MYCOBACTERIUM TUBERCULOSIS

Anushi JainRoll No. : 08

Paper IIMsc II

INTRODUCTION

• Mycobacteria are slender rods.

• They sometimes show branching, filamentous forms resembling fungal mycelium.

• They are aerobic, non-motile, non capsulated and non-sporing , usually having slow growth.

• They are acid fast bacilli.• Mycobacteria can be

classified as Mycobacterium I or Mycobacterium II.

• The genus Mycobacteria contains three groups : obligate parasites, opportunistic pathogens and saprophytes.• Obligate Parasites : this includes :-1. Mycobacterium tuberculosis complex : M. tuberculosis, M. bovis, M. africanum, M. microti, M. canetti, M. caprae, M. pinnipeddi .2. Mycobacterium leprae• Opportunistic pathogens : These are the non-

tuberculous mycobacteria (NTM). They are categorized as photochromogens, scotochromogens, non-photochromogens and rapid growers.• Saprophytes : Saprophytic mycobacterium are isolated

from many sources. These includes M. phlei (from grass) and M. smegmatis (from smegma).

MYCOBACTERIUM TUBERCULOSIS• M. tuberculosis and M. bovis are the

human tuberculosis causing mycobacteria.• M. tuberculosis was discovered in 1882

by Robert Koch.

MORPHOLOGY • M. tuberculosis is a straight or slightly

curved rod whereas M. bovis is straight and short.

• 3 x 0.3 μm in size.• They can occur singly, in pairs or as small

clumps.• They are gram positive.• They are filamentous, club shaped and

branching. • They are acid and alcohol fast.

CULTURAL CHARACTERISTICS

• The bacilli have slow growth, with a generation time of 14-15 hours in vitro.• the colonies maybe appear after 2weeks and may take

upto 8 weeks.• The optimum temperature is 37°c and the growth does

not occur below 25°c or above 40°c.• Optimum pH is 6.4-7.0• M. tuberculosis is eugonic whereas M. bovis is dysgonic.• The addition of 0.5% glycerol improves the growth of M.

tuberculosis but have no effect on or even impair the growth of M. bovis whereas sodium pyruvate helps the growth of both types.

• Several media, both solid and liquid, can be used for growth of tubercle bacilli.

Solid Media :• The most widely used solid media is

Lowenstein-Jensen (LJ).• On solid media, M. tuberculosis forms

dry, rough, raised, irregular colonies with a wrinkled surface.• The colonies are creamy white,

becoming yellowish or buff-coloured on further incubation.• Other media that can use are

Petragnini media, Dorset media, Tarshis medium, Loeffler media or Pawlowsky media.

Liquid Media

• The most liquid media used are Dubos’s, Middlebrooks's, Proskauer and Beck’s, Sula’s and Sauton’s.• In liquid media, the

growth begins at the bottom, creeps up and forms prominent pellicle at the surface.

RESISTANCE

• Mycobacteria are not completely heat resistance, being killed at 60°c for 10-15 minutes.• Cultures may be killed by exposure to direct sunlight for

two hours, but the bacteria survive in sputum for 20-30 hours and may remain viable in droplet nuclei for 8-10 days.• They are resistance to chemical disinfectants like 5%

phenol, 15% Sulphuric acid, 3% nitric acid, 5% oxalic acid and 4% sodium hydroxide.• They are sensitive to formaldehyde and glutaraldehyde.• They can be destroyed by 80% ethanol in 2 minutes and

by iodine in 5 minutes.

Host Range

• M. tuberculosis causes natural infection in humans, other primates, dogs and other animals in close contact with humans.

• It is highly infectious for guinea pigs ands hamsters.• M. bovis is more pathogenic for animals.• It causes tuberculosis in cattle, carnivores, dogs and cats,

swine, parrots and other birds of prey.• It is moderately pathogenic for rats.• ‘African strains’ or M. africanum was isolated from some

parts of South Africa, showing intermediate properties between human and bovine types.

• ‘Asian Type’ of tubercle bacilli was isolated from South India, where of low virulence for guinea pigs.

ANTIGENIC PROPERTIESMany antigens have been identified in the mycobacterial cell wall.They constituents of lipids, proteins, polysaccharides.1. Lipids : • The cell wall is rich in mycolic acid, which plays an important

role in pathogenesis.• Mycolic acid is also responsible for the acid fastness of the

bacteria and cellular tissue reactions of the body.2. Proteins : • They are responsible for the tuberculin reaction.• They induce delayed type hypersensitivity.3. Polysaccharides :• Their role in pathogenesis is not certain. • They induce delayed type hypersensitivity.

BIOCHEMICAL REACTIONS

NIACIN TEST :• Niacin is detected by

addition of 10% cyanogen bromide and 4% aniline in 96% ethanol to a suspension of the culture.• Positive reaction – Canary

yellow colour.• M. tuberculosis – Positive.• M. bovis – Negative.

Aryl Sulphatase Test

• The bacilli is grown in a medium containing 0.001M tripotassium phenolphthalein disulphate.• 2N NaOH is added, drop

by drop.• Positive reaction – Pink

colour.• Only positive for atypical

mycobacteria.

Catalase-Peroxidase Test

• Equal volumes of H2O2 and 0.2% catechol in distilled water are added to 5ml of test culture and allowed to stand.• Effervescence – Catalase

positive.• Browning – Peroxidase positive.• Atypical mycobacteria – Catalase

positive and Peroxidase negative.• M. tuberculosis – Catalase

negative and Peroxidase positive.

Nitrate Reduction Test

• Sulphanilamide and n-naphthyl-ethylene is added to bacterial suspension in nitrate medium.• Positive reaction – Red

colour.• M. tuberculosis –

Positive.• M. bovis – Negative.

Amidase Test

• Five amides are tested, namely, acetamide, benzamide, carbamide, nicotinamide and pyrazinamide.

• A 0.00165M solution of amide is incubated with bacillary suspension at 37°c and 0.1ml of MnSO4.4 H2O, 1.0ml of phenol solution and 0.5ml of hypochlorite solution are added.

• Positive reaction – blue colour.

Pyrazinamide Test

• Enzyme pyrazinamidase hydrolyses pyrazinamide to ammonia and pyrazionic acid which is detected by adding ferric ammonium sulphate.• Positive reaction – Pink

colour band.• M. tuberculosis – Positive.• M. bovis – Negative.

Neutral Test• Virulent strains of tubercle

bacilli can bind to neutral red in alkaline buffer solution, virulent strains cannot bind.

Tween 80 Hydrolysis test• Tween 80 is added to the

medium.• Positive reaction – Colour

change from yellow to red at pH 7.

Inhibition by thiophene-2 carboxylic acid (T2H) test can also performed.

TYPING METHODSPhenotypic

• Bacteriophage Typing : Tubercle bacilli has been classified into four phages type : A, B, C and a intermediate between A and B, designated I.• Bacteriocin Typing : M.

tuberculosis can also be typed by means of bacteriocin produced by rapidly growing mycobacteria.

Molecular Typing• IS6110 RFLP Typing :

Restriction endonuclease treatment yields nucleic acid fragments of varying lengths, the pattern of which are strain specific. • Spoligotyping (spacer

oligotyping) : This is based on polymorphism in the direct repeat (DR) locus.

THANK YOU