1. Corynebacteria

2. Gram positive rods Spore-forming AEROBIC ANAEROBIC Non spore-forming
Genus: Bacillus
B. anthracis
B. cereus
B. subtilis
ANAEROBIC
Genus: Clostridium
C. tetani
C. botulinum
C. difficile
C. perfringens
Non spore-forming
AEROBIC
Corynebacteria
C. diphtheriae
diphtheroids
Listeria monocytogenes
ANAEROBIC
Lactobacillus spp.

3. Corynebacterium spp
Gram positive bacilli, with characteristic morphology (club shaped and beaded)
Non motile
Non spore forming
Non capsulated
Non--hemolytic on sheep blood agar
Facultative anaerobic
C. diphtheriae is fastidious while diphtheriods are non-fastidious
Catalase positive
Oxidase negative

4. Species of Corynebacteria
Corynebacterium diphtheriae
Other Significant Corynebacterium species
C. xerosis
C. pseudodiphtheriticum
C. pseudotuberculosis
C. jeikeium, (skin)
C. ulcerans
Normal flora of RT, urethra, vagina, Skin

5. Introduction – C. diphtheriae
Diphtheros – leather (tough, leathery pseudomembrane)
Also known as Klebs–Loeffler bacillus
Causes Diphtheria

6. Important features of C. diphtheriae
Slender Gram positive bacilli
Pleomorphic, non motile, non sporing
Chinese letter or Cuneiform arrangement
Stains irregularly, tends to get easily decolorised
May show clubbing at one or both ends - Polar bodies/ Metachromatic granules/ volutin or Babes Ernst granules
Metachromatic Granules:
made up of polymetaphosphate
Bluish purple color with Loeffler’s Methylene blue
Special stains

8. Virulence factor Exotoxin – Diphtheria toxin: Protein in nature
very powerful toxin
Responsible for all pathogenic effects of the bacilli
Produced by all the virulent strains
Two fragments A & B

9. Mechanism of Action of Diphtheria Toxin:
Mechanism of Action of Diphtheria Toxin: Inhibition of Protein Synthesis

10. Epidemiology
Habitat – nose, throat, nasopharynx & skin of carriers and patients
Spread by respiratory droplets, usually by convalescent or asymptomatic carriers
Incubation period of diphtheria – 3 to 4 days

11. Diphtheria Site of infection
Faucial (palatine tonsil) – commonest type
Laryngeal
Nasal
Otitic
Conjunctival
Genital
Cutaneous – usually a secondary infection on pre-existing lesion, caused by non toxigenic strains

12. Pathogenesis & Clinical Manifestations
Human Disease
Usually begins in respiratory tract
Virulent diphtheria bacilli lodge in throat of susceptible individual
Multiply in superficial layers of mucous membrane
Elaborate toxin which causes necrosis of neighboring tissue cells
Inflammatory response eventually results in pseudomembrane (fibrinous exudate with disintegrating epithelial cells, leucocytes, erythrocytes & bacteria)
Usually appears first on tonsils or posterior pharynx and spreads upward or down
In laryngeal diphtheria, mechanical obstruction may cause suffocation
Regional lymphnodes in neck often enlarged (bull neck)

13. Complications of diphtheria
Mechanical complications are due to the pseudomembrane, while the systemic effects are due to the toxin.
Asphyxia – due to obstruction of respiratory passage
Toxic myocarditis
Congestive heart failure
Postdiphtheritic paralysis – occurs in 3rd or 4th week of disease, spontaneous recovery
Sepsis – pneumonia & otitis media

15. Laboratory Diagnosis Specimen – swab from the lesions Microscopy
Gram stain: Gram +ve bacilli, chinese letter pattern
Immunofluorescence
Albert’s stain for metachromatic granules

16. Biochemical Reaction
All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)

17. Laboratory Diagnosis
Culture – isolation of bacilli requires media enriched with blood, serum or egg
Blood agar
Loeffler’s serum slope – rapid growth, 6 to 8 hrs
Tellurite blood agar – tellurite is reduced to tellurium, gives gray or black color to the colonies

18. Growth of diphtheria bacilli
Blood agar
Tellurite blood agar
Loeffler’s serum slope

19. 3 biotypes of C. diphtheriae are characterized on BTA
i.e. Gravis, mitis and intermedius biotypes
The most severe disease is associated with the gravis biotype
Colony of gravis biotype is large, non-hemolytic & grey.
Colonies of mitis biotype are small, hemolytic and black
Colonies of intemedius biotype are intermediate in size, non-hemolytic with black center & grey margin.

20. Laboratory Diagnosis Biochemical reactions
Ferments sugar with acid formation but not Gas ferments: glucose, galactose, maltose and dextrin, but not ferment sucrose.
Resistant to light, desiccation and freezing
Sterilization: sensitive to heat (destroyed in 10mins at 58°C or 1min in 100°C), chemical disinfectants

21. Laboratory Diagnosis Virulence tests - Test for toxigenicity
Invivo tests – animal inoculation (guinea pigs)
Subcutaneous test
Intracutaneous test
Invitro tests
Elek’s gel precipitation test
Tissue culture test

22. Detection of toxin: Elek’s Test
Principle:
It is toxin/antitoxin reaction
Toxin production by C.diphtheriae can be demonstrated by a precipitation between exotoxin and diphtheria antitoxin
Procedure:
A strip of filter paper impregnated with diphtheria antitoxin is placed on the surface of serum agar
The organism is streaked at right angels to the filter paper
Incubate the plate at 37C for 24 hrs

23. Filter paper saturated with diphtheria antitoxin
Resuls:
After 48 hrs incubation, the antitoxin diffusing from filter paper strip and the toxigenic strains produce exotoxin, which diffuses and resulted in lines four precipitation lines radiating from intersection of the strip and the growth of organism
Lines of precipitations
Inoculated M.O.
Positive Elek’s Test

24. Laboratory Diagnosis Virulence tests - Invitro tests
Tissue culture test - incorporation of bacteria into agar overlay of eukaryotic cell culture monolayers.
Result: toxin diffuses into cells and kills them

25. Treatment
specific treatment must not be delayed if clinical picture suggests of diphtheria
rapid suppression of toxin-producing bacteria with antimicrobial drugs (penicillin or erythromycin)
early administration of antitoxin: 20,000 to 1,00,000 units for serious cases, half the dose being given IV 25

26. Prophylaxis 1.active immunization (vaccination)
DPT - triple vaccine given to children; contains diphtheria toxoid, Tetanus toxoid and pertussis vaccine
Schedule i) Primary immunization - infants and children - 3 doses, 4-6 weeks interval
- 4th dose after a year
- booster at school entry
ii) Booster immunization - adults -Td toxoids used (travelling adults may need more)

27. Prophylaxis
Passive immunization ADS (Antidiphtheritic serum, antitoxin) - made from horse serum
- 500 to1000 units subcutaneously
Combined immunization First dose of adsorbed toxoid + ADS, to be continued by the full course of active immunisation

28. CONTROL isolate patients treat with antibiotics actively
complete vaccination schedule should be used with booster every 5 years

29. Listeria
Listeria is a genus of bacteria that contains 10 species, each containing two subspecies. Listeria species are gram-positive, rod-shaped, facultatively anaerobic, and non spore-forming. The major human pathogen in the Listeria genus is L. monocytogenes. It is usually the causative agent of the relatively rare bacterial disease listeriosis, a serious infection caused by eating food contaminated with the bacteria. The disease affects pregnant women, newborns, adults with weakened immune systems, and the elderly.

30. Listeria monocytogens gram staining method

31. Listeriosis is a serious disease for humans; the overt form of the disease has a case-fatality rate of about 20%. The two main clinical manifestations are sepsis and meningitis. Meningitis is often complicated by encephalitis, when it is known as meningoencephalitis, a pathology that is unusual for bacterial infections

32. Treatment
In non-invasive listeriosis, the bacteria often remain within the digestive tract, causing mild symptoms lasting only a few days and requiring only supportive care. Muscle pain and fever in mild cases can be treated with pain relievers, and diarrhea and gastroenteritis can be treated with medications if needed.
In invasive listeriosis, the bacteria have spread to the bloodstream and central nervous system. Treatment includes intravenous delivery of high-dose antibiotics and in-patient hospital care