MICROBIOLOGY LECTURE 8 – Mycobacteria 6.

Other immunoreactive components

Notes from Lecture a. Wax D & muramyldipeptide (MDP)
USTEMED ’07 Sec C - AsM  peptidoglycans (cell wall)
 adjuvant activity
Characteristics:  induce a cell-mediated immune response
- aerobic, slightly curved or straight rods against the protein
- 0.2 to 0.6 um wide; 1.0 to 10 um long b. Trehalose-6-6’-dimycolate
- cell wall: multilayered complex lipids  cord factor
- common properties with Corynebacterium and  immunoreative properties
Nocardia:
 adjuvant activity
o produce mycolic acid
 elicits extensive pulmonary granulomas
o ~guanine + cytosine content (G+C)
 anti-tumor properties
c. Sulfatides (Trehalose 2’-sulfates esterified with fatty
Mycobacterium tuberculosis
acids)
Tuberculosis  sulfur-containing glycolipids (sulfolipids)
- An ancient disease  can replace cord factor as a component of
- Seen in stone age skeletons and early Egyptian an oil-BCG cell wall or endotoxin
mummies preparation causing tumor regression
- 1882 – Koch’s discovery
- Koch’s postulates: Determinants of Pathogenicity
o An organism associated with clinical disease - Cord factor
o Isolated it in pure culture - Sulfatides
o Reproduces the disease in animals
Epidemiology of MTb
o Recovered the bacillus in pure culture from
- TB is a global problem
the experimental animal - 8 to 10 million new cases worldwide
- 3 million deaths worldwide
Morphology of MTb - Yearly decline in TB incidence ended in 1984 when the
- Slender, straight or slightly curved rod with rounded HIV infection increased in number
ends - Philippine statistics: FHSIS-DOH 2001
- 0.3 to 0.6 um width x 1-4 um length
- true branching in old cultures and smears from o Respiratory TB: 6th leading cause of
caseous lymph nodes morbidity: 11-,841 cases and rate of
- acid fast: lipid-barrier (mycolic acid) and carbolfushin 142.2/100,000 population
dye complex o TB meningitis: 466 cases and rate of
- hydrophobic surface layers trap the dye 0.6/100,000 population
o Other forms of TB: 11,494 cases and rate of
Mycobacterial Cell Wall 14.7/100,000 population
- 60% lipid including mycolic acids - Transmission
- backbone: covalent structure consisting of 2 polymers o Inhalation of dried residues of droplets
covalently linked by phosphodiester bonds:
containing tubercle bacilli
o peptidoglycan
o Droplet nuclei (1 to 10 um) can reach the
o arabinogalactan
alveoli and initiated infection
- Tuberculous infection vs. Tuberculous disease:
MTb Physiology
Tuberculin Clinical
- Cultural characteristics:
test symptoms
o Strictly aerobic
Tuberculous infection + -
o Most spp. Grow slowly with generation times
Tuberculous disease + +
8 to 24 hrs.
o Grow fairly on simple artificial media except
- risk factors for the development of Tuberculous
M. leprae disease:
- Species are differentiated by: o intrinsic characteristics of the individual –
o Rate and optimal temperature of growth
age, sex, body build, & genetic
o Production of pigments susceptibility
o Biochemical tests (niacin, catalase test, o use of adenocorticosteroids &
urease test, etc) immnunosuppressive agents
o hematologic diseases
Antigenic Structure of Mycobacteria o reticuloendothelial disease
1. Old tuberculin (OT)
o Diabetes mellitus
o 1881 – described by Koch by boiling a 6-week old
o Silicosis
broth culture
o HIV infection
o Heat–stable protein is the active component
Pathogenesis of TB
2. Purified Protein Derivative (PPD)
o Partially purified preparation of OT prepared by
ammonium sulfate fractionation Inhaled MTb bacilli
o PPD-S: adopted by WHO for skin testing

3. Purified Antigens:
o Recombinant DNA techniques & antigen alveoli
expression in E.coli
o Affinity purification of antigen preparations using
monoclonal antibody immunosorbent columns
Multiply in the pulmonary epithelium or macrophages
4. Polysaccharides:
2-4 weeks
o Protein-free polysaccharides (arabinogalactans &
arabinomannans) Bacilli are destroyed by immune system
i. Immunogenic
ii. Give precipitin reactions with antisera
iii. Active in C’fixation &
hemmaglutination reactions Survivors

5. Phosphatidyl Inositol Mannosides (PIMS) blood

o Lipoteichoic acid-like polymers with a role in
Extrapulmonary
macrophage recognition
sites
o Associated with cross protective immunity

Bacterial sulfolipids inhibit fusion of phagocytic

vesicles with lysosojmes

Clinical Manifestations
- Primary Tuberculosis
o No previous contact with the organisms
o 95% of cases are arrested
o positive tuberculin test
o chest x-ray: pulmonary nodule & some
fibrosis (Ghon complex)
o 10% develop clinical Tb later in life
- Primary disease:
1. Initial phase
o Primary Tb
 – mild or asymptomatic and results
in exudative lesions with PMN and
fluid accumulation around the
bacilli
 cell-mediated immunity and
hypersensitivity rxn
2. tubercle formation
o granulomatous lesion
 central area of large,
multinucleate giant cells
(macrophage syncytia) with
tubercle bacilli, an id-zone of pale
epithelioid cells, and a peripheral
collar of fibroblasts and
mononuclear cells
o Alcoholism
o Advanced age
o Severe stress
o Immunosuppressive tx
o DM, AIDS
- Chronic Pulmonary Tuberculosis
o Insidious onset of fever, fatigue, anorexia,
night sweats, and wasting
o Cough and sputum
o Hemptysis and chest pain
- Extrapulmonary Tuberculosis
o Miliary lesions in the bones, joints, GIT,
meninges, lymph nodes and peritoneum
o AIDS patients with Tb progress into severe
and unusual manifestations
Tuberculin Test
- delayed hypersensitivey to M. tuberculosis protein
antigens
- Mantoux test: PPD
o 0.1 mL of 5 TU of PPD-S
o Activity is expressed as tuberculin units (TU)
o Intradermal injection (48-72 hrs)
induration
o Positive reaction 4-6 weeks after initial
contact with bacilli
- Tine Test: multiple puncture method
o For screening only

Primary Tuberculosis

Lesion arrest Lesion breaks

down

Fibrosis & Caseous matrials

cacification Liver, spleen, Cavity formation
kidneys, bone, Spread of
meninges infection

Viable, non- - Interpretations of the Mantoux skin test for

proliferating Bacilli is Tuberculosis
organisms Miliary Tb dispersed in the
lymph and
blood stream

- Stages in the Pathogenesis of tuberculosis

- Mantoux test (tuberculin skin test)

- Reactivation of Tuberculosis
Due to:
o Impairment in immune status
o Malnutrition
 Colonies of M. Tb on Middlebrook 7H11
agar viewed microscopically. Note the
beginning of the cording characteristics

- Conditions affecting the tuberculin reaction:

o Negative – active TB in suppressed cell-
mediated immunity Colonies of M. tuberculosis (3 to 4
weeks old) on Middlebrook 7H11 agar.
o Cross-reaction may be observed with other Colonies have a rough appearance and
spp. of Mycobacteria exhibit cording, exemplified by the
darker areas
Laboratory Identification
- Identification of M. tuberculosis in clinical specimens:
o Ziehl-Neelsen stain
o Kinyoun stain
o Fluorescent acid fast dye (auramine-
rhodamine) of sputum, bronchial washings, M. Tuberculosis colonies on
urine, spinal fluid sediment, biopsy material Lowenstein-Jenssen agar
8 weeks of incubation
M. tuberculosis stained with Kinyoun acid-fast stain

Treatment of MTb
- Multidrug therapy: First line drugs
o Isoniazid
o Rifampin
o Ethambutol
M. tuberculosis stained with fluorochrome stain
o Streptomycin
o Pyrazinamide
- Multidrug resistance (MDR)
- Drugs used in the treatment of tuberculosis
Drug Daily Adult Dosage Major Toxicity
First-line
agents

Isoniazid 300 mg orally or im Hepatitis, neurophathy

Rifampin 600 mg orally or iv Hepatitis, flulike
- Molecular techniques: syndrome
1. Amplified MTb direct test
o makes copies of 16S ribosomal RNA and Pyrazinamide 1.5-2.5 g orally Hepatitis,
detected by genetic probe hyperuricemia
2. PCR
o amplifies small portion of target DNA Ethambutol 15-25 ng/kg orally Optic neuritis
o facilitates DNA finger printing of specific Streptomycin 0.5-1 g im Vestibular dysfunction,
strains deafness, renal (rare)
Second line
Laboratory Report of Acid Fast Bacilli agents
Number of Bacilli Report
0 No AFB seen Cycloserine 250-500 mg twice a Seizures, psychiatric
1-2/300 fields Doubtful; request another specimen day orally symptoms, CNS
1-9/100 fields + dysfunction
1-9/10 fields ++
1-9/field +++ Ethionamide 250-500 mg twice a Nausea, vomiting,
>9/field ++++ day orally hepatitis psychiatric
symptoms
Culture of MTb
- Lowenstein –Jensen (L-J) medium Capreomycin 0.5-1g im, then 1 g Deafness, vestibular
o egg- potato- base media 2-3 times a day dysfunction, renal
damage
- Middlebrook 7H-10
o agar- base media
Kanamycin
- 5% to 10% CO2
- 3-6 weeks incubation
- 12 B vial for Bactec MTb 460- radiometric and semi- Prevention of MTb
- INH prophylaxis
automated method of TB culture
o No protection to uninfected person after
M. Tuberculosis on L-J agar slant M. Tb on Middlebrook 7H11 agar treatment is stopped
(with casein hydrolysates): cream - BCG vaccination
colored, dry and wrinkled o Useless after the patient has been infected
with tubercle bacilli

Mycobacterium bovis
 - causes clinical illness similar to that caused by M.
tuberculosis
- milk is the common vehicle
- primary lesion in the cervical or intestinal lymph
nodes

Mycobacteria other than Tuberculosis (MOTT)

- non-tuberculous Mycobacteria (NTM)

o Runyon Classification of NTM:
 Photochromogens
pigment following
light
Scotochromogens
- M. scofulaceum
o chronic cervical adenitis in children
– develop o resistant to antituberculous drugs
exposure to o surgical excision of infected cervical nodes
o habitat are raw milk, soil, water, dairy
products
- M. szulgai
o Cervical adenitis in children
o Habitat is water and soil

Scotochromogen M. gordonae with yellow colonies

Photochromogens – unpigmented when grown in the dark (A) and

develop pigment after light exposure (B)

 Scotochromogens – develop
Non-photochromogens
pigment in the dark or light
- M.avium-intracellulare complex (MAC or MAI)
o In patients without AIDS:
 Pulmonary infections in patients
with preexisting pulmonary
disease; cervical lymphadenitis;
disseminated disease in
immunocompromised, HIV-
negative patients
o In patients with AIDS:
Scotochromogens – pigmented in the dark © and does not intensify after
exposure to light (D)  Disseminated disease;
environmental sources are natural
water
 Non-Photochromogens – non-
pigmented regardless of grown in - M. ulcerans
the dark or light o Indolent cutaneous and subcutaneous
infections
o Infections occur in tropical or temperate
climates
o Has not been isolated from the environment

Different colony morphology seen on culture of one

strain of M. avium complex

Nonphotochromogens – non-pigmented when grown in

the dark (E) and after light exposure (F)

 Rapid-growers – colonies of NTM

that appear on solid media in less
than 7 days

Photochromogens
- M. kansasii
o Chronic pulmonary disease; extra-pulmonary
diseases (cervical lymphadenitis &
cutaneous disease) Rapid Growers
o 3% of clinical illness known as TB
o cross-reactive to PPD - M. abscesus
o Disseminated disease in
o sensitive to standard antituberculous drugs
immunocompromised patients, skin and soft
such as rifampin tissue infections, pulmonary infections,
o habitat is tap water postoperative infections

- M. marium - M. fortuitum
o Cutaneous disease o postoperative infections infections in breast
o Natural reservoir is fresh water and augmentation andmedian sternotomy; skin
saltwater as a result of contamination from and soft tissue infections
infected fish and other marine life
- M. chelonae
M. kansasii colonies exposed to light o Skin and soft tissue infections, postoperative
wound infections, keratitis
 Smooth, multilobated colonies of M. fortuitum on
Lowenstein-Jenssen medium

Fite-Faraco stain: single AFB in

a nerve of patient with
indeterminate leprosy

Borderline leprosy: Lesions are

inflamed and succulent with
central clearing, producing a
“punched out” appearance, or
may appear as plaques or bands
with peripheral edges fading into
normal surrounding skin. Central
sensory deficits if present.

Mycobacterium Leprae

H & E stain: Epithelioid granuloma

with few scattered lymphocytes
and a clear subepidermal zone

Borderline leprosy: lesions are

multiform, from borderline
tuberculoid appearance to more
infiltrated nodules and plaques,
with some loss of sensation at the
center.

Classification of Leprosy BL: thick erythematous plaques on

the nose , right cheek and chin
- clinical significance: with some lesions appearing
o chronic granulomatous condition of nodular.
peripheral nerves and mucocutaneous
tissues, particularly the nasal mucosa

Tuberculoid Progression Leptromatous

Leprosy leprosy
Of disease
Borderline leprosy: Uniformly
- laboratory identification and symmetrically
o cannot be cultured on artificial media distributed, infiltrated
o can be grown in the footpads of mice and maculopapular lesions. No
armadillo sensory impairment.
o acid-fast bacilli from nasal mucosa and
other infeted areas in lepromatous leprosy
o histopath and clinical findings in tuberculoid
leprosy

Indeterminate leprosy: on the Borderline leprosy: In Fite-

extensor suface of extremities. Faraco stain AFB is present
in moderate numbers seen
Lesion is single, faintly within a nerve
hypochromic macule with ill-
defined borders; slightly
erythematous with hypoesthetic
areas in some parts. At times
sensation is intact

Indeterminate leprosy in children:

solitary, ill-defined,
Hypopigmented macules, partially
anesthetic. May progress to either
tuberculoid or lepromatous forms.
Tuberculoid leprosy: small ,single, circinate lesion
with pink, elevated, finely granular,well-defined
border. Central hypopigmented macule was
insensitive to touch & pain. Associated with enlarged
peripheral nerves.
 Tuberculoid leprosy: H&E stain of
an epithelioid granuloma with
thick zone of lymphocytes
destroying a nerve which is
unrecognizable

Far advanced nodular lepromatous

leprosy. Diffuse infiltration with
nodules over the eyebrows,
cheeks, ear lobes, nose, and chin.

Arm of patient with lepromatous

leprosy: multiple typical nodules
skin. Central sensory deficits if
present.

Lepromatous leprosy:
Diffuse infiltration, madarosis, and
loss of eyelashes(diffuse
lepromatosis)

H & E stain of skin section with

active Lepromatous leprosy: highly
vacuolated foam cells, normal
appearing nerves, histiocytic
granuloma.

Fite-Faraco stain in Lepromatous

leprosy: enormous numbers of
AFB, in huge clumps, termed
globi.

Treatment and prevention

- sulfones: Dapsone
- Rifampin
- Clofozamine
- For erythema nodosum leprosum:
o Thalidomide
o TNF-α inhibitor

-fin-

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