support for microbiology laboratories for 2nd year medical students

1. Laboratory diagnosis in infections
produced by the genera:
Vibrio, Campylobacter, Helicobacter,
Pseudomonas
http://www.slideshare.net/DanaSinzianaBreharCi/vibrio-cam

2. Genus Vibrio
• Family Vibrionaceae:
– Genera:
• Vibrio – species clinically significant for human pathology:
– Vibrio cholerae
– Vibrio parahaemolyticus
– Vibrio vulnificus
• Aeromonas
• Plesiomonas

3. Vibrio cholerae
Classification (~structure of ”O”
antigen):
2 serogroups:
• O1 (epidemic cholera):
– 3 subtypes:
• Ogawa
• Inaba
• Hikoshima
– 2 biotypes:
• Classic
• El Tor
• Non-O1
(non-epidemic cholera-like illness)
Clinical forms:
- Diarrhoeic disease (favourable
evolution)
- Severe forms with acute
dehydration (choleric
enterotoxin)

4. Vibrio cholerae
- Collection and transport of specimens -
• Faeces – as early as possible (within the first 24 hours
after onset of symptoms)
• Vibrios - High sensitivity to dryness – inoculation onto
transportation media (Cary Blair and alkaline peptone
water – pH 8.6)

5. Vibrio cholerae
- Direct examination -
• Macroscopic exam of stool:
watery (~60/day), (”rice water
diarrhoea”)
• Microscopy: wet mount under
dark fileld microscopy: motile
comma-shaped vibrios actively
moving on the microscopic
field

6. Vibrio cholerae
- Gram stained smear -
• Gram negative, comma-
shaped bacilli

7. Vibrio cholerae
- Cultivation and Isolation -
• Enrichment liquid medium: alkaline peptone water –
incubation at 37°C, 6 hours
• Reinoculation on solid selective media:
– TCBS agar (Thiosulphate Citrate Bile salts Sucrose agar):
sucrose fermentation (yellow colonies)
– Blood agar: complete hemolysis
– MacConkey: lactose-negative colonies

8. Vibrio cholerae
• TCBS agar (thiosulphate
citrate bile salts sucrose agar):
sucrose fermentation (large,
yellow colonies)

9. Vibrio cholerae on blood agar:
medium size colonies (2-3 mm) with complete
hemolysis

10. Vibrio cholerae on MacConkey agar
• moderate size (1-3
mm), lactose-
negative / slightly
pink colonies
(resembling
”late”/”slow” lactose
fermentation)

11. Vibrio cholerae:
biochemical tests identification

12. Vibrio cholerae
Antigenic structure based identification
• Serogroups
– (O1/nonO1)
• Serotypes
– (Ogawa/Inaba)
• Biotypes
– (Classic/El Tor)

13. Vibrio cholerae
- Antibiotic sensitivity -
• High sensitivity to tetracyclines, nalidixic acid, norfloxacin
• Antimicrobial sensitivity testing – required to monitor
acquired resistance of certain strains

14. Vibrio parahemolyticus
• Clinical significance:
– acute gastroenteritis after
consumption of contaminated
seafood
– skin infections, conjunctivitis
(occupational related –
workers manipulating
contaminated seafood)

15. Vibrio parahemolyticus
Cultivation and Isolation
TCBS agar:
sucrose is NOT
fermented
(green colonies)

16. Vibrio vulnificus
• Clinical significance:
– primary sepsis following
severe, purulent infection of
skin lesions (occupationally
related: workers manipulating
contaminated seafood, fish)
• Bacteriological diagnosis:
– similar steps as above
– Important differential
character: lactose
fermentation! (”the lactose-
positive vibrio”): RED
colonies on MacConkey

17. Genus Campylobacter
• Clinical significance: acute diarrhoeal disease; risk
factors: long term hospitalization, long term antibiotic
treatments (disbalance of intestinal microbiota)
• 11 species:
– C.jejuni, C.coli – involved in human infections
• Microscopy:
– Gram negative bacilli, encurved/S-shaped, motile (single polar
flagellus)
• Cultivation:
– special media + microaerophilia (anaerobic jar/candle
jar/anaerobic kits)

18. Campylobacter – Gram staining
• Gram negative bacilli,
spiralated/S-
shaped/encurved/”seagull
wings”

19. Campy selective medium:
chocolate agar + antibiotics
• normal fecal flora inhibited
(trimethoprim, vancomycin,
and polymyxin B)
• prepared, stored, dispensed
and packaged under oxygen-
free conditions
• Small (1 mm), non-hemolytic
colonies, white/grey, Oxydase
positive
• Gram stained smears from
colonies: Gram negative,
spiralated/S-shaped bacilli
• Antimicrobial sensitivity must
be tested

20. Genus Helicobacter
• Clinical significance:
– gastro-duodenal ulcer, gastritis (Helicobacter pylori)
• Collection of specimens:
– gastric mucosa fragments (endoscopy, gastric lavage)
– transport media (e.g. ”Portagerm pylori”, BioMerieux), at +4°C
• Microscopy:
– Gram stained smear: Gram negative bacilli, encurved/spiraled,
4-6 polar flagella
• Cultivation:
– selective, blood containing media + antibiotics e.g. Helicobacter
pylori agar; at least 3 days incubation!!
– Microaerophilic incubation* (see next slide)

21. *Definition of terms:
bacterial growth in relation with respiratory
processes (use of O2, CO2)
• Obligate aerobes need oxygen because they cannot ferment or
respire anaerobically (e.g. Mycobacterium tuberculosis)
• Obligate anaerobes are poisoned by oxygen (e.g. Clostridium tetani,
Clostridium difficile)
• Facultative anaerobes can grow with or without oxygen (e.g.
Staphylococcus, Streptococcus, E.coli)
• Microaerophiles need some amount of oxygen but are poisoned by
high concentrations of oxygen (e.g. Campylobacter, Helicobacter,
Neisseria gonorrhoeae)

22. Helicobacter pylori – Gram stained smears

23. Helicobacter pylori on blood agar
• non-hemolytic, small (1-2 mm)
colonies after 3 days of
microaerophilic incubation
• Oxydase-positive

24. Genus Pseudomonas
• Gram negative bacilli, obligate aerobic, motile,
nonsporulating, do not ferment glucose, oxydase positive
• Pseudomonas aeruginosa a.k.a. pyocyanic bacillus
• Habitat:
– water, soil, air, human skin;
– humid conditions - biofilm e.g. in hospitals (toilets, humidifiers,
respirators, plants, etc); human carriers

25. Pseudomonas aeruginosa
• Clinical significance:
– Comensal, facultatively pathogen; colonizes mucous
membranes e.g. mere isolation in throat swab in the absence of
relevant clinical context does not mean etiologic diagnosis but
rather positive selection after antibiotic treatment
– opportunistic pathogen = infects impaired tissues & organs
(tissues with lesions, patients with decreased immunity, etc);
involved in hospital acquired infections e.g. eye infections,
infection of burns/surgical wounds, UTI, lung infections,
meningitis (by iatrogenic inoculation), sepsis

26. Pseudomonas aeruginosa infections:
surgical wound, eye, burns

27. Pseudomonas aeruginosa
- Bacteriological diagnosis -
• Collection of specimens:
– depending on site of infection (pus, wound secretion, CSF, etc)
• Microscopy:
– Gram stained smear: only relevant for naturally sterile
specimens (e.g. CSF): high number of PMNs + Gram negative
bacilli
• Cultivation:
– nonfastidious germ; grows on any medium
– Naturally sterile specimens (CSF): blood agar
– Faeces: selective media for enterobacteria

28. Pseudomonas aeruginosa
• Green colonies on agar
(secretion of pigments:
e.g. pyocyanin)
• There are also
achromogenic strains!

29. Pseudomonas aeruginosa on blood agar
• metallic sheen

30. Pseudomonas aeruginosa
- Biochemical tests -
• API 20NE – identification system for non-fastidious, non-enteric,
Gram negative bacilli
OR
• API 20E

31. Pseudomonas aeruginosa
- Antimicrobial susceptibility testing -
• Required due to rapid acquisition of antibiotic resistance
(especially in case of ”hospital strains” – sometimes
highly resistant)