In the name of In the name of GodGod

ByBy: Dr. : Dr. S. S. Khoramrooz, Ph.D.S. S. Khoramrooz, Ph.D.

Department of Microbiology, Faculty of Medicine, Department of Microbiology, Faculty of Medicine,

Yasuj University of Medical Sciences, Yasuj, IranYasuj University of Medical Sciences, Yasuj, Iran

Enterobacteriacae identification

Yasouj University of Medical ScienceYasouj University of Medical Science

Department Department OfOf

MicrobiologyMicrobiology

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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)

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Classification of EnterobacteriaceaeClassification of Enterobacteriaceae

There are several selective and differential media used to isolate distinguishes between LF & LNFThe most important media are:

MacConkey agarEosin Methylene Blue (EMB) agarSalmonella Shigella (SS) agarIn addition to Kiligler Iron agar (KIA)

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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

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Growth of Enterobacteriaceae on MacConkey agar

Uninoculated plateLactose non feremtersSalmonella, Shigella,

Proteus

Lactose feremtersE. coli, Citrobacter

Klebsiella, Enterobacter

Colorless colonies Pink colonies

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Kligler Iron AgarLactose Fermentation

Glucose fermentation

Gas Production (H2 & CO2 )

H2S Production

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glucoselactoseFerrous sulfatepH indicator: phenol red

Kligler Iron Agar (KIA)

• Proteins

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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 +

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Reaction on KIA

Result

Example

Butt color

Slant color

H2S

Red Red NegativeAlk/Alk/-

(No action on sugars)

Non fermenter e.g.

Pseudomonas

Yellow Red

Negative A/Alk/- (Glucose fermented

without H2S)

LNF e.g. Shigella

Yellow Red

Positiveblack in

butt

A/Alk/+ (Glucose fermented

with H2S)

LNF e.g. Salmonella &

Proteus

Yellow YellowNegative

A/A/- (three sugars are

fermented)

LF e.g. E. coli, Klebsiella,

Enterobacter

Result

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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.

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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.

Tryptophaneamino acids

Tryptophanase Indole + Pyurvic acid + NH3

Kovac’s Reagent

Red color in upper organic layer` 13

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 (-).

Positive teste.g. E. coli

Negative teste.g. Klebsiella

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IMViC testMethyl Red-Voges Proskauer (MR-VP) Tests

Glucose

Acidic pathway

Mixed acids pH less than 4.4

Methyl Redindicator

Red color

Principle

MR positiveE. coli

Or Neutral pathway

Acety methyl carbinol(ACETOIN)

solution Asolution B

Pink colorVP positiveKlebsiella

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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.

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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.

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IMViC test: MRVP test

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

Method

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IMViC test: MR/VP testResults

Methyl Red test Voges-Proskauer test

Red: Positive MR (E. coli)

Yellow or orange: Negative MR (Klebsiella)

Pink: Positive VP (Klebsiella)

No pink: Negative VP (E. coli) 19

Citrate Utilization TestCitrate Utilization TestPrinciple:Citrate Na2CO3

Alkaline,↑pH

Blue colour

Bromothymol blue

Simmone’s Citrate media

Positive test: Klebsiella, Enterobacter, Citrobacter

CO2 + Na + H2OPyruvate

Positive test

Negative test: E. coli

Contains Citrate as a sole of C source

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Citrate Utilization Test

Incubate at 37°C for 24 hours.

MethodMethod

Streak a Streak a Simmon's Citrate agar slant with slant with

the organism the organism

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Citrate Utilization Test

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

ResultResult

PositiveKlebsiella, Enterobacter

NegativeE. coli 22

Urease Test

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

UreaUrease

CO2 + NH3H2O

NH4 OH ↑ in pH

Phenol Red

PinkPositive test

Streak a urea agar tube with the organism

incubate at 37°C for 24 h

Method

PrincipPrincipllee

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Urease Test

• 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

Result

Positive test Negative test

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MotilityFrom left to right:+ – +

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SIM Sulfide, Indole, Motility

04/18/23 S. S. Khoramrooz 26

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.

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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.

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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.

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Amino Acid Decarboxylation1

Lysine → Cadaverine

Ornithine → Putrescine

Arginine → Citrulline → Ornithine → Putrescine1Conversion of arginine to citrulline is a dihydrolase reaction

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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.

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Amino Acid Decarboxylation

Lys Orn Arg

Klebsiella + – –

Enterobacter +/– + +/–

Escherichia + +/– –/+

Salmonella + + +

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Amino Acid Decarboxylation

Lys Orn Arg

E. aerogenes + + –

E. cloacae – + +

P. mirabilis – + –

P. vulgaris – – –

Shigella D – + –

Shigella A-C – – –

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IPViC Reactions for Initial Grouping of the Enterobacteriaceae

• Indole

• Phenylalanine deaminase

• Voges-Proskauer

• Citrate

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Initial Grouping of the Enterobacteriaceae (VP=Voges Proskauer, PDA=Phenylalanine

Deaminase)

GENERA VP PDA

Klebsiella POSITIVE NEGATIVE

Enterobacter POSITIVE NEGATIVE

Serratia POSITIVE NEGATIVE

Hafnia POSITIVE NEGATIVE

Pantoea POSITIVE NEGATIVE

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Initial Grouping of the Enterobacteriaceae

GENERA VP PDA

Proteus1 NEGATIVE POSITIVE

Morganella NEGATIVE POSITIVE

Providencia NEGATIVE POSITIVE

1Proteus mirabilis: 50% of strains VP positive37

Initial Grouping of the Enterobacteriaceae

GENERA VP PDAEscherichia NEGATIVE NEGATIVEShigella NEGATIVE NEGATIVEEdwardsiella NEGATIVE NEGATIVESalmonella NEGATIVE NEGATIVECitrobacter NEGATIVE NEGATIVEYersinia NEGATIVE NEGATIVE

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Initial Grouping of the Enterobacteriaceae1

GENERA INDOLE CITRATE

Escherichia POSITIVE NEGATIVE

Shigella Yersinia

POSITIVE2

POSITIVE3 NEGATIVE NEGATIVE

Edwardsiella POSTIVE NEGATIVE

1VP negative, PDA negative 2Shigella groups A, B, and C variably positive for indole production (25-50%), group D Shigella negative. 3Yersinia enterocolitica 50% positive

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Initial Grouping of the Enterobacteriaceae1

GENERA INDOLE CITRATE Salmonella NEGATIVE POSITIVE2

Citrobacter NEGATIVE POSITIVE

1VP negative, PDA negative 2Salmonella serotype Paratyphi A and Typhi negative

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Key Characteristics of the Enterobacteriaceae

TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

E coli

A/A + + + + + +/

/ +

Shi A-C

Ak/A /

+ Shi D

Ak/A + +

Ed Ak/A + + + + + +

Sal Ak/A + + + + + + +/

Cit A/A

Ak/A

+ + + + +/ + /

+ +/

Yer A/A + +/

+/

RT (1) +

(1) RT=room temperature 41

Key Characteristics of the Enterobacteriaceae

TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

Kle pne

A/A + + + + + + Kleoxy

A/A + + + + + + + En aer

A/A + + + + + + + En cloa

A/A + + + + +/ + + + Serr (1)

A/A + + + + + + + Haf Ak/

A + + + + + + Pan A/A

Alk/A

+ /+ +/ /+ +/ /+ /+

(1) Produces DNase, lipase, and gelatinase 42

Key Characteristics of the Enterobacteriaceae

TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

Prot mira

Ak/A + + +/ +/ + + +s +

Prot vulg

A/A +/ + + /+ + + +s Mor Ak/

A + + + + + + Prov

Ak/A + + + + +

s = swarming motility

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Biochemical Characteristics of Escherichia coli and Shiglla

E. coli E. coli O157:H7 ShigellaTSI A/Ag A/Ag Alk/ALactose + + –ONPG + + –/+1

Sorbitol + – +/–Indole + + +/–Methyl red + + +VP – – –Citrate – – –Lysine + + –Motility + + –

1Shigella sonnei (group D) ONPG +

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Biochemical Characteristics of Salmonella Most Serotypes Typhi Paratyphi

ATSI Alk/A Alk/A Alk/A

H2S (TSI) + + (weak) –Citrate + – –Lysine + + –Ornithine + – +Dulcitol + – +Rhamnose + – +Indole – – –Methyl red + + +VP – – –

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Xylose Lysine Deoxycholate (XLD) Agar: Composition

• Xylose 0.35%• Lysine 0.5%• Lactose 0.75%• Sucrose 0.75%• Sodium chloride 0.5%• Yeast extract 0.3%• Sodium deoxycholate 0.25%• Sodium thiosulfate• Ferric ammonium citrate• Agar 1.35%• Phenol red• pH = 7.4

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).

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.

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.

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.

Hektoen Enteric (HE) Agar: Composition

• Peptone 1.2%• Yeast extract 0.3%• Bile salts 0.9%• Lactose 1.2%• Sucrose 1.2%• Salicin 0.2%• Sodium chloride 0.5%• Ferric ammonium citrate• Acid fuchsin• Thymol blue• Agar 1.4%• pH = 7.6

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.

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Some strains appear mucoid

Particularly common in patients with cystic fibrosis

Some strains produce diffusible pigments

Pyocyanin [blue]

Fluorescein [yellow]

Pyorubin [redbrown]

Pseudomonas aeruginosa

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Laboratory Diagnosis

CultureGrow easily on common isolation media such as blood agar and MacConkey37-42C

Identification

The colonial morphology (e.g., colony size, hemolytic activity, pigmentation, odor)

Biochemical tests (e.g., positive oxidase reaction)

+

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The End

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