Biochemical Tests Enterobacteriaceae Dr.T.V.Rao MD 1.

Biochemical TestsEnterobacteriaceae

Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

Tests To KnowCommon Study Tests

Indole

Methyl Red/Voges Proskauer

Citrate

H2S production in SIM

Urea hydrolysis

Motility

Lactose fermentation

Sucrose fermentation

Glucose fermentation & gas productionDr.T.V.Rao MD 2

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 3

Initial Grouping of the Enterobacteriaceae

GENERA VP PDA

Proteus1 NEGATIVE POSITIVE

Morganella NEGATIVE POSITIVE

Providencia NEGATIVE POSITIVE

1Proteus mirabilis: 50% of strains VP positiveDr.T.V.Rao MD 4

Initial Grouping of the Enterobacteriaceae

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

Dr.T.V.Rao MD 5

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

Dr.T.V.Rao MD 6

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 Dr.T.V.Rao MD 8



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

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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 Shigella

E. coli E. coli O157:H7 Shigella

TSI A/Ag A/Ag Alk/A

Lactose + + –

ONPG + + –/+1

Sorbitol + – +/–

Indole + + +/–

Methyl re + + +

VP – – –

Citrate – – –

Lysine + + –

Motility + + –

1Shigella sonnei (group D) ONPG +

Dr.T.V.Rao MD 11

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 – – –Dr.T.V.Rao MD 12

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

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

Indole TestHow 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 15

Reading the Result

Indole

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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 Water.Dr.T.V.Rao MD 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. Dr.T.V.Rao MD 18

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.

Methyl Red: left – and right + VP: left + and right – Dr.T.V.Rao MD 19

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.

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Citrate TestHow 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.

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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 22

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 23

Urease-Producing Enterobacteriaceae

Proteus

Morganella

Providencia rettgeri

Klebsiella pneumoniae

Klebsiella oxytoca

Enterobacter cloacae

Yersinia enterocolitica

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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 25

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.

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Reactions for Identification of Genera and Species1

Decarboxylation of amino acids

Motility

Urease activity

Hydrogen sulfide (H2S) production1Voges-Proskauer, phenylalanine

deaminase, indole, and citrate reactions are

useful to both cluster Enterobacteriaceae

and identify to genus and species. Dr.T.V.Rao MD 27

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 28


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 29

Amino Acid Decarboxylation1

Lysine → Cadaverine

Ornithine → Putrescine

Arginine → Citrulline → Ornithine → Putrescine

1Conversion of arginine to citrulline is a dihydrolase reaction

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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.

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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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Lys Orn Arg

Klebsiella + – –

Enterobacter +/– + +/–

Escherichia + +/– –/+

Salmonella + + + Dr.T.V.Rao MD 33


Lys Orn Arg

E. aerogenes + + –

E. cloacae – + +

P. Mirabilis – + –

P. vulgaris – – –

Shigella D – + –

Shigella A-C – – _

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H2S-Producing Enterobacteriaceae

SalmonellaEdwardsiellaCitrobacterProteus

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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.

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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 sensitivity2Salmonella-Shigella3Xylose-lysine-deoxycholate

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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.

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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 39

Additional Biochemical Reactions for the Enterobacteriaceae1

Fermentation of mannitol, dulcitol, salicin, adonitol, inositol, sorbitol, arabinose, raffinose, rhamnose, maltose, xylose, trehalose, cellobiose, alpha-methyl –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 pigment1JJ Farmer, Enterobacteriaceae: Introduction and

Identification, ASM Manual, 8th Edition (2003). Dr.T.V.Rao MD 40

Programme Created for Medical and Paramedical students in

Microbiology

Email

[email protected]

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References

www.slideshare.net

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Biochemical Tests Enterobacteriaceae Dr.T.V.Rao MD 1.

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