•
Obligate aerobe.
•
Rods.
•
Non spore forming .
•
Non capsulated .
•
Acid fast bacteria
.

Mycobacteria
•
Cell wall contains high lipid content ( 40-60 %), which is responsible for their staining &cultural characters .
• Not classified as either
Gram-positive or
Gram-negative as they do not have the chemical characteristics of either.
•
Acid fast Stained by
Ziehl- Neelsen stain.

1.Members of M. tuberculosis.
Cause tuberculosis in human .
(M tuberculosis, M bovis. )
2. Non tuberculous species ( Atypical mycobacteria ) .:-
Virtually all other species :- Examples :- M.Avian
intracellulare ;. M.kansasii ;M. scroflaceum.
3..M.leprea : Cause Leprosy in man .
The causative agent of leprosy.

• The etiologic agent of tuberculosis (TB) in humans.
* 33 % world wide infetion .
• Humans are the only reservoir for the bacterium.

General Characteristics of the organism .

Facultative intracellular parasite, usually of macrophages .

High lipids ; 60 % content in the cell wall of M. tuberculosis causes :
•
Impermeability to Ordinary stains & dyes .
• Resistance to many antibiotics .
•
Resistance to acids & alkalis .
Stained by strong basic dyes using heat . Once stained it resist decolorization even usin g strong acids and alcohol so they called acid and alcohol

Morphology
•
Small , straight or slightly curved bacilli , non motile , non sporulated .
• Occur singly, in pairs or in masses .
•
Acid & alcohol fast ( resist decolurization with acid & alcohol) .
• Not stained by Gram .
Mycobacteria. Different types of structure

Cultural characters
•
Obligate aerobe .
•
5 to 10% CO2 , enhances growth .
• No growth on ordinary media.
• Optimum temperature is
37 ºC (strict mesophile ).
•
No growth below 30 ºC or above 39 ºC .
•
Very slow grower , may require 4 to 6 weeks to get visual colonies

Media used to grow M.TB
•
An egg based medium
Lowenstein-Jensen &
Dorest egg media .
•
An agar based medium
Middle brook's medium .

Tuberculosis
The source : ishuman and transmited by respiratory droplets . Which inhaled to settle in the alveoli .
•
Affects the lower respiratory system .
•
Characterized by:

Pathogenesis
• Tuberculous mycobacteria enter the alveoli by airborne transmission .
• They resist destruction by alveolar macrophages & multiply, forming the primary lesion or tubercle( gohans focus ).
•
They then spread to regional lymph nodes ( gohons triad ), enter the circulation, & reseed the lungs.
• Tissue destruction results from cell-mediated hypersensitivity .

Pathogenesis

Host Defenses
• Acquired resistance is mediated by T lymphocytes , which lyse infected macrophages directly or activate them via soluble mediators (e.g gamma interferon) to destroy intracellular bacilli.
•
Antibodies play no protective role .

• Sputum, bronchial or gastric washings.
•
At least
of sputum are required for diagnosis .

2.Direct smear
•
Prepared from sputum either direct or after liquefaction by
N-acetyl –L cysteine or by other methods .
• Stained by carbol fuchsin
(Ziehl-Neelsen) or fluorochrome stain .
•
By Ziehl-Neelsen(Z-N) stain
,acid-fast bacilli appear pink against blue background.
•
By fluorochrome stain , the bacilli appear as bright yellow fluorescent against dark background .

Acid fast bacilli , Z-N stain

Acid fast bacilli, Z-N stain

Acid fast bacilli, Z-N stain

•
•
•

Disadvantages of direct smear
•
Non sensitive ,the sensitivity range is 25 to 75%.
•
At least 5000-10,000 bacilli per ml of sputum are needed to detect positive smear .
•
Non specific ; Other acid-fast bacilli are similar to M.TB. in stained smear & should be ruled out by other methods .
•
A positive stain & negative culture may be caused by non viable organisms, which can occur in persons receiving anti tuberculous medication.

•
Sputum sample is treated with NaOH , to kill other contaminating bacteria (decontamination ) but does not kill the M.TB. because M.TB. is resistant to alkali .
•
Sputum is inoculated in selective medium containing antimicrobial agents as (Lowenstein-
Jensen ).
•
Cultures are incubated at 35°C to 37°C in an atmosphere of 5 to 10% CO2.

•
All cultures should be examined weekly for 4- 8 weeks
•
Colonies on L-J medium are, raised , rough , confluent, grayish & dry (eugenic growth)
.
•
Culture is highly sensitive & specific .
•
Allows visualization of colony morphology & pigmentation, which is useful for distinguishing colonies of M.TB from those of some non tuberculos mycobacteria .

Colonies of M.TB. on L-J medium

Colonies on L-J medium

Rapid culture method (BACTEC
System)
• The media used are broth media containing radio-labeled palmitic acid as the sole carbon source.
•
As M.TB. multiplies, it utilizes the palmitic acid & release radiolabeled CO2.
• Using the BACTEC system,
M.TB. growth can be detected in
9-16 days .

4.Intradermal skin test
(Tuberculin test)
Priciple
Individual previously infected with tubercle bacilli will develop hypersensitivity to proteins of
M.TB. which develops 6-8 weeks after infection.
Intradermal injection of PPD into a previously infected, hypersensitive person results in the delayed (48-72 hr) appearance of an indurated reaction.

Tuberculin test
• Performed by using old tuberulin or PPD , injected either intradermal or by multipuncture
•
Mantoux test is the standard tuberculin test .
•
It requires the intradermal injection of ( 0.1 ml ) containing
( 5 tuberculin units ) of PPD .
• The transverse diameter of induration is measured 48 to 72 hours later .

Method of Tuberculin test

Measurment of Tuberculin test

Tuberculin test (Interpretation)
Positive
Diameter of the induration is 10 mm or greater
.
There is erythema , swelling & induration .
Positive reactions means previous exposure to M.TB.
Negative reaction
Induration less than 5 mm .

•
It is impossible to distinguish between active disease & past infection by a positive tuberculin test.
•
Recent conversion of the reaction from negative to positive needs clinical attention.

Positive reaction in absence of previous exposure to M.TB .
May be due to prior exposure or infection with other mycobacteria or vaccination with BCG.

reaction in spite of
to M.TB .
• Occurs in persons with impaired
response & other conditions such as late stages of tuberculosis , malnutrition & steroid therapy

Recent methods for diagnosis
DNA probes and gas-liquid chromatography
Rapid, specific & sensitive methods for identification of mycobacteria after sufficient growth is present on medium.
PCR :
•
Useful for direct detection of mycobacteria in clinical specimen within 24 hours or less .
•
It is rapid, specific & sensitive
.

Diagnosis of renal tuberculosis
•
Sample: early morning urine of at least 3 successive days .
•
Centrifugation of urine &preparation of smear from the deposit .
•
Staining of the smear by Z-N stain , decolourized by both acid &alcohol to differentiate M.TB from
M.smegmatis
which is found normally in the genitalia & is acid fast but non alcohol fast .
•
Culture of deposit after decontamination on L-J medium .

Diagnosis of tuberculous meningitis
•
C.S.F is collected by lumbar puncture .
•
C.S.F is less turbid than that in meningococcal meningitis & contains excess of lymphocytes
•
Direct film is prepared from the deposit after centrifugation (Diagnostic) .
•
The deposit is cultured directly on L-J medium or
Dorest egg medium without decontamination .

Mycobacterium bovis
• Similar to M.TB in morphology & culture characters.
•
Differs in being shorter & thick .
•
On L-J medium , gives poor growth as glycerol does not favour its growth (dysgonic) .
•
Pyruvic acid favours its growth.

Mycobacterium bovis
•
Causes TB in cows & in humans.
•
Both cows & humans can serve as reservoirs.
•
Humans can be infected by consumption of non pasteurized milk.
•
This may lead to the development of extra pulmonary TB .

Treatment
•
Tuberculosis is usually treated with four different antimicrobial agents .
•
The course of drug therapy usually lasts from 6-
9 months.
•
The most commonly used drugs are rifampin , isoniazid , pyrazinamide , & ethambutol , or streptomycin .

•
Living attenuated vaccine prepared from bovine strain ( bacillus of Calmette &
Guerin) .
•
Prepared by repeated subculture of bovine strain for 250 times on glycerol-potato-bile medium .
•
Given by intradermal injection .

BCG vaccine
•
Given to all infants in the first year of life & to tuberculin negative adults .
•
BCG vaccinated person will be converted from negative tuberculin reactor to positive reactors
(tuberculin conversion) .
•
Should not be given to tuberculin positive persons .

Non tuberculous Mycobacteria
•
Previously referred to as "atypical" mycobacteria
•
Widely distributed in natural sources .
•
Non virulent to guinea pigs .
•
Not transmitted from human to human, but is acquired from natural sources .
•
Are important opportunistic pathogens in immuno compromised patients .

Non tuberculous Mycobacteria
Causes
• pulmonary disease by M kanssasii and M avium-intracellulare,
•
Disseminated infection : In immunocompromised patients, caused by the M aviumintracellulare complex
•
Cervical lymphadenitis by M scrofulaceum
• Granulomatus skin lesions & soft tissue infections by M marinum
(swimming pool granuloma) or M
ulcerans.

Non tuberculous Mycobacteria
•
Resistant to the usual anti -tuberculosis drugs.
•
No vaccine is available.
•
Diagnosis requires culture & identification.

Classification
•
Non tuberculous mycobacteria are classified by pigmentation in the light or dark & by growth rate into 4 groups ( The
Runyon groups ).
•
Species in groups I to III are slow growers further characterized by pigment production

The Runyon groups
•
Group I : Photochromogens pigmented only when exposed to light.
•
Group II: Scotochromogens
Form pigment in the dark.
•
Group III: Non chromogens are non pigmented .
•
Group IV : are rapid growers .

Differences between M.tuberculosis & Nontuberculous Mycobactemria
Character
Acid fast
M.tuberculosis
Non tuberculous mycobacteria
Acid fast Acid fast
Growth on L-J medium
Temperature
Growth
Pigment
P-nitrobenzoic acid
Pathogenicity to guinea pig
Anti tuberculous drugs
Transimmion
Human infection grow
Strict mesophile
Slow
Non chromagen
Inhibit growth
Pathogenic
Sensitive
Human to human
Tuberculosis grow
Grow at 22,37 & 45 ºC .
Slow or rapid
Non chromagen,photo, or scoto chromagen
No effect .
Non pathogenic
Resistant
Not lymphadenitis ,skin
&pulmonary

•
Causes leprosy.
• Humans are the only natural host for
• Structure M. leprae is similar to other mycobacteria

Mycobacterium leprae
Morphology : acid fast bacilli ,found in large masses or single, straight or slightly curved . Strict aerobic
;prefer cool temoerature 30 c . So it infect superficial cool areas . Skin and nerves .
• Stained by modified Ziehl- neelsen stain which is modified by using 5%
H2So4 for decolourization, It is less acid fast than M.TB .
•
Cannot be cultivated in vitro & it multiplies very slowly in vivo .

Clinical Manifestations
Two clinical forms
1.lepromatous
•
Granulomatus nodules in skin , peripheral nerves, mucous membranes & organs. ( Leonine face )
•
Loss of specific cellmediated immunity.
2.Tuberculoid :- ( Claw hand ) .
•
Skin and nerve lesions .
•
Intact cell-mediated immunity .

Diagnosis
•
Specimens
Nerve , skin lesions biopsies & nasal scrapings .
•
Film
Stained by modified Z-N stain to show acid fast bacilli in masses or single .
•
Culture
M .leprae cannot be cultured .

The lepramin skin test
•
A heat-killed suspension of M leprae is injected into skin of the patient
•
Has little diagnostic value but will provide information of prognostic importance about the immune status of the individual.
The PCR technique
•
By which very small amounts of M leprae DNA can be detected directly in clinical specimens .
May be useful in diagnosis .