but not by serum. Subsequently, Koch found that the injection of a culture filtrate into an animal that had been previously infected with tubercle bacilli evoked an allergic response.

VI. IMMUNE RESPONSE TO TUBERCULOSIS INFECTION (cont)

A. The Koch Phenomenon (cont): Koch called this culture filtrate TUBERCULIN, and since it takes this kind of reaction several hours to develop, it has been referred to as DELAYED TYPE HYPERSENSITIVITY (presently called Type IV Reaction) The proteins in the culture filtrate that evoke this Type IV response have been partially purified and are available commercially as PURIFIED PROTEIN DERIVATIVE (PPD), whereas Kochs original preparation is called OLD TUBERCULIN (OT).

VII. MYCOBACTERIUM LEPRAE

The etiologic agent of leprosy is morphologically similar to M.tuberculosis. The organism has never been grown on artificial media. It is an obligate intracellular parasite. The generation time has been estimated to be 13 days. More recently, the organism has been cultivated in the foot pad of mice and the ninebanded armadillo. An antigen preparation for skin testing, called LEPROMIN, is available.

Epidemiology
Leprosy is apparently transmitted when exudate of mucous membrane lesions and sin ulcers reach broken skin. It is not a highly contagious disease and patients need not be isolated. Young children and adult males seem to be more susceptible. The incubation period is estimated to range from a few months to 30 years or more. In the U.S., leprosy occurs usually in persons from Puerto Rico, the Philippines, Mexico, Cuba and Samoa.

Epidemiology (cont)
The national leprasorium is located in Carville, Louisiana. Over two hundred new cases are diagnosed each year in the U.S. As of October 17, 1992, there were 126 cases reported for the year.

Pathogenesis
The organisms have a predilection for skin and nerves. In the CUTANEOUS form of the disease, large, firm nodules are distributed widely, and, on the face, they create a characteristic LEONINE appearance. In the NEURAL form, segments of peripheral nerves are involved, leading to localized patches of anesthesia.

Phases of Leprosy
In either the cutaneous or neural forms, three phases may be identified: 1. Lepromatous 2. Tuberculoid 3. Intermediate

Phases of Leprosy: Lepromatous

This is the progressive and malignant form of the disease, resulting in death in untreated cases. The organisms are found in essentially every organ of the body. The prognosis is poor.

Phases of Leprosy: Tuberculoid

This form of the disease is frequently a self-limiting disease that may even regress spontaneously. The major characteristic in this form of the disease is a normal cellular response mounted against the organisms. Organisms are extremely rare and may not be seen in biopsies.

Phases of Leprosy: Intermediate

Organisms are seen in areas of necrosis but are rare elsewhere. The long-range outlook is fair. Shifts from one phase to another do occur.

ACTINOMYCETES

Fungus-like Bacteria

I. PERSPECTIVE
Actinomycetes are the most abundant organisms in the soil. They break down organic material and are exceedingly important for their roles in the cycles of nature.

I. PERSPECTIVE (cont)
The actinomycetes, despite their outward mold-like appearance, are true bacteria:  They contain muramic acid in their cell walls  They lack mitochondria  They contain 70S ribosomes  They lack a nuclear envelope  They are inhibited or killed by many antibacterial antibiotics  Are motile forms possessing a flagella

Actinomyces
These gram-positive organisms are anaerobic or microaerophilic. They do not form spores or capsules. They need rich media for growth. A.israelii is usually responsible for the disease in humans, whereas A.bovis causes the disease in cattle.

Actinomyces: Pathogenesis
A.israelii and A.bovis are quite regularly isolated as part of the normal flora in the oral cavity and the alimentary tract of humans and cattle, respectively. They are more frequently commensuals than pathogenic. A.israelii can be cultured from the majority of human tonsils and scraping of the gums and teeth.

Actinomyces: Pathogenesis
The conditions that lead the organism to become invasive may include:  Trauma (including dental surgery)  Aspiration of a heavily contaminated tooth  Detached bits of dental tartar  Human bites

Actinomyces: Pathogenesis
Actinomycosis is characterized by chronic, destructive abscesses of connective tissue. Wherever the lesions occur, they are basically the same. Abscesses expand into contiguous tissues and eventually form BURROWING SINUSES to the skin surface, where they discharge purulent discharge. Actinomycosis occurs in three locations: Cervicofacial, Thoracic, and Abdominal.

Actinomyces: Cervicofacial
Initial symptoms include minor pain, together with a hard nodular or lumpy swelling of the jaw. Later, the swelling softens, the organisms spread and eventually extend through the skin, forming multiple draining sinuses. The skin abscesses discharge a purulent material containing micro-colonies of A.israelii. Because of their yellow color of these colonies, they are called SULFER GRANULES and their presence in draining abscesses provides good presumptive evidence of a diagnosis of actinomycosis.

Actinomyces: Thoracic
Thoracic disease may occur as a primary lesion in the lung after aspiration, or it may result from either a cervicofacial or an abdominal infection.

Actinomyces: Abdominal
The primary source of abdominal actinomycosis is an infected appendix, but infection may also follow penetrating abdominal wounds, perforated intestinal ulcers, or even blunt trauma without apparently perforation. Penetration of the abdominal wall, yielding draining abscesses containing sulfer granules provide means of diagnosis.

Nocardia
In contrast to Actinomyces, Nocardia species are inhabitants of soil. They are aerobic, grow on simple media, and over a wide range of temperature. Nocardia are gram-positive and partially acid-fast. There are two species which are pathogenic for humans: N.asteroides and N.brasillensis. Two diseases to note: Pulmonary Nocardiosis and Mycetoma.

Nocardia: Pulmonary Nocardiosis

The majority of cases of pulmonary nocardiosis are caused by N.asteroids. It occurs most frequently as a pulmonary disease in immunocompromised or debilitated persons following inhalation of the organism. Lesions superficially resemble the cavities of chronic tuberculosis. Neither the characteristic granuloma of tuberculosis nor the burrowing sinuses of actinomycosis are seen.

Nocardia: Mycetoma
Different species of Nocardia are associated with mycetoma in different parts of the world (e.g. N.brasillensis in Mexico). Mycetomas are chronic subcutaneous infections in which abscesses extend by destruction of soft tissues and bone to eventually erupt through the skin. These abscesses are clinically very similar to those of actinomycosis. Granules are present to help in the isolation and identification of the etiologic agent.

Streptomyces
The genus Streptomyces includes an extremely large group of organisms distributed worldwide. Unlike members of the genera Actinomyces and Nocardia, they form long chains of aerial spores called CANDIDA. Species of this genus are only rarely pathogenic, but occasional cases of mycetoma have been attributed to these organisms.

Streptomyces (cont)
Members of this genus have achieved extreme prominence as a result of their ability to produce antibiotics. This genus and its various species produce more than 90% of the therapeutically useful antibiotics. Streptomycin and actinomycin were the first to be isolated and characterized, but since 1940, over 500 different antibacterial compounds, including most of the antibiotics used today, have been isolated and characterized from members of the genus Streptomyces.