The EnterobacteriaceaeBiochemical Properties

Dr. John R. WarrenDepartment of PathologyNorthwestern University

Feinberg School of MedicineJune 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 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.

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-dimethylamino- benzaldehyde, 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 CEscherichia 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

I P Vi CEschericia + – – –Shigella +/– – – –Yersinia +/– – – –Edwardsiella + – – –Salmonella – – – +Citrobacter – – – +Klebsiella +/– – + +Enterobacter – – + +Serratia – – + +Proteus +/– + +/– +Morganella + + – +Providencia – + – +

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.

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

Lys Orn Arg

Klebsiella + – –

Enterobacter +/– + +/–

Escherichia + +/– –/+

Salmonella + + +

Amino Acid Decarboxylation

Lys Orn Arg

E. aerogenes + + –

E. cloacae – + +

P. mirabilis – + –

P. vulgaris – – –

Shigella D – + –

Shigella A-C – – –

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 sensitivity2Salmonella-Shigella3Xylose-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)

GENERA VP PDA

Klebsiella POSITIVE NEGATIVE

Enterobacter POSITIVE NEGATIVE

Serratia POSITIVE NEGATIVE

Hafnia POSITIVE NEGATIVE

Pantoea POSITIVE NEGATIVE

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

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

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

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

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

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

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 +

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 – – –

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).

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.

Koneman’s 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.