MYCOBACTERIUM

ELIZABETH P. QUILES, M.D.,FPASMAP

Department of Microbiology
College of Medicine
Our Lady of Fatima University
GENERAL CHARACTERISTICS
❖ Rod-shaped, aerobic bacilli
❖ Neither gram positive or negative
❖ Hydrophobic → due to high lipid content of the
cell wall
❖ Acidfast → due to mycolic acid
❖ Do not stain readily → once stained →
resistant to
decolorization with acid or alcohol → ACIDFAST
❖ Decolorizer → acid alcohol (95% ethyl alcohol
+ 3% HCl)
CLASSIFICATION
(BASED ON PIGMENTATION)

❖ PHOTOCHROMOGENS produce pigment in

light but not in darkness

❖ SCOTOCHROMOGENS produce pigment in

the dark

❖ NONCHROMOGENS non pigmented

RANYOUN CLASSIFICATION
❖ TB COMPLEX
M. tuberculosis
M. africanum
M. bovis

❖ PHOTOCHROMOGENS
M. asiaticum M. simiae
M. kansasii M. marinum
❖ SCOTOCHROMOGENS

M. flavescens M. gordonae
M. scrofulaceum M. szulgai

❖ NON CHROMOGENS

M. avium M. celatum
M. haemophilium M. gastri
M. ulcerans M. xenopi
❖ RAPID GROWERS
M. fortuitum
M. chelonei
M. smegmatis
M. flavescens
MYCOBACTERIA THAT INFECT HUMANS
HUMAN PATHOGENS
M. tuberculosis
M. leprae
M. bovis

POTENTIALLY PATHOGENIC
M. avium complex
M. kansasii
M. fortuitum
MYCOBACTERIUM TUBERCULOSIS
❖ Acidfast bacilli, arranged singly or in groups
❖ Obligate aerobes
❖ Growth enhanced by increased O2 tension
❖ Long, slender rods, beaded
❖ Much granules seen in gram staining
❖ Champion slow grower
generation time = 13-15 hrs
doubling time = 18 hrs
6-8 weeks = before reported as negative culture
❖ Survive in the environment for long period of time
CONSTITUENTS OF THE CELL
WALL

A. LIPIDS
1. MYCOLIC ACID
2. WAXES
3. PHOSPHATIDES
❖ Mycolic acid + muramyl dipeptide (from
peptidoglycan) →
granuloma formation
B. PROTEINS
a. elicit the tuberculin reaction
b. elicit formation of antibodies

C. POLYSACCHARIDES
a. induce immediate type of hypersensitivity

b. serve as antigens in reactions with sera of

infected persons
VIRULENCE FACTORS

1. CORD FACTOR (trehalose-6,6’-dimycolate)

a. responsible for “serpentine cord” formation
(bacilli arranged in parallel chains)
b. inhibits migration of PMN leukocytes
c. elicits granuloma formation
d. serve as immunologic adjuvant
2. SULFATIDES
a. synergistically potentiate toxicity of cord
factor
b. promote survival of bacilli inside
macrophages

❖ No toxin produced
PATHOGENICITY AND PATHOGENESIS
❖ Among the top 10 disease killers in the
Philippines
❖ Predisposing factors:
1. chronic fatigue
2. sedentary life
3. malnutrition
4. poor housing conditions
5. occupational
MODES OF TRANSMISSION

❖ Inhalation of droplet nuclei

❖ Ingestion of unpasteurized milk or milk
products
❖ Kissing
❖ Fomites
❖ Skin contact
PRINCIPAL TYPES OF LESIONS
1. EXUDATIVE TYPE
❖ Consists of inflammatory reaction, PMN
leukocytes &
later monocytes around the tubercle bacilli
❖ Particularly seen in lung tissue
❖ May be absorbed or lead to massive necrosis
of tissue or
may develop into productive type
❖ Tuberculin test (+)
2. PRODUCTIVE TYPE
❖ Chronic granuloma
❖ Consists of 3 zones:
a. central area of multinucleated giant cells
containing tubercle bacilli
b. midzone of epitheloid cells
c. peripheral zone of fibroblasts, lymphocytes &
monocytes
❖ Peripheral zone → develop fibrous tissue;
central area →
caseation necrosis → TUBERCLE → break into
bronchus
& empty contents → CAVITATION
❖ Healing by fibrosis or cavitation
❖ The production, development & healing or
progression are
determined by:
a. no. of bacilli in the inoculum
b. resistance & hypersensitivity of the host
SPREAD IN THE HOST

1. Direct extension
2. Through the lymphatics and then the
bloodstream →
organs → miliary distribution
3. Via the bronchi & gastrointestinal tract
PRIMARY INFECTION

❖ 1st contact with tubercle bacilli

❖ Acute exudative lesion develops →
lymphatics &
regional LN → often heals rapidly
❖ LN undergoes massive caseation → calcifies
❖ Tuberculin test (+)
❖ Most often involve the base of the lung
REACTIVATION

❖ Tubercle bacilli survived the primary lesion

❖ Characterized by chronic tissue lesions,
tubercle formation,
caseation & fibrosis
❖ Regional LN are only slightly involved without
caseation
❖ Located at the apex of the lung
IMMUNITY

❖ HUMORAL IMMUNITY
antibodies produced are non-protective
❖ CELLULAR IMMUNITY
activated CD4 & TH1 release large amounts of
gamma interferon → activate macrophage →
destroy intracellular bacilli
❖ BCG VACCINATION
on absolute immunity only relative immunity
TUBERCULIN TEST
ANTIGENS
Old tuberculin (OT)
Purified Protein Derivative (PPD)
OLDER ANTIGENS
Old filtrate (BF)
Bacillary emulsion (BE)
Tuberculin residuum (TR)
Synthetic old tuberculin trichloroacetic
precipitate (SOTT)
METHODS

Mantoux – intradermal
Tine – disposable kit
Koch – subcutaneous
Volmer – patch
Von Pirquet – cutaneous
Moro - percutaneous
CLINICAL FINDINGS

EARLY MANIFESTATIONS
❖ Afternoon rises in body temperature
❖ Fatigue
❖ Weakness
❖ Loss of appetite
❖ Weight loss
FAR ADVANCED
❖ Chronic cough with hemoptysis
DIAGNOSIS

SPECIMENS
❖ Fresh sputum
❖ Gastric washings
❖ Urine
❖ CSF
❖ Blood
❖ Biopsy material
SPECIMEN SELECTION & COLLECTION
❖ Volume
❖ No. of specimens

one specimen/day for 3-5 days

❖ Timing

early morning specimens

CULTURE

1. Lowenstein Jensen medium – egg-

based

2. Middlebrook medium – agar-based

❖ Incubate at 35-37 C in 5-10% CO2 for up to 8

weeks
STAINING:
❖ ACIDFAST STAIN
2 methods:
1. Ziehl Neelsen method – hot method
- counter stain → methylene blue
2. Kinyoun method – cold method
- counter stain → malachite green

❖ FLUOROCHROME STAIN
BIOCHEMICAL TESTS:
Niacin test – (+)
Nitrate reduction – (+)
Catalase test – (-)
PCR (Polymerase Chain Reaction)
55-90% sensitive; 99% specific
ANTIBIOTIC SUSCEPTIBILITY TESTING
very important due to increasing number of
multi-drug resistant
strains
OTHER TESTS:
Gas Chromatography
Mycodot – rapid method 70% sensitivity; 96%
specificity
Nucleic acid hybridization excellent specificity &
sensitivity
very expensive
requires technical expertise
TREATMENT

1st Line anti-TB drugs

❖ INH, Rifampicin, ethambutol, pyrazinamide

❖ Used for 6 months to 1 year
❖ DOTS (Direct Observation) → useful to
ensure patient
compliance
2nd Line anti-TB drugs

❖ Cycloserine, ethionamide, PAS, Viomycin,

capreomycin,
fluorquinolones, kanamycin, rifambutin
❖ Streptomycin → not used anymore because
of 8th nerve
deafness
MYCOBACTERIUM LEPRAE
❖ Formerly known as Hansen’s bacillus
❖ Acidfast bacilli arranged singly or in pallisade
❖ Located intracellularly in foam cells or lepra cells
(endothelial cells of blood vessels or in
mononuclear cells)
❖ Humans are the natural hosts
❖ Cannot be grown in artificial laboratory medium
❖ Can only be grown in footpads of mice,
armadillo & chimpanzee
LEPROSY
❖ Incubation period: short (few days) up to 40
years
❖ Onset is insidious
❖ Involves the cooler tissue of the body → skin,
superficial
nerves, nose, pharynx, larynx, eyes & testicles
❖ MOT: prolonged contact and exposure
SKIN LESIONS: may occur as
❖ Pale, anesthetic macular lesions 1-10 cm in
diameter
❖ Discrete erythematous, infiltrated nodules 1-
5 cm in diameter
❖ Diffuse skin infiltration
NEUROLOGIC DISTURBANCES:
❖ Nerve infiltration & thickening → anesthesia,
neuritis,
paresthesia, trophic ulcers, bone resorption &
shortening of
digits
❖ Disfigurement (leonine facies) – due to skin
infiltration &
nerve involvement
❖ Saddle nose – due to bone resorption
DIAGNOSIS

❖ Clinical findings
❖ Acidfast staining of skin lesions or nasal
scrapings
❖ Lepromin test for tuberculoid leprosy
❖ No serologic test is useful
TREATMENT

❖ Dapsone (diaminophenylsulfone) – mainstay

of therapy
❖ Emerging resistance → drug combination →
dapsone,
rifampicin & clofazimine
❖ 2 years duration
PREVENTION

❖ Isolation of patients
❖ For exposed children & household members
→
chemoprophylaxis → dapsone
CORYNEBACTERIUM
DIPHTHERIAE
❖ Gram positive bacilli, club-shaped
❖ Arranged in pallisades or V or L formations →
“Chinese
character” appearance
❖ Metachromatic granules → Babes Ernst
granules
❖ Humans are the only natural hosts
❖ Transient flora of the nasopharynx
❖ Not all strains are virulent → lyzogenized by
tox+ gene →
virulent
❖ The DNA that codes for the toxin is part of
the genetic material
of a temperate bacteriophage → during
lyzogeny of the virus
→ DNA of virus integrates into the bacterial
chromosome →
toxin synthesis
VIRULENCE FACTOR

Diphtheria toxin →Exotoxin

Fragment A → cleaves nicotinamide from NAD
→transfers ADP-ribose to EF-2 → inactivation of
EF-2 ADP ribosylation of elongation factor →
inhibits protein synthesis
Fragment B → mediates binding with the
glycoproteins receptors on the cell membrane
DIPHTHERIA
MOT:
❖ Airborne droplets
MANIFESTATIONS:
❖ Fever, sore throat
❖ Pseudomembrane – thick, gray, adherent
membrane over
the tonsils and throat
❖ Massive enlargement of cervical lymph
nodes → “Bull neck” appearance
COMPLICATIONS

1. Extension of the membrane into the larynx &

trachea →
airway obstruction
2. Myocarditis with arrhythmias & circulatory
collapse
3. Recurrent laryngeal nerve palsy
DIAGNOSIS

❖ SPECIMEN: throat swab

❖ CULTURE:
1. Loeffler’s medium
2. Tellurite plate – reduces elemental tellurium
within
the organism → gray black color of the medium
→ (+)
1. blood agar plate
PATHOGENICITY TEST – toxin production
1. Animal inoculation (Rabbit pathogenicity
test)
2. gel diffusion precipitin test (ELEK test)
❖ STAINING
1. Gram stain
2. L.A.M.B. (Loeffler’s alkaline methylene blue)
–
metachromatic granules
TREATMENT

1. ANTITOXIN – should be given immediately →

neutralize
unbound toxin
2. ANTIBIOTICS – Penicillin or erythromycin →
inhibit
growth of organism, reduce toxin production,
decrease incidence of chronic carriers
PREVENTION

IMMUNIZATION
> DIPHTHERIA TOXOID (usually in combination
with
tetanus toxoid & killed pertussis organism)
- 3 doses (2, 4 & 6 months)
- booster at 1 & 6 years of age
- booster every 10 years
> does not prevent nasopharyngeal carriage
LISTERIA MONOCYTOGENES
❖ Short, gram positive bacilli
❖ Arranged in V or L formations
❖ Exhibits tumbling end to end movement at
22-28 C but not at 37 C
❖ Produce a zone of beta hemolysis
❖ Serotypes Ia, Ib and IVb – 90% of
humanisolates
❖ Serotype IVb → cause epidemic of listeriosis
associated with cheese from unpasteurized milk
VIRULENCE FACTORS
❖ Internalin – cell wall surface protein →
interacts with E-
cadherin → promotes phagocytosis into the
epithelial
cells → production of listeriolysin O →
promotes
proliferation of the bacteria
❖ Produce siderophores → obtain iron from
transferrin →Iron
is an important virulence factor
CLINICAL FINDINGS
GRANULOMATOSIS INFANSEPTICA
❖ Perinatal human listeriosis
❖ May be an intra-uterine infection
❖ Early-onset syndrome → sepsis & death
before or after
delivery
❖ Late-onset syndrome → meningitis (birth to
3rd week of life)
ADULT INFECTIONS:
❖ Meningoencephalitis
❖ Bacteremia
❖ Focal infection – rare

▪ Occur commonly in immunocompromised

individuals
DIAGNOSIS

SPECIMEN: blood or CSF

CULTURE:
❖ Mueller-Hinton agar
❖ Identification enhanced when grown on agar
containing
sheep blood → beta zone of hemolysis
TREATMENT

❖ Ampicillin – combined with

- erythromycin
- IV trimethoprim-sulfamethoxazole
- gentamycin – but does not enter host cells