MycobacteriaceaeMycobacteriaceae Aerobic Gram-Positive Bacilli Aerobic Gram-Positive Bacilli

Form Filaments Form Filaments Stain Acid-fast Stain Acid-fast

MycobacteriaceaeMycobacteriaceae Aerobic Gram-Positive Bacilli Aerobic Gram-Positive Bacilli

Form Filaments Form Filaments Stain Acid-fast Stain Acid-fast

FATHER FATHER DAMIENDAMIEN

(1840 – 1889) (1840 – 1889) Lived and died Lived and died for victims of for victims of

Hansen’s Hansen’s disease disease (leprosy)(leprosy)

on Molokai, on Molokai, HawaiiHawaii

Mycobacterium: Genera • “fungus” “small rod”• Aerobic, fastidious, slow growing

– Cell division 12-24 hr.– Lab culture 1-2 months

• Large lipid content in cell wall, resistant to– Disinfectants– Detergents– Common antibiotics– Lab basic stains

• ~100 species, many isolated in humans• Important human pathogens:

– M. tuberculosis – “small swelling”– M. avian-intracellulare – “birds”; TB-like illness,

common in AIDS patients– M. leprae – “scaly skin”; Hansen’s disease

Nontuberculosis Mycobacteria: Runyon

Classification• Based on growth rate, pigmentation• Non-Runyon Group: M. tuberculosis, M.

leprae • Group I: Slow-Growing Photochromagen

– No pigment grown in dark– Photoactivated pigment upon exposure to light

• Group II: Slow-Growing Scotochromogen– Yellow, orange pigments grown in light or dark– Pigment deepens in two weeks

• Group III: Slow-Growing Nonphotochromagen– Produce white, yellow pigment– Pigment not intensify upon exposure to light– M. avian complex

• Group IV: Rapid Grower– Colonies in seven days

Mycobacterium: Staining• G(+) bacilli, slender, branched

filaments; stain poorly because of lipids (mycolic acid, waxD) in cell wall

• Acid-fast stain– Presumptive diagnosis mycobacterial

disease– Heating for stain penetration of high lipid

cell wall– Stain penetrates, binds to mycolic acid, not

remove with acid-alcohol treatment– Not easily decolorize, stain holds “fast”)

Acid-Fast Stain • Ziehl-Neelsen stain

– Carbol fuchsin - heat to penetrate– Acid-alcohol - decolorize– Methylene blue - counterstain

• Kinyon stain– Carbol fuchsin – no heat, higher

phenol allow penetration– Acid-alcohol - decolorize– Methylene blue - counterstain

• Fluorochrome stain– Auramine-rhodamine – stain,

phenol– Acid-alcohol - decolorize– Acridine orange – counterstain– UV microscope scan slide high

dry, detect AFB by fluorescence– Read stained slide easier, faster

Mycobacterium: Lab Culture

• Work under biosafety cabinet• Specimen of choice

– Patient coughs up sputum from lung– NaOH digest, decontaminate organic

debris, RT normal flora– N-acetyl-L-cystine to liquify

• Enrich, selective media– Egg or agar based– Antimicrobial agents - malachite green,

cyclohexamide, nalidixic acid• Lowenstein-Jensen medium – egg

based, colonies 18-24 days• Middlebrook 7H10, 7H11 medium –

agar based, colonies 12-14 days

Mycobacterium tuberculosis: Lab Culture

• Solid media - rough, dry, granular, non-pigmented, buff color colony

• Liquid media - contains Tween 80, albumin, faster growth

• RT grow best 5-10% CO2 , 370 C• Skin lesion grow best, 30-330 C• RT culture 6-8 weeks before

discard as negative• Skin lesion culture 12 weeks• CDC desires more timely

report, possible with new DNA amplification methods of ID

Mycobacterium: Lab ID • Rate of growth• Culture temperature• Pigmentation and photoreactivity• Biochemical testing: niacin

production, nitrate reduction, catalase at 680 C, tween hydrolysis, arylsulfatase production, tellurite reduction, salt tolerance, pyrazinamidase production

• Test of choice - DNA amplification; routinely done in PH lab, rapidly ID species

Mycobacterium tuberculosis

• Human only natural reservoir• Worldwide - third of population infected

– ~2 billion people– ~8 million new cases/year– ~2-3 million deaths/year

• USA ~10 million infected– Since 1985, dramatic increase number

cases/year– Infections in homeless, drug and alcohol

abusers, prisoners, AIDS patients– After 1992, now slowly decline due to

increase PH prevention programs

M. tuberculosis: Virulence Factors

• Cord factor – cell wall glycolipid– Serpentine growth (filaments, cords), grow in

close parallel arrangement– Toxic to leukocytes, anti-chemotactic – Role in development of granulomatous

lesions• Iron capturing ability – required for

survival inside phagocytes• Sulfolipids - prevent phagosome-

lysosome fusion (important in intracellular survival)

• Tissue damage - no known bacterial toxin or enzyme implicated; host immune response thought responsible, by inflammation, cell-mediate immunity (CMI)

M. tuberculosis: Transmission

• Close contact - person-to-person• Inhalation - infectious aerosols

into alveolar spaces• Exposure to few organisms (10-

200) may establish “infection” (elicit immune response, no disease)

• Humans very susceptible to infection, but remarkably resistant to tuberculosis disease

Primary Tuberculosis• Exposure - bacilli reach alveoli,

ingested by macrophage• MO multiply - cause chemotactic

response, recruits macrophages, T cells

• Enzymes, cytokines release - start inflammatory response, wall off MOs (tubercle formation)

• Inflammatory response also causes lung damage

• Small number MO - no tissue damage• Large number MO - CMI response

results in tissue necrosis• Patient becomes PPD skin test(+)

Early Tubercle

Reactivation Tuberculosis • Few weeks macrophages die - release

bacilli, form caseous center in tubercle• In healthy individuals - disease usually

arrested, lesions calcified • Tubercle bacilli - may remain dormant

in lesion; later reactivation of disease• Host defenses fail - mature tubercle

form; caseous center enlarge, liquify to form tuberculous cavity where bacilli multiply outside macrophage

Mature Tubercle

Extrapulonary Tuberculosis

• Tubercle ruptures - release bacilli; disseminate throughout lung, circulatory, lymphatic system

• Miliary (Extrapulmonary) TB – spread to lymph nodes, pleura, many other organs:– Progressive form of disease– Weight loss, coughing with blood, loss of

vigor (old name consumption)

Tuberculosis: Infection and Disease

• In 3-6 weeks - patient’s CMI activated, bacteria replication stops

• Within 2 years - 5% patients progress to active disease

• Sometime later in life - 5-10% patients develop active disease

• AIDS patient, TB infected:– Due to M. avian– 10% develop active disease within 1 year– 2x more likely to spread, rapidly progress

to death– Impaired CMI unable to arrest infection

M. tuberculosis: Treatment • Slow growth of MO, chronic infection

require 6-9 months drug treatment • Combination of drugs to prevent

emergence of resistant strains• Current recommended drugs:

isoniazid (INH), ethambutol, pyrazinamide, rifampin

• Drug resistance– 1990 report multidrug-resistant M. tb

(MDR-TB) in AIDS patients, homeless in N.Y., Miami

– In developing countries, extensively drug-resistant TB (XDR-TB), resistance to second line drugs, potentially untreatable

M. tuberculosis: Prevention

• BCG (bacille Calmette-Guerin) Vaccine– Attenuated M. bovis– Used where TB high– Reduce TB if vaccinated young

age• Tuberculin skin test

– PPD (purified protein derivative M. tb) injected under skin

– Test host CMI response– Delayed-type hypersensitivity

reaction (>10 mm induration), 48 hr., if previous or current infection; not necessarily active disease

• Control disease– PH surveillance– Drug treatment and intervention– Case monitoring, prevent

transmission

M. avium-intracellular Complex

• Common in soil, water, food• Before HIV - transient colonization in

patients with compromised pulmonary function (bronchitis, obstructed pulmonary disease, previous infection); ~pulmonary TB

• After HIV - USA most common mycobacteria disease in AIDS patients

• Infection disseminated - all organs, large number MO

M. avium-intracellular Complex

• Transmission – ingestion of contaminated food or water, not person-to-person

• Greatest risk for infection are immunocompromised

• Multiply in localized lymph nodes, spreads to disseminated disease

• Impair organ function due to replication MOs, host immune response

• MO ubiquitous and control of exposure difficult

Mycobacterium leprae: Hansen’s Disease

• “to peel” “leprosy”• ~12 M cases worldwide (Africa, Asia,

Latin America); rare USA• Reservoir of MO in armadillo• Does not grow in cell-free culture• Transmission by person-to-person,

direct contact, inhale infectious aerosols

• Requires prolonged, intimate contact for transmission

• Two clinical forms of disease:– Tuberculoid– Lepromatous

Hansen’s Disease • Mycobacteria obligate intracellular

parasites in histiocytes, Schwann cells, epitheloid structures - called Lepra cells

• Incubation period 2-4 years• Clinical manifestations depend upon

adequacy of host CMI response• Like TB - many infected, few develop

clinical symptoms of disease• Subclinical/Tuberculoid – infection

contained by CMI• Lepromatous - large number bacilli in

sputum, nasal secretion, skin

Hansen’s Disease: Tuberculoid

• Patient - strong CMI, weak antibody response

• Lesions - skin, peripheral nerves, few in number; raised, erythematous margin, flat center

• Nerve damage due to CMI response - loss sensation of touch, temperature; pain within lesion

• Skin biopsy reveals many lymphocytic, epithelial cells, but no AFB

• Infectivity, transmission low• Lepromin skin test (+)

Hansen’s Disease: Lepromatous

• Patient - strong antibody response, defective CMI (“foamy” macrophage, few lymphocytes, numerous bacilli)

• Involve all areas of skin; waxy, nodular appearance, may thicken and fold

• Destruction of cutaneous nerves, eyebrows, eyelashes, nasal septum

• Skin biopsy reveals lymphocytes, many Lepra cells packed with AFB

• Infectivity high• Lepromin skin test is (-)

M. leprae: Treatment and Prevention

• Tuberculoid form – rifampicin, dapsone; 6 months

• Lepromatous – rifampicin, dapsone, clofazimine; minimum 1 year

• Control by prompt recognition and treatment of infected patients

• Lepromin skin test is similar to TB skin test

The Gifts of Civilization: Germs and Genocide In

Hawaii • O. A. Bushnell, University of Hawaii

Press. 1993• Hawaii isolated, difficult to reach by sea• 1778 – “discovered” by Captain Cook• Estimate ~1 million Hawaiian

inhabitants• 1832 – census ~130,000• 1900 - ~30,000• Why decline?• Infectious diseases upon immune naive

population i.e. STD, plague, cholera, TB, Hansen’s disease from Chinese immigrants, smallpox, chickenpox, measles, mumps, rubella;

FATHER FATHER DAMIEN DAMIEN(1840 – (1840 – 1889)1889)

FATHER FATHER DAMIEN DAMIEN(1840 – (1840 – 1889)1889)

The man who lived and The man who lived and died for the victims of died for the victims of

Hansen’s disease (leprosy)Hansen’s disease (leprosy)

Damien Born in Belgium 1840

• He was an ordinary boy, brother, and priest.

• So what made him different?

• Damien arrives Hawaiian Islands 1864

• He agreed to do a job that no one else would do.

• He lived and worked for a group of people who had been sent away from their homes to a remote Hawaiian island, Molokai

• They were outcasts because they had Hansen’s Disease.

Hansen’s Disease

• A scaly skin disease.

• A chronic disease caused by a bacteria.

• In those days there was no cure for Hansen’s Disease.

• The outside world did not like to think about this awful disease, and chose to forget the people who suffered from it.

• Damien gave back these people hope and pride.

• He loved them like a family and in the end he died as one of them, a victim of Hansen’s Disease.

• Damien’s life and death forced people to face the problem of Hansen’s Disease.

• He said to the world that work needed to be done – and quickly!

• Today, a cure for Hansen’s Disease has been found.

• We no longer have to fear the disease or the people who suffer from it.

• In 2009, for Father Damien’s many contributions to society, he was Canonized Saint Damien by the Vatican.

Class Assignment• Textbook Reading

– Chapter 26 Mycobacterium Tuberculosis– Omit: Clinical Significance and

Differentiation of Nontuberculosis Mycobacterium

• Key Terms• Learning Assessment Questions

Case Study 7: Mycobacterium

• A 35-year-old man with a history of intravenous drug use entered the local health clinic with complaints of a dry, persistent cough; fever; malaise; and anorexia.

• Over the preceding 4 weeks, he had lost 15 pounds and experienced chills and sweats.

• A chest radiograph revealed patchy infiltrates throughout the lung fields.

Case Study 7: Mycobacterium

• Because the patient had a nonproductive cough, sputum was induced and submitted for bacterial, fungal, and mycobacterial cultures, as well as examination for Pneumocystis organisms.

• Blood cultures and serologic tests for HIV infection were performed.

• The patient was found to be HIV positive. • The results of all cultures were negative

after 2 days of incubation; however, cultures were positive for M. tuberculosis after an additional week of incubation.

Case Study 7: Questions

• 1. What is unique about the cell wall of mycobacteria, and what biologic effects can be attributed to the cell wall structure?

• 2. Why is M. tuberculosis more virulent in patients with HIV infection than in non-HIV-infected patients?

• 3. What is the definition of a positive skin test (PPD) result for M. tuberculosis?

• 4. Why do mycobacterial infections have to be treated with multiple drugs for 6 months or more?