This document discusses the laboratory diagnosis of infections caused by various Gram-positive bacilli, including Corynebacterium, Listeria, Erysipelothrix, and Bacillus. It provides details on specimen collection, microscopic examination, culture methods and media, and biochemical testing for identifying these bacteria, with a focus on Corynebacterium diphtheriae, Listeria monocytogenes, and Bacillus anthracis. Vaccination is emphasized as an important prevention method for diphtheria.

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