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Microbiology - Lecture 15 - Mycobacteria and Actinomycetes

Make sure you know the objectives.
Distinguish between active and latent tuberculosis, and between an infection and disease.

Develop a treatment plan for a patient with tuberculosis taking into consideration all important factors (active vs latent, HIV status, drug resistance etc etc).

Evaluate the results of PPD skin test.

Differentiate between M. tuberculosis and the atypical mycobacteria.

Classify the actinomycetes by microbiological characteristics, epidemiology, pathogenesis and site of infection.
How are mycobacteria species divided?
Mycobacteria species are divided into slow-growing and fast-growing species.

Generally, the fast-growing species are non-pathogenic.
Name six mycobacteria species.
1. M. tuberculosis - the causative agent of tuberculosis.

2. M. bovis - bovine tuberculosis. Historically caused by contaminated milk, but now rare due to pasteurization.

3. M. leprae - leprosy. The first major bacterial human pathogen to be described, but still cannot be cultured outside of an animal.

4. Atypicals - such as M. avium, M. intracellulare (called MAC or MAI), M. kansasii and M. marinum. These species frequently infect patients with AIDS.

5. M. fortuitum - fast-grower.

6. M. smegmatis - nonpathogenic but fast-growing. Will not see in clinic.
What are the one line defining characteristics of mycobacterium?
Slow growing, obligate aerobic (found in lung often), thin bacilli, non-motile, non-spore forming (without oxygen go dormant), and acid fast.
What is the doubling time of M. tuberculosis?
M. tuberculosis has a doubling time of around 18 hr as compared to 30 min for many other bacteria.

Therefore it can take 3-10 weeks to get visible growth from a clinical specimen.
Note on cell wall.
Mycobacteria cell walls contain a wide array of different compounds which create a waxy fatty barrier of low permeability.

Because of this cell wall, mycobacteria tend to be more resistant to antibiotics and chemical agents. Very resistant to drying.

Many of these compounds are serologically active, triggering an immune system response. Many strains lacking these compounds are avirulent.
What are mycolic acids? What is a Arabinogalactan polymer?
Some important basic cell wall compounds are:

Mycolic acids - long-chain branched fatty acids.

Arabinogalactan polymer - unique polysaccharide.
What is Mycolyl-AG?
a. Arabinogalactan substituted by mycolic acids.

b. The major cell wall polysaccharide of TB.

c. Covalently attached to peptidoglycan.
What is Wax D?
a. Peptidoglycolipid - Muramyl dipeptide

b. Induces delayed type hypersensitivity to almost any antigen with which it is mixed. Freund's adjuvant.

c. Responsible in part for the induction of delayed type hypersensitivity to tuberculoprotein seen in positive skin test.
What is Cord factor?
a. Special mycoside (trehalose dimycolate) missing from some avirulent strains.

b. Associated with cording, which is due to bacilli aggregating in long serpentine chains with their long axes parallel.

c. Toxic to mice.

d. Inhibits leukocyte migration and causes chronic granulomas.
What is Lipoarabinomannan?
a. Cell wall associated glycolipid.

b. Anchored in the cell membrane but extends all the way to the surface.

c. Inhibits gamma-interferon activation of macrophages, induces TNF alpha release.
Which is true about mycobacteria cell walls?

A. They contain many different lipids specific to mycobacteria
B. It makes mycobacteria resistant to many antibiotics and dyes
C. It is responsible for the acid fast characteristic of mycobacteria
D. None of the above
E. All of the above
E, all are true.
What is the epidemiology of where TB is found?
1. All 50 states, but 60% of all cases are in 7 states, including California (Florida, Georgia, Texas, Illinois, New Jersey, New York).

2. Mostly urban areas.

3. Foreign-born people, where TB is 4-6X higher than for US-born people.

4. Minorities. Compared to Caucasians:
a. Asian Americans are 16X as likely to have TB.
b. African Americans are 8X.
c. Hispanic and Native Americans are 5X.
d. Socioeconomic rather than racial differences may account for these differences.

5. People with HIV - In the US, 26-38% of TB cases are estimated to be linked to HIV.

6. People in close contact with those who have TB.
Who is at risk of getting M. tuberculosis infection (not TB)?
1. People in close contact with people who have TB. Households.

2. Employees and residents of congregate settings. Nursing homes, prisons.

3. Foreign-born people, particularly from high incidence countries.

4. Low income individuals.

5. Some racial/ethnic populations.

6. Drug users.
Who is at risk of developing tuberculosis (the disease)?
1. People with the HIV virus.

2. People recently infected (within 2 years) with M. tuberculosis.

3. People with other medical conditions that increase the risk, diabetes, end stage renal disease, immunosuppression.

4. Drug users.

5. People with a history of inadequately treated TB.
What are virulence factors TB?
a. No exotoxin or endotoxins

b. Cell wall

c. Host factors
Why is chemotherapy difficult for TB?
Facultative intracellular pathogen - mycobacteria principally reside intracellularly in monocytes, reticuloendothelial cells, and giant cells.

This makes chemotherapy difficult.
Of those who are exposed to TB, what percent develop infection? What percent do not develop infection?
22% - infection.

78% - no infection.
Describe what happens when people are exposed to TB and are not infected.
1. TB is an airborne infection.

2. Infected individuals transmit the disease by discharging particles contaminated with bacteria into the air.

3. Small droplets reach the alveoli.

4. Bacilli are taken up by alveolar macrophages and are usually inhibited or killed. This is typically the end of it.
Describe what happens when people are exposed to TB and are infected.
1. Sometimes bacteria continue to divide and are released when the macrophage dies. Important factors are:
a. Number of bacilli taken in
b. Virulence of the bacilli
c. Host resistance

2. From there they spread to the regional lymph nodes and from there to the lungs, bones, liver, kidneys and brain.

3. These bacilli attract macrophages from tissues.

4. Exudative lesion

5. 4-6 weeks from original exposure, the person will test positive on the tuberculin skin test.

6. Not infectious
Describe trafficking of M. tuberculosis in a macrophage.
TB bacilli gets endocytosed by the macrophage and it enters into a phagosome.

A lysosome does not fuze with the phagosome and the phagosome is not acidified. This allows TB bacilli to live and then start dividing and bidding off bacilli.
Of those who get a TB infection, what percent develop primary active TB? What percent develop latent TB?
5% develop primary active TB.

95% develop latent TB.
What type of necrosis do you have a latent TB?
Caseous necrosis develops.

a. Center of lesion of giant cells (fused macrophages) containing live tubercle bacilli. Both of these are being killed.

b. Surrounded by partly activated macrophages and lymphocytes.

c. Bacteria are unable to grow due to lack of O2, or presence of toxic products.

Strong cell-mediated immunity develops, and activated macrophages surround and wall off lesions.
How is latent TB viable?
Viable bacteria are still present, often for decades.

Reactivate at a rate of anywhere from 2-23% in a lifetime. Often 10% in lifetime.

a. Highest reactivation percentage is in the first 2 years after exposure.

b. Predisposing factor possibly associated with deterioration of general health of person due to another disease - such as chronic alcoholism, malignancy, or diabetes - or some temporary suppression of cell-mediated immune response.

c. HIV 5-10% reactivation/year.
HIV 100X
Diabetes 3X.
What happens to patient who develop primary active TB?
1. A small number of patients do not develop adequate acquired immunity, the infection progresses, and tuberculosis ensues.

2. They are infectious at this stage through coughing. Patients are placed in isolation!

3. Tubercles develop - Productive lesion.

4. Necrosis continues, weight loss and death.

5. Tissue damaging response is largely responsible for clinical manifestations.

6. Humoral antibody development does not alter the course of the disease.
What are tubercles that develop in TB?
Tubercles - Productive lesion

a. Can be visible to naked eye.

b. Liquefaction of lesions which is ideal for bacilli growth.

c. Mostly free bacilli at this stage.

*In the lung these are seen as open cavities.
What type of light kills TB?
UV light.
Note on TB granuloma.
Macrophages will surround and try to wall off TB. So get caseous granuloma.

This is 4-6 weeks after exposure.

Will NOT test positive with PPD, but not contagious.

This is end of log growth of bacteria, this is due to cell mediated immunity.

This is why TB is bad for AIDS patients.
Where in the lung and in which populations do you see primary active TB?
Lower lung, see in children.
Where in the lung and in which populations do you see secondary active TB?
Upper lung, see in adults.
Describe tubercle discharging.
The caseous center of a granuloma will become liquid and is more of tissue damaging than macrophage activating.

The center will then discharge into the bronchial tree.

Now patient is infected because are coughing and spreading the bacteria.

The TNF-alpha will organize granuloma. So anti-TNF therapy can activate TB!
What are the common sites of TB infection?
1. Pulmonary tuberculosis (73% of cases are exclusively this)

2. Extrapulmonary tuberculosis. This is rarely contagious (except laryngeal TB).
a. Lymphatic is most common. Scrofula

b. Bone/spine - Pott's disease

c. Miliary tuberculosis -Very severe disease, patient's acutely ill, with fever, shortness of breath, or even acute respiratory failure.

From most to least common sites:
-Lymphatic system
-Genitourinary systems
-Central nervous system
-Bones and joints
-Disseminated (miliary TB)
What are the six steps to diagnosing TB?
Six steps in diagnosis of TB:
1. Medical history: close contact with TB patient?
2. Physical examination
3. X-ray
4. Tuberculin Skin Test
5. Bacteriologic
6. Newer Tests
What factors in the physical exam are you looking for in TB?

Chronic productive cough for 3 or more weeks

Low-grade fever

Night sweats - wetting the sheets

Weight loss and loss of appetite

What are the size measurements for PPD reactions?
Reading skin test - depends on size of induration and risk factors.

a. A reaction which is <5 mm in size is a negative reaction.

b. >5 mm is doubtful but can be positive for some high-risk people.

c. >10 mm is positive for most people with medium risk of exposure. Treatment!

d. >15 mm or greater is positive who have no risk factor. Treatment!

In the state of California >10 mm requires treatment.
What two things can cause a false-positive PPD reaction?
1. Non-tuberculosis mycobacteria

2. BCG vaccination
What four things can cause a false-negative PPD reaction?
Anergy - decrease in TB skin reaction due to immune suppression like in HIV.

Recent TB infection

Very young age (<6 months old)

Overwhelming TB disease
What does the Tuberculin Skin Test detect?
a. Used to detect prior infection with M. tuberculosis.

b. PPD (purified protein derivative) obtained by purification of the filtrate from 6-week old heat killed cultures of M. tuberculosis.

c. Usually 0.1 cc containing 5 TU (tuberculin units) injected intracutaneously in the forearm about 4 inches below the elbow, not over a vein.

a. Individuals having no contact with M. tuberculosis have no reaction.

b. Individuals infected with M. tuberculosis develop induration, edema, erythema in
48-72 hours.
What does a positive PPD mean?
Positive test indicates that the individual has been infected in the past and continues to carry viable bacteria.

It does not necessarily mean they have active tuberculosis.

An X-ray is the usual follow-up.
What does a definitive diagnosis of TB require? How does it work?
Definitive diagnosis requires isolation of M. tuberculosis from clinical specimens. Although patient history, physical exam, X-ray, etc. can all suggest active TB, the only reliable diagnosis must rely on acid fast stain or culture.

Very important to distinguish M. tuberculosis from other mycobacteria.

First step is usually acid fast staining.

Traditional acid fast stain or flurochrome
stain. Presumptive ID.

Cultivation from sputum using selective Lowenstein-Jensen media. BACTEC system takes about 9-14 days for detection.

Tests for drug resistance/susceptibility are always performed.
What are newer tests for TB other than PPD?
a. BACTEC - detects radioactive CO2 from a tagged lipid indicating growth. Takes about 9 - 14 days for detection.

b. Whole Blood gamma-IFN release assay - QuantiFERON-TB Gold and T-SPOT. TB test. Indirect tests for exposure to M. tuberculosis. Measures the CMI response to two mycobacterial peptides - specific for M. tuberculosis.
-Good in low incidence settings and less cross reactivity with BCG vaccine, but more expensive.

c. PCR based tests are being developed and some are currently available. Very good for smear-positive cases (up to 80%). Also other molecular probes.
Can you isolate M. tuberculosis from latent TB?
No, only from active TB.
What are the six CDC classifications for TB?
0 - no TB exposure, not infected.

1 - TB exposure, no evidence of infection.

2 - TB infection, no disease.

3 - TB, clinically active.

4 - TB, not clinically active.

5 - TB suspected. Diagnosis pending.

Note: A suspected case must not remain in class 5 for more than 3 months. All class 3 or 5 should be reported to local and state officials and CDC.
What is the most important in TB treatment?
Effective chemotherapy is the most important aspect of successful TB therapy. All other factors (rest, climate, diet, psychological state, and extent of disease) are of secondary importance if the chemotherapeutic regimen is adequate.
What antibiotics are used in TB?
Antibiotics - RIPE

a. Isoniazid - (INH) Inhibits synthesis of mycolic acids of the cell wall.

b. Rifampin family - (RIF) Inhibits RNA polymerase. Some HIV drugs interfere with this antibiotic. Given along with Vitamin B6 (pyridoxine)

c. Pyrazinamide - (PZA) Collapses the proton gradient.

d. Ethambutol - (EMB) Inhibits synthesis of mycolyl-AG.

e. Streptomycin - (SM) aminoglycoside that interferes with translation.
What is the treatment for active TB?
All forms of active tuberculosis are best treated with a multiple drug regimen - due both to antimicrobial synergy and to avoid development of antibiotic resistance (high number of bacteria).

a. Several drug combinations have been used involving isoniazid, rifampin, pyrazinamide, and streptomycin or ethambutol.

b. One current regiment for active TB involves administering INH and RIF concurrently for 6 months with pyrazinamide during the first 2 months.

c. Streptomycin or ethambutol is usually added until drug sensitivity is verified.

d. Because of the length of time and complexity of treatment many people do not complete treatment. DOT (Directly Observed Therapy) is often used.
What is the treatment for latent TB?
Latent TB is usually treated with 9 months of INH, provided active TB is ruled out first.

Treatment significantly prevents the progression to active disease.
What is MDR TB?
MDR (Multi Drug Resistant) strains are resistant to at least INH and RIF.

No change in virulence but since initial treatment may not be adequate patients may be infectious for a longer period of time.

Treatment may take 3 years.
What is XDR TB?
XDR - Extensively drug resistant - resistant to a fluoroquinolone, INH, RIF, and at least
one of three injectable drugs (i.e., amikacin, kanamycin, or capreomycin).

Treatment may take 3 years.
What is the BCG vaccine?
Attenuated strain of bovine tuberculosis M. bovis, Bacillus of Calmette, and Guérin.
When is the BCG vaccine used?
US Public Health service does not recommend its routine use because:

a. It is not effective (anywhere from 0-80% effective against pulmonary TB). However it does confer protection against serious forms of childhood TB.

b. It interferes with the TB skin test.

c. It can cause problems in people with weakened immune systems (HIV).

Rarely used in the US but it is used in other countries.

Treatment of bladder cancer!

If someone has been vaccinated with BCG assume exposure to TB.
What HIV considerations do you make with TB?
1. HIV is the strongest known risk factor for progressing from latent to active disease.

2. HIV kills CD4+ T-helper cells which allows M. tuberculosis to grow unchecked.

3. HIV-positive persons with TB are not considered any more infectious.

4. HIV has changed the classic clinical presentation with extrapulmonary TB much more common.

5. HIV can interfere with tuberculin skin test.

6. Protease inhibitors used to treat HIV interfere with antibiotics used to treat TB.

7. Increase in length of treatment.
What is Hansen's disease?
What are MAC or MAI?
M. avium-intracellulare complex are very hard to differentiate

Not spread person to person, present in the environment. Isolation not needed.

Rare in immunocompetent people

Disseminated MAC is a common opportunistic infection of bacterial origin in AIDS patients.

In HIV patients TB often appears first, followed by MAC as lymphocyte count drops.

Diagnosis is made by culturing MAC from blood or tissue.

MAC are generally resistant to first-line antibiotics.
What is leprosy? How is it spread?
1. Rare in the US ~100 cases/year. 1-3 million cases/year worldwide.

2. Mostly in California, Hawaii and Texas; mostly in immigrants.

3. M. leprae can not be grown in vitro, but in foot pads of mice and armadillos.

4. Grows best at 30°C and prefers cooler parts of the body.

5. Infects Schwann cells of peripheral nervous system.

6. Two forms:
a. Tuberculoid - benign and non-progressive slow. Few bacilli visible in tissue. Will see granulomas on histology.

b. Lepromatous -malign and progressive. Usually due to severe problems with cellular immunity. Numerous bacilli visible in tissue. This one produces LEPERS!

7. Means of transmission is respiratory in nasal secretions.

8. Diagnosis is usually by acid fast staining of skin or nasal scrapings along with the lepromin skin test.

9. Effective drug treatment with dapsone or rifampin, but it takes several years
What is MOTT?
MOTT - mycobacterium other than TB.

1. M. kansasii - can look like TB and cause a chronic lung disease.

2. M. marinum - swimming pool granulomas, pathogen of fish and frogs, skin lesions.

3. M. fortuitum- skin lesions and on prosthetic devices. Very drug resistant. Rapid grower.
What are Actinomycetes? Give the one line characteristics.
Bacteria, not fungi, but form long branching filaments like fungi. Grow end to end and separate. - hyphae.

Gram Positive.

Non-spore forming.
What are the two Actinomycetes we will be concerned with?
1. Actinomyces israelii

2. Nocardia asteroides
What is the microbiology of Nocardia with respect to gram stain and culture?
Non-spore-forming Gram positive bacilli that often form branched chains or filaments.

Delicate, irregularly stained or beaded appearance.

Partially acid fast.

Where is Nocardia found and what distinctive features does it have?
1. Found in soil and water

2. Distinctive features:
a. Abscess formation
b. Spread from lungs to other organs
What are the clinical aspect of Nocardia?
Opportunistic infection.

Starts as chronic lobar pneumonia
-Can mimic TB

Often spreads
-To CNS most common: brain abscesses
-To kidneys
-To skin
What is the differential?

49 yr old renal transplant recipient

CC: Fever, cough, chest pain x 2 weeks

Meds: Prednisone, mycophenolate
Nocardia asteroides
What is the differential?

CC: Tooth pain, weight loss, fever, swelling around jaw, discharge of pus.

Meds: None.
Actinomyces israelii
What is the epidemiology of Nocardia?
1. No person-to-person transmission.

2. Opportunistic infection in patients with impaired cell-mediated immunity (immunosuppression, solid organ transplantation).
What diagnostic tests can you run for Nocardia?
1. Sputum, pus, CSF for Gram stain, acid-fast stain.

2. But Nocardia isolated from sputum may reflect colonization and not infection.
What is the treatment for Nocardia?
1. Trimethoprim-sulfamethoxazole.

2. Surgical excision and drainage may be required.
What is the one line characteristics for actinomyces israelii?
1. Gram strain - gram positive

2. Culture - Anaerobe (or microaerobic)
What is the pathogenesis of actinomyces israelii?
1. Infection is usually from trauma that inoculates the bacteria into the tissue. LIke dental trauma. Bacteria is normal flora of oral cavity. Found around face and neck, see in boxers.

2. Three distinctive features:
a. Sinus (= fistula) formation

b. Crossing of tissue planes with spread to contiguous tissue

c. Sulfur granules in tissue: 1mm yellowish granules consisting of macrophages, fibrin and bacteria
What are the clinical aspects of actinomyces israelii?
1. Most common presentation: cervicofacial actinomycosis ("lumpy jaw):

a. Swollen, erythematous mass in the jaw with draining fistulas. Called lumpy jaw. Very noticeable/disfiguring.

b. Spreads to contiguous tissue and bone in the head and neck

2. Two other presentations:

a. Thoracic actinomycosis: pneumonia with fever, cough, purulent sputum, but with sinus tracts through chest wall, invasion of ribs

b. Abdominal actinomycosis: often after ruptured appendix or an ulcer
What is the epidemiology of actinomyces israelii?
No person-to-person transmission: the organism is part of the normal flora in humans.
What are diagnostic tests for actinomyces israelii?
Pus from draining sinus, sputum: Examine for sulfur granules Gram stain and culture
What is the treatment for actinomyces israelii?
1. Penicillin (long course)
2. Surgical excision and drainage may be required
Actinomyces and Nocardia - Compare and contrast.
These are environmental organisms that cause disease under certain circumstances:

- Actinomyces: inoculation across mucosal barrier.

- Nocardia: defects in cell-mediated immunity.

No person-to-person transmission

Anaerobe (actinomyces) vs. aerobe (Nocardia).