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Description : Major Biochemical Reactions for Identification of the Enterobacteriaceae

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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 (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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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 <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.
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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 (VP=Voges Proskauer, PDA=Phenylalanine 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 O157:H7 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. Koneman’s 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.