microbiology lecture pptt for medicine studentsmicrobiology lecture pptt for medicine students

1. The causative agents of life-
threatening infections. Biological
properties of causative agents of
cholera, plague and anthrax.
Pathogenesis, laboratory diagnostics
and prevention of diseases caused by
them.

2. TAXONOMIC POSITION OF THE
PATHOGENIC VIBRIOS
• Family Vibrionaceae
• Genus Vibrio
• Medical important species:
• V.cholerae
• V.parahaemolyticus
• V.vulnificus
• V.alginolyticus

3. Classification of the vibrios
1. Biochemical classification by Heiberg (1934)
• Heiberg (1934) classified vibrios into six groups
based on the fermentation of mannose, sucrose
and arabinose.
• V.cholerae belongs to Group I (man+, suc+, ara -)
2. Serological classification
• All vibrios possess common H-antigen and group-
specific O-Ag (139 serogroups)
• V.cholerae belongs to O1 group

4. VIBRIO CHOLERAE
Morphology
Gram negative short, slightly
curved rod (comma shaped) about
1.5 μm x 0.2-0.4 μm in size
In stained films of mucous flakes
from acute cholera cases, the
vibrios are seen arranged in
parallel rows, as the 'fish in
stream' appearance.
• It is actively motile, with a
single polar flagellum (detection
of motility – wet mount
technique, phase contrast
microscopy).

5. Cultural characteristics
• The cholera vibrions is strongly aerobic
• Temperature range of 16-40°C (optimum 37 °C).
• Growth is better in an alkaline medium the range of pH
being 6.4-9.6 (optimum 8.2)
• On nutrient agar:
colonies are moist, translucent, round disks, about 1-2 mm
in diameter, with a bluish tinge in transmitted light.
• In peptone water:
growth occurs as a fine surface pellicle

6. Special media
1) Alkaline peptone water at pH 8.6;
2) Monsur's taurocholate tellurite peptone water at pH 9.2.
3) Alkaline bile salt agar (BSA) pH 8.2.
4) Monsur's gelatin taurocholate trypticase tellurite agar
(GTTA) medium:
small, translucent colonies with, a greyish black centre and a
turbid halo.
5) TCBS medium (thiosulfate, citrate, bile salts and sucrose):
large yellow convex colonies which may become green on
continued incubation

7. Growth of V.cholerae on the TCBS

8. Resistance
• Cholera vibrios are susceptible:
• to heat
• drying and acids.
• It resist high alkalinity.
• They are destroyed at 55 °C in 15 minutes.
• They are killed in a few minutes in the gastric juice
of normal acidity but they may survive for 24 hours
in achlorhydric gastric juice.
• Survival in water is influenced by its pH,
temperature, salinity, presence of organic pollution
and other factors.

9. Classification of the V. cholerae
• According to their biological properties V.cholerae
is divided into 2 biovars:
• V. cholerae b/v classical
• V. cholerae b/v El-Tor

10. Distinguishing features of biovars
Tests b/v
classical
b/v El
Tor
Agglutination of fowl
erythrocytes
- +
Lysis of sheep erythrocytes - +
Growth in the presence of
polymixin B
- +
Sensitivity to phages Mukerjee`s
group IV
phage (C)
Mukerjee`s
group V
phage (El -
Tor)
Acetoin production in the Voges-
Proskauer test
- +

11. Antigenic structure
• According to structure of the O1-Ag species V.
cholerae is subdivided into 3 serotypes:
• Ogawa (AB)
• Inaba (AC)
• Hikojima (ABC)

12. Factors of virulence
• Exotoxin (choleragen, cholera enterotoxin,
cholera toxin, CT, or CTX).
causes prolonged activation of cellular adenylatecyclase and
accumulation of cAMP, leading to outpouring into the small
intestinal lumen, of large quantities of water and
electrolytes and the consequent watery diarrhea.
• Endotoxin.
• Adherence factors (pili)
• Proteolytic enzymes (gelatinase, mucinase)

13. Worldwide spreading of 7th pandemic

16. Epidemiology and pathogenesis
Cholera is an exclusively human disease.
Source of infection: sick person , carrier
Route of transmission: fecal-oral
Ways of transmission:
1. Through contaminated water
2. Through contaminated food.
3. with dirty hand (domestic spread of infection)
Incubation period – 3-5 days
Site of affection: small intestine (mild form), small
intestine and stomach (severe form)

17. Laboratory diagnosis
Specimens: Watery stool, rectal swab, water,
food, vomiting
Primary diagnosis: microscopy
Final diagnosis: culture isolation
Retrospective diagnosis: serology

18. Express methods of laboratory diagnosis
• For rapid diagnosis, the characteristic motility
of the vibrio and its inhibition by antiserum
can be demonstrated under the dark field or
phase contrast microscope, using cholera
stool from acute cases
• Immunofluorescence

19. Methods
Microscopy:
1. Stained smears by Gram
2. Wet drop smears to determine
vibratory motility

20. Bacteriological method
The major steps are:
1. Inoculation of the collected samples into alkaline
peptone water and spread a large loop of feces
over a plate of TCBS medium.
2. After incubation for 5 h subculture from first
peptone water is transmitted into second alkaline
PW and on the second plate of TCBS agar.
1) Microscopy of wet smears from PW,
2) agglutination with O-1 antiserum.

21. Bacteriological method
3. Final identification (Heinberg’s tests, biovar detection,
serological and phage typing)

22. Prophylaxis
• General measures
• Purification of water supplies
• Better provision for sewage disposal
• Infected patients should be isolated, their
excreta disinfected
• Contacts and carriers are followed up, given
with antibiotics (tetracycline)

23. Specific measures
• Killed parenteral vaccine – composed of
equal number of Inaba and Ogava strains
• Killed oral vaccine – B subunit whole cell
vaccine. The vaccine contains cholera toxin B
subunit, heat killed classical vibrio and
formalin killed El- Tor vibrio
• Live oral vaccine – recombinant DNA vaccine

24. Treatment
• Oral rehydration therapy
• Antibiotics (tetracycline)

25. Vibrio cholerae

26. Pathogenic Yersinia
Taxonomy and classification
Family: Enterobacteriaceae
Genus: Yersinia
Medical important species:
1. Y.pestis – causative agent of a plague
2. Y. pseudotuberculosis – causative agent of
fatal typhoid like illness
3. Y.enterocolitica – causative agent of enteric
and systemic diseases in animal and
human beings

27. Morphology:
It is Gram-negative
bacteria, 1.5 x 0.7μm
It is non-motile
Shape: short, ovoid
bacteria
Staining: it is bipolar
stained with
methylene blue
It forms microcapsule
in the host’s tissue

28. Cultivation
It is facultative anaerobe
It can grow at wide range of pH (4-10) and
temperature (25 0С) onto the ordinary
nutrient media (MPA and MPB)

29. Solid media:
R-form colonies with
compact centers and flat
transparent (later
opaque) lacy edges.
Nutrient broth:
flocculent growth occurs
on the bottom and
delicate pellicle onto the
surface with hanging
threads (stalactite
growth).

30. Cultural characteristics

31. Resistance and sensitivity
It is sensitive to the heat, sunlight, drying and
chemical disinfectants.
But it can survive for several months and
even multiply in the soil of rodent burrows
(saprophytic form).

32. Antigen structure and virulent factors
1.Fraction I (F-I) antigen is a heat labile
envelope protein (it inhibits phagocytosis)
2.V and W antigens (inhibitors of the
intracellular killing of the bacteria)
3.Virulent enzymes: coagulase and
fibrinolysin
4.Plague toxins: “murine toxin” inhibits the
cell respiration (exotoxin); and endotoxin

33. Epidemiology of the plague
The plague is zoonotic
disease.
The source of infection is
wild or domestic
rodents.
Among the rodents the
plague is transmitted
by rat fleas (vectors).

34. Epidemiology

35. Epidemiology of the plague
The major route of transmission among
humans:
1.By biting of infected fleas
2.By skinning and handling of carcasses of
infected wild animals
3.By inhalation of the dried flea feces or
respiratory droplets from person with
pneumonic form
4.By drinking of polluted water

37. Pathogenesis of the plague
Incubation period is 2-5 days.
Clinical forms :
1. Bubonic form (hemorrhagic
inflammation of the lymph
nodes draining the site of
entry of the bacteria)
2. Pneumonic form
(hemorrhagic pneumonia)
3. Enteric form
4. Septicemia (primary and
secondary)

38. Septic form of “black death”

39. Laboratory diagnostics
Clinical samples: bubon extract, sputum,
section tissue of the spleen, liver;
carcasses of the wild rodents
Primary diagnosis:
1. Microscopy of the stained smears with
methylene blue
2. Immunofluorescence microscopy
3. Ring precipitation test for revealing of the
Ag in the putrefied carcasses

40. Immunofluorescence

41. Laboratory diagnostics
For final laboratory
diagnosis next methods
are used:
1.Culture method
(identification is based
onto the cultural and
biochemical properties,
and sensitivity to the
plague phage)
2.Biological method

42. Specific prevention:
Live attenuated or killed
vaccine prepared from
Girard`s EV strain is used at
foci of the plague
Post vaccination immunity
lasts about 6-12 months
Chemoprophylaxis is given for
all contact persons
(tetracycline is administered
orally for 5 days)

43. Bacillus anthracis
Family: Bacillaceae
Genus: Bacillus
Medical important species: B. anthracis (causative agent of
anthrax)

44. Morphology
It is large Gram (+) rods arranged in chains
It is non-motile
It forms capsule into the tissue
It is spore-forming (the spore is central and
formed into the soil)

45. B.anthracis

46. Cultivation
It is aerobe and facultative anaerobe and easily cultivated
onto the ordinary nutrient media
Nutrient media:
1. Nutrient agar (colony has “Medusa head” appearance)
2. Blood agar – non-hemolytic colonies
3. Gelatin stab culture (“inverted fir tree”)
4. Penicillin media (“string of pearls”)

47. Colonies of B.anthracis

48. Resistance
The vegetative form is killed at 600C in 30 min
The spore form is high resistant and killed by
autoclaving
Animal products are disinfected by
2% formaldehyde for 20 min at 30-400C;
for animal hair and bristles the 0.25% solution
is used at 600C for 6 hrs

49. Antigen structure and virulent factors
Antigens:
1. Capsular antigen (polypeptide)
2. Cell wall antigen (polysaccharide)
3. Somatic antigen (heat labile protein)
4. Exotoxin consists from edema factor (OF), protective
antigen (PA) and lethal factor (LF). All factors form
anthrax toxin factor and act toxically together only

50. Epidemiology
The source of infection is ill animal (cattle,
sheep, horses, and swine).
Human may be infected by:
1.Direct contact with
contaminated hair, bristles
and carcasses of animal
2. Inhalation of the dust or
wool from infected animal
3. Ingestion of contaminated cooked meat

51. Pathogenesis There are three form of anthrax in
humans that can lead to fatal
septicemia:
1. Cutaneous form (malignant
pustule is covered with black
scar)
2. Pulmonary anthrax (wool
sorter`s disease)- hemorrhagic
bronchopneumonia
3. Intestinal anthrax – violent
enteritis with bloody diarrhea
4. Septicemia

52. Pathogenesis of anthrax

54. Cutaneous form of anthrax

55. Laboratory diagnostics
Clinical samples: pustule discharge, sputum,
blood
1. Microscopy of the smear stained by Gram,
by Romanovsky-Giemsa;
Immunofluorescence
2. Culture method

56. Laboratory diagnostics 3. Experimental infection of
white mouse or guinea pig
4. Retrospective diagnose is
made by serology and
allergic skin test
5. To reveal contamination of
the raw animal products
with spores of B.anthracis
Ascoli`s thermoprecipitin
test is used

57. Prevention
Animals and human are protected by active
immunization
Immunization is possible with alive attenuated
Sterne vaccine (animals) or STI vaccine (human).
They include non-capsulated avirulent mutant strain
The post vaccinal immunity lasts for 6-12 months