MYCOBACTERIA

Dr. Sudheer Kher

KEYWORDS
Acid Fast Ziehl-Neelsen Stain Mycolic acids Tuberculosis (Kochs disease)

M. tuberculosis M. bovis M. leprae

PPD Tuberculin BCG Polymerase chain reaction Runyon groups

Tubercle Lowenstein Jensen medium

Classification of Mycobacteria
1.

a) b) c) d) e)
2.

Tubercle bacilli

4.

a) b)
3.

Lepra bacilli

Human MTB Bovine M. bovis Murine M. microti Avian M. avium Cold blooded M. marinum Human M. leprae Rat M. leprae murium

a) b) c) d)
5.
6.

Atypical Mycobacteria (Runyon Groups)

Photochromogens Scotochromogens Nonphotochromogens Rapid growers

Johnes bacillus
Saprophytic mycobacteria

M. paratuberculosis

a) b)

Mycobacteria causing skin ulcers

M. ulcerans M. belnei

a) b) c) d) e)

M. butyricum M. phlei M. stercoralis M. smegmatis Others

Tuberculosis (TB, Consumption, Kochs Disease)

M. tuberculosis major human disease healthy people problems association with AIDS multiple drug-resistance Chronic disease Prolonged treatment

General characters of the genus

Slender rods Resist staining but once stained, resist decolorization by dilute mineral acids; hence called ACID FAST BACILLI (AFB)

Aerobic, Non-motile, Non-sporing, Noncapsulated. Growth generally slow Genus includes

Obligate parasites Opportunist pathogens Saprophytes

Mycobacterium tuberculosis
One of the most serious infectious diseases in the developing world One third of worlds population infected with M.

tuberculosis Thirty million people have active disease Nine million new cases occur Three million people die of the disease, each
year.

Mycobacterium tuberculosis (MTB)

Morphology
Ziehl Neelsen stain Once stained by Carbol fuchsin, resist decolorization by 20% Sulphuric acide and absolute alcohol. Acid & Alcohol Fast (AFB) Fluorescent dyes like Auramine O or Rhodamine also stain and the decolorization is resisted. Reason for Acid & Alcohol fastness
Presence of unsaponifiable wax Mycolic acid Semi permeable membrane around the cell Property of cell wall and related to integrity of the cell wall

Staining may be uniform or granular

MTB : Cultural characters

Grow

slowly. Generation time 14-15 hrs Colonies appear after 2 weeks or at 6-8 weeks MTB - Obligate aerobe

MTB grows more luxuriantly (eugonic) than M. bovis (dysgonic). Addition of 0.5% Glycerol supports growth of human strains. No effect or inhibitory effect on bovine strains.

MTB : Culture media

Solid media
Egg containing
Lowenstein-Jensen Medium Petragnini medium Dorset

Liquid media
Dubos Middlebrooks Proskauer & Becks Sulas Sautons

Blood containing
Tarshis

Serum containing
Loefflers serum slope

Potato containing
Pawlowskys

Resistance
Not specifically resistant to heat. 60 C x 20 min destroys. In sputum can survive 20-30 hrs Relatively resistant to disinfectants. Survives exposure to

5 % Phenol 15 % Sulphuric acid 3 % Nitric acid 5 % Oxalic acid 4 % NaOH

Biochemical reactions

Niacin test Human MTB produces niacin when grown in egg medium. Aryl Sulphatase test Enzyme Aryl sulphatase formed by only atypical mycobacteria.

Biochemical reactions
Neutral red test Virulent strains of tubercle bacilli bind neutral red in alkaline solution while avirulent strains can not. Catalaseparoxidase test Most atypical mycobacteria are strongly catalase positive while MTB is only weakly positive. MTB is strongly peroxidase positive while atypical mycobacteria are negative. Nitrate reduction test Positive in MTB and negative in M. bovis

Mycobacterium tuberculosis (MTB)

Morphology
Straight or slightly curved rods

Modes of infection
1- Droplet infection Person to person by inhalation aerosols Mycobacterium tuberculosis (Pulmonary tuberculosis) 2- Ingestion of milk Infected cattle Mycobacterium bovis (Intestinal tuberculosis) 3- Contamination of abrasion Laboratory workers

(Skin infection)

Pathogenesis of tuberculosis
infects lung distributed within macrophages

facultative intracellular pathogen inhibits phagosome-lysosome fusion resists lysosomal enzymes

Other minor pathogenesis factors tuberculosis

mycobactin siderophore cord factor damages mitochondria

Cell-mediated immunity tuberculosis

infiltration
macrophages lymphocytes

granulomas tubercles

Classical tubercular lesion Granuloma with typical Langhans giant cells, epithelioid cells, lymphocytes and fibrosis.

There are multiple light areas (opacities) of varying size that run together (coalesce). Arrows indicate the location of cavities within these light areas. The appearance is typical for chronic pulmonary tuberculosis.

Tuberculosis
Clinical picture :
* * * * * Low grade fever Weight loss Night sweats Fatigue Cough & haemoptysis

Laboratory diagnosis

M. tuberculosis

Acid fast bacteria in sputum Culture on L J media Biochemical identification Antibiotic sensitivity test Tuberculin test PCR

Lowenstein Jensen Medium

Selective. Always in screw capped bottle. Bluish Green. Contains Egg protein Solidifying agent Mineral salts Mg sulphate, Mg citrate Asparagine Malachite Green Selective agent Sterilized by - Inspissation

Laboratory diagnosis -

tuberculosis

skin testing
delayed hypersensitivity tuberculin protein purified derivative, PPD

X-ray

Tuberculin Test (Mantoux test)

Delayed hypersensitivity skin test to assay: cell mediated immunity to tubercle bacillius Material: A purified protein derivative (PPD) Dose : 0.1 ml of (PPD) is injected intradermal Reading : Positive test is defined as - Induration equal or greater than 10 mm - Develop 48-72 hours after injection

Positive skin test tuberculosis

indicates exposure to organism does not indicate active disease

Tuberculin Test
Interpretation: * A positive test indicates previous exposure and carriage of T.B. * A negative tuberculin test excludes infection in suspected persons * Tuberculin positive persons may develop reactivation type of T.B. * Tuberculin negative persons are at risk of gaining new infection * False positive reactions are mainly due to: - Infection with nontuberculous mycobacteria * False negative reactions may be due to: - Sever tuberculosis infection (Miliary T.B.) - Hodgkins disease - Corticosteroid therapy - Malnutrition - AIDS * Children below 5 years of age with no exposure history: - Positive test must be regarded suspicious

Laboratory Diagnosis
Demonstration of bacilli Culture & isolation or Animal inoculation Demonstration of hypersensitivity to tubercular protein Serological tests limited value

Specimen
* According to site of infection : - Sputum - Urine - Gastric lavage - Blood - Body fluids - Tissue biobsy

* Specimens need appropriate processing Liquefaction with N-acetyl-L- cysteine Decontamination with NaOH Centrifugation

Sputum

Laboratory Diagnosis

Pulmonary TB
Specimen
Sputum Early morning, if scanty 24 hrs, three consecutive day samples. Laryngeal swabs or gastric lavage in children. Grading 1+ -> 3-9 bacilli in entire smear 2+ -> 10 or more in entire smear 3+ -> 10 or more bacilli seen in most oil immersion fields

Microscopy See at least 100 field / 10 minutes.

Culture & AST Animal inoculation Animal of choice Guinea Pig.

Laboratory Diagnosis

Extra -Pulmonary TB
Specimen
CSF in suspected meningitis Pleural fluid & other exudates 2-3 days urine in renal TB Biopsy material.

Concentration methods
Purpose Homogenization & Concentration in sputum & other specimens. Methods

Concentration methods

Useful for microscopy, culture & animal inoculation

Petroffs method
Most widely used Equal volumes of Sputum + 4% NaOH incubated at 37C X 20 min.Centrifuge at 3000 rpm X 30 min. Sediment neutralized by N/10 HCl. Can be used for smear, culture, animal inoculation

Simpler method
To avoid centrifugation & Neutralization Equal volumes of sputum + solution of Cetrimoniom bromide 20 g + NaOH 40 g in one litre. Allow to stand for five minutes. Inoculate Acid Buffered LJ Medium with swab.

Laboratory diagnosis M. tuberculosis (culture)

grows very slowly several weeks non-pigmented colonies niacin production
*differentiates from other mycobacteria

Tuberculosis

polymerase chain amplification

rapid diagnosis

Pathogenesis
* Inhalation of tubercle bacilli

* They multiply in the alveolar macrophages

* An early tubercle (granuloma) is formed

Pathogenesis
* Lesions, healing or progression of infection depend upon 1- Dose of infecting mycobacteria 2- Resistance and hypersensitivity of host * Virulence : Glycolipids on the outer surface of bacteria - Enhance granuloma formation - Inhibit migration of polymorphnuclear leucocytes - Help survival of tubercle bacilli inside macrophages

Pathogenesis
A- Primary infection: * An exudative lesion : - spread to regional lymph nodes - A scar of healing may later calcify (Ghon focus) - Lymph nodes caseate and then calcify - Bacilli in the lesion slowly die - Tuberculin test becomes positive - The person immune & hypersensitive

Pathogenesis
B- Reactivation type :
* Activation of tubercle bacilli due to immunity * Formation of tubercles that caseate - fibrosis - open into a bronchus (open tuberculosis) * Tubercle bacilli erode a blood vessels - Infect any organ (Miliary T.B.)

Treatment
Chemoprophylaxis INH for one year Domicilliary treatment preferred Drugs

Rifampicin Isoniazide Pyrazinamide Streptomycin Ethambutol Ethionamide Thiacetazone Paraminosalicylic acid Cycloserine

Bactericidal

Bacteriostatic

Treatment
Short term chemotherapy of six months is sufficient Problem area Development of resistance by mutant selection

Solution Treatment by two to three drug combination, adequate treatment.

Treatment
Use multiple drug therapy to prevent emergence of resistant mutants * Long duration treatment (6-18 months)

* Four drugs are usually started in initial therapy due to: - Intracellular location of bacilli - Slow growth rate of bacilli - Caseous material blocks penetration of drugs - Some bacilli persist in a metabolically inactive state
* Sputum becomes non-infective 2-3 weeks after starting therapy

Treatment
Drugs used :
1- First line drugs : - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective): - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin * Noncompliance (failure to complete the course): Directly observed therapy (DOT) Health care workers observe the medication

Immuno-prophylaxis
Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG). The strain causes self limited lesion and induces hypersensitivity & immunity. Coverts tuberculin negative person to positive reactor. Immunity lasts for 10-15 years. Immunity 60-80%

BCG
Given at birth without tuberculin testing Protects against TB, the disease runs milder course in protected, prevents skeletal, meningeal & miliary forms. Also found useful in leprosy, leukaemias and other malignancies by non-specific stimulation of RE system.