Mycobacteria Aerobic Rod shaped Nonsporeforming (Nonsporogenous) Non-encapsulated Nonmotile Acid-fast bacilli Growth in simple artificial media or a more complex

medium (Lowenstein-Jensen medium) Cell wall

complex lipids (mycolic acids) acid-fastness hydrophobic cell surface resistant to chemical clumped slow disinfectants, strong growth uptake of acids & alkalis nutrients slow growth

• obligate aerobe• “just like us”• Use glycolysis, Kreb’s TCA cycle and electron

transport chain with oxygen as final electron acceptor

• Most commonly affects the lungs• acid-fast rods

• Due to high content of mycolic acid, long chain linked fatty acids and cell wall lipids (also, antibacterial drugs find it hard to penetrate mycobacteria bec. of this)

• With certain growth pattern (parallel) which is known as serpentine cording

Mycobacterium tuberculosis

Types Mycobacterium tuberculosis Mycobacterium bovis Mycobacteria associated w/ Nontuberculous Infections Mycobacterium ulcerans Mycobacterium leprae

Bases for the differentiation of different species: a. Rate of growth

Rapid growers <7 days MTB are slow growers compared to atypical

mycobacteria.

b. Optimal temperature for growth = 35 - 37°C

c. Pigment production MTB – no pigment! Photochromogen – ‐ develop yellow pigment when

exposed to constant light source; nonpigmented in dark

Scotochromogen – produce yellow tro orange pigments in dark; prigments intensify to orange or red when exposed to constant light source for 2 weeks

Non(photo)chromogen – nonpigmented or light tan or buff‐colored colonies

d. Biochemical tests 1. Niacin test

- Produced by all Mycobacterium species - MTB lacks enzyme that converts free niacin to

niacin ribonucleotide - Test determines accumulation of free niacin

(lack of enzyme) in the reagent strips - Useful in determining MTB which are niacin (+)

2. Nitrate reduction - (+) MTB - Presence of nitroreductase (convert nitrates to

nitrites)

3. Heat‐stable Catalase - (- ) MTB complex

A. Morphology and Identification-In tissue = thin and filamentous rods; in artificial media, coccoid and filamentous forms can be seen.

-Cannot be classified as either gram-positive or gram negative. Once a mycobacterium is stained with a basic dye, it cannot be decolorized even without iodine (the mordant).

B. Culture1. Semisynthetic Agar media – Middlebrook 7H10

and 7H11. Need large amount of inocula. Usually used for observing colony morphology, susceptibility testing, and with added antibiotics and malachite green, as selective media.

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MYCOBACTERIA

2. Inspissated egg media – Lowenstein-Jensen. Usually used for selective media.

3. Broth media - Middlebrook 7H9 and 7H12. These support the growth of small inocula.

C. Growth Characteristics-Obligate aerobes

-Increased CO2 tension enhances growth

-Doubling time of tubercle bacilli is about 18 hours

-Proliferates at 22-33 degrees Celsius

D. Reaction to Physical and Chemical Agents-Tend to be more resistant to chemical agents

-Resistant to dying and survive long in dried sputum

Mycobacterium Tuberculosis• major human disease (pulmonary tuberculosis)

• WHO in 2001 ~3M mortality rate and > 3.8M new

cases 90% from developing countries person to person contact (respiratory

droplets)*TB is chronic infection since carrier chronically carries the bacterium

• problems defining disease for AIDS because they are

immunocompromised with low levels of CD4+ T cells (200 is considered low=high susceptibility to M. tb)

multiple drug-resistance

M. avium-intracellulare complex (MAC)• healthy people

infection almost never • people with AIDS

major bacterial opportunist (treatment should raise CD4 levels above 100 in 6 months)

• multiple drug-resistance

M. bovis • Mode of Transmission: ingestion of contaminated milk

(gastrointestinal tb)• rarely seen in US• pathogenic to humans

• given as vaccine (BCG vaccine=attenuated)*agent of tuberculosis in cattles

M. leprae• leprosy/Hansen’s disease• major disease of third world eps. in rural communities• transmition in unclear but believed to be thru soil,

inhalation and insects• Phenolic glycolytic 1 in cell wall specific for M. leprae• rare in US • infects upper respiratory system, skin, testis, peripheral

nerves*cannot be cultured in vitro

Transmission – M. tuberculosis• M. tuberculosis causes disease

healthy individuals • transmitted by airborne droplets

Commonly through coughing When inhaled, they go to alveoli and gets

phagocytized by macrophages where they are able to survive and multiply then are carried to lymph nodes

Tissue hypersensitivity and cell mediated immunity (CD4+ T cells associated with MHC II) controls the multiplication of M. tb

Mycobacterium tuberculosis in the lung

Pathogenesis of TuberculosisA. Facultative Intracellular Growth

• infects lung (post. portion of apices where it is highly oxygenated)

Lower part of the upper lung Upper part of the lower lobe – tb infiltrates

evident in x-rays• 1st exposure – no specific immunity• Extrapulmonary sites of tb scrotum, bones, kidney,

liver• distributed within macrophages• Multiply and survival

inhibits phagosome-lysosome fusion resists lysosomal enzymes

B. Cell-mediated immunity

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• infiltration macrophages - primary cells infected by M.

tuberculosis lymphocytes – for tissue hypersentivity and cell

mediated immunity (there would be Langhan’s cells and epithiliod cells = granulomas with caseation necrosis, also due to high antigen load)

• Granulomas Result of the hypersensitivity that is part and

parcel of the host immune response• Tubercles

Soft: granuloma with caseation necrosis Hard: no caseation necrosis

Key factors in the pathogenesis of TB• Cord factor: a glycolipid found in the cell walls of the

bacteria importance in virulence, being toxic and

inducing granulomatous reactions• Lipoarabinomannan (LAM): inhibits macrophage

activation• Secretion of TNF-ɑ : causes fever, tissue damage and

weight loss• Complement activated on the surface of M.tb opsonize

and facilitate its uptake by macrophage complement receptor

manifestations of the disease are not due to organism but due to the immune response of the body

Other minor pathogenesis factors tuberculosis • mycobactin

– siderophore – cord factor – damages mitochondria

Laboratory Diagnosis1. Skin testing

delayed hypersensitivity tuberculin Purified protein derivative (PPD) – for

screening, (+) indicates an induration (there would be a wheal) of >10mm, provided that there is no history of BCG vaccination (false positive = BCG vaccination); derived from old cox tuberculin (extract of culture of M. tb) that uses 5 tuberculin units that contains 0.001 PPDs proteins and is administered by Mantoux test on volar surface of forearm, creating a wheal, that should be read 48-72 hours after the test (delayed hypersensitivity test)

Positive skin test –tuberculosis• indicates exposure to organism• does not indicate active disease

Disease is indicated by clinical symptoms and positive sputum and culture

2. X-ray Usually chest, strong presumptive evidence Typical pattern: radio-opaque infiltrates in the

apices, hilar lymph adenopathy, in children (primary complex) with patchy infiltrates with hilar lymph adenopathy (90% can contain the infection and the lesion will be calcified or fibrotic)

3. AFB smears• primary tool using sputum sample (collected early

morning, 3 samples)• Not a 100% accurate*Carbolfuschin binds to the mycolic acid of the cell wall

*Ziehl neelsen (hot method) and Kinyoun (cold method)

4. Culture (gold standard)• grows very slowly• In solid (agar based) and liquid media• 2-3 weeks before colonial growth• non-pigmented colonies, sensitive to isoniacin• niacin production

differentiates from other mycobacteria, (+) for M. tb and not for M. bovis

5. Polymerase chain reaction for amplification• Rapid and sensitive

A few as 10 TB bacilli in the sputum will generate a positive result

• Very expensive6. Nucleic acid method,

using a probe

Antimicrobial treatment• extensive time periods (e.g. 9 months)• organism grows slowly, or dormant• two or more antibiotics

– e.g. rifampin and isoniazid – resistance minimized

Isoniazid, rifampin, pyrazinamide, and ethambutol are administered for 2 months followed by 4 months of isoniazid and rifampin (unless susceptibilities indicate drug resistance).

Vaccination• BCG vaccine

an attenuated strain of M. bovis not effective

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Can’t protect against infection but can reduce the incidence of tb meningitis in children that is why it is still given

Can be used as an adjuvant • in US

incidence is low vaccination not practiced immunization interferes with diagnosis

Mycobacterium Leprae

Leprosy (Hansen's Disease)• causative agent: M. leprae • Mode of transmission: aerosols from lesions in the Upper

Resp. Tract (but transmission thru soil is investigated)• Incubation may take 5-6 weeks or even years (as long 40

years)• Said to be the “least communicable disease”, children

are commonly affected• chronic disease

disfigurement• rarely seen in the U.S. • common in third world

millions of cases • infects the skin

low temperature (in earlobes and tip of nose)

• Affects the peripheral nerve and skin, resulting in disabling deformities

Produces ulcers, resorption of bone worsened from careless use of hands (nerve

damage=numbness from stimuli), amputation of digits

Disease Pattern of LeprosyTuberculoid Lepromatous

few organisms in the lesion few organisms

active cell-mediated immunity immunosuppression

there is follicular granuloma no granuloma formation

Early involvement of the nerves

“leonine facies”Peripheral nerves are affected late

Asymmetrical peripheral nerve involvement

Symmetrical involvement of the nerves

Production of M. leprae antigens and pathogenesis studies • in vitro

unculturable • in vivo growth

low temperature armadillo mouse footpad

Diagnosis - Leprosy • lepromin

skin testing Use of whole inactivated organism, like PPD Not a useful diagnostic test since it presents

false (+)• acid-fast stains

skin biopsies Using FITE stains

• clinical picture

Atypicals - mycobacteria (including M. avium) other than M. tuberculosis and M. leprae• infect compromised host• ubiquitous/environmental mycobacteria (found

everywhere)• not transmitted man -man

• healthy people

Mycobacterium avium and AIDS

Atypical Mycobacterial Diseases • tuberculosis-like (pulmonary tb)• leprosy-like (skin lesions look alike)

Mycobacteria and AIDS• M. avium is much less virulent than M. tuberculosis

does not infect healthy people infects AIDS patients (<100 CD4 count)

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• M. tuberculosis infection infects healthy people infects AIDS patients

* earlier stage of disease* more systemic

Clinical Features with AIDS • systemic disease (versus pulmonary)

greater in AIDS AIDS px with MAC have Normal chest x-ray

• lesions often lepromatous

Antibiotic Therapy• selected primarily for M. tuberculosis • if M. avium suspected other antibiotics included

Other atypical• pigmented or not• pigmentation

light dark

• growth fast slow

Clinical Disease• Cutaneous disease

• M. marinum (from pools and ponds)• M. ulcerans

• Pulmonary disease nodules bronchiectasis chronic pneumonitis cavitary

Atypical Mycobacteria that can produce pulmonary manifestations

MACM. kansasii *yellow bacillus due to carotene pigmentM. xenopi M. malmoense M. abscesses

MAC (M. avium complex)Secondary disease of px with

COPDTBCystic fibrosis

Treatment• MAC

Clarithromycin (Claricin)Ethambutol (with or without) RifabutinEcithromycin (prophylaxis for 12 months if CD4 count continues to go down)

• NTM & HIVGlucocorticoids Immunosuppresives Lymphoma & leukemia Heritable disorder-IFN gamma production

Disseminated in HIV px (asides from M. tb)MAC/M. kansasii

Mycobacterial Species Identification• Cellular fatty acid profiles (liquid chromatography)• Lysis centrifugation method (highly specific for

mycobact in HIV px)• Mycolic acid profiles (liquid chromatography)• Genetic markers (nucleic acid hybridization method)• Ernest-Ranian classification (colonial characteristics

of atypical mycos)

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Type Tuberculoid LepromatousImmunity - Strong cell - mediated immune response;

delayed type hypersensitivity- Severely depressed immunity; ↓ in delayed

type hypersensitivity- Erythema nodosum

Lepromine Test (+) positive (-) negativeClincal Manifestations

- large maculae – skin, nose, ears and tests), in superficial nerve endings

- Neuritis- Severe asymmetric nerve involvement of

suddent onset- Benign and non progressive- Lesions: heavy infiltration by

lymphocytes, giant and epitheloid cells- Few bacilli in lesion (Paucibacillary)

- large numbers present- Reticuloendothelial system and immunity ↓- Abundant AFB in the skin (multibacillary)- Slow symmetric nerve involvement- With nodular lesion- Progressive and malign- Cont. bacteremia- Facial disfugurement

CMI Intact cell mediated Immunity Deficient cell mediated ImmunityLab diagnosis - Rare AFB in clinical samples;

- skin biopsies show mature granulomas in the dermis (epithelioid cells, giant cells & many lymphocytes);

- Diagnosis depends on clinical findings & histology of biopsy material

- many acid-fast bacilli in skin scrapings from nasal mucosa & other infected areas; skin biopsies show no epithelioid cells & giant cells, rare lymphocytes & histiocytes w/ a foamy appearance

Treatment Dapsone and Rifampin ClofazamineLong term antibiotic therapy

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