1_Mycobacterium

8/14/2019 1_Mycobacterium

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Chapter 12Chapter 12

MycobacteriaMycobacteria

( P124 )( P124 )

P DF " p d f F a c t o r y Pr o " www. f i n e p r i n t . c o m. c n
http://www.fineprint.com.cn/http://www.fineprint.com.cn/


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Acid-fast bacilli, slender, straight orslight curved; aerobic; growing

with branching tendency.



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Acid-fast bacilli: They are neither gram-

positive nor gram-negative. They are

stained poorly by dyes used in Gram

stain. Although they are not stained

readily, once stained, they resist

decolorization by acid or alcohol, and

are therefore called acid-fast bacilli.



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Types of Mycobacterium

M. tuberculosis-----tuberculosis

M. leprae-----leprosy

Atypical mycobacteria (such as M.kansasii)-----tuberculosislike disease, but are less

frequent pathogens.

Rapidly growing mycobacteria-----such asM.chelonei. Occasionally cause disease in

immunocopromised human.



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M.tuberculosis (MTB)

(human tubercle bacillus)

This organism causes tuberculosis. Worldwide,

M.tuberculosis causes more deaths than any

other single microbial agent. Approximately

one third of the worlds population is infected

with this organism. Each year, it is estimated

that 3 million people die of tuberculosis and 8million new cases occur. WHO name March

24th as tuberculosis day.



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1. Important properties

(1) Morphology and structure

Shape: thin, straight or slightly curved rod, cord

arrangement.Acid-fast stain----red (Ziehl-Neelsen stain shown

as red rods against a deep sky-blue background).

Lipid-rich cell wall: thick, complex, lipid-rich cellwall, which is relative with its staining and

pathogenesis.



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(2) Culture

Obligate aerobes: This characteristic explains itspredilection for causing disease in highly oxygenated

tissues such as the upper lobe of the lung.

Special nutrient requirement: The usual medium is

Lowenstein-Jensen medium, which contain whole

eggs (yolk), glycerol, potate, malachite green (a dye,

which can inhibit the unwanted normal flora present in

sputum specimens).



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Slow growth: MTB grows slowly, and its

generation time (doubling time) is 18h~24h. Becauseof this slow growth, cultures of clinical specimens

must be held for 6-8 weeks before recorded as

negative.

Colony: Rough colony , like cauliflower.

In liquid media: form pellicle on surface of liquid

media. There are two reason: the cell wall contains

lots of lipid; it is aerobe, and the surface of the liquid

media has enough oxygen.



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Colony of MTB: Rough colony , like cauliflower



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(3) Resistance

Resistant to drying (especially in sputum, it can

survive in dried expectorated sputum for 6-8 months;

this property may be important in its transmission by

aerosol.More resistant to chemical disinfectant than other

nonspore-forming bacteria. For example, MTB is

relatively resistant to acids and alkalis.

Alkalis(4%NaOH)----is used to concentrate clinical

specimens; it destroys unwanted bacteria, human

cells, and mucus but not the organism.



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Alkalis(4%NaOH)----is used to concentrate clinical

specimens;

It destroys unwanted bacteria, human cells, and

mucus but not the organism.

thick sputum thin sputum4%NaOH



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wet heat (moist heat---60 30min, 70 3min).

disinfectants---alcohol, glutaraldehyde, formaldehyde

Sensitive to:

drugs---rifampin, streptomycin, isoniazid, etc.

UV(ultraviolet)---The material that the tuberculosis

patients used can be sterilized by UV.



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(4) Variationdrug resistance, toxicity (BCG) variation.

Drug resistance variation: Some strains of

MTB will become to be resistant to some

antimycobacterial drug, such as isoniazid. Some

strains are resistant to multiple antibiotics (called

multiple-drug-resistant, or MDR strains), havebecome a worldwide problem.



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Virulent variation ------BCG

BCG (Bacillus Calmette-Gurin)---- BCG is a live

attenuated vaccine which contain a strain of live,

attenuated Mycobacterium bovis. After M. bovis has

been cultured on nutrient media which containglycerol, bile, potate to multiply 230 generations for

13 years, the M. bovis lost its pathogenicity, but keep

the antigenicity of mycobacterium. It was discovered

by Calmette and Gurin, so called BCG. BCG is avaccine widely used for the protection against

tuberculosis.



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The degree of protection of BCG is only partial and

not 100%.

BCG is considered efficacious in young children but

not so in adults.



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2. Pathogenesis

MTB is the pathogen of tuberculosis, which

has no exotoxin, no endotoxin, or no invasive

enzymes.

(1) Pathogenic substance

1) Lipids

2) Proteins



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1) Lipids

most important pathogenic substance.

Possess 60% of the cell-wall dry weight, thecontent of lipids closely related to its virulence.

Kinds of lipids----mycolic acid, cord factor,

wax D, phosphatides and sulfatides.



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Mycolic acid: It is a long-chain fatty acid, whichcontribute to the organisms acid-fast property.

Wax D: one of the active components in Freunds

adjuvant, which is used to enhance the immune responseto many antigens in experimental animals.

Phosphatides: which play a role in caseation necrosis

and can inhance the proliferation of mononuclear and

inhance the macrophage transform into epithelioid cells.

Tubercle



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Cord factor: It cause the MTB be cord arrangementand also associated with virulence:

Inhibit migration of leukocyte to form chronic granuloma.

Bind to mitochondrial membranes, cause functional

damage to respiration and oxidative phosphorylation.



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Sulfatides:

which can prevent the phagosome from fusing with

lysosome, thereby allowing the organism to escape the

degradative enzymes in the lysosome. So MTB can

survive and multiply within a non-activated phagocytic

cell.

MTB is intracellular organism.



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2) Proteins Protein antigens: such as old tuberculin, purifiedprotein derivative, etc---when some of those proteins

are combined with waxes, they can elicit delayed

hypersensitivity.

These protein can also elicit the formation of a

variety of antibodies. Some kinds of antibody is

protective Ab, such as anti-Ag85B.



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(2) Lesions

Lesions are dependent on the presence of the organism

and the host response, and there are two types of lesions.

Exudative lesions, which consist of an acute

inflammatory response and occur chiefly in the lungs atthe initial site of infection.

Granulomatous lesions, which consist of a central

area of giant cells containing tubercle bacilli surrounded

by a zone of epithelioid cell (progress to Tubercles).

A tubercle is a granuloma surrounded by fibrous

tissue that has undergone central caseation necrosis.

Tubercle heal by fibrosis and calcification.



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(3) Spread of oragnisms in the Host

MTB spread in the host by direct extension,

through the lymphatic channels and bloodstream,and via the bronchi and gastrointestinal tract.



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(4) Intracellular site of growth

Once mycobacteria establish themselves in tissue,

they reside principally intracellularly in monocytes,

reticuloendothelial cells and giant cells.

The intracellular location is one of the features that

makes chemotherapy difficult and favors microbial

persistence.



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The primary infection occurred tuberculin-negative

in early life, usually in children.

The bacilli was inhaled into alveolar cave. Thesebacilli are engulfed by alveolar marophages in which

they replicate to form the initial lesion. Some bacilli

carried in macrophage to the hilar lymph nodes where

additional foci of infection develop.

The initial lesion together with the enlarged hilar

lymph nodes form primary complex.

Types of Pulmonary tuberculosis:

Primary infection and post-primary infection.



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initial lesion

enlarged hilar lymph nodes

All the lesion is exudative lesion. With the establishing

of CMI, these exudative lesion will be replaced byfibrous tissue and calcification tissue.



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The reactivation is usually caused by tubercle bacillithat have survived in the primary lesion.

Post-primary infection

After the primary infection, if the hosts are infected

by MTB out of the host once again, it is reinfection.

Reactivation and reinfection



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These differences between primary infection and

post-primary infection are attributed to difference ofresistance and hypersensitivity induced by the first or

secondary infection of the host with tubercle bacilli.



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3. Immunity and hypersensitivity

(1) Immunity

(2) Hypersensitivity

(3) Tuberculin test



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(1) Immunity

The main anti-infectious immunity is cellular immunity.

CD4-positive T cells are sensitized by primaryinfection. When they encounter the MTB again, they will

become activation, and release some cytokine, such as

(interleukin-2, IL-2), and -Interferon.

After recovery from the primary infection, resistance to

the MTB is mediated by cellular immunity.

Macrophages: which can be activated by -Interferon,then kill the organism in the cell. It is very important that

the activation of macrophages by -Interferon in the host

defense against MTB.



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(2) Hypersensitivity

In the course of primary infection, the host acquires

cellular immunity to the tubercle bacilli and also acquires

hypersensitivity to the MTB. This is made evident by the

development of a positive tuberculin reaction.

Hypersensitivity and resistance appear to be distinct

aspects of related cell-mediated reactions (cellularimmunity).



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(3) Tuberculin test

Tuberculin test----is a skin test, and can detect the

prior infection by a positive result, which is due to a

delayed hypersensitivity reaction.

Reagent----PPD (purified protein derivative).

Principle: delayed hypersensitivity reaction.

MTB special media for 6-8 weeks TCA precipitation PPD



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Tuberculin test

Result and interpretation



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Tuberculin test

Application

Basis of BCG inoculation, detect immunity effect.

Diagnosis for young children tuberculosis.

Epidemiological investigation.

Cellular immunity test of patients with tumor.



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3. Bacteriological diagnosis

Specimens: sputum, pus, CSF(cerebrospinal fluid), urine.

Direct smear---acid-fast stain (Ziehl-Neelsen stain)---

Microscopic examination.

Observe the acid-fast bacteria.

Culture---After digestion of the specimen by treatment

with NaOH and concentration by centrifugation, the

material is cultured on special media, such as Lowenstein-

Jensen agar, for up to 8 weeks.Observe the rough colony , which is like cauliflower.

Rapid diagnosis

to detect its DNA by PCR (Polymerase chain reaction).



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Treatment

Multiple-drug therapy is used to prevent theemergence of drug resistant mutants during a long

duration (6- or 9-month) of treatment.

Isoniazid, rifampin, and pyrazinamide.

DOT (directly observed therapy)

Because the long duration of treatment, noncompliance

lead to failure of patients to complete the full course of

therapy, which allow the resistant organisms to survive.One approach to the problem of noncompliance is

directly observed therapy (DOT), in which health care

workers observe the patient taking the drug.



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Mycobacterium leprae (P128)

M. leprae cause leprosy.

Leprosy: chronic infectious disease, involving

skin, mucosa, peripheral nerve and organs, by

way of skin to skin contact or respiratory

route.



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1. Biological characteristics

Acid-fast rods, predominant in "foam cells" .

M.leprae has not been grown in the laboratory,either on artificial media or in cell culture.

It can be grown in the mouse footpad or in the

armadillo, but it grows very slowly.

Humans are the nature hosts.The optimal temperature for growth (300C) is lower

than body temperature; it therefore grows

preferentially in the skin and superficial nerves.



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Acid-fast rods (red) in "foam cell"



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2. Transmission

Infection is acquired by prolonged contact with

patients with lepromatous leprosy, who discargeM.leprae in large numbers in nasal secretions and from

skin lesions.



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3. Pathogenesis

The organism replicates intracellularly.

There are two distinct forms of leprosy----

Tuberculoid type and Lepromatous type.

In tuberculoid leprosy, the cellular immunity

response to the organism limits its growth, very few

acid-fast bacilli are seen, granulomas containing giant

cells form, and the lepromin skin test (similar to the

tuberculin test) is positive.



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In lepromatous leprosy, the cellular immunity

response to the organism is poor, the skin and mucous

membrane lesions contain large numbers of organisms,

foamy histiocytes (called foam cells) rather than

granulomas are found, and lepromin skin test result is

negative.



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Tuberculoid Lepromatous

Cellular immunity High Very low

Humoral immunity Low High

Lepromin skin test Positive Negative

Table: The difference between two types of leprosy



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4. Laboratory diagnosis

1) Pathogen examination

In lepromatous leprosy, the bacilli are easilydemonstrated by performing an acid-fast stain of skin or

nasal scrapings. Lipid-laden macrophages (foam cells)containing many acid-fast bacilli are seen in the skin.

In the tuberculoid form, very few organisms are seen

and the appearance of typical granulomas is sufficient

for diagnosis.

2) Lepromin test---- which is like tuberculin test.



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5. Prevention and control

1) Prevention----no vaccine.

Isolation of all lepromatous patients, coupled with

chemoprophylaxis with dapsone for exposed

children, is required.

2) Chemotherapeutics----dapsone

The mainstay of therapy is dapsone. Treatment isgiven for at least 2 years or until the lesions are free

of organisms.

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