Enterobacteriaceae

The Enterics Family Enterobacteriaceae often referred to as “enterics” Four major features: All ferment glucose (dextrose) All reduce nitrates to nitrites All are oxidase negative Many found in the intestines of human or other mammals Varying pathogenicity: Commensals, opportunists or pathogens

Laboratory Diagnosis of Enterics Sugar fermentation Phenol red broth Triple Sugar Iron (TSI) Methyl red - Vogues Proskauer SIM Break down of tryptophan and cysteine; motility Citrate utilization Urease production Phenylalanine deaminase Decarboxylase tests

Phenol red broth Phenol red broth Allows determination of carbon source preferred and metabolism (Oxidation or fermentation) Contains simple carbon sources: Peptone (protein  amino acids) Desired sugar added Contains a pH indicator Phenol red Yellow - acid pH - Fermentation Orange - neutral pH - Oxidation Red - alkaline pH - Oxidation

Phenol Red Broths Yellow (acid) + gas Yellow (acid) no gas Fermentation of sugar Yellow (acid) no gas Fermentaion of sugar Orange (neutral) no gas Oxidation of sugar Red (alkaline) no gas Oxidation of proteins Uninoculated

TSI — Three Sugars and Iron Red slant/Red butt No fermentation Protein catabolism Red slant/Yellow butt Only glucose fermentation Yellow slant/yellow butt Lactose and/or sucrose fermentation

Methyl Red Test Test for fermentation with acid accumulation Carbon Sources: Glucose and proteins Glucose  pyruvate  lactic and/or acetic acid + CO2 Indicator -methyl red MR +: red (pH < 5.2) MR - : Yellow (pH > 5.2) Neutral Acid

- + Voges-Proskauer Test Used to determine fermentation with the production of neutral end products Glucose  pyruvate  acetoin  butanediol Reagents Acetoin + -naphthol + KOH + O2  Red color - +

Sim Test : Sulfide, Indole, Motility Sulfide: Identifies bacteria capable of metabolizing cysteine Cysteine  H2S; H2S + FeSO4  Black precipitate Indole: Identifies bacteria capable of metabolizing tryptophan Convert tryptophan to indole and pyruvic acid by using the enzyme tryptophanase Kovac’s reagent reacts with indole to give a red color

Non-motile Non inoculated Indole + - H2S and motile

Citrate Utilization Sodium citrate as a sole carbon source pH indicator - bromothymol blue. Sodium citrate → Pyruvic acid + Oxalacetic acid + CO2 Excess sodium from Sodium Citrate + CO2 + H2O → Na2CO3 (pH : green → blue) + -

Urease Test Used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease The pH indicator, phenol red, is used to detect the breakdown of urea and the production of ammonia As the alkaline end products build, NH4(CO3)2, phenol red turns to pink + -

Phenylalanine Deaminase Test Used to identify bacteria possessing the enzyme phenylalanine deaminase Phenylalanine deaminase converts phenylalanine to phenylpyruvic acid + NH3 Ferric chloride (FeCl3) reacts with the phenylpyruvic acid and changes color from yellow to green + -

Decarboxylase Test Contains glucose, test amino acid and pH indicator Fermentation of glucose generates acids (turns medium yellow) and activates decarboxylase If decarboxylase is present it generates alkaline products and changes pH indicator to purple (positive). If no decarboxylase present then media remains acid and yellow (negative)

Ornithine Decarboxylase Broth Interpretation Tubes 1 –aa & +aa: Acid reaction - glucose fermentation; no decarboxylation Tube 2 -aa: Acid reaction - glucose fermentation Tube 2 +aa: Acid reaction - glucose fermentation followed by decarboxylation (alkaline reaction) - aa +aa -aa +aa 1 2

Lysine Decarboxylase Lysine iron agar slants Contain lysine, glucose, peptones, bromcresol purple (pH indicator), sodium thiosulfate Lysine decarboxylation is an anaerobic process which occurs in the butt Decarboxylation of lysine produces an amine end-product which reacts with the pH indicator to give a purple color in the butt Negative decarboxylation: yellow butt

Lysine Deaminase Lysine iron agar slants Lysine deamination is an aerobic process which occurs on the slant Lysine deamination generates ammonia which reacts with pH indicator to produce a dark red color on the slant Negative deamination: purple slant

Lysine Iron Agar slants Purple slant & butt Decarboxylase positive Purple slant & yellow butt Decarboxylase negative Purple slant & black butt Red slant & yellow butt Deaminase positive

Nitrate Reductase Test Used to detect the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or some other nitrogenous compound, such as molecular nitrogen (N2) using the enzyme nitrate reductase

Nitrate Reductase Step 1: Test for nitrite NO3- + 2 H+ + 2 e-  H2O + NO2-  Nitrate Nitrite NH3 or N2 Step 1: Test for nitrite NO2- + sulfanilic acid and alpha naphthylamine  HNO2 Red color indicates presence of Nitrite Nitrate was reduced Yellow color indicates absence of Nitrite Nitrate was not reduced Nitrate was reduced to Nitrite which was further reduced + ?

Nitrate Reductase (Cont’d) Interpretation of ambiguous result after step 1 Step 2: Test for the presence of nitrate NO3- + Zn (s)  NO2- + - Red color indicates presence of Nitrate Nitrate was not reduced by bacteria Nitrate was reduced by Zn Yellow color indicates absence of Nitrate Nitrate was reduced to Nitrite which was further reduced