Corynebacterium diphtheriae

Corynebacterium Corynebacterium diphtheriaediphtheriae

Morphology

•Another characteristic is the presence of metachromatic

granules (RNA and polymetaphosphate) in bacterial cells.

The granules are bluish-purple with methylene blue, and

dark purple by Albert staining method.

methylene blue stain

metachromatic granules

Albert stain

Culture•Aerobic and facultative anaerobic, growing well at 37C on blood- or serum-containing medium.

•Loeffler’s serum slant: not a selective medium but gives abundant growth and typical morphology of the bacillus.

•Blood tellurite agar: a selective medium because tellurite can suppress the growth of normal flora in throat, and the colonies are black-colored.

• Pediatric

• Symptoms:

formation of a false membrane in the

throat

high fever

difficulty in breathing

Pseudomembrane

• Diphtheria is a typical toxigenic infectious disease. Diphtheria toxin is the major virulent factor.

•Effective antitoxin therapy and successful toxoid vaccine available.

• A rare disease, still occur in non-immunized populations.

• High death rate.

Virulent factor: diphtheria toxin

The toxin encoding gene (tox) is carried byβ-corynebacteriophage only the bacillus infected by the phage and committed lysogenic conversion produce diphtheria toxin

The regulation of tox gene expression is mediated by an iron-activated inhibitor (DtxR) which is chromosomally encoded by C.

diphtheria

EM of ß-corynebacteriophage carrying tox gene

◆ many exotoxins are called as A-B type toxins

because they consist of A and B subunits. The B

subunit generally mediates the toxin complex molecule

to adhere and then enter the host cell. A subunit

provides the toxic activity.

ToxicToxicBindingBinding

AA

Cell surfaceCell surface

BB

Diphtheria toxin is an A-B type toxin

A diphtheria toxin molecule contains 535 amino acids with subunit A and subunit B.

Subunit B contains receptor binding and transmembrane domains. Subunit A contains catalytic domain. It inactivate elongation factor 2 (EF2). Thus the protein synthesis is suppressed.

The toxin is very toxic to humans and many animals (e.g.,monkey, rabbit, guinea-pig). Particularly harmful to heart and central nervous system. The lethal dose is as low as 100 to 150 ng/kg.

• Corynebacterium diphtheriae can cause pharyngitis which leads to formation of thick grey membrane called pseudo-membrane. The pseudo-membrane is composed of fibrin, dead epithelial cells, bacteria and neutrophils. It exfoliates easily and then blocks the airway which results in hypoxia ( 缺 氧 ) and suffocation ( 窒息 ).

• As a result of the action of diphtheria toxin to peripheral motor neurons and myocardium, life-threatening systemic complications (principally loss of motor function and congestive heart failure) may develop.

Clinical ManifestationsClinical Manifestations

Diagnosis of diphtheria requires laboratory confirmation of toxigenic Corynebacterium diphtheriae.It is usually determined by in vitro tests such as Elek immunodiffusion test.

Immunoprecipitation line

Diphtheria patients must be promptly treated with antitoxin to neutralize circulating (free) diphtheria toxin. Antitoxin cannot neutralize the toxicity of exotoxin that has bound to the host cells.

Antibiotics (penicillin and erythromycin) are used as part of the treatment.

Diphtheria toxoid is a component of the DPT vaccine, which includes diphtheria toxoid, killed whole cell pertussis （ Bordetella pertussis ） and tetanus toxoid （ Clostridium tetani ） .

MycobacteriaMycobacteria

Acid-fast bacilliMycobacteria do not stain readily. However, if these bacteria are once stained by Ziel-Neelsen Acid-Fast Stain method, they are able to resist decolorization by acid or alcohol.

Among the species of Mycobacteria, M. tuberculosis is the most important pathogen that causes human tuberculosis. M. bovis is a zoonotic causative agent of cattle tuberculosis. It also causes human tuberculosis. M. leprae is the causative agent of leprosy which is still present in the third world.

Morphology and culture

• Mycobacterium tuberculosis has the typical morphology and acid-fast property of Mycobacteria.

•Lowenstein-Jensen medium is the widely used medium for cultivating Mycobacterium tuberculosis. In this medium, malachite green ( 孔雀绿 ) is included to inhibit other bacteria.

•The growth rate is slower than most other bacteria. The doubling time is about 18 hours.

The colonies are formed after 3-6 weeks of incubation on Lowenstein-Jensen medium. The colonies are buff colored and dry breadcrumb-like.

VariationDrug resistance variation: Many clinical isolates of Mycobacterium tuberculosis are drug-resistant. Virulence variation: BCG (Bacillus Calmette-Guerin) is the human tuberculosis vaccine. It is a preparation of live attenuated Mycobacterium bovis.L-form: a form that lacks the cell wall

Virulent factorsProduce no endotoxin, exotoxin or invasive enzymes

The pathogenicity is due to cell wall components, and substances inducing immunopathological reactions.

Lipids in the cell wallCord factor: lipoarabinomannan, damages mitochondriaPhosphatide: causes the formation of tubercule and granulomaSulfatides: inhibits phagosome-lysosome fusionWax D: serves as an “adjuvant” that induce hypersensitive reaction.

Virulent factors

Proteinse.g., Tuberculin mixed with wax D can induce hypersensitive reaction.

PolysaccharideBound to wax D to induce the infiltration ( 浸润 ) of inflammatory cells.

Virulent factors

PathogenesisPathogenesisMycobacterium tuberculosis enters alveoli by airborne

transmission

It resists destruction by alveolar macrophages and propagate

To spread to lymph nodes

Cell-mediated immune response stops cycle of destruction and spread.

To enter bloodstream and seed other organs

To reseed the lungs

To cause primary tuberculosis

Tissue destruction

results from cell-mediated

hypersensitivity.

To lead to post-primary tuberculosis

• Mycobacterium tuberculosis is transmitted

through droplet and airborne dust containing

the microbe.

• Mycobacterium tuberculosis has multiple

routes invading human body, such as

respiratory tract, intestinal tract and skin, to

cause various tuberculosis.

• Among the different tuberculosis, pulmonary

tuberculosis is the most common form.

Transmission and infection

• Bacteriological examination: the definitive

diagnosis for tuberculosis based on the

presence of acid-fast bacteria in the sputum

(pulmonary tuberculosis) and in other clinical

samples.

• Tuberculin skin test: is a method of

identifying persons who once infected with

Mycobacterium tuberculosis.

• Tuberculin skin test is performed by an

intradermal injection of 5 tuberculin units / 0.1 ml

of purified protein derivative (PPD) into forearm.

The result (diameter of induration around the site

of injection) is read 48 to 72 hours after the

injection.

•A tuberculin reaction of >= 5 mm of induration is

classified as positive result. However, a positive

test only means to exposure once to the

microorganism but does not certainly indicate

active disease.

• Prevention: Vaccination of BCG vaccine.

• Treatment: Antibiotics used to treat

tuberculosis such as streptomycin, isoniazid,

rifampin etc., are quite different from those

used to treat other bacterial infectious

diseases. Clinically, several anti-tuberculosis

drugs must be simultaneously applied to

avoid drug resistance and to increase

efficiency of therapy.