Definition:Definition:

Generally, these microorganisms are

characterized by their special reaction to

stain.

They are difficult to be stained by ordinary

stain due to the high content of lipoid

substances in the cell wall.

However, once they stained they resist

decolorization even with acid, thus they called

acid fast bacilli when stained with Zhiel -

Neelsen stain.

The acid fastness is due to the high lipid

content (about 60%) of their cell wall.

They contain N-glycolylmuramic

(mycolic) acid instead of N-

acetylmuramic acid.

They are Gram +ve but stained poorly.

Non spore forming, non motile and

non capsulated.

This group includes both pathogenic and

non pathogenic bacteria.

ClassificationClassification

Mycobacterium tuberculosis

Atypical Mycobacterium

M. leprae

Saprophytic Mycobacterium

Mammalian type

Avian type

Reptilian or cold blood type

•Human.

•Bovine.

•Murin type.

Morphology:Morphology:

Acid Fast beaded bacilli arranged in bundles.

Non spore forming.

Non motile.

Non capsulated.

Culture characteristics:Culture characteristics:

Require Dorset’s egg media.

Can grow on in presence of Malachite green

(Löwenstein-Jensen media) as selective

agent.

Strictly aerobic.

Grow very slow, thus require 6-8 weeks

before discarding the culture as negative.

Biochemical Reactions:Biochemical Reactions:

Niacin test:

Positive in human type.

Negative in bovine type.

Epidemiology:Epidemiology:

Tuberculosis still remains among the most

important communicable diseases in the

world today with an estimated 50 million

active cases of who 3 million will die

annually from its effects.

In Egypt, the prevalence rate is about 1.7

% of which two thirds are active cases.

The death rate is about 15/100,000.

There are mainly two modes of infection;

either by droplet (airborne)

infection and this is generally by human type

or by consumption of contaminated milk

or milk products and this is usually by

bovine type.

Human type: causes pulmonary tuberculosis

mainly by droplet infection from an open case.

Bovine type: is attracted by the ingestion of

contaminated milk or milk products.

The bacilli will reach to the cervical glands.

Also, the organism may reach to the mesenteric

lymph nodes causing lymphadenitis.

From the lymph glands the bacilli may reach to

many organs causing generalized miliary

tuberculosis.

Primary tuberculosis; may occur at any

epithelial site (at any part of the body);

however, it is most commonly restricted to the

lung TB.

Pathogenicity:Pathogenicity:

Mycobacteria survive after ingestion by

macrophages and behave as

facultative intracellular organisms.

The infected cells express histocompatibility

complex (MHC)-associated bacterial peptides

that trigger T cells responses.

Activated CD4+ and TH1 cells release large

amounts of interferon- (IFN-), which

activates the infected macrophages.

The activated macrophages, in turn,

destroy the intracellular Mycobacterium.

The major pathogens are Mycobacterium

tuberculosis, the causative agent of

tuberculosis, and Mycobacterium leprae,

the cause of leprosy.

Atypical mycobacterium, such as

Mycobacterium avium-intracellulare

complex and Mycobacterium kansasii,

can cause tuberculosis disease but less

frequent pathogens.

Rapidly growing mycobacteria, such as

Mycobacterium chelonei, are

saprophytes that occasionally cause

human disease in immunocompromised

hosts.

In human, the tuberculosis is either:

Pulmonary type.

Extra-pulmonary type.

Determinants of Pathogenicity:Determinants of Pathogenicity:

Cording factor is a glycolipid derivative of

mycolic acid that is present on the outer

surface of M. tuberculosis. The glycolipid

inhibits migration of polymorphonuclear

(PMN) leucocytes and elicits granuloma

formation. Also, it is immunogenic.

Sulfatides permits the bacteria to survive

in the macrophages.

Antibacterial resistance; by mutation.

Clinical picture:Clinical picture:

The typical symptoms include; fever,

fatigue, night sweats, and weight loss.

Many organs can be involved. Miliary

tuberculosis (spread via blood) is

characterized by multiple disseminated

lesions that resemble millet seeds.

Tuberculous meningitis and tuberculous

osteomyelitis are important

disseminated forms.

Laboratory Diagnosis:Laboratory Diagnosis:Direct Methods: The specimen. Direct Examination (Zhiel-Neelsen stain). Methods Depend on Isolation: Culture. Biochemical reactions. Animal inoculation.Methods other than isolation: Tuberculin (Mantoux) Test. Chromatographic Analysis. Molecular Methods.

Direct Methods: The specimen:

Sputum: in case of pulmonary

tuberculosis.

Pus: in case of skin type.

Urine: in case of urinary tuberculosis.

CSF or blood: in case of meningitis.

Blood: in case of miliary tuberculosis.

Stool: in case of intestinal tuberculosis.

Special Remarks:

For sputum: It's better to take the morning

sample and for 3 successive days. The

sample requires decontamination process

and may be concentration.

Pus: could be treated as sputum.

Urine: is treated as above and in direct

examination acid and alcohol must be used to

avoid the false +ve result due to presence of

the saprophytic mycobacterium in the

smegma.

CSF: it's a normal sterile liquid, thus direct

examination and culture are done directly

from the deposit after centrifugation

without the need of decontamination

process.

Stool: treated as urine.

Direct Examination:

A film is prepared and suspected specimen

and is stained with Ziehl - Neelsen stain.

The appearance of acid-fast bacilli

having the morphology of TB (tubercle

bacilli) is almost diagnostic of tuberculosis

infection.

Alternatively, the bacilli could be detected

with auramine stain and visualized by

fluorescence microscope.

Methods Depend on Isolation:

Culture:

May be positive even when the direct

test is negative and this could be

attributed to the presence of very few

numbers of bacilli in the sample.

The sample (from non sterile sites) is

undergone a process of decontamination

and concentration.

Culture is done on Löwenstein-Jensen or

Middlebrook medium and incubates aerobically

at 37 C and examined weekly up to 8 weeks

before considering it negative.

Colonies are dry and warty appearance and

buffy in color (human type) or soft and flat

colonies (bovine type).

Alternatively, culture could be made in liquid

BACTEC medium, in which radioactive

metabolites are used and growth can be

detected by the production of radioactive CO2

in shorter time.

Biochemical reactions:

Niacin test is +ve in case of human type and -

ve in case of bovine type.

Catalase -ve.

Animal inoculation:

The prepared sample for culture may also be

injected into two guinea pigs. One of these

animals is killed after 4-6 weeks and examined

for the typical lesions of tuberculosis and

also by film stain and culture. The other

animal is left under observation.

Methods other than isolation:

Tuberculin (Mantoux )Test:

The immunological base of this test is the type IV

hypersensitivity or delayed type which

depends on cell mediated immune reaction.

This test was firstly described by Koch who used

the crude extract of TB (tubercle bacilli) known as

Koch's Old Tuberculin (K.O.T).

Recently, a purified protein derivative (PPD)

is used instead.

Tuberculin test is performed by intradermal

injection of the PPD (5 IU) in one forearm.

In case of +ve tuberculin, a red and indurated

area (10 mm in diameter) appears after 2-3

days. The induration is very important.

Significance of tuberculin test:

Tuberculin + ve; means a case or previously

exposed to TB or vaccinated. Since by adulthood

as many as 80% of Egyptians are

positive, thus the test is of great diagnostic value

(together with the clinical symptoms) in childhood.

Tuberculin -ve; means neither a case nor

having immunity.

Tuberculin test is used before BCG

vaccination, which should be given only

to tuberculin -ve people. If it is given to

tuberculin +ve individual, it may

provoke a virulent unwanted reaction.

Bacillus Calmette–Guérin is a vaccine

against tuberculosis that is prepared from

a strain of the attenuated (weakened) live

bovine tuberculosis bacillus,

Mycobacterium bovis, that has lost its

virulence in humans by being specially

subculture in an artificial medium for 13

years, and also prepared from

Mycobacterium tuberculosis.

Albert Calmette Camille Guérin

Chromatographic analysis

Depend on the analysis of fatty acids by

Gas chromatography or HPLC.

Molecular Methods:

These methods are based on the detection of

a specific gene(s) of the TB. Of these

methods:

DNA probe method (Nucleic acid

hybridization).

Nucleic acid probes are commercially available.

The detection is done within 2-3 hours.

PCR method.

It is a rapid and sensitive method and

does not require the presence of the

organism but only a specific DNA

fragments to be present.

The limitations of these two methods

are:

1.The cost, technical expertise.

2.The unavailability of the organism to do

antibiotic sensitivity testing.

Prevention and Control: Prevention and Control:

1. B.C.G. vaccine; it is a living attenuated

vaccine derived from M. bovis. Attenuation is

obtained by repeated subcultring (about 250

times) on unsuitable medium containing

glycerol, potato and bile.

2. Hygienic measures, etc.

3. Eradication of infected animals (tuberculin +ve)

by slaughtering.

4. Good nutrition and effective pasteurization of

milk.

Treatment:Treatment:

Because of drug resistance, antibiotic

sensitivity test should be

performed.

Resistance by mutation.

General Features:General Features:

Acid fast bacilli differ from the typical TB in being

non pathogenic to guinea pig.

They can be treated with antibiotics and they

are resistant to anti-tuberculous drugs.

They are widely distributed in the environment.

They form smooth and pigmented colonies.

They are classified according their culture

characteristics into:

Atypical or Anonymous Atypical or Anonymous MycobacteriumMycobacterium

Slow Growing

Rapid Growing

Photochromogenic

Scotochromogenic

Non Chromogenic

Slow growing:Slow growing:

A. Photochromogenic:

Produce yellow pigment when the

growth is being exposed to light.

Example: M. kansasii, M. marinum.

B. Scotochromogenic:

Produce orange color chiefly in the dark.

Example: M. scrofulaceum.

C. Non Chromogenic:

Produce no pigments.

Example: M. avium -intracellular

complexes.

Rapid growing:Rapid growing:

Grow within few days and they can grow in

ordinary media.

They can produce several types if infection

even mimics TB in symptoms.

Examples:

M. fortuitum -chelonei complex.

M. smegmatis is a rapidly growing mycobacterium

that is not associated with human disease. It is

part of the normal flora of smegma, the material that

collects under the foreskin of the penis.

Typical mycobacterium

Atypical mycobacterium

Culture Characteristics - Produce dry, warty and buff

colonies (human type) or soft

white colonies

(bovine type).

- Require enriched media.

- Give smooth and

pigmented colonies.

- Can grow on ordinary

media.

Catalase test -ve Strongly +ve

Niacin test +ve (human type)-ve (bovine type) +ve

Pathogenicity to guinea pigs Pathogenic Non pathogenic

Susceptibility to anti-tuberculous normally susceptible Not

Morphology:Morphology:

They are acid and alcohol fast bacilli when

stained with modified Ziehl- Neelsen

stain (5% H2 SO4).

They may appear beaded but coarser than

TB.

They arrange in masses or groups and

mostly intracellular.

Non spore forming.

Non motile.

Culture:Culture:

They cannot grow on any artificial

media or cell culture.

It can be grown in the mouse footpad or in

the armadillo.

Humans are the natural host.

The optimum temperature for growth is 30

°C. It is lower than body temperature; it

thus grows preferentially in the skin and

superficial nerves.

Epidemiology:Epidemiology:

Transmission: Infection is acquired by

prolonged contact with patients with

lepromatous leprosy, who discharge M.

leprae in large numbers in nasal

secretions and from skin lesions.

The incubation period is extremely long,

lasting from several months to 20 years.

The disease occurs worldwide, with most

cases in tropical regions of Asia and

Africa.

Pathogenicity:Pathogenicity: Leprosy in man occurs in three clinical forms:

The nodular or lepromatous type:

- In which the organisms produce nodules and

form granulation in skin, mucous

membrane and internal organs.

Tuberculoid type.

In this case the nerve endings are usually

affected with paralysis or loss of sensation

of the affected area.

Mixed type.

Laboratory Diagnosis:Laboratory Diagnosis: The specimen:

Collected from any ulcerated nodules of

the skin or from the nasal secretion as

well as by gentile scraping of the

nasal septum. Direct Examination:

Film stained with modified Ziehl

Neelsen stain, the presence of acid and

alcohol fast bacilli, usually in groups,

intracellular is diagnostic.

Indirect allergic test or Lepromin test:

It is similar to tuberculin test; however,

the results are unreliable.

Immunization:Immunization:BCG can also be given to protect against

leprosy especially for individuals between

10- 29 years old.

Treatment:Treatment:Dapsone in combination with

clofazimine and rifampin.

Prevention and Control: Prevention and Control: No vaccine is available.

Isolation of cases and their treatment for

2 years or until the

disappearance of symptoms.

Chemoprophylaxis with dapsone of

exposed children.

General Features:General Features:

They are true bacteria (related to

corynebacteria and mycobacteria), but

they form long branching filaments that

resemble the hyphae of fungi.

They are microaerophilic or anaerobic

on primary isolation.

Nocardia are aerobic organism.

Actinomyces Nocardia

Staining characters

Gram-positive Weakly Gram-positive and acid-fast

Oxygen requirement

Strict or facultative anaerobes

Strict Aerobes

Epidemiology Part of normal flora (oropharynx, gastrointestinal tract)

Prevalent in soil; occasionally found in sputum of normal

individuals.Clinical

disease Produce

abscesses with yellow

granules (sulfur granules)

Cause subcutaneous and

pulmonary infection

Treatment Penicillin G Sulfonamides

General Features:General Features:

Gram-positive bacteria which grow in

filaments that readily break up into rods and

may show branching.

Non-motile.

Non-spore-forming.

Non-acid fast.

In tissue, colonies develop to show the

diagnostic yellow "sulfur granules",

which is visible by naked eye and is found

in pus discharge through draining sinuses

Culture characteristics:Culture characteristics:A washed, crushed sulfur granule

should be used for culture.

Grow on blood or serum glucose agar

anaerobically. Incubation at 37° C for at

least 7 days.

The growth is enhanced by 5% CO2.

Colonies are small, cream, adherent

and nodular.

Pathogenesis and Clinical disease:Pathogenesis and Clinical disease: Actinomyces is endogenous in origin and

results in a chronic granulomatous

infection with abscess formation.

Profuse pus discharges by draining

through sinuses.

Infection probably starts after local

trauma, e.g. the extraction of

carious teeth, appendectomy.

Intra-utrine contraceptive devices; are

often colonized with Actino israelii.

The significance of this uncertain but,

perhaps in association with other

organisms, the organism may cause low-

grade intra-utrine infection.

Treatment:Treatment:Penicillin, licomycin or tetracyclines.

Nocardia asteroidsNocardia asteroidsPathogenesis and Clinical Pathogenesis and Clinical

disease:disease:Generally cause granulomatous

suppurative infections.

It affects lungs with secondary brain

abscess.

It is opportunistic pathogen and affects

usually immunocompromized

patients.

Nocardia madurae and N. brasiliensisNocardia madurae and N. brasiliensisAffect subcutaneous tissues; “madura

foot" or mycetoma which is a tropical form

of nocardiosis and affects foot and

produces chronic discharging sinuses.

Treatment:Treatment:Surgical treatment.

Trimethoprim-sulfamethoxazole.