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The Enterobacteriaceae Biochemical Properties

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Title: The Enterobacteriaceae Biochemical Properties


1
The EnterobacteriaceaeBiochemical Properties
  • Dr. John R. Warren
  • Department of Pathology
  • Northwestern University
  • Feinberg School of Medicine
  • June 2007

2
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

3
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.

4
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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6
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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8
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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10
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.

11
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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13
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

14
IMViC Reactions
  • I M Vi C
  • Escherichia coli
  • Edwardsiella tarda
  • Proteus vulgaris
  • Klebsiella pneumoniae
  • Klebsiella oxytoca
  • Enterobacter spp.
  • Serratia marcescens
  • Citrobacter freundii
  • Citrobacter koseri

15
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

16
IPViC Reactions
  • I P Vi C
  • Eschericia
  • Shigella /
  • Yersinia /
  • Edwardsiella
  • Salmonella
  • Citrobacter
  • Klebsiella /
  • Enterobacter
  • Serratia
  • Proteus / /
  • Morganella
  • Providencia

17
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.

18
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.

19
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.

20
Amino Acid Decarboxylation1
  • Lysine ? Cadaverine
  • Ornithine ? Putrescine
  • Arginine ? Citrulline ? Ornithine ? Putrescine
  • 1Conversion of arginine to citrulline is a
    dihydrolase reaction

21
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.

22
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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24
Amino Acid Decarboxylation
  • Lys Orn Arg
  • Klebsiella
  • Enterobacter / /
  • Escherichia / /
  • Salmonella

25
Amino Acid Decarboxylation
  • Lys Orn Arg
  • E. aerogenes
  • E. cloacae
  • P. mirabilis
  • P. vulgaris
  • Shigella D
  • Shigella A-C

26
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.

27
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.

28
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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30
Urease-Producing Enterobacteriaceae
  • Proteus
  • Morganella
  • Providencia rettgeri
  • Klebsiella pneumoniae
  • Klebsiella oxytoca
  • Enterobacter cloacae
  • Yersinia enterocolitica

31
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.

32
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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34
H2S-Producing Enterobacteriaceae
  • Salmonella
  • Edwardsiella
  • Citrobacter
  • Proteus

35
IPViC Reactions for Initial Grouping of the
Enterobacteriaceae
  • Indole
  • Phenylalanine deaminase
  • Voges-Proskauer
  • Citrate

36
Initial Grouping of the Enterobacteriaceae
(VPVoges Proskauer, PDAPhenylalanine Deaminase)
37
Initial Grouping of the Enterobacteriaceae
38
Initial Grouping of the Enterobacteriaceae
39
Initial Grouping of the Enterobacteriaceae1
40
Initial Grouping of the Enterobacteriaceae1
41
Key Characteristics of the Enterobacteriaceae
42
Key Characteristics of the Enterobacteriaceae
43
Key Characteristics of the Enterobacteriaceae
44
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

45
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

46
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).

47
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)

48
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.

49
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.
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