Laboratory diagnosis gram positive and gram negative bacilli

presentation of Gram positive and negative rods (Enterobacteria) - to be used by dental medicine students as a support for microbiology laboratories

1. Laboratory diagnosis of
Gram positive and Gram negative bacilli

2. Gram positive and Gram negative bacilli
Genera:
•Corynebacterium
•Bacillus
•Listeria
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. 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)
•Cultivation:
–Blood agar
–Selective media with tellurite (Tinsdale, Gundel-Tietz)
–Highly selective Loffler medium

6. Corynebacterium diphtheriae: encurved
rods, ”swolen” ends

7. Corynebacterium diphtheriae – Gram stain

8. ”Daisy flower” – Corynebacterium

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

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

11. Survival outside host
(continued)
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).

12. 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

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

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

15. Family Enterobacteriaceae
•Gram negative rods, non-spore forming, non-fastidious
•Wide spread in the environment (plants, soil, water,
human and animal intestines, on mucous membranes,
etc)
•Clinical significance:
–intestinal and extraintestinal infections
•Collection of specimens in intestinal infections:
–stool (faeces)
•Collection of specimens in extraintestinal infections:
–Urine, ear secretion, wound exudate, CSF, etc

16. Collection of stool (faeces)
•Disposable stool collection containers (simple / with
transportation medium Carry Blair: non-nutritive medium
which prevents overgrowth of Enterobacteriaceae but
preserves viable enteric pathogens (Salmonella,
Shigella, Vibrio, etc)

17. Collection of urine
When?:
- in the morning (first miction)
How?:
-clean uro-genital area
-eliminate first flow
-collect middle flow in
sterile container
Send to lab immediately or store
at 2-8°C

18. Collection of cerebrospinal fluid (CSF)
Lumbar punction (spinal tap)
•patient lies on the side, knees pulled up toward
chest, chin tucked downward
•back cleaned and disinfected (iodine) + health
care provider injects local anesthetic into lower
spine
•spinal needle inserted into lower back area
•needle properly positioned, CSF pressure
measured and sample collected in sterile tube
•needle removed, area cleaned, bandage placed
over puncture site

19. Genus Yersinia
Species:
•Yersinia enterocolitica:
–colonizes the intestines; only some strains are pathogenic – may
cause diarrhoea + apendicitis-like symptoms
•Yersinia pestis (causative agent of plague)
–reservoir of germs: rodents (rats)
–interpersonal transmission (human to human)
–Routes of infection:
•Vectors: Flea bites →skin lesions (inflammation, necrosis, purulent
secretion) + swollen lymph nodes (buboes) = bubonic plague
→sepsis
•Airborne: Inhalation →pneumonia = pulmonary plague

20. Left: Oriental rat flea (vector of Y.pestis)
Right, upper image: Y.pestis infected flea bite
Right, lower image: swollen lymph nodes (buboes)

21. Genus Salmonella
Clinical significance:
•1. Food poisoning - infection limited to the intestine
without going through the intestinal barrier
•2. Enteric fevers – systemic infections (typhoid fever,
paratyphoid fevers)

22. Genus Salmonella (continued)
Biological specimens:
-Blood for hemoculture (enteric fevers)
-Stool for coproculture (enteric fevers, food poisoning,
asymptomatic carriers)
-Urine for uroculture (enteric fevers)
Culture media:
-MacConkey: semitransparent colonies, colourless,
lactose-negative
-Istrati-Meitert/ADCL: transparent, colonies with black
center = production of H2S

23. Genus Salmonella
Salmonella: transparent colonies with black centre
Klebsiella: pink, mucoid colonies

24. H2S production on Hektoen agar
(Salmonella colonies)

25. Genus Shigella
•Clinical significance:
–dysentery = multiple diarrhoeic, bloody / purulent stools, fever,
dehydration
•Culture media:
–MacConkey – white/colourless colonies (Lactose-negative)
–Istrati-Meitert/ADCL – no H2S

26. Shigella on Endo agar – no H2S production

27. Genus Escherichia; Species: E.coli
-Facultatively pathogenic
-Enteric infections produced by 5 E.coli groups:
-EPEC = Entero-Pathogenic E.coli
-ETEC = Entero-Toxigenic E.coli
-EIEC = Entero-Invasive E.coli
-EHEC = Entero-Hemorrhagic E.coli
-EAEC = Entero-Adherent E.coli

28. MacConkey agar
Left: not inoculated; Right: E.coli (Lactose positive,
red colonies)

29. MacConkey agar:
Lactose positive colonies
E.coli (red); Klebsiella (pink)

30. Genus Klebsiella
•Facultatively pathogenic: Colonizes the respiratory
mucosa and the intestine
•In immunosuppresed patients (premature infants, elderly
people) – potential for severe infections (pneumonia,
sepsis, meningitis)
•Hospital acquired infections: surgical wound infections,
urinary infections, sepsis
•Involvement in diarrhoeic diseasae - debated

31. Klebsiella – blood agar (non hemolytic
mucoid colonies)

32. Klebsiella: Mucous colonies

33. Klebsiella – pink colonies (Lactose positive)
on MacConkey agar

34. Genus Proteus
•Facultatively pathogenic
•Colonizes the intestines
•May cause infections in case of immunesuppression e.g.
urinary infections, synusitis, otitis, meningitis + hospital
acquired infections
•Cultivation: characteristic ”invasion” of the culture
medium – the swarming phenomenon – favours biofilm
formation → catheter infections

35. Swarming of Proteus on culture plates
(concentric growth rings)