MYCOBACTERIA

Mycobacteria are slender rods – sometimes show

branching filamentous forms resembling fungal
mycelium.
In liquid cultures form mould like pellicle hence
the name ‘mycobacteria’ meaning fungus like
bacteria.
Do not stain readily, but once stained, resist
decolourisation with dilute mineral acids So
called Acid fast bacilli or AFB.
Aerobic, non motile, noncapsulated, nonsporing.
More than 50 species of Mycobacteria identified.
Mycobacteria that infect humans are:
M.tuberculosis
Non- tuberculous mycobacteria
M.leprae
 M.tuberculosis
 Tuberculosis transmitted by aerosols from an infected
individual. Inhaled bacteria penetrate to alveoli and are
ingested by alveolar macrophages. Bacteria grow intracellularly
and slowly.
Morphology
Straight or slightly curved rods, occurring singly, in pairs or as
small clumps.
Nonmotile and non capsulated.
Acid fast bacilli This property due to the presence of fatty
acids in their cell wall called Mycolic acid.
They are difficult to stain– if stained difficult to decolourise with
acids like 20% sulphuric acid and are called acid fast bacilli. For
this staining technique called Ziehl Neelsen staining technique is
used. This helps for demonstrating mycobacteria in clinical
specimens.
Cultural Characteristics:
Bacilli grow slowly. Generation time 14-15 hrs. Colonies appear in
about 2 weeks to 8 weeks.
Optimum temp 37⁰C.
pH – 6.4-7.0
Obligate aerobe.
M.tuberculosis grow luxuriantly in culture as compared to M.bovis
which grow sparsely. Therefore they were termed as eugonic and
dysgonic respectively.
Addition of 0.5%glycerol and sodium pyruvate improves the
growth.
Tubercle bacilli do not have exacting growth requirements but are
highly susceptible to traces of toxic substances like fatty acid in
culture media. This toxicity is neutralized by serum albumin or
charcoal. Koch originally grew the bacillus on heat coagulated
bovine serum.
Both liquid and solid media used for tubercle bacilli:
Solid media:
Most widely employed media for routine culture is Lowenstein-
Jensen(LJ) media. Consist of hens eggs, mineral salt solution,
asparagine and malachite green. Last one act as a selective
agent inhibiting other bcteria.
Colonies are dry, rough, raised, irregular with a wrinkled surface.
They are creamy white becoming yellowish or buff coloured on
further incubation. Other solid media containing egg
(Petragnini,Dorset),blood (Tarshis),serum (Loeffler)or
potato(Pawlowsky)
Liquid media:
Dubos, Middle brooks, Proskauer and Becks,Sulas’s and
Sauton’s.
Liquid media not used for routine culture but for sensitivity
testing, chemical analysis and preparation of antigens and
vaccines.
In liquid media growth begins at the bottom, creeps up
the sides and forms a prominent surface pellicle which
extend along the sides above the medium.
Diffuse growth obtained in Dubos medium containing
Tween -80(sorbitan monooleate)
Virulent strains produce long serpentine cords in liquid
media, while avirulent strains grow in a more dispersed
manner.
TUBERCULOSIS
Source of infection:
M.tuberculosis causes natural infection in humans.
Source of infection is usually an open case of pulmonary
tuberculosis. One open case of tuberculosis in India may infect an
average of 25 contacts before death or cure.
Mode of infection is direct inhalation of bacilli contained in the
droplet nuclei of expectorated sputum.
Coughing, sneezing and speaking release numerous droplets- as
many 3000 infectious nuclei per cough.
Strains of tubercle bacilli isolated from parts of Africa, that show
properties of human and bovine types are called African strains or
M.africanum.
Asian types is name given to strains of tubercle bacilli originally
isolated from South India, susceptible to hydrogen peroxide and
isoniazide sensitive.
Majority of inhaled bacilli are arrested by natural
defenses of upper respiratory tract. Bacilli reaching the
lungs are ingested by alveolar macrophages.
 Factors that influence the outcome of infection are:– the
number and virulence of the infecting bacilli and host
factors including genetic susceptibility, age,
immunocompetence, stress, nutrition and coexisting
illness.
Immunology:
Tubercle bacilli do not contain or secrete a toxin. Exact
basis of virulence is not known, but seems to be related
to their ability to survive and multiply in macrophages.
The only specific immune mechanism effective is the cell
mediated type.
Pathology
The essential pathology in tuberculosis is the production
in infected tissues of a charactristic lesion, the tubercle.
This is an avascular granuloma composed of a central zone
containing giant cells, with or without caseation and a
peripheral zone of lymphocytes and fibroblasts.
Tubercle lesions are primarily of 2 types:
Exudative and productive.
Exudative type: this is an acute inflammatory reaction
with accumulation of edema fluid, polymorpho-nuclear
cells. This is seen when bacilli are many and virulent and
host response is more in the nature of DTH than of
protective immunity.
Productive: this type of lesion is predominantly cellular ,
associated more with protective immunity than DTH.
Classification of Tuberculosis:
Depending on the time of infection and the type of
response tuberculosis is classified into 2 types: primary
tuberculosis and post primary tuberculosis.
Primary tuberculosis:
Initial infection by tubercle bacilli in a host.
In endemic countries like India this occurs in young
children.
Bacilli engulfed by the alveolar macrophages multiply and
give rise to a subpleural focus of tuberculous pneumonia,
commonly located in the lower lobe or lower part of the
upper lobe(Gohn focus). The hilar lymph nodes are
involved. The ghon focus together with the hilar lymph
node constitute the primary complex.
This occurs 3-8 weeks from the time of infection and is
associated with the development of tuberculin
hypersensitivity.
The lesion heals spontaneously in 2-6 months, leaving
behind a calcified nodule. Sometime a few bacilli may
survive in the healed lesion and remain latent.
In children's with impaired immunity or other risk factors,
primary lesion may enlarge and cause miliary or
meningeal or other forms of disseminated tuberculosis.
Post –primary (secondary or adult)tuberculosis:
This is due to reactivation of latent infection or
exogenous reinfection and differs from the primary type.
It mainly affects the upper lobes of the lungs. The lesions
undergo necrosis and tissue destruction, leading to
cavitation.
Lymph node involvement is not common. The necrotic
materials break out into the airways, leading to
expectoration of bacteria laden sputum- which act as the
main source of infection to contacts.
In immunodeficient people cavity formation is
uncommon and leads to disseminated lesions in lungs
and other organs.
Laboratory Diagnosis:
1. Demonstrating the bacillus in the lesions by microscopy.
2. Isolating it in culture by transmitting the infection to
experimental animals.
3. Demonstrating hypersensitivity to tuberculoprotein.
4. Using molecular diagnostic methods.
Specimen collection depends on the site of lesion –
pulmonary or extrapulmonary.
Pulmonary Tuberculosis:
1. Specimen collection:
Sputum: Best collected in the early morning before any
meal. If sputum is scanty, a 24 hr sample may be tested.
Sputum sampling on 3 days increases the chance of
detection.
Sputum not available – Laryngeal swabs or bronchial
washings may be collected.
In small children who tend to swallow the sputum –
gastric lavage can be examined.
Direct sputum sample smears maybe prepared from the
thick part of the sputum in the peripheral laboratories
and stained.
2. Decontamination and concentration of specimens:
Specimens from non sterile sites and sputum need prior
treatment so that microorganisms other than
mycobacteria may not overgrow during prolonged
incubation.
Petroff’s method: simple method, used widely.
Sputum is incubated with equal volume of 4 % NaOH
solution at 37⁰C with frequent shaking till it becomes
clear. Then centrifuged at 3000 rpm for 20 min.
Sediment neutralised with N/10 HCl.
This method do not kill the bacilli.
NALC combined with 2% NaOH:
Better than petroffs method. N acetyl cysteine is used
for liquefaction of sputum. NaOH kills the
contaminating bacteria. Sample is concentrated by
centrifugation.
3. Microscopy:
 Staining:
a) Ziehl Neelsen staining technique:
 Direct or concentrated smears of sputum can be used.
 Sputum microscopy is the most reliable single method
in diagnosis and control of tuberculosis.
 Smear prepared from thick purulent portion of sputum.
 Flood the smear with carbol fuschin and heat gently for
5-7 minutes without letting stain boil and become dry.
 Wash it off with gently flowing tap water.
 Add 20% sulphuric acid in 95% ethanol or 3% HCl in
95% ethanol and leave it for 1-2 minutes. Repeat this
step until no more stain comes off.
Wash off with water.
Flood the smear with methylene blue dye and leave it for
1 minute and wash with water.
Air dry and examine the smear under the oil immersion
objective.
Acid-fast bacteria retain the primary dye, carbol-fuschin,
and stain pink.
Non-acid fast bacteria take up the methylene blue dye
and appear blue.
b) Auramine rhodamine:
Smears are stained with auramine phenol or auramine
rhodamine fluorescent dyes and when examine under
ultraviolet illumination, bacilli will appear as bright rods
against a dark background.
c) Kinyoun’s modification of acid fast staining:
This is a cold method where heating of the stain is not
employed.
Increasing concentration of phenol acid and increasing
duration of staining is used instead.
4. Culture:
Solid media: Concentrated specimen is inoculated on to 2
bottles of LJ medium. Cultures examined for growth after
incubation at 37ᵒC. Examine the cultures at least twice
weekly. A negative report is given after 8-12 weeks.
Growth observed is smeared for AFB staining.
For routine purpose a slow growing , nonpigmented ,
niacin positive , acid fast bacilli is taken as M.tuberculosis.
Confirmation by detailed biochemical studies.
Liquid Media: Middlebrook medium is used.
d)Automated system:
Used more commonly and requires less time.
5. Animal Inoculation:
Concentrated material is inoculated intramuscularly into
the thigh of 2 healthy guniea pigs. Animals are weighed
before inoculation and at intervals thereafter.
Progressive weight loss is an indication of infection.
Infected animals shows positive tuberculin test.
One animal killed after 4 weeks and autopsied. If it shows
no evidence of Tb then the other autopsied after 8
weeks.
7. Molecular methods:
PCR and ligase chain reaction(LCR) are used.
8.Immunodiagnosis:
Serological tests are not useful in diagnosis. Demonstration
of hypersensitivity to tuberculoprotein (tuberculin test) is a
standard procedure.
Tuberculin Skin test( Mantoux test)
Mantoux first described this test.
Tuberculin is partially purified extract of M.tuberculosis
proteins(PPD/ purified protein derivative).
Principle of TST is that PPD evokes a delayed
hypersensitivity reaction when injected intradermally.
0.1 ml of PPD containing 5 Tuberculin units is injected on
forearm. Site is examined 48-72 hrs later and induration is
measured.
• Induration of diameter 10 mm or more is considered as
positive.
• 5mm or less is considered as negative and 6-9 mm equivocal.
• TST is used to diagnose:
• children's with active TB
• to diagnose latent TB
• to select susceptible cases or as an indication of successful
vaccination.
• Intrepretation:
• Positive: Indicates hypersensitivity to tuberculoprotein,
denoting infection with tubercle bacillus or BCG immunisation
with recent or past , with or without clinical diseases.
• Test becomes positive 4-6 weeks after infection or
immunisation.
Negative: Persons who have never had contact with
tubercle bacilli are tuberculin negative.
 False negative results may occur in certain situations like
miliary tuberculosis, convalescence from some viral
infections like measles , severe mal nutrition,
immunosuppresive therapy.
False negative results may also occur due to inactive PPD
preparations and improper injection techniques.
Prophylaxis:
General measures : Adequate nutrition, good housing
and health education.
Immunoprophylaxis:
BCG Vaccine
A live attenuated vaccine developed by Calmette and
Guerin ( 1921).
Live attenuated strain of M.bovis.
Following vaccination immunity lasts for 10-15 years.
Similar to immunity provided by natural infection.
BCG vaccine administrated intradermally on the
deltoid immediately after birth or as early as possible
thereafter before the age of 12 months.
Treatment
Antituberculous drug are of 2 types: bactericidal &
bacteriostatic.
Bactericidal drugs like rifampicin(R) and
pyrazinamida(Z) are called sterilizing drugs because
they are able to effectively kill the bacilli in the
lesions.
Other bactericidal drugs like Isoniazid (H) is effective
only against replicating bacilli & Streptomycin (S) only
against extracellular bacilli – so cannot sterilize
lesions.
Bactericidal drugs along with bacteriostatic drug
ethambutol (E) constitute the first line drugs.
The current practice is DOTS ( Directly observed
Treatment Short Course).
Short regimens of 6-7 months – effective & convenient.
Schedule-
- A combination of four drugs (HRZE) given 3 times a
week- during an initial intensive phase of 2 months.
- Followed by 4-5 months of continuing phase with only
2 drugs given 3 times a week.
• Drug therapy and Drug resistance
• Main problem of chemotherapy is drug resistance – due
to mutation in tubercle bacilli.
• MDR- Tb or multi drug resistant TB is defined as
resistance of M.tuberculosis to both isoniazid(INH) and
rifampicin (RIF)
MDR is a globel problem affecting both poor and rich
nations alike.
XDR strains: these are extensively resistant strains. It is
defined as MDR plus resistance to fluroquinolones and
one of the second injectables : amickacin ,capreomycin ,
kanamycin.
RNTCP: Revised national tuberculosis control
programme: This is a national tuberculosis control
programme introduced in 1993 in India.