support for 2nd year medical students

1. Laboratory diagnosis of infections caused by
Gram positive bacilli
Corynebacterium, Listeria, Erysipelothrix,
Bacillus

2. Gram positive and Gram negative bacilli
Genera:
• Corynebacterium
• Listeria
• Erysipelothrix
• Bacillus
Family Enterobacteriaceae
Genera:
A. Highly pathogenic:
• Yersinia
• Salmonella
• Shigella
A. Facultatively pathogenic:
• E.coli
• Klebsiella
• Proteus
• Enterobacter
• Serratia
• Citrobacter

3. Genus Corynebacterium
Species Corynebacterium diphtheriae
- high pathogenicity
- Clinical significance: diphtheria = disease produced by
the diphtheric toxine
- → symptoms at the entry gate: sore throat, adherent
membrane (pseudomembrane) on tonsils, pharynx,
nasal cavity
- → general toxic symptoms: fever (hematogenic difusion
of diphtheric toxin)

4. Left: diphtheric pseudomembrane on tonsils
Right: diphtheric skin lesion

5. Genus Corynebacterium
Other pathogenic species (zoonosis)
Producers of diphteric toxin:
• Corynebacterium ulcerans – infection of cattle – mastitis;
infection transmissible to humans via infected milk
• Corynebacterium pseudotuberculosis – comensal of
animals (horses, sheep) – transmissible to humans via
direct contact or contaminated milk

6. Species Corynebacterium diphtheriae
Laboratory diagnosis
• Colection of specimens: throat / nasal / wound swab
• Microscopy:
– Gram positive bacilli, aspect of ”Chinese letters” / capital letters
– low value (C.diphtheriae – similar to other comensal
corynebacteria in the throat – “diphteroid bacilli”)
• Cultivation:
– Blood agar
– Selective media with tellurite (Tinsdale, Gundel-Tietz)
– Highly selective Loffler medium

7. Corynebacterium diphtheriae: Gram+,
encurved rods, ”swolen” ends

8. Corynebacterium diphtheriae – Gram+,
encurved rods, “swollen” ends

9. Corynebacterium diphtheriae
- Cultivation -
• Blood agar:
nonhemolytic, white-grey
colonies, striated margins
(”daisy flower”)

10. Corynebacterium diphtheriae
- Cultivation - continued
• Tinsdale medium (tellurite):
small, black colonies + brown
surrounding area (reaction
between H2S produced by
bacteria and potasium tellurite
in medium)

12. Corynebacterium diphteriae on Tinsdale
agar: black colonies with brown halo

13. Left: Corynebacterium diphtheriae on blood agar
Right: Corynebacterium diphtheriae on tellurite

14. Corynebacterium diphteriae
- Cultivation - continued
• Loffler medium – high
selectivity: white, creamy
colonies on slant

15. Biochemical tests: Urease test
(medium: urea + phenol red indicator)
• Principle: urease
producing germs
decompose urea in the
culture medium: PINK
• Corynebacterium
diphteriae – NEGATIVE
TEST – tube on the right
• diphteroid bacilli (normal
flora) – POSITIVE TEST
– tube in the middle

16. Toxigenic vs Nontoxigenic strains
- Toxigenesis tests -
A. In vivo: experimental disease
in Guinnea pigs
B. In vitro: Elek´s test:
• Elek agar inoculated with
streaks of bacterial culture;
• strip of sterile filter paper
impregnated with antidiphteric
antitoxin placed perpendicular
to bacterial streaks;
• incubate 24 hours;
• POSITIVE test = lines of Ag-
Ab precipitate = strain is
toxigenic (important
differentiation from diphteroids
– normal flora of the pharynx)

17. Elek test - continued
Elek agar: agar + bovine serum
1 and 4 – Elek (+) = double radial
immunodiffusion – precipitation
lines in each angle formed by
bacterial streak and strip
impregnated with diphteric
antitoxin = toxin producing
strains
2 and 3 = Elek (-) – nontoxigenic
strains

18. Corynebacterium diphteriae
Antimicrobial sensitivity
• Sensitive to penicillin, vancomycin, erythromycin – given asap,
based on clinical suspicion – before lab confirmation!
• + treatment must include Diphteria ANTITOXIN !!
• Lack of/ delayed treatment may cause death (effects of toxin –
inhibition of protein synthesis; organ necrosis – e.g. heart, liver,
kidneys; neurologic lesions)
IMPORTANT: Diphteria – vaccine-preventable disease!!
• Vaccination protocols e.g.
– trivalent vaccines: (Diphteria, Tetanus, Pertusis);
– pentavalent vaccines (Hemophilus influenzae type B, Pertusis, Tetanus,
Diphteria, Hepatitis B)

19. Gram positive and Gram negative bacilli
Genera:
• Corynebacterium
• Listeria
• Erysipelothrix
• Bacillus
Family Enterobacteriaceae
Genera:
A. Highly pathogenic:
• Yersinia
• Salmonella
• Shigella
A. Facultatively pathogenic:
• E.coli
• Klebsiella
• Proteus
• Enterobacter
• Serratia
• Citrobacter

20. Genus Listeria
Common characters:
• Gram positive, short bacilli
(rods) /cocobacilli, aerobic,
non-spore forming, length: 0.5-
1 µm/diameter: 0.5 µm,
rounded ends
• Arrangement in palisades / V-
shape / chains
• Mobility due to peritrichous
flagella (flagella around the
rod) – up to 5

21. Genus Listeria
- Clinical significance -
• Natural habitat: soil, water, sewage, plants, foods (raw,
undercooked)
• Species involved in human pathology: Listeria monocytogenes,
Listeria seeligeri, Listeria ivanovii
• Transmission via contaminated food (milk, diary products, poultry
meat) – food poisoning
• Transplacentar transmission → meningitis, sepsis in newborns
• Immunosuppressed patients (HIV, blood malignancies): meningitis,
encephalitis, sepsis
• Occupational infection in veterinarians, slaughterhouse workers
(from infected animals/animal tissues): pulmonar infections

22. Genus Listeria
- Laboratory diagnosis -
• Collection of specimens: CSF, blood, amniotic fluid,
respiratory secretions, food samples, etc.
• Microscopic examination: difficult to differentiate from:
– corynebacteria,
– Hemophilus influenzae (excessive decoloration of smear),
– streptococci (short rods/coccobacilli with rounded ends
sometimes resemble ovoid cocci)

23. Listeria – Gram stained smear

24. Genus Listeria
- Laboratory diagnosis – continued
Cultivation:
• Normally sterile collection sites (CSF, blood):
– Inoculation in liquid media (enriched nutrient broth), 35°C, 5-7
days (daily examination for bacterial growth)
– Reinoculation from turbid broth tubes on blood agar, incubation
for 2 more days
• Nonsterile collection sites (faeces, respiratory secretions,
foods):
– Initial inoculation on enrichment media
– Reinoculation on selective media (e.g. with antibiotic content)

25. Genus Listeria
- Laboratory diagnosis – continued
Colonial characters:
• Blood agar: small (up to 1 mm), round, smooth,
translucent colonies, discrete hemolysis (sometimes
under the colony, visible only after picking up the colony
with loop)
• Selective media (e.g. Oxford agar): black colonies, black
halo (formation of iron compounds)

26. Listeria colonies on blood agar

27. Listeria monocytogenes on Oxford agar
• Black colonies, black
halo

28. Listeria:
main bacteriological diagnosis elements

29. Listeria
- Biochemical tests -
• CAMP test – POSITIVE for
L.monocytogenes
• (enhanced hemolysis in the
area where Listeria streak
meets S.aureus streak)
• CAMP test is negative for
nonpathogenic Listeria strains

30. Gram positive and Gram negative bacilli
Genera:
• Corynebacterium
• Listeria
• Erysipelothrix
• Bacillus
Family Enterobacteriaceae
Genera:
A. Highly pathogenic:
• Yersinia
• Salmonella
• Shigella
A. Facultatively pathogenic:
• E.coli
• Klebsiella
• Proteus
• Enterobacter
• Serratia
• Citrobacter

31. Genus Erysipelothrix
• Common characters: Gram
positive bacilli, non spore
forming, tendency to form long
filaments
• Clinical significance: zoonosis;
human diseases occur as
work-related infections
(veterinarians, butchers,
fishermen): inflammatory skin
lesions (erysipeloid)

32. Gram positive and Gram negative bacilli
Genera:
• Corynebacterium
• Listeria
• Erysipelothrix
• Bacillus
Family Enterobacteriaceae
Genera:
A. Highly pathogenic:
• Yersinia
• Salmonella
• Shigella
A. Facultatively pathogenic:
• E.coli
• Klebsiella
• Proteus
• Enterobacter
• Serratia
• Citrobacter

33. Genus Bacillus
Species Bacillus anthracis
• Large, Gram positive rods (10
µM),
• straight cut ends,
• aerobic,
• disposed in chains
• Spore forming

34. Bacterial survival outside
host
Spores: reproductive structures adapted
for longtime survival in unfavourable
conditions
(etymology: ancient Greek spora = seed)
Bacterial spores - outer layer of keratin resistant to chemicals, staining and
heat → bacterium able to stay dormant for years, protected from
temperature differences, absence of air, water and nutrients
Spore forming bacteria:
• Clostridium spp (e.g. Clostridium difficile, Clostridium tetani);
• Bacillus spp (B. anthracis).

35. Bacillus anthracis – Gram staining
• Gram positive, long
bacilli, squared ends,
endo-spores visible in the
middle of bacterial cells –
spores are resistant to
staining
(magnification 1500X)

36. Bacillus anthracis (continued)
• High pathogenicity
• Disease = zoonosis (infection of animals AND humans)
• Clinical forms:
– Cutaneous anthrax – spores enter the body via skin lesions
– Pulmonary anthrax – inhalatory infection
– Digestive anthrax – ingestion of infected undercooked meat
– + biological weapon (inhalatory infection) – agent of bioterrorism

37. Left: anthrax skin lesion
Right: ”anthrax attack” letter (1 week after 9/11)

38. Bacillus anthracis: cultivation
• Nonfastidious germ; grows
well on blood agar
• Colonial characters: large (2-5
mm) white colonies, non-
hemolytic, irregular margins,
sometimes comma shaped
(lower image), ground-glass
aspect
(above details visible under
magnifying glass)

39. Bacillus anthracis on blood agar