The Enterobacteriaceae

Biochemical Properties
Dr. John R. Warren
Department of Pathology
Northwestern University
Feinberg School of Medicine
June 2007

Major Biochemical Reactions

for Identification of the
Enterobacteriaceae

Voges-Proskauer fermentation reaction

Phenylalanine deaminase activity
Indole production from tryptophan
Utilization of citrate as a single carbon
source

Butylene Glycol Pathway of

Glucose Fermentation
Glucose fermentation requires the reoxidation
of NADH generated by fermentation back to
NAD. This is accomplished by the reduction of
pyruvic acid to a variety of metabolic
pathways.
In the butylene glycol pathway of glucose
fermentation this occurs by the reduction and
condensation of pyruvic acid to acetoin and
butylene glycol.

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 redcolored 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.

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.

Indole Reaction
Enterobacteriaceae that possess tryptophanase can
utilize tryptophan by deamination and hydrolytic
removal of the indole side chain.
Free indole is detected by p-dimethylaminobenzaldehyde, whose aldehyde group reacts with
indole forming a red-colored complex.
Production of indole from tryptophan is an
important biochemical property of Escherichia coli,
many strains of group A, B, and C Shigella,
Edwardsiella tarda, Klebsiella oxytoca, and Proteus
vulgaris.

Citrate Utilization
Citrate is utilized by several of the
Enterobacteriaceae as a single carbon
source. To test this ability bacteria are
incubated in medium that contains only
citrate as a source of carbon.
Ammonium phosphate is available as a
nitrogen source.

Citrate Utilization
Enterobacteriaceae that can utilize citrate will
extract nitrogen from ammonium phosphate
releasing ammonia. Ammonia produces an
alkaline pH shift, and the indicator
bromthymol blue turns blue from its green
color at neutral pH.
Citrate utilization is a key biochemical
property of Salmonella, Citrobacter, Klebsiella,
Enterobacter, and Serratia.

IMViC Reactions
I = indole production from tryptophan
M = methyl red test in which acidification of
glucose broth (pH<4.4) due to formation of
mixed carboxylic acids (lactic, acetic, formic)
from pyruvate results in pH indicator methyl red
turning red
Vi = positive Voges-Proskauer test due to
formation of acetoin from pyruvate in glucose
broth
C = ability to utilize citrate as single carbon
source

IMViC Reactions
I M
Vi
C
Escherichia coli
Edwardsiella tarda
Proteus vulgaris
Klebsiella pneumoniae
Klebsiella oxytoca
+
Enterobacter spp.

Serratia marcescens
Citrobacter freundii
Citrobacter koseri

+
+
+

+
+

+
+
+

+
+
+

+
+
+
+
+

IPViC Reactions
I = indole production from tryptophan
P = phenylpyruvic acid production from
phenylalanine
Vi = positive Voges-Proskauer test due to
formation of acetoin from pyruvate in
glucose broth
C = ability to utilize citrate as single
carbon source

IPViC Reactions
Eschericia
Shigella
Yersinia
Edwardsiella
Salmonella
Citrobacter
Klebsiella
Enterobacter
Serratia
Proteus
Morganella
Providencia

I
+
+/
+/
+

+/

+/
+

+
+
+

Vi

+
+
+
+/

+
+
+
+
+
+
+
+

Reactions for Identification of

1
Genera and Species

Decarboxylation of amino acids

Motility
Urease activity
Hydrogen sulfide (H2S) production

Voges-Proskauer, phenylalanine
deaminase, indole, and citrate reactions are
useful to both cluster Enterobacteriaceae
and identify to genus and species.
1

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.

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.

Amino Acid Decarboxylation1

Lysine Cadaverine
Ornithine Putrescine
Arginine Citrulline Ornithine
Putrescine
1Conversion of arginine to citrulline is a
dihydrolase reaction

Amino Acid Decarboxylation

Tube Amino Acid Color Interpretation
Base None
Yellow Broth acidified1
1
Lysine
Purple Positive
2
Ornithine Yellow Negative
3
Arginine
Yellow Negative
1Indicates organism is a viable glucose fermenter,
and pH of broth medium sufficiently acidified
to activate decarboxylase enzymes.

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.

Amino Acid Decarboxylation

Klebsiella
Enterobacter

Lys
+
+/

Orn

Arg

+/

Escherichia
Salmonella

+
+

+/
+

/+
+

Amino Acid Decarboxylation

Lys
Orn
E. aerogenes
E. cloacae
P. mirabilis
P. vulgaris
Shigella D
Shigella A-C

Arg
+
+

+
+
+

Bacterial Motility
Many but not all Enterobacteriaceae
demonstrate flagellar motility.
Motility can be measured by use of
<0.4% semisolid (soft) agar or
microscopic examination of drops of
broth containing bacteria and hanging
from cover slips.
Shigella and Klebsiella are non-motile,
and Yersinia is non-motile at 35oC but
motile at 22o-25oC.

Motility Agars
Sulfide-indole-motility (SIM) is a semisolid
motility agar that contains peptonized iron for
detection of H2S and tryptophan for indole
production.
Pure motility agar lacks an H2S indicator and
tryptophan for indole production, and contains
tetrazolium salts that are reduced to red
formazan complexes to enhance visual
assessment of motility.

Urease Reaction
Urease hydrolyzes urea releasing ammonia
which alkalinizes the medium by forming
ammonium carbonate, and the pH
indicator phenol red becomes red.
Proteus, Morganella, and Providencia are
strong urease producers, Klebsiella a weak
urease producer, and Yersinia enterocolitica
frequently a urease producer.

Urease-Producing
Enterobacteriaceae

Proteus
Morganella
Providencia rettgeri
Klebsiella pneumoniae
Klebsiella oxytoca
Enterobacter cloacae
Yersinia enterocolitica

Hydrogen Sulfide (H2S)

In presence of H+ and a sulfur source
(sodium thiosulfate, sulfur-containing amino
acids and proteins) many Enterobacteriaceae
produce the colorless gas H2S.
For detection of H2S a heavy-metal (iron or
lead) compound is present that reacts with
H2S to form black-colored ferrous sulfide.

Systems for H2S Detection1

Lead acetate paper
SIM tube (peptonized iron)
Hektoen and SS2 agar (ferric ammonium
citrate)
XLD3 agar (ferric ammonium citrate)
Triple-sugar-iron agar (ferrous sulfate)
1In order of decreasing sensitivity
2Salmonella-Shigella
3Xylose-lysine-deoxycholate

H2S-Producing
Enterobacteriaceae

Salmonella
Edwardsiella
Citrobacter
Proteus

IPViC Reactions for Initial

Grouping of the
Enterobacteriaceae

Indole
Phenylalanine deaminase
Voges-Proskauer
Citrate

Initial Grouping of the Enterobacteriaceae

(VP=Voges Proskauer, PDA=Phenylalanine
Deaminase)

Initial Grouping of the

Enterobacteriaceae
GENERA

VP

PDA

Proteus1

NEGATIVE

POSITIVE

Morganella

NEGATIVE

POSITIVE

Providencia

NEGATIVE

POSITIVE

1Proteus

mirabilis: 50% of strains VP positive

Initial Grouping of the

Enterobacteriaceae
GENERA
Escherichia
Shigella
Edwardsiella
Salmonella
Citrobacter
Yersinia

VP
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE

PDA
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE

Initial Grouping of the

1
Enterobacteriaceae

Initial Grouping of the

1
Enterobacteriaceae

Key Characteristics of the

Enterobacteriaceae

Key Characteristics of the

Enterobacteriaceae

Key Characteristics of the

Enterobacteriaceae

Biochemical Characteristics of
Escherichia coli and Shiglla
TSI
Lactose
ONPG
Sorbitol
Indole
Methyl red
VP
Citrate
Lysine
Motility
1

E. coli
A/Ag
+
+
+
+
+

+
+

E. coli O157:H7
A/Ag
+
+

+
+

+
+

Shigella sonnei (group D) ONPG +

Shigella
Alk/A

/+1
+/
+/
+

Biochemical Characteristics of
Salmonella
TSI
H2S (TSI)
Citrate
Lysine
Ornithine
Dulcitol
Rhamnose
Indole
Methyl red
VP

Most Serotypes
Alk/A
+
+
+
+
+
+

Typhi
Alk/A
+ (weak)

Paratyphi A
Alk/A

+
+
+

Additional Biochemical Reactions

for the Enterobacteriaceae1
Fermentation of mannitol, dulcitol, salicin, adonitol,
inositol, sorbitol, arabinose, raffinose, rhamnose,
maltose, xylose, trehalose, cellobiose, alpha-methyl Dglucoside, erythritol, melibiose, arabitol, glycerol,
mucate, and mannose
Utilization of malonate, acetate, and tartrate
Gelatin hydrolysis, esculin hydrolysis, lipase, and
DNase
Growth in KCN
Yellow pigment
1JJ Farmer, Enterobacteriaceae: Introduction and
Identification, ASM Manual, 8th Edition (2003).

Methodology of Microbial
Identification
Manual (broth and agar reaction tubes)
Packaged (strips or panels of minaturized
reaction cupules or wells containing
colorimetric or fluorometric substrates) (API
20E-bioMerieux; MicroScan-Dade Behring;
Sensitire-TREK)
Automated (panels or cards with minaturized
wells or chambers with colorimetric or
fluorometric reactions instrument-recorded
automatically) (VITEK 2-bioMerieux;
MicroScan Walkaway; Sensititre Automated)

Recommended Reading
Winn, W., Jr., Allen, S., Janda, W.,
Koneman, E., Procop, G., Schreckenberger,
P., Woods, G.
Konemans Color Atlas and Textbook of
Diagnostic Microbiology, Sixth Edition,
Lippincott Williams & Wilkins, 2006:
Chapter 6. The Enterobacteriaceae.

Recommended Reading
Murray, P., Baron, E., Jorgensen, J., Landry,
M., Pfaller, M.
Manual of Clinical Microbiology, 9th
Edition, ASM Press, 2007:
Nataro, J.P., Bopp, C.A., Fields, P.I., Kaper, J.B., and
Strockbine, N.A. Chapter 43. Escherichia, Shigella, and
Salmonella.
Wanger, A. Chapter 44. Yersinia.
Abbott, S.L. Chapter 45. Klebsiella, Enterobacter,
Citrobacter, Serratia, Plesiomonas, and other
Enterobacteriaceae.