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Dr. John R. Warren Department of Pathology Northwestern University Feinberg School of Medicine June 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.
• Acetoin and butylene glycol are detected by oxidation to diacteyl at an alkaline pH, and the addition of α -naphthol which forms a redcolored 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.
• The deamination of phenylalanine is an important biochemical property of Proteus. .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. Morganella. and Providencia. Phenylpyruvic acid is detected by addition of ferric chloride that forms an intensely dark olive-green colored complex when binding to phenylpyruvic acid.
whose aldehyde group reacts with indole forming a red-colored complex. • Free indole is detected by p-dimethylaminobenzaldehyde. . • Production of indole from tryptophan is an important biochemical property of Escherichia coli. and C Shigella. B.Indole Reaction • Enterobacteriaceae that possess tryptophanase can utilize tryptophan by deamination and hydrolytic removal of the indole side chain. Klebsiella oxytoca. and Proteus vulgaris. Edwardsiella tarda. many strains of group A.
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 • Citrate is utilized by several of the Enterobacteriaceae as a single carbon source. .
.Citrate Utilization • Enterobacteriaceae that can utilize citrate will extract nitrogen from ammonium phosphate releasing ammonia. • Citrate utilization is a key biochemical property of Salmonella. Klebsiella. Ammonia produces an alkaline pH shift. and the indicator bromthymol blue turns blue from its green color at neutral pH. Enterobacter. and Serratia. Citrobacter.
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 .4) due to formation of mixed carboxylic acids (lactic.IMViC Reactions • I = indole production from tryptophan • M = methyl red test in which acidification of glucose broth (pH<4. acetic.
Serratia marcescens Citrobacter freundii Citrobacter koseri I + + + – + – – – + M + + + – – – – + + Vi – – – + + + + – – C – – – + + + + + + .IMViC Reactions Escherichia coli Edwardsiella tarda Proteus vulgaris Klebsiella pneumoniae Klebsiella oxytoca Enterobacter spp.
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 Eschericia Shigella Yersinia Edwardsiella Salmonella Citrobacter Klebsiella Enterobacter Serratia Proteus Morganella Providencia I + +/– +/– + – – +/– – – +/– + – P – – – – – – – – – + + + Vi – – – – – – + + + +/– – – C – – – – + + + + + + + + .
indole.Reactions for Identification of 1 Genera and Species • Decarboxylation of amino acids • Motility • Urease activity • Hydrogen sulfide (H2S) production Voges-Proskauer. and citrate reactions are useful to both cluster Enterobacteriaceae and identify to genus and species. 1 . phenylalanine deaminase.
pyridoxal.Amino Acid Decarboxylation • Enterobacteriaceae contain decarboxylases with substrate specificity for amino acids. . peptone and beef extract. • Moeller broth contains glucose for fermentation. and the pH indicator bromcresol purple. an amino acid. and are detected using Moeller decarboxylase broth overlayed with mineral oil for anaerobiosis.
ornithine. and arginine are utilized. A base broth without amino acid is included in which glucose fermentation acidifies the broth. .Amino Acid Decarboxylation • If an Enterobacteriaceae contains amino acid decarboxylase. amines produced by decarboxylase action cause an alkaline pH. turning the bromcresol purple yellow. • Lysine. and bromcresol purple turns purple.
Amino Acid Decarboxylation1 Lysine → Cadaverine Ornithine → Putrescine Arginine → Citrulline → Ornithine → Putrescine 1 Conversion 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 1 Indicates organism is a viable glucose fermenter. . and pH of broth medium sufficiently acidified to activate decarboxylase enzymes.
Proteus mirabilis. and Salmonella.Amino Acid Decarboxylation • Decarboxylation patterns are essential for the genus identification of Klebsiella. Enterobacter. • Decarboxylation patterns are also essential for the species identification of Enterobacter aerogenes. and Shigella sonnei. Escherichia. . Enterobacter cloacae.
Amino Acid Decarboxylation Klebsiella Enterobacter Escherichia Salmonella Lys + +/– + + Orn – + +/– + Arg – +/– –/+ + .
Amino Acid Decarboxylation E. mirabilis P. cloacae P. aerogenes E. vulgaris Shigella D Shigella A-C Lys + – – – – – Orn + + + – + – Arg – + – – – – .
. • 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. and Yersinia is non-motile at 35oC but motile at 22o-25oC. • Shigella and Klebsiella are non-motile.Bacterial Motility • Many but not all Enterobacteriaceae demonstrate flagellar motility.
and contains tetrazolium salts that are reduced to red formazan complexes to enhance visual assessment of motility. . • Pure motility agar lacks an H2S indicator and tryptophan for indole production.Motility Agars • Sulfide-indole-motility (SIM) is a semisolid motility agar that contains peptonized iron for detection of H2S and tryptophan for indole production.
• Proteus. and the pH indicator phenol red becomes red.Urease Reaction • Urease hydrolyzes urea releasing ammonia which alkalinizes the medium by forming ammonium carbonate. Morganella. Klebsiella a weak urease producer. and Providencia are strong urease producers. and Yersinia enterocolitica frequently a urease .
Urease-Producing Enterobacteriaceae • • • • • • • Proteus Morganella Providencia rettgeri Klebsiella pneumoniae Klebsiella oxytoca Enterobacter cloacae Yersinia enterocolitica .
• For detection of H2S a heavy-metal (iron or lead) compound is present that reacts with H2S to form black-colored ferrous sulfide.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.
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) 1 In order of decreasing sensitivity 2 Salmonella-Shigella 3 Xylose-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 Klebsiella .
Initial Grouping of the Enterobacteriaceae GENERA Proteus 1 VP NEGATIVE NEGATIVE NEGATIVE PDA POSITIVE POSITIVE POSITIVE Morganella Providencia 1 Proteus mirabilis: 50% of strains VP positive .
Initial Grouping of the Enterobacteriaceae GENERA Escherichia Shigella Edwardsiella Salmonella Citrobacter Yersinia VP NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE PDA NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE .
Initial Grouping of the 1 Enterobacteriaceae GENERA Escherichia I P .
Initial Grouping of the 1 Enterobacteriaceae GENERA Salmonella Citrobacter I N N .
Key Characteristics of the Enterobacteriaceae TSI E A/A coli Shi AAk/ A ON GAS H2S + + − − − − .
Key Characteristics of the Enterobacteriaceae TSI Kle pne Kle oxy A/A A/A ON GAS H2S + + − + + − .
Key Characteristics of the Enterobacteriaceae TSI Prot Ak/ mir A a Prot A/A ON GAS H2S − + + .
coli A/Ag + + + + + – – + + E. coli O157:H7 A/Ag + + – + + – – + + Shigella Alk/A – –/+1 +/– +/– + – – – – Shigella sonnei (group D) ONPG + .Biochemical Characteristics of Escherichia coli and Shiglla TSI Lactose ONPG Sorbitol Indole Methyl red VP Citrate Lysine Motility 1 E.
Biochemical Characteristics of Salmonella TSI H2S (TSI) Citrate Lysine Ornithine Dulcitol Rhamnose Indole Methyl red VP Most Serotypes Alk/A + + + + + + – + – Typhi Alk/A + (weak) – + – – – – + – Paratyphi A Alk/A – – – + + + – + – .
esculin hydrolysis. . sorbitol. ASM Manual. cellobiose. acetate. and tartrate • Gelatin hydrolysis. and DNase • Growth in KCN • Yellow pigment 1 JJ Farmer. adonitol. inositol. arabinose. Enterobacteriaceae: Introduction and Identification. glycerol. dulcitol. lipase. salicin. and mannose • Utilization of malonate. alpha-methyl –Dglucoside. xylose. raffinose. arabitol. rhamnose. erythritol. 8th Edition (2003). mucate. maltose.Additional Biochemical Reactions for the Enterobacteriaceae1 • Fermentation of mannitol. melibiose. trehalose.
MicroScan Walkaway. Sensitire-TREK) • Automated (panels or cards with minaturized wells or chambers with colorimetric or fluorometric reactions instrument-recorded automatically) (VITEK 2-bioMerieux. Sensititre Automated) . MicroScan-Dade Behring.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.
The Enterobacteriaceae. Allen. P.. S. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. W. Lippincott Williams & Wilkins. W. . G... 2006: • Chapter 6. Jr. Schreckenberger. Janda.. Sixth Edition. Koneman. Woods. E. Procop.. G..Recommended Reading Winn..
. J. Jorgensen.. A. C. 9th Edition. P. • Abbott. Citrobacter. Pfaller. and Strockbine. and other Enterobacteriaceae. N.. Shigella. Landry. Chapter 45.A. Fields. Chapter 43.Recommended Reading Murray. Yersinia.I.L.. Enterobacter. M. J. ASM Press. M. Baron. 2007: • Nataro. E..P.A.. Escherichia.. Serratia. Kaper.B. Klebsiella. • Wanger. . Bopp. J. Plesiomonas. Chapter 44. Manual of Clinical Microbiology. and Salmonella. P.. S.
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