Mycobacteria

 

Tuberculosis is caused by Mycobacterium tuberculosis.

History

Early 1800's - tuberculosis (TB) epidemic in US and Europe. Death rate ~1% in some cities. As living conditions began to improve, TB incidence decreases.

1950's - cure discovered for TB. Public health programs aimed at eliminating TB initiated. Numbers of TB cases begin to decline.

Early 1970's - most anti-TB programs dismantled as TB cases continue to decline.

1984 - beginning of increase in TB cases; continues to present. Why?

Increase in immigration (25% of cases imported)

Crowded conditions; substance abuse; AIDS

Increase in drug-resistant TB

1990 - first outbreaks of multidrug resistant M. tb reported in AIDS patients and homeless - New York City and Miami

1997 – worldwide

17 million total deaths due to infectious diseases

3 million deaths from TB/year

10 million new cases of TB/year

affects 1/3 of the world’s population

Characteristics

obligate aerobe

nonmotile

nonsporeforming

nonencapsulated

rod-shaped, slender, bent

**facultative intracellular pathogen

humans only natural reservoir

animal models available

acid-fast staining characteristics

GP type of cell wall:

thick PG layer + arabinogalactan + lipoarabinomannan + mycolic acids

Mycobacteria spp. range from widespread innocuous saprobic inhabitants of soil and water to those species responsible for tuberculosis and leprosy.

M. tuberculosis

Clinical

M. tb is an important pathogen even in developed countries such as the US. Tuberculosis is a chronic infectious disease. The TB bacillus can infect any organ of the body but is usually associated with the lungs.

In most scenarios, only 10-20% of an exposed population comes down with TB. However, the disease is especially prevalent in crowded urban areas and in certain urban groups (American Indians and Eskimos) that have a high genetic susceptibility.

The extent of the disease dependent on:

immunity of the host

infecting dose of bacilli.

1. inhalation of bacteria

2. phagocytosed by alveolar macrophages in lung

3. bacterial multiplication proceeds and spreads to the regional lymph nodes.

4. Bacteremia spreads bacilli to all parts of the body.

5. Bacteremia cleared by RES; bacterial multiplication continues in the lung (also kidneys, vascular skeletal areas, lymph nodes)

6. After 3-4 weeks, cellular immunity develops. Activated macrophages limit further bacterial growth and reduce the number of organisms in both primary and metastatic foci

Of those infected:

5 to 15 percent develop primary tuberculosis. These include the very young or adults who are immunocompromised or malnourished.

85 to 95 percent wall off the infection, but some viable bacilli are retained which can later be reactivated.

Symptoms: includes fever, coughing (bloody sputum), weight loss, malaise. Progressive irreversible lung destruction occurs. If the bacteria escape from the lungs, they may cause systemic disease affecting any area of the body. Much of tissue damage - a result of cell-mediated immune responses of the host.

The systemic form of the disease is almost always fatal. The course of the disease is slow, with a decline in health over several years.

Persons infected with HIV (AIDS virus) are extremely susceptible to tuberculosis. HIV-infected patients are 100 - 300 times more likely to develop tuberculosis than those not infected by the virus. This is associated with their poor cell-mediated responses. In AIDS patients, the disease if more rapid (months), and the fatality rate is nearly 80%.

 

Transmission: aerosol droplets and sputum from individuals with open pulmonary lesions. The organism can survive in moist or dried sputum for up to 6 weeks. Transmission occurs when there is frequent and prolonged close contact between a susceptible person and a person with an active case of TB.

Patients receiving effective chemotherapy rapidly lose their infectiousness for other persons.

 

M. tb Virulence

no known toxins or exoenzymes

a. Survival and growth inside monocytes and macrophages. Mechanism controversial, but most likely: inhibits phagolysosomal fusion

b. Wall structure

The complex, lipid-rich cell wall of mycobacteria makes it resistant to many disinfectants and protects it from the effects of phagolysosomal components - responsible for M. tb survival within the environment and macrophages.

Backbone of CW:

peptidoglycan and arabinogalactan -> esterified to mycolic acids

Mycolic acids: unique to the cell walls of mycobacteria, nocardiae, and corynebacteria. They are large (C60-C90 for M.tb), saturated, -alkyl, -hydroxyl fatty acids found in both waxes (esters of fatty acids with fatty alcohols) and glycolipids (lipid linked to carbohydrates).

 

Cord factor: a mycoside of 6,6'-dimycolate of trehalose (dimer of 1,1 linked glucose – nonreducing sugar).

Virulent strains of M tb grow as intertwining serpentine cords (aggregates of bacilli). This trait correlates with the content of a surface lipid called cord factor which contributes to the hydrophobic character of the organism.

it is more abundant in virulent strains; cells with cord factor extracted are nonvirulent

it is toxic: causes profound disturbances of microsomal enzymes, mitochondria, and lipid metabolism in the livers of mice.

mice are protected by immunization with cord factor.

 

Lipoarabinomannan (CW glycolipid) suppresses T cell proliferation; may interfere with interferon-gamma secretion.

 

 

Staining: Gram-stain reagents cannot penetrate the mycobacterial cell wall because of lipid surrounding the PG. ~60% of the dry weight of cell wall is lipid. Special methods must be used to promote penetration of the dyes to stain mycobacteria:

Ziehl-Neilsen acid fast stain

staining the sample with carbolfuchsin (red)

decolorize in 95% ethanol-3% HCl

counterstain is either malachite green or methylene blue.

"Acid-fast" bacteria appear red against a blue or green background.

The acid-fast direct smear on a clinical sample is limited by the necessity for at least 5000 AFB/ml in a concentrated specimen for a positive smear. Smears also cannot differentiate between M. tb and other Mycobacterium sp. that can cause pulmonary infections.

c. iron chelating components:

exochelin is an extracellular soluble compound that robs iron from ferritin (storage form of iron in the mammalian cell)

mycobactins are cell associated and transport the iron through the mycobacterial cell wall.

 

 

 

Immunity

Humans are very susceptible to tuberculous infection but remarkably resistant to tuberculous disease.

Antibodies are produced in response to tuberculous infection, but they appear to play no beneficial role in host defense. Antibodies to M. tb are not effective at clearing the microbe because GP cells are not lysed by Ab + complement. Organisms are facultative intracellular parasites - survive within macrophages that are not activated.

 

Resistance to tuberculosis is related to the ability of macrophages to kill the bacilli or to prevent them from multiplying. The main defense in the lung is the alveolar macrophage. M. tb has evolved the ability to survive and grow in resting macrophages.

Two to 4 weeks after infection, immune CD4+ T helper lymphocytes elaborate cytokines that activate the circulating macrophages, rendering them capable of bacterial killing.

Healthy adult exposed to relatively low numbers of bacteria: activated macrophages generally appear early enough to stop the infection before appreciable damage to the lung occurs. Such people become skin test positive, but do not develop symptoms of TB.

 

T cells, fibroblasts, and macrophages may eventually wall off the growing lesion with a thick fibrin coat: the walled-off lesion = tubercle. Tubercles eventually calcify - visible on Xray. Some walled-off lesions may contain live bacteria. M. tb may survive for decades in such lesions.

 

Later in life, suppression of the immune system (cancer, drugs, AIDS) may allow the bacteria to break out of the lesions and begin to grow again: reactivation TB.

 

In the absence of a rapid and effective T cell response, the focus of damaged tissue grows. Erosion of tissue ----> spread.

 

 

Lab diagnosis of M. tb:

easiest a fastest way to a positive diagnosis: demonstration of acid-fast bacilli in stained smears of sputum. Culture is necessary since it can detect fewer organisms than a stained smear; cultural characteristics distinguish tubercle bacilli from other acid fast bacilli; tests for drug sensitivities should be done.

doubling time of 12-24 hours. As a result colonies do not appear on culture media until 2-4 weeks following inoculation. Other mycobacteria (from the environment) grow more rapidly (colonies in 2-3 days), but not compared to other bacteria like E. coli that has a 20 min doubling time.

 

M. tb is differentiated from other mycobacteria by its lack of pigmentation, presence of corded, serpentine colonies, and other biochemical tests.

 

Tuberculin skin test

PPD (purified protein derivative):

 

Positive test: redness, swelling and induration at the injection site. The average diameter of induration at the injected site is measured at 48h, and a reaction of 10 mm or more is positive.

The test measures delayed-type hypersensitivity to TB. It reveals present or past infection with the TB bacillus (or BCG vaccine), not necessarily the presence of active disease since reactivity may persist for a lifetime. A person remains skin test positive as long as viable organisms remain in the body.

If a person is infected with M. tb and the organisms are completely eliminated, the tuberculin reaction will eventually become negative. Same for a person vaccinated with BCG but never infected with tubercle bacilli.

A positive skin test is followed by chest x-ray and exam of sputum smears and cultures for M. tb. Only 3-5% of people who become skin test positive actually develop active TB.

 

Therapy

M. tb is naturally resistant to many antibiotics - lipid-rich OM. Effective barrier to keep antibiotics out of the cell.

Thus, most commonly used antibiotics are ineffective against M. tb.

Exceptions: rifampin, Sm, some new fluoroquinolones.

 

First-line antituberculosis drugs:

1. isoniazid (INH-isonicotinic acid hydrazide) - inhibits mycolic acid synthesis; bactericidal; nontoxic; cheap; oral administration

2. rifampin - bactericidal; nontoxic; oral administration; inhibits transcription by binding to ( subunit of DNA-dependent RNA polymerase.

3. pyrazinamide - ? unknown mechanism of action; bactericidal

4. streptomycin - bactericidal; parenteral administration; binds to 30S ribosomal subunit

5. ethambutol - bacteriostatic, inhibits arabinogalactan synthesis; affects synthesis of mycolic acids as well.

Second-line antituberculosis drugs:

1. ethionamide - bacteriostatic, inhibits mycolic acid synthesis

2. cycloserine - bacteriostatic; inhibits peptidoglycan synthesis by inhibiting D-alanine racemase and D-ala-D-ala synthetase.

3. para-aminosalicylic acid - bacteriostatic; inhibits folic acid biosynthesis (structural analog of PABA).

 

When these drugs are used singly, resistance occurs at a relatively high frequency. Therefore, combinations of drugs are used to treat TB, typically INH and rifampin and pyrazinamide and either ethambutol or Sm.

The chronic nature of the disease requires that therapy be continued for long periods of time (6 to 9 mo) to minimize the rate of relapse; combination therapy with two or more drugs delays the emergence of resistant organisms. Drugs should be able to penetrate macrophages.

 

Resistance to anti-TB drugs

M. tb becomes resistant to drugs because

treatment regimens are inadequate

patients fail to adhere to treatment regimens

Mechanisms of action of antitubercular drugs: little is known

Mechanisms of resistance to these drugs: little is known.

a. Some strains of isoniazid resistant M. tb have a deletion or missense mutation in the katG gene (encoding catalase-peroxidase activity). Reintroduction of katG on a plasmid makes the cell isoniazid susceptible. ?katG product activates isoniazid to a more toxic form, able to exert antibacterial activity (inhibits mycolic acid synthesis). Antibiotic resistance is due to loss of a functional gene - unusual! Does not account for INH-resistance in all strains.

b. isoniazid - Gene inhA – an enoyl acyl reductase that is involved in fatty acid synthesis. Maybe this is the cellular target of INH in the mycobacterial cell. Mutation in inhA conferred resistance to isoniazid and ethionamide.

c. Rifampicin - alteration in  subunit of RNA polymerase.

 

 

Prevention:

1. Chemoprophylaxis is a mainstay of TB control in the US. Isoniazid daily for 9 to 12 months is effective and well tolerated. The regimen is recommended for individuals with recent skin test conversion or household contacts of TB patients.

 

 

2. TB vaccine: Calmette and Guerin subcultured a Mycobacterium bovis strain >200X to develop the attenuated BCG (Bacillus Calmette Guerin) strain in 1921.

In >120 countries (excluding the US), humans are immunized against tuberculosis with this live, attenuated (multiplies to only a limited extent in vivo) strain of M. bovis.

BCG can be administered im and is safe enough to give infants. Billions of doses have been administered world-wide. It is one of the safest vaccines known. Cheap: 10 cents/dose. It is effective in children, but it does not prevent against reactivation of a previous tuberculosis infection. It cannot be used in immunocompromised patients.

If effectiveness differs due to genetic differences in different populations. Reported ranges are from 0-80% protection for at period of 5 to 10 years. Average: 50% protective.

The BCG vaccine is not recommended in the US because: