The Enterobacteriaceae Biochemical Properties

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The EnterobacteriaceaeBiochemical Properties

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

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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

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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.

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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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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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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.

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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.

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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.

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IMViC Reactions

• I indole production from tryptophan
• M methyl red test in which acidification of
  glucose broth (pHlt4.4) 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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IMViC Reactions

• I M Vi C
• Escherichia coli
  
• Edwardsiella tarda
  
• Proteus vulgaris
  
• Klebsiella pneumoniae
  
• Klebsiella oxytoca
• Enterobacter spp.
• Serratia marcescens
  
• Citrobacter freundii
• Citrobacter koseri
  

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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

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IPViC Reactions

• I P Vi C
• Eschericia
• Shigella /
• Yersinia /
• Edwardsiella
• Salmonella
• Citrobacter
• Klebsiella /
• Enterobacter
• Serratia
• Proteus / /
• Morganella
• Providencia

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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.

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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 ? Putrescine
• 1Conversion of arginine to citrulline is a
  dihydrolase reaction

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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.

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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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Bacterial Motility

• Many but not all Enterobacteriaceae demonstrate
  flagellar motility.
• Motility can be measured by use of lt0.4
  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.

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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.

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Urease-Producing Enterobacteriaceae

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

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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 sensitivity
• 2Salmonella-Shigella
• 3Xylose-lysine-deoxycholate

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H2S-Producing Enterobacteriaceae

• Salmonella
• Edwardsiella
• Citrobacter
• Proteus

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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
(VPVoges Proskauer, PDAPhenylalanine Deaminase)
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Initial Grouping of the Enterobacteriaceae
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Initial Grouping of the Enterobacteriaceae
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Initial Grouping of the Enterobacteriaceae1
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Initial Grouping of the Enterobacteriaceae1
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Key Characteristics of the Enterobacteriaceae
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Key Characteristics of the Enterobacteriaceae
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Key Characteristics of the Enterobacteriaceae
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Biochemical Characteristics of Escherichia coli
and Shiglla

• E. coli E. coli
  O157H7 Shigella
• TSI A/Ag A/Ag Alk/A
• Lactose
  
• 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 A
• TSI Alk/A
  Alk/A Alk/A
• H2S (TSI)
  (weak)
• Citrate
  
• Lysine
  
• Ornithine
  
• Dulcitol
  
• Rhamnose
  
• Indole
  
• Methyl red
  
• VP
  

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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 pigment
• 1JJ Farmer, Enterobacteriaceae Introduction and
  Identification, ASM Manual, 8th Edition (2003).

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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)

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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.

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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.