Biochemical Tests In Enterobacteriaceae 91m2t

Biochemical Tests Enterobacteriaceae Dr.T.V.Rao MD

Dr.T.V.Rao MD

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Tests To Know Common Study Tests Indole Methyl Red/Voges Proskauer Citrate H2S production in SIM Urea hydrolysis Motility Lactose fermentation Sucrose fermentation Glucose fermentation & gas production Dr.T.V.Rao MD

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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

Dr.T.V.Rao MD

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Initial Grouping of the Enterobacteriaceae GENERA

VP

PDA

Proteus1

NEGATIVE

POSITIVE

Morganella

NEGATIVE

POSITIVE

Providencia

NEGATIVE

POSITIVE

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Proteus mirabilis: 50% of strains VP positive Dr.T.V.Rao MD

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Initial Grouping of the Enterobacteriaceae GENERA Escherichia Shigella Edwardsiella Salmonella Citrobacter Yersinia

VP NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE Dr.T.V.Rao MD

PDA NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE 5

Initial Grouping of the Enterobacteriaceae1 GENERA

INDOLE

CITRATE

Escherichia

POSITIVE

NEGATIVE

Shigella Yersinia Edwardsiella

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POSITIVE 3 POSITIVE POSTIVE

NEGATIVE NEGATIVE NEGATIVE

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

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Initial Grouping of the 1 Enterobacteriaceae GENERA Salmonella Citrobacter

INDOLE NEGATIVE NEGATIVE

CITRATE POSITIVE2 POSITIVE

1

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

Dr.T.V.Rao MD

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Key Characteristics of the Enterobacteriaceae TSI

ON

E coli

A/A

Shi AC Shi D Ed

Ak/ A

/ + +   +    + + +/  + /     +       

Sal Cit

Ak/ A Ak/ A Ak/ A

A/A Ak/ A Yer A/A

+   +

GAS H2S

 + + +

 + + +

VP

   

+   

(1) RT=room temperature

IND

 +  

CIT

  + +

PDA UR

LYS OR

AR

    +    + + +  +/ + + +    +/ +/ /   +  + 

 

+/  Dr.T.V.Rao MD

MO

+/ 

RT (1)

 +  8

Key Characteristics of the Enterobacteriaceae Kle pne Kle oxy En aer En cloa Serr (1) Haf Pan

TSI

ON

GAS H2S

A/A

+ + + + + + +

+ + + + + +

A/A A/A A/A A/A Ak/ A A/A Alk/ A

/+

      

VP

IND

CIT

PDA UR

MO

LYS OR

+ + + + + +

 +    

+ + + + + 

     

  + + + + 

+ + +  + + 

+ +  +/

 

+/ /+ +/ /+ /+

  + + + + 

AR

   +   

(1) Produces DNase, lipase, and gelatinase Dr.T.V.Rao MD

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Key Characteristics of the Enterobacteriaceae Prot mir a Prot vulg Mor Pro v

TSI

ON

Ak/ A

 + +

A/A

 +/ +  + /+ + + +s     +   +  + + +  +     + + + + +   

Ak/ A Ak/ A

GAS H2S VP

+/

IND CIT

PDA UR



+ + +s  + 

+/

MO

LYS OR

AR

s = swarming motility Dr.T.V.Rao MD

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Biochemical Characteristics of Escherichia coli and Shigella E. coli

E. coli O157:H7 A/Ag

Shigella

TSI

A/Ag

Lactose

+

+

–

ONPG

+

+

–/+1

Sorbitol

+

–

+/–

Indole

+

+

+/–

Methyl re

+

+

+

VP

–

–

–

Citrate

–

–

–

Lysine

+

+

Motility

+

+

Dr.T.V.Rao MD

Alk/A

– –

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Biochemical Characteristics of Salmonella Most Serotypes TSI

Alk/A

Typhi

Paratyphi A

Alk/A

Alk/A

H2S (TSI)

+

+ (weak)

–

Citrate

+

–

–

Lysine

+

+

–

Ornithine

+

–

+

Dulcitol

+

–

+

Rhamnose

+

–

+

Indole

–

–

–

Methyl red

+

+

+

VP

–

–

–

Dr.T.V.Rao MD

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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 Dr.T.V.Rao MD

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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 Dr.T.V.Rao MD 14 oxytoca, and Proteus vulgaris.

Indole Test How to Perform Test: Inoculate Tryptone broth with inoculating loop.

Property it tests for: This test is performed to help differentiate species of the family Enterobacteriaceae. It tests for the bacteria species’ ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one.

Media and Reagents Used: Tryptone broth contains tryptophan. Kovac’s reagent—contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol—yellow in color.

Reading Results: Kovac’s reagent reacts with indole and creates a red color at the top part of the test tube. Dr.T.V.Rao MD

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Reading the Result

Indole

Dr.T.V.Rao MD

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Methyl Red/Voges Proskauer (MR/VP) How to Perform Tests: Inoculate 2 glucose broths with inoculating loop. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube.

Properties they test for: Both tests are used to help differentiate species of the family Enterobacteriaceae. MR—tests for acid end products from glucose fermentation.

VP—tests for acetoin production from glucose fermentation.

Media and Reagents Used: Glucose Broth Methyl Red indicator for acid Voges Proskauer reagents—A: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide, & Deionized Dr.T.V.RaoWater. MD

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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. Dr.T.V.Rao MD

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MR/VP continued Reading Results: MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production)

VP—A + result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change.

Dr.T.V.Rao Methyl Red: left – and right + MD

VP: left + and right –

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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. Dr.T.V.Rao MD

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Citrate Test How to Perform Test: Inoculate slant with inoculating loop.

Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon and ammonium as sole nitrogen source.

Media and Reagents Used: Simmon’s Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicator—bromthymol blue. Dr.T.V.Rao MD

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Citrate Test Reading Reading Results: A + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a – result remains green Left positive and right negative. Dr.T.V.Rao MD

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IMViC Reactions I

M

Vi

C

Escherichia coli

+

+

–

–

Edwardsiella tarda

+

+

–

–

Proteus vulgaris

+

+

–

–

Klebsiella pneumoniae –

–

+

+

Klebsiella oxytoca

+

–

+

+

Enterobacter spp.

–

–

+

+

Serratia marcescens

–

–

+

+

Citrobacter freundii

–

+

–

+

Citrobacter koseri

+

+

–

+

Dr.T.V.Rao MD

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Urease-Producing Enterobacteriaceae Proteus Morganella

Providencia rettgeri Klebsiella pneumoniae

Klebsiella oxytoca Enterobacter cloacae

Yersinia enterocolitica Dr.T.V.Rao MD

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Urea Hydrolysis How to Perform Test: Inoculate Urea broth with inoculating loop.

Property it tests for: This test is done to

determine a bacteria’s ability to hydrolyze urea to make ammonia using the enzyme urease.

Media and Reagents Used: Urea broth contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator. Dr.T.V.Rao MD

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Urease Test Reading Results: Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made. Dr.T.V.Rao MD

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Reactions for Identification of Genera and Species1 Decarboxylation of amino acids Motility

Urease activity Hydrogen sulfide (H2S) production 1Voges-Proskauer,

phenylalanine

deaminase, indole, and citrate reactions are useful to both cluster Enterobacteriaceae

and identify to genus and species. Dr.T.V.Rao MD

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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. Dr.T.V.Rao MD

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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. Dr.T.V.Rao MD

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Amino Acid

1 Decarboxylation

Lysine → Cadaverine

Ornithine → Putrescine Arginine → Citrulline → Ornithine → Putrescine 1Conversion

of arginine to citrulline is a dihydrolase reaction Dr.T.V.Rao MD

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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. Dr.T.V.Rao MD

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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. Dr.T.V.Rao MD

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Amino Acid Decarboxylation Lys

Orn

Arg

–

–

Enterobacter +/–

+

+/–

Escherichia

+

+/–

–/+

Salmonella

+

+

+

Klebsiella

+

Dr.T.V.Rao MD

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Amino Acid Decarboxylation Lys

Orn

Arg

E. aerogenes +

+

–

E. cloacae

–

+

+

P. Mirabilis

–

+

–

P. vulgaris

–

–

–

Shigella D

–

+

–

Shigella A-C –

–

_

Dr.T.V.Rao MD

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H2S-Producing Enterobacteriaceae Salmonella Edwardsiella Citrobacter Proteus Dr.T.V.Rao MD

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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. Dr.T.V.Rao MD

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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 Dr.T.V.Rao MD

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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. Dr.T.V.Rao MD

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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. Dr.T.V.Rao MD

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Additional Biochemical Reactions 1 for the Enterobacteriaceae Fermentation of mannitol, dulcitol, salicin, adonitol, inositol, sorbitol, arabinose, raffinose, rhamnose, maltose, xylose, trehalose, cellobiose, alphamethyl –D-glucoside, 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). Dr.T.V.Rao MD

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Programme Created for Medical and Paramedical students in Microbiology Email

[email protected] Dr.T.V.Rao MD

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