Mycobacterium

Dr. Ng Woei Kean

Ph.D. (Molecular Medicine)
 General Characteristics
• Mycobacterium is an aerobic, non-spore forming
non-motile bacilli.
• It is not easily stained by gram method
• It resists decolouration by acid or alcohol. So, it
is called acid fast.
• It resist dryness & survives for long period in
dried materials.
• Mycobacterium tuberculosis is UV and heat
sensitive.
• Two hours of sunlight will kill it.
• But, if it is mixed with sputum, survival is up to
20-30 hours.
 Classification of
Mycobacterium
1. MTB complex :
M. tuberculosis
M. bovis
M. microti
M. africana
2. Mycobacterium avium complex (MAC):
M. avium
M. intracellulare
M. paratuberculosis
M. lepraemurium
• 3. Mycobacteria other than tuberculosis (MOTT) OR
NTM:
• - Colony characters different from M.
tuberculosis
- Lack guinea pig pathogenecity
- Isoniazid (INH) resistant
- Catalase positive
4. Noncultivatable NTM
M. leprae
Runyon classified MOTT into several groups based
on colony characters & pigment production.
 Classification of Mycobacterium
Group Name
Runyon Gr I M. kansasii, M. marinum
(Photochromogen) M. simiae

Runyon Gr II M.scrofulaceum, M. szulgai,

(Schotochromogen) M. flavescens, M. godonae

Runyon Gr III MAC, M. ulcerans, M. gastri

(Non-
Photochromogen)
M. fortuitum, M. abscesus
Runyon Gr IV M. chelonae, M. smegmatis
(Rapid
growers)
 Diseases
• Mycobacterium primarily causes infection of the
lungs.
• But other organs are also affected.
• Among all mycobacteria, M. tuberculosis is the
most common mycobacteria responsible for
human diseases.
 Special Cellular Structures of
M. tuberculosis
• Cell wall contains large amount of glycolipids.
• Lipid fraction of cell wall contains 3 major
components:
(a) Mycolic acid - long chain fatty acids.
(b) Wax D - the major component of Freund's
complete adjuvant
(c) Cord factor - Trehalose-6-dimycolate
• Arabinoglactan - this links peptidoglycan layer to
mycolic acid.
• Other glycolipids
- lipoarabinomannan
- phospatidylinositol mannosides
• Proteins
Cell wall structure of Mycobacterium
 Virulence Properties of Cell
Wall Components
Mycolic acid
• Complexed with peptidoglycan causes
granuloma.
• Prevents complement deposition.
• Pevent killing inside macrophage
Cord factor
• Inhibits migration of WBC.
• Causes granuloma.
• Acts as adjuvants

Protein
• Induces tuberculin skin reaction
• Overall the high lipid contents of
mycobacterial cell wall is associated with :
- Impermeability to dyes
- Resistance to many antibiotics
- Resistance to killing by acid and alkali
- Resistance osmotic lysis
- Resistance to killing by macrophage
Tuberculosis is passed person-to-person, by inhaling droplets of
Mycobacterium tuberculosis that are in the air. When a person
coughs, sneezes, or speaks droplets are released into the air and
others can become infected. Human can also get Tuberculosis from
skin-to-skin contact. This type of contraction is more rare but is
possible; Individual can contact Tuberculosis from flesh wounds,
tattoos and non-sterilized instruments.
Pathogenesis
MTB enters into the body by inhalation (or ingestion)

Bacilli enters into alveolar macrophage

Move to the bronchial lymph nodes

Macrophage present MTB bacilli to T cells

DTH and CMI develops No

CMI

Disease is contained Progress to

death Bacteria live but do not replicate
• The infection is characterized by formation of
granuloma and central caseation.
• Caseation: Incomplete necrosis of tissue
resulting in solid or semisolid acellular and
amorphous materials.
• Formation of Ghon’s complex following primary
infection.
 Tuberculin Test (Mantoux Test)
• It is a hypersensitivity skin test which indicate
delayed hypersensitivity or cell-mediated
immune response to certain antigenic
components (proteins) of Mycobacterium
following infection with MTB.
• The hypersensitivity is characterized by
development of skin erythema and induration
(firmness of skin).
• A purified protein derivative (PPD) is employed
as the test antigen.
• 5 TU (tuberculin units) of PPD in a 0.1 ml
volume is intracutaneously injected in
the forearm.
• Formation of indurations is read at 48 or 72
hours.
Positive Reaction
• 10 mm indurations after 48-72 hours in a person
with normal immune system
OR
• 5 mm induration after 48-72 hours in those with
HIV infection
Interpretation
• 10 mm indurations - 90% infected.
• 15-20 mm indurations - almost all are infected.
• 5-10 mm indurations - suspicious for MTB
infection (in contact of active cases or may be
due to BCG).
• False positive
- infection with non-tuberculous mycobacteria
• False negative
- Normally 20% may be negative with active TB
- If done within 6-8 weeks of infection
- Disseminated tuberculosis
- Protein malnutrition
- Viral infection
- Sarcoidosis
- Steroid
- Diseases of reticuloendothelial system
 Diagnosis
• Specimens
Sputum: 5-10 ml early morning samples
Urine: 100 ml early morning samples
CSF: 5 ml
Plural or Peritoneal fluids: 100 ml
Pus: as much as possible
Tissue: adequate
• Three consecutive samples
required.
• Detection of TB bacilli by :
- Culture and
- Acid-fast bacili (AFB) or Ziehl–Neelsen
staining
Culture
• Primary Media: Lowenstein-Jensen (LJ) media;
7H10 and 7H11 (Middlebrook medium)
• Selective media with antibiotics: Gruft
modification of L-J media; Selective 7H10 and
7H11.
• Special media: BACTEC 14C Palmitic acid; MGIT
media (fluorescence media)
• Duration of incubation - up to 8 weeks
 Molecular methods
• Detection of MTB specific nucleic acid by PCR
or probe/hybridization
 Vaccination
BCG vaccine (Bacillus of Calmette and Guerin)
• Live attenuated vaccine
• A Mycobacterium bovis of low virulence is used
as vaccine.
• Its efficacy in preventing pulmonary TB is
doubtful.
• BCG is effective in preventing extra pulmonary
infection.