1. Enterobacteriacae identification
In the name of God
Department Of
Microbiology
Yasouj University of Medical Science
Enterobacteriacae identification
By: Dr. S. S. Khoramrooz, Ph.D.
Department of Microbiology, Faculty of Medicine,
Yasuj University of Medical Sciences, Yasuj, Iran

2. Characters of Enterobacteriaceae
All Enterobacteriaceae
Gram-negative rods
Ferment glucose with acid production
Reduce nitrates into nitrites
Oxidase negative
Facultative anaerobic
Motile except Shigella and Klebsiella
Non-capsulated except Klebsiella
Non-fastidious
Grow on bile containing media (MacConkey agar)

3. Classification of Enterobacteriaceae
Klebsiella, Enterobacter
E. coli, Citrobacter,
Lactose fermenters
Non-lactose fermenter
Salmonella, Shigella
Proteus, Yersinia
There are several selective and differential media used to
isolate distinguishes between LF & LNF
The most important media are:
MacConkey agar
Eosin Methylene Blue (EMB) agar
Salmonella Shigella (SS) agar
In addition to Kiligler Iron agar (KIA)

4. Tests To Know Case Study Tests Indole Methyl Red/Voges Proskauer
Citrate
H2S production in SIM
Urea hydrolysis
Motility
Lactose fermentation
Glucose fermentation & gas production
Decarboxylation of amino acis
Fermentation of sugars
Reaction on selective media

5. Growth of Enterobacteriaceae on MacConkey agar
Colorless colonies
Pink colonies
Lactose non feremters
Salmonella, Shigella,
Proteus
Lactose feremters
E. coli, Citrobacter
Klebsiella, Enterobacter
Uninoculated plate

6. Kligler Iron Agar
Lactose Fermentation Glucose fermentation Gas Production (H2 & CO2 ) H2S Production

7. Kligler Iron Agar (KIA)
Proteins
glucose
lactose
Ferrous sulfate
pH indicator: phenol red

9. Red/Red Alkaline /Alkaline K/K Lactose -/Glucose – Yellow/Yellow Acid/Acid A/A Lactose +/Glucose + Red/Yellow Alkaline/Acid K/A Lactose -/Glucose + Gas - H2S - Red/Yellow Alkaline/Acid K/A Lactose -/Glucose + Gas + H2S - Red/Yellow Alkaline/Acid K/A Lactose -/Glucose + Gas - H2S + Red/Black Alkaline/Acid K/A Lactose -/Glucose + Gas + H2S +

10. Result Example Result Reaction on KIA H2S Slant color Butt color
Non fermenter e.g. Pseudomonas
Alk/Alk/-
(No action on sugars)
Negative
Red
LNF
e.g. Shigella
A/Alk/-
(Glucose fermented without H2S)
Yellow
e.g. Salmonella & Proteus
A/Alk/+
(Glucose fermented with H2S)
Positive
black in butt LF
e.g. E. coli, Klebsiella, Enterobacter
A/A/-
(three sugars are fermented)

12. IMViC Test Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests:
The following four tests comprise a series of important determinations that are collectively called the IMViC series of reactions
The IMViC series of reactions allows for the differentiation of the various members of Enterobacteriaceae.

13. IMViC: Indole test Principle
Certain microorganisms can metabolize tryptophan by tryptophanase
The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia
The presence of indole is detected by addition of Kovac's reagent.
Tryptophanase
Tryptophane
amino acids
Indole + Pyurvic acid + NH3
Kovac’s Reagent
Red color in upper organic layer`

14. IMViC: Indole test Result:
A bright pink color in the top layer indicates the presence of indole
The absence of color means that indole was not produced i.e. indole is negative
Special Features:
Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-).
Negative test
e.g. Klebsiella
Positive test
e.g. E. coli

15. IMViC test Methyl Red-Voges Proskauer (MR-VP) Tests
Principle
Glucose
Acidic pathway Or
Neutral pathway
Acety methyl carbinol
(ACETOIN)
Mixed acids
 pH less than 4.4
solution A
solution B
Methyl Red
indicator
VP positive
Klebsiella
MR positive
E. coli
Red color
Pink color

16. Butylene Glycol Pathway of Glucose Fermentation
In the butylene glycol pathway
pyruvic acid to acetoin and butylene glycol.
Acetoin and butylene glycol are detected by oxidation to diacteyl at an alkaline pH.
and the addition of -naphthol which forms a red-colored complex with diacetyl.
Important biochemical property used for the identification of Klebsiella, Enterobacter, and Serratia.

17. Voges-Proskauer Reaction
Acetoin and butylene glycol are detected by oxidation to diacteyl at an alkaline pH, and the addition of -naphthol which forms a red-colored complex with diacetyl.
The production of acetoin and butylene glycol by glucose fermentation is an important biochemical property used for the identification of Klebsiella, Enterobacter, and Serratia.

18. IMViC test: MRVP test Method
Inoculate the tested organism into MRVP broth
Incubate the tubes at 37°C for 24 hours
For methyl red:  Add 6-8 drops of methyl red reagent.
For Voges-Proskauer:  Add 12 drops of solution A (-naphthol), mix, 4 drops of Solution B (40% KOH), mix

19. IMViC test: MR/VP test Results Voges-Proskauer test Methyl Red test
Pink: Positive VP (Klebsiella)
Red: Positive MR (E. coli)
No pink: Negative VP (E. coli)
Yellow or orange: Negative MR (Klebsiella)

20. Citrate Utilization Test
Principle:
Citrate
Na2CO3
Pyruvate
CO2 + Na + H2O
Alkaline,↑pH
Simmone’s Citrate media
Contains Citrate as a sole of C source
Bromothymol blue
Positive test
Blue colour
Positive test: Klebsiella, Enterobacter, Citrobacter
Negative test: E. coli

21. Citrate Utilization Test
Method
Streak a Simmon's Citrate agar slant with
the organism
Incubate at 37°C for 24 hours.

22. Citrate Utilization Test
Result
Examine for growth (+)
Growth on the medium is accompanied by a rise in pH to change the medium from its initial green color to deep blue
Positive
Klebsiella, Enterobacter
Negative
E. coli

23. Urease Test Principle Method NH4 OH Urea CO2 + NH3
Urea agar contains urea and phenol red
Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3
Ammonia combines with water to produce ammonium hydroxide, a strong base which ↑ pH of the medium.
↑ in the pH causes phenol red r to turn a deep pink. This is indicative of a positive reaction for urease
Urease
H2O
Urea
CO2 + NH3
NH4 OH
↑ in pH
Phenol Red
Method
Pink
Positive test
Streak a urea agar tube with the organism
incubate at 37°C for 24 h

24. Urease Test
Result
If color of medium turns from yellow to pink indicates positive test.
Proteus give positive reaction after 4 h while Kelebsiella and Enterobacter gave positive results after 24 h
Positive test
Negative test

25. Motility
From left to right:
– +

26. SIM Sulfide, Indole, Motility
4/16/2017
S. S. Khoramrooz

27. Proteus species A exhibits characteristic “swarming”
B shows urease positive on right
Proteus species

28. Phenylalanine Deaminase Reaction
Enterobacteriaceae utilize amino acids in a variety of ways including deamination.
Phenylalanine is an amino acid that forms the keto acid phenylpyruvic acid when deaminated.
Phenylpyruvic acid is detected by addition of ferric chloride that forms an intensely dark olive-green colored complex when binding to phenylpyruvic acid.
The deamination of phenylalanine is an important biochemical property of Proteus, Morganella, and Providencia.

29. Amino Acid Decarboxylation
Enterobacteriaceae contain decarboxylases with substrate specificity for amino acids, and are detected using Moeller decarboxylase broth overlayed with mineral oil for anaerobiosis.
Moeller broth contains glucose for fermentation, peptone and beef extract, an amino acid, pyridoxal, and the pH indicator bromcresol purple.

30. Amino Acid Decarboxylation
If an Enterobacteriaceae contains amino acid decarboxylase, amines produced by decarboxylase action cause an alkaline pH, and bromcresol purple turns purple.
Lysine, ornithine, and arginine are utilized.
A base broth without amino acid is included in which glucose fermentation acidifies the broth, turning the bromcresol purple yellow.

31. Amino Acid Decarboxylation1
Lysine → Cadaverine
Ornithine → Putrescine
Arginine → Citrulline → Ornithine → Putrescine
1Conversion of arginine to citrulline is a dihydrolase reaction

32. Amino Acid Decarboxylation
Decarboxylation patterns are essential for the genus identification of Klebsiella, Enterobacter, Escherichia, and Salmonella.
Decarboxylation patterns are also essential for the species identification of Enterobacter aerogenes, Enterobacter cloacae, Proteus mirabilis, and Shigella sonnei.

33. Amino Acid Decarboxylation
Lys Orn Arg
Klebsiella – –
Enterobacter /– /–
Escherichia /– –/+
Salmonella

34. Amino Acid Decarboxylation
Lys Orn Arg
E. aerogenes –
E. cloacae – + +
P. mirabilis – + –
P. vulgaris – – –
Shigella D – + –
Shigella A-C – – –

35. IPViC Reactions for Initial Grouping of the Enterobacteriaceae
Indole
Phenylalanine deaminase
Voges-Proskauer
Citrate

36. Initial Grouping of the Enterobacteriaceae (VP=Voges Proskauer, PDA=Phenylalanine Deaminase)

37. Initial Grouping of the Enterobacteriaceae

38. Initial Grouping of the Enterobacteriaceae

39. Initial Grouping of the Enterobacteriaceae1

40. Initial Grouping of the Enterobacteriaceae1

41. Key Characteristics of the Enterobacteriaceae

42. Key Characteristics of the Enterobacteriaceae

43. Key Characteristics of the Enterobacteriaceae

44. Biochemical Characteristics of Escherichia coli and Shiglla
E. coli E. coli O157:H Shigella
TSI A/Ag A/Ag Alk/A
Lactose –
ONPG –/+1
Sorbitol – /–
Indole /–
Methyl red
VP – – –
Citrate – – –
Lysine –
Motility –
1Shigella sonnei (group D) ONPG +

45. Biochemical Characteristics of Salmonella
Most Serotypes Typhi Paratyphi A
TSI Alk/A Alk/A Alk/A
H2S (TSI) (weak) –
Citrate – –
Lysine –
Ornithine –
Dulcitol –
Rhamnose –
Indole – – –
Methyl red
VP – – –

46. Xylose Lysine Deoxycholate (XLD) Agar: Composition
Lactose %
Sucrose %
Sodium chloride %
Yeast extract %
Sodium deoxycholate %
Sodium thiosulfate
Ferric ammonium citrate
Agar %
Phenol red
pH = 7.4

47. XLD Agar: Growth of Salmonella
Salmonella selective due to bile salt.
Xylose fermentation (except Salmonella serotype Paratyphi A) acidifies agar activating lysine decarboxylase.
With xylose depletion fermentation ceases, and colonies of Salmonella (except S. Paratyphi A) alkalinize the agar due to amines from lysine decarboxylation.
Xylose fermentation provides H+ for H2S production (except S. Paratyphi A).

48. XLD Agar: Appearance of Salmonella
Ferric ammonium citrate present in XLD agar reacts with H2S gas and forms black precipitates within colonies of Salmonella.
Agar becomes red-purple due to alkaline pH produced by amines.
Back colonies growing on red-purple agar-presumptive for Salmonella.

51. XLD Agar: Growth of Escherichia coli and Klebsiella pneumoniae
Escherichia coli and Klebsiella pneumoniae are
lysine-positive coliforms that are also lactose and sucrose fermenters.
The high lactose and sucrose concentrations result in strong acid production, which quenches amines roduced by lysine decarboxylation.
Colonies and agar appear bright yellow. Neither Escherichia coli nor Klebsiella pneumoniae produce H2S.

52. XLD Agar: Growth of Shigella and Proteus
Shigella species do not ferment xylose, lactose, and sucrose, do not decarboxylate lysine, and do not produce H2S. Colonies appear colorless.
Proteus mirabilis ferments xylose, and thereby provides H+ for H2S production.
Colonies appear black on an agar unchanged in color (Proteus deaminates rather than decarboxylates amino acids).
Proteus vulgaris ferments sucrose, and colonies appear black on a yellow agar.

55. Hektoen Enteric (HE) Agar: Composition
Peptone %
Yeast extract %
Bile salts %
Lactose %
Sucrose %
Salicin %
Sodium chloride %
Ferric ammonium citrate
Acid fuchsin
Thymol blue
Agar %
pH = 7.6

56. HE Agar: Growth of Enteric Pathogens and Commensals
High bile salt concentration inhibits growth of gram-positive and gram-negative intestinal commensals, and thereby selects for pathogenic Salmonella (bile-resistant growth) present in fecal specimens.
Salmonella species as non-lactose and non-sucrose fermenters that produce H2S form colorless colonies with black centers.
Shigella species (non-lactose and non-sucrose fermenters, no H2S production) form colorless colonies.
Lactose and sucrose fermenters (E. coli, K. pneumoniae) form orange to yellow colonies due to acid production.

59. Pseudomonas aeruginosa
Some strains appear mucoid
Particularly common in patients with cystic fibrosis
Some strains produce diffusible pigments
Pyocyanin [blue]
Fluorescein [yellow]
Pyorubin [redbrown]

60. + Laboratory Diagnosis Culture Identification
Grow easily on common isolation media such as blood agar and MacConkey
37-42C
Identification
The colonial morphology (e.g., colony size, hemolytic activity, pigmentation, odor) +
Biochemical tests (e.g., positive oxidase reaction)

63. The End