Biochemical Tests Enterobacteriaceae Dr.T.V.Rao MD Dr.T.V.Rao MD

Tests To Know Common Study Tests Indole Methyl Red/Voges Proskauer Citrate H2S production in SIM Urea hydrolysis Motility Lactose fermentation Sucrose fermentation Glucose fermentation & gas production Dr.T.V.Rao MD

Initial Grouping of the Enterobacteriaceae (VP=Voges Proskauer, PDA=Phenylalanine Deaminase) Dr.T.V.Rao MD

Initial Grouping of the Enterobacteriaceae Dr.T.V.Rao MD

Initial Grouping of the Enterobacteriaceae Dr.T.V.Rao MD

Initial Grouping of the Enterobacteriaceae1 Dr.T.V.Rao MD

Initial Grouping of the Enterobacteriaceae1 Dr.T.V.Rao MD

Key Characteristics of the Enterobacteriaceae Dr.T.V.Rao MD

Key Characteristics of the Enterobacteriaceae Dr.T.V.Rao MD

Key Characteristics of the Enterobacteriaceae Dr.T.V.Rao MD

Biochemical Characteristics of Escherichia coli and Shigella E. coli E. coli O157:H7 Shigella TSI A/Ag A/Ag Alk/A Lactose + + – ONPG + + –/+1 Sorbitol + – +/– Indole + + +/– Methyl re + + + VP – – – Citrate – – – Lysine + + – Motility + + – 1Shigella sonnei (group D) ONPG + Dr.T.V.Rao MD

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 – – – Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

Indole Test How to Perform Test: Inoculate Tryptone broth with inoculating loop. Property it tests for: This test is performed to help differentiate species of the family Enterobacteriaceae. It tests for the bacteria species’ ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one. Media and Reagents Used: Tryptone broth contains tryptophan. Kovac’s reagent—contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol—yellow in color. Reading Results: Kovac’s reagent reacts with indole and creates a red color at the top part of the test tube. Dr.T.V.Rao MD

Reading the Result Indole Dr.T.V.Rao MD

Methyl Red/Voges Proskauer (MR/VP) How to Perform Tests: Inoculate 2 glucose broths with inoculating loop. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube. Properties they test for: Both tests are used to help differentiate species of the family Enterobacteriaceae. MR—tests for acid end products from glucose fermentation. VP—tests for acetoin production from glucose fermentation. Media and Reagents Used: Glucose Broth Methyl Red indicator for acid Voges Proskauer reagents—A: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide, & Deionized Water. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

Methyl Red: left – and right + MR/VP continued Reading Results: MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production) VP—A + result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change. VP: left + and right – Methyl Red: left – and right + Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

Citrate Test How to Perform Test: Inoculate slant with inoculating loop. Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon and ammonium as sole nitrogen source. Media and Reagents Used: Simmon’s Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicator—bromthymol blue. Dr.T.V.Rao MD

Citrate Test Reading Reading Results: A + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a – result remains green Left positive and right negative. Dr.T.V.Rao MD

IMViC Reactions I M Vi C Escherichia coli + + – – Edwardsiella tarda + + – – Proteus vulgaris + + – – Klebsiella pneumoniae – – + + Klebsiella oxytoca + – + + Enterobacter spp. – – + + Serratia marcescens – – + + Citrobacter freundii – + – + Citrobacter koseri + + – + Dr.T.V.Rao MD

Urease-Producing Enterobacteriaceae Proteus Morganella Providencia rettgeri Klebsiella pneumoniae Klebsiella oxytoca Enterobacter cloacae Yersinia enterocolitica Dr.T.V.Rao MD

Urea Hydrolysis How to Perform Test: Inoculate Urea broth with inoculating loop. Property it tests for: This test is done to determine a bacteria’s ability to hydrolyze urea to make ammonia using the enzyme urease. Media and Reagents Used: Urea broth contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator. Dr.T.V.Rao MD

Urease Test Reading Results: Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

Amino Acid Decarboxylation1 Lysine → Cadaverine Ornithine → Putrescine Arginine → Citrulline → Ornithine → Putrescine 1Conversion of arginine to citrulline is a dihydrolase reaction Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

Amino Acid Decarboxylation Lys Orn Arg Klebsiella + – – Enterobacter +/– + +/– Escherichia + +/– –/+ Salmonella + + + Dr.T.V.Rao MD

Amino Acid Decarboxylation Lys Orn Arg E. aerogenes + + – E. cloacae – + + P. Mirabilis – + – P. vulgaris – – – Shigella D – + – Shigella A-C – – _ Dr.T.V.Rao MD

H2S-Producing Enterobacteriaceae Salmonella Edwardsiella Citrobacter Proteus Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

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). Dr.T.V.Rao MD

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References www.slideshare.net Dr.T.V.Rao MD