1. Corynebacterium diphtheriae

8. 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

9. Culture
Aerobic and facultative anaerobic, growing well at 37C 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.

10. Pediatric
Symptoms:
formation of a false membrane in the throat
high fever
difficulty in breathing

11. Pseudomembrane

12. Diphtheria is a typical toxigenic infectious disease
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.

13. 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

14. EM of ß-corynebacteriophage carrying tox gene

15. ◆ 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.
Toxic
Binding A
Cell surface B

16. 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.

17. The toxin is very toxic to humans and many animals (e. g
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.

18. Clinical Manifestations
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.

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

20. Immunoprecipitation line

21. 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 ）.

22. Mycobacteria

27. Acid-fast bacilli
Mycobacteria 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.

29. Among the species of Mycobacteria, M
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.

30. 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.

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

32. Variation
Drug 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

33. Virulent factors Produce no endotoxin, exotoxin or invasive enzymes
The pathogenicity is due to cell wall components, and substances inducing immunopathological reactions.

35. Virulent factors Lipids in the cell wall
Cord factor: lipoarabinomannan, damages mitochondria
Phosphatide: causes the formation of tubercule and granuloma
Sulfatides: inhibits phagosome-lysosome fusion
Wax D: serves as an “adjuvant” that induce hypersensitive reaction.

37. Virulent factors Proteins Polysaccharide
e.g., Tuberculin mixed with wax D can induce hypersensitive reaction.
Polysaccharide
Bound to wax D to induce the infiltration (浸润) of inflammatory cells.

38. Tissue destruction results from cell-mediated hypersensitivity.
Pathogenesis
Mycobacterium tuberculosis enters alveoli by airborne transmission
It resists destruction by alveolar macrophages and propagate
Tissue destruction results from cell-mediated hypersensitivity.
To cause primary tuberculosis
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 lead to post-primary tuberculosis

39. Transmission and infection
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.

40. 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.

41. 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.

43. What is it?
The Mantoux tuberculin skin test (TST) is the standard method of determining whether a person is infected with Mycobacterium tuberculosis. Reliable administration and reading of the TST requires standardization of procedures, training, supervision, and practice.
How is the TST Administered?
The TST is performed by injecting 0.1 ml of tuberculin purified protein derivative (PPD) into the inner surface of the forearm. The injection should be made with a tuberculin syringe, with the needle bevel facing upward. The TST is an intradermal injection. When placed correctly, the injection should produce a pale elevation of the skin (a wheal) 6 to 10 mm in diameter.
How is the TST Read?
The skin test reaction should be read between 48 and 72 hours after administration. A patient who does not return within 72 hours will need to be rescheduled for another skin test.
The reaction should be measured in millimeters of the induration (palpable, raised, hardened area or swelling). The reader should not measure erythema (redness). The diameter of the indurated area should be measured across the forearm (perpendicular to the long axis).
How Are TST Reactions Interpreted?
Skin test interpretation depends on two factors:
Measurement in millimeters of the induration
Person’s risk of being infected with TB and of progression to disease if infected
Classification of the Tuberculin Skin Test Reaction
An induration of 5 or more millimeters is considered positive in-HIV-infected persons-A recent contact of a person with TB disease -Persons with fibrotic changes on chest radiograph consistent with prior TB-Patients with organ transplants-Persons who are immunosuppressed for other reasons (e.g., taking the equivalent of >15 mg/day of prednisone for 1 month or longer, taking TNF-a antagonists)An induration of 10 or more millimeters is considered positive in-Recent immigrants (< 5 years) from high-prevalence countries-Injection drug users-Residents and employees of high-risk congregate settings-Mycobacteriology laboratory personnel-Persons with clinical conditions that place them at high risk-Children < 4 years of age - Infants, children, and adolescents exposed to adults in high-risk categoriesAn induration of 15 or more millimeters is considered positive in any person, including persons with no known risk factors for TB. However, targeted skin testing programs should only be conducted among high-risk groups. What Are False-Positive Reactions?
Some persons may react to the TST even though they are not infected with M. tuberculosis. The causes of these false-positive reactions may include, but are not limited to, the following:
Infection with nontuberculosis mycobacteria
Previous BCG vaccination
Incorrect method of TST administration
Incorrect interpretation of reaction
Incorrect bottle of antigen used
What Are False-Negative Reactions?
Some persons may not react to the TST even though they are infected with M. tuberculosis. The reasons for these false-negative reactions may include, but are not limited to, the following:
Cutaneous anergy (anergy is the inability to react to skin tests because of a weakened immune system)
Recent TB infection (within 8-10 weeks of exposure)
Very old TB infection (many years)
Very young age (less than 6 months old)
Recent live-virus vaccination (e.g., measles and smallpox)
Overwhelming TB disease
Some viral illnesses (e.g., measles and chicken pox)

44. 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.