support for medical students, 2nd year of study, microbiology laboratories

1. Laboratory diagnosis in infections produced by
Legionella, Haemophilus, Bordetella

2. Genus Legionella
• Gram negative bacilli, aerobic, filamentous, long, motile,
variable no of flagella, nonsporulating
• Habitat: water i.e.
– natural aquatic/humid environment (thermal waters, sewage
waters, ground water (low concentration) + potting mixes;
– artificial systems - biofilm on water distribution systems (tap &
shower water, toilet flush), air conditioning systems (cooling
towers), whirlpools
• Bacterial growth and multiplication favoured by:
– Temperature: 32-42°C
– Association with other aquatic species (Amoebae, Algae, other
bacteria = biofilm)

3. Genus Legionella
- Clinical significance -
• Transmission: inhalation of contaminated water
droplets (AEROSOLS):
– Cooling towers, hot and cold water systems, spa
baths, clinical humidifiers, natural warm spas/hot
springs, sprays in display cabinets (fruits, vegetables,
flowers, etc), indoor fountains, etc
• Human pathology: over 20 species - most
frequent:
– L.pneumophila serogroup 1-6 (~90%) + L.micdadei,
L.longbeachae, etc

4. Legionelosis / Legionaires‘ disease
• 1976: epidemic among participants to the annual
meeting of the American Legion held in Belevue-
Stratford Hotel in Philadeplhia
• 221 cases of pneumonia (34 deaths)
Main clinical forms:
- Pneumonia – possible severe evolution in elderly or
other immunocompromised hosts
- Pontiac fever – flu-like symptoms, less severe

5. Legionelosis
- Laboratory diagnosis -
Specimen Test
Sputum,
broncho-
alveolar fluid,
pleural fluid,
lung biopsy
- Microscopy: only relevant in normally sterile
specimens (e.g. pleural fluid)
-Culture:
- BCYE (buffered charcoal yeast extract) agar, CO2
atmosphere, slow growth (3-10 days)
Urine ELISA; immunochromatography: L. pneumophila
antigen
Blood (serum) ELISA: IgM and IgG antibodies
WATER Culture – BCYE + supplements

6. Identification of infection source
• Ideally, clinical cases should be linked to the source of
infection i.e. contaminated water
• NO interpersonal transmission!
• The only transmission route: inhalation of contaminated
aerosols from natural or artificial water sources
• Identification of contaminated waters should be followed
by decontamination of water source:
– Hiperchlorination
– Water temperature outside interval which favours growth of
Legionella spp: cold water under 20°C and hot water over 50°C
– Avoid stagnant water (”dead legs”, lateral pipes, storage tanks
with ”dead zones”)

7. Detection and enumeration of Legionella in
water samples
• Water samples – filtered through membrane (0.2 µm
pore size) – filtrate eluted in sterile distiled water
• Inoculation on buffered charcoal yeast extract (BCYE)
agar containing L-cysteine and a source of iron
• Colonial characters: white, purple to blue, or lime green;
may fluoresce under ultraviolet light; ground glass or
opalescent under microscope
• No growth in the absence of cysteine

8. Legionella colonies on BCYE agar
• Small, white, colonies
(become grey in a few
days)
• Fluorescent under UV
light
• Identification by latex
agglutination from
colonies

9. Genus Haemophilus
• Gram negative cocco-bacilli (may also appear as
filamentous bacilli), imobile, nonsporulating,
encapsulated (invasive infections)
• Clinical significance:
– comensals of the respiratory tract
– infections:
• meningitis (Haemophilus influenzae) – VACCINE preventable
• STI: soft chancre (Haemophilus ducreyi)

10. Genus Haemophilus
- Bacteriological diagnosis -
• Collection of specimens: CSF, nasopharyngeal exudate,
chancre secretion
• Microscopy: Gram stained smears – Gram negative
cocco-bacilli / filamentous / long / short bacilli (high
polymorphism); encapsulated (pathogenic strains)
• Culture:
– chocolate agar
– blood agar (further inoculated with Staphylococcus strain –
”satellitism phenomenon”: Haemophilus colonies will grow
around Staphylococcus colonies)

11. Satellitism of Haemophilus spp on
Staphylococcus spp
• Staphylococcus
aureus produce NAD
(nicotinamide adenine
dinucleotide) as
metabolic by-product
• Haemophilus spp may
grow on sheep blood
agar very close to the
colonies of
Staphylococcus
aureus

12. Genus Haemophilus
- Bacteriological diagnosis - continued
Identification:
• Satelliting of Staphylococcus (see above)
• Biochemical tests (API NH)
• Antigenic structure based identification (agglutination
tests): for encapsulated strains – polysacharide capsular
Ag → 6 serotypes of Haemophilus influenzae (a, b, c, d,
e ,f) – the most frequent: type b (Hib)
Antimicrobial susceptibility testing – mandatory due to
emergence of resistant strains (penicillins, tetracyclines,
cephalosporins)

13. Genus Bordetella
• Gram negative cocco-bacilli
• Clinical significance: Bordetella pertusis (whooping
cough)
• Collection of specimens:
– Expectoration onto culture plate (Bordet-Gengou medium)
– Nasal exudate collected with dacron swab (cotton is toxic for
Bordetella)
– Specimens transported asap to lab

14. Genus Bordetella
- Bacteriological diagnosis -
• Microscopy: Gram negative coccobacilli; +
immunofluorescence
• Culture: Bordet-Gengou medium (charcoal, blood,
amidon, albumin), incubation in humid atmosphere,
35°C, at least 7 days – colonies like ”mercury drops”
• Biochemical characters: API 20E + additional tests
• Antimicrobial sensitivity: Erythromycin (no routine testing
required)
• VACCINE PREVENTABLE
– In Romania: Diphteria-Tetanus-Pertusis vaccines given at the
ages of 2, 4, 6, 13 months, 4 years (DTP)