Lectures 18 and 19: Mycobacterium

Mycobacteria Stains

Mycolic acid cell wall

Carbol-fuchsin lipid soluble stain: Red

Auramine-rhodamine stain: Binds to mycolic acids and fluoresces under UV light → yellow/orange rods

Mycobacteria Virulence Factors

Mycolic Acids and Lipids:

Invade macrophages and dendritic cells

Mask PAMPs in cell wall to avoid innate immune recognition

Cord Factor

Elicits pro-inflammatory cytokines (TNF) from macrophages that promote granuloma formation and tissue necrosis

Causes serpentine growth in vitro

Causes weight loss and wasting in patients

Inhibits phagolyosome fusion

Mycobacteria Overview

Aerobic, non-motile, rods

Slow growing

Löwenstein–Jensen or Middlebrook medium used for cultivation

Mycobacteria Infection

Infectious respiratory particles that have escaped mucociliary clearance in upper airway

Does not require direct deposition

Inhaled bacteria reach the alveoli and are engulfed by patrolling macrophages

Cell envelope lipids (Sulfatides) inhibit the fusion of the phagosome with acidic lysosomes → allowing for survival inside macrophages

Replicate inside the phagosome within macrophages

Mtb-infected macrophages and dendritic cells carry the infection to other locations within the lungs and local lymph nodes

Hematogenous dissemination also occurs and may seed virtually any organ

Granuloma Formation

Dendritic cells carrying Mtb migrate to local lymph nodes and present antigens to T cells

Mtb-specific T cells traffic to granuloma -delayed, takes weeks!

T cells produce IFN-γ that activate macrophages within the developing granuloma to kill Mtb

Macrophages differentiate into epithelioid structures that enclose the infected cells

Some macrophages turn into lipid-laden foamy macrophages that can sustain live Mtb

Risk factors for Disease Progression

T cell immunity- HIV/AIDS

Age: 3 high-risk periods - 1) infancy, 2) adolescents (15-25), 3) elderly

Forms of immunodeficiency

Active Pulmonary TB signs & symptoms

Prolonged fever, night sweats, cough, anorexia & weight loss (consumption)

Supraclavicular adenopathy, chest pain, hemoptysis (less common)

Subset develop extrapulmonary disease (Miliary TB, TB lymphadenitis)

Tuberculin Skin Test

T cell-mediated Type IV hypersensitivity reaction (delayed) to Mtb antigens

DCs with Tuberculin purified protein derivative (PPD) migrate to lymph node, activate Mtb-specific T cells

T cells migrate to injection and release cytokines to produce the induration & redness

Measure the diameter of the induration

No risk factors, bump diameter must be >15 mm to be positive

HIV, a bump diameter of 5 mm or greater is positive

False Negatives:

Immune compromised including infants < 6 months

Very recent infection (3-9 weeks)

False positives:

Bacillus Calmette-Guérin (BCG) vaccine (attenuated M. bovis)

Infection with non-TB Mycobacteria

Bacillus Calmette-Guérin (BCG) vaccine

Effectively prevents disseminated (miliary) TB disease in infants

Usually given to newborns

Decreases progression to TB disease in infants and children

Does not prevent infection with Mtb

Does not prevent future cases of adult pulmonary TB

Interferon-Gamma Release Assay

Blood test

Measure the amount of IFN-γ produced by Mtb-specific T cells in response to TB antigens

No false positive with BCG vaccine

Only way to show definitive infection

Culture

Acid-fast bacilli (AFB) stains on sputum or tissue may show Mtb - early indication

Growth on Mtb medium (~4-6 weeks)

Rifampin

Used for treating LTBI and active disease

Lipophilic so it diffuses well into Mtb

Binds to bacterial RNA polymerase, preventing transcription of DNA to RNA

Highly bactericidal

Isoniazid

Used for treating LTBI and active disease

Diffuses well into host cells and into Mtb

Inhibits enzymes important for producing mycolic acids in the cell wall

Rapid killing (bactericidal) for bacteria that are metabolically active; slower killing for non-dividing bacteria

Thus need long treatments

Pyrazinamide

Used for treating active disease

May disrupt the plasma membrane

Can kill in acidic environments (i.e. vacuole in macrophages)

Effective at slowly killing dividing/non-dividing bacilli

Decreases duration of treatment by 2 months when combined

M. bovis is intrinsically resistant to PZA

Ethambutol

Used for treating active disease when resistance to INH is suspected

Mechanism of action is to inhibit cell wall synthesis by blocking arabinogalactan polymerization (sugar layer)

Side Effect: Can cause optic neuritis

Non-Tuberculosis Mycobacteria Overview

Free-living saprophytes ubiquitous in the environment

Inhabit water and soil

Opportunistic pathogens -transmission occurs through exposure to contaminated aerosols, dust particles, or water

NTM Clinical Manifestations

Three main types of Diseases:

1) Pulmonary: Older adults with underlying lung disease: Consolidation with cavitation, Bronchiectasis, Small nodules and cavities

2) Cervical lymphadenitis: Young children, non-painful, unilateral enlarged cervical lymph nodes sometimes with erythema - afebrile. Typically firm on palpation (can be fluctuant)

3) Skin and soft tissue: Chronic papules and ulcers from direct inoculation

Less Commonly:

Disseminated mycobacteriosis in HIV/AIDS

Other organs

Nocardia

Found in soil

Aerobe, catalase +

Gram-positive and forms long, branching, filaments resembling fungi

Acid-fast (weak)

Pulmonary nocardiosis

Infections in immunocompromised - presents as acute respiratory illness

Subset have disseminated disease to CNS, skin, joints

TST test is negative

Multifocal lung consolation predominant finding on CT

Sulfonamides

SMX inhibits dihydropteroate synthase, thus inhibiting folate synthesis

Trimethoprim - inhibits bacterial dihydrofolate reductase

Cause sequential block of folate synthesis