Enterobacteriacae identification In the name of God Department Of Microbiology Yasouj University of Medical Science Enterobacteriacae identification By: Dr. S. S. Khoramrooz, Ph.D. Department of Microbiology, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran

Characters of Enterobacteriaceae All Enterobacteriaceae Gram-negative rods Ferment glucose with acid production Reduce nitrates into nitrites Oxidase negative Facultative anaerobic Motile except Shigella and Klebsiella Non-capsulated except Klebsiella Non-fastidious Grow on bile containing media (MacConkey agar)

Classification of Enterobacteriaceae Klebsiella, Enterobacter E. coli, Citrobacter, Lactose fermenters Non-lactose fermenter Salmonella, Shigella Proteus, Yersinia There are several selective and differential media used to isolate distinguishes between LF & LNF The most important media are: MacConkey agar Eosin Methylene Blue (EMB) agar Salmonella Shigella (SS) agar In addition to Kiligler Iron agar (KIA)

Tests To Know Case Study Tests Indole Methyl Red/Voges Proskauer Citrate H2S production in SIM Urea hydrolysis Motility Lactose fermentation Glucose fermentation & gas production Decarboxylation of amino acis Fermentation of sugars Reaction on selective media

Growth of Enterobacteriaceae on MacConkey agar Colorless colonies Pink colonies Lactose non feremters Salmonella, Shigella, Proteus Lactose feremters E. coli, Citrobacter Klebsiella, Enterobacter Uninoculated plate

Kligler Iron Agar Lactose Fermentation Glucose fermentation Gas Production (H2 & CO2 ) H2S Production

Kligler Iron Agar (KIA) Proteins glucose lactose Ferrous sulfate pH indicator: phenol red

Red/Red Alkaline /Alkaline K/K Lactose -/Glucose – Yellow/Yellow Acid/Acid A/A Lactose +/Glucose + Red/Yellow Alkaline/Acid K/A Lactose -/Glucose + Gas - H2S - Red/Yellow Alkaline/Acid K/A Lactose -/Glucose + Gas + H2S - Red/Yellow Alkaline/Acid K/A Lactose -/Glucose + Gas - H2S + Red/Black Alkaline/Acid K/A Lactose -/Glucose + Gas + H2S +

Result Example Result Reaction on KIA H2S Slant color Butt color Non fermenter e.g. Pseudomonas Alk/Alk/- (No action on sugars) Negative Red LNF e.g. Shigella A/Alk/- (Glucose fermented without H2S) Yellow e.g. Salmonella & Proteus A/Alk/+ (Glucose fermented with H2S) Positive black in butt LF e.g. E. coli, Klebsiella, Enterobacter A/A/- (three sugars are fermented)

IMViC Test Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests: The following four tests comprise a series of important determinations that are collectively called the IMViC series of reactions The IMViC series of reactions allows for the differentiation of the various members of Enterobacteriaceae.

IMViC: Indole test Principle Certain microorganisms can metabolize tryptophan by tryptophanase The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia The presence of indole is detected by addition of Kovac's reagent. Tryptophanase Tryptophane amino acids Indole + Pyurvic acid + NH3 Kovac’s Reagent Red color in upper organic layer`

IMViC: Indole test Result: A bright pink color in the top layer indicates the presence of indole The absence of color means that indole was not produced i.e. indole is negative Special Features: Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-). Negative test e.g. Klebsiella Positive test e.g. E. coli

IMViC test Methyl Red-Voges Proskauer (MR-VP) Tests Principle Glucose Acidic pathway Or Neutral pathway Acety methyl carbinol (ACETOIN) Mixed acids  pH less than 4.4 solution A solution B Methyl Red indicator VP positive Klebsiella MR positive E. coli Red color Pink color

Butylene Glycol Pathway of Glucose Fermentation In the butylene glycol pathway 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 -naphthol which forms a red-colored complex with diacetyl. Important biochemical property used for the identification of Klebsiella, Enterobacter, and Serratia.

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.

IMViC test: MRVP test Method Inoculate the tested organism into MRVP broth Incubate the tubes at 37°C for 24 hours For methyl red:  Add 6-8 drops of methyl red reagent. For Voges-Proskauer:  Add 12 drops of solution A (-naphthol), mix, 4 drops of Solution B (40% KOH), mix

IMViC test: MR/VP test Results Voges-Proskauer test Methyl Red test Pink: Positive VP (Klebsiella) Red: Positive MR (E. coli) No pink: Negative VP (E. coli) Yellow or orange: Negative MR (Klebsiella)

Citrate Utilization Test Principle: Citrate Na2CO3 Pyruvate CO2 + Na + H2O Alkaline,↑pH Simmone’s Citrate media Contains Citrate as a sole of C source Bromothymol blue Positive test Blue colour Positive test: Klebsiella, Enterobacter, Citrobacter Negative test: E. coli

Citrate Utilization Test Method Streak a Simmon's Citrate agar slant with the organism Incubate at 37°C for 24 hours.

Citrate Utilization Test Result Examine for growth (+) Growth on the medium is accompanied by a rise in pH to change the medium from its initial green color to deep blue Positive Klebsiella, Enterobacter Negative E. coli

Urease Test Principle Method NH4 OH Urea CO2 + NH3 Urea agar contains urea and phenol red Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3 Ammonia combines with water to produce ammonium hydroxide, a strong base which ↑ pH of the medium. ↑ in the pH causes phenol red r to turn a deep pink. This is indicative of a positive reaction for urease Urease H2O Urea CO2 + NH3 NH4 OH ↑ in pH Phenol Red Method Pink Positive test Streak a urea agar tube with the organism incubate at 37°C for 24 h

Urease Test Result If color of medium turns from yellow to pink indicates positive test. Proteus give positive reaction after 4 h while Kelebsiella and Enterobacter gave positive results after 24 h Positive test Negative test

Motility From left to right: + – +

SIM Sulfide, Indole, Motility 4/16/2017 S. S. Khoramrooz

Proteus species A exhibits characteristic “swarming” B shows urease positive on right Proteus species

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.

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.

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.

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

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.

Amino Acid Decarboxylation Lys Orn Arg Klebsiella + – – Enterobacter +/– + +/– Escherichia + +/– –/+ Salmonella + + +

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

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)

Initial Grouping of the Enterobacteriaceae

Initial Grouping of the Enterobacteriaceae

Initial Grouping of the Enterobacteriaceae1

Initial Grouping of the Enterobacteriaceae1

Key Characteristics of the Enterobacteriaceae

Key Characteristics of the Enterobacteriaceae

Key Characteristics of the Enterobacteriaceae

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 +

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 – – –

Xylose Lysine Deoxycholate (XLD) Agar: Composition Lactose 0.75% Sucrose 0.75% Sodium chloride 0.5% Yeast extract 0.3% Sodium deoxycholate 0.25% Sodium thiosulfate Ferric ammonium citrate Agar 1.35% Phenol red pH = 7.4

XLD Agar: Growth of Salmonella Salmonella selective due to bile salt. Xylose fermentation (except Salmonella serotype Paratyphi A) acidifies agar activating lysine decarboxylase. With xylose depletion fermentation ceases, and colonies of Salmonella (except S. Paratyphi A) alkalinize the agar due to amines from lysine decarboxylation. Xylose fermentation provides H+ for H2S production (except S. Paratyphi A).

XLD Agar: Appearance of Salmonella Ferric ammonium citrate present in XLD agar reacts with H2S gas and forms black precipitates within colonies of Salmonella. Agar becomes red-purple due to alkaline pH produced by amines. Back colonies growing on red-purple agar-presumptive for Salmonella.

XLD Agar: Growth of Escherichia coli and Klebsiella pneumoniae Escherichia coli and Klebsiella pneumoniae are lysine-positive coliforms that are also lactose and sucrose fermenters. The high lactose and sucrose concentrations result in strong acid production, which quenches amines roduced by lysine decarboxylation. Colonies and agar appear bright yellow. Neither Escherichia coli nor Klebsiella pneumoniae produce H2S.

XLD Agar: Growth of Shigella and Proteus Shigella species do not ferment xylose, lactose, and sucrose, do not decarboxylate lysine, and do not produce H2S. Colonies appear colorless. Proteus mirabilis ferments xylose, and thereby provides H+ for H2S production. Colonies appear black on an agar unchanged in color (Proteus deaminates rather than decarboxylates amino acids). Proteus vulgaris ferments sucrose, and colonies appear black on a yellow agar.

Hektoen Enteric (HE) Agar: Composition Peptone 1.2% Yeast extract 0.3% Bile salts 0.9% Lactose 1.2% Sucrose 1.2% Salicin 0.2% Sodium chloride 0.5% Ferric ammonium citrate Acid fuchsin Thymol blue Agar 1.4% pH = 7.6

HE Agar: Growth of Enteric Pathogens and Commensals High bile salt concentration inhibits growth of gram-positive and gram-negative intestinal commensals, and thereby selects for pathogenic Salmonella (bile-resistant growth) present in fecal specimens. Salmonella species as non-lactose and non-sucrose fermenters that produce H2S form colorless colonies with black centers. Shigella species (non-lactose and non-sucrose fermenters, no H2S production) form colorless colonies. Lactose and sucrose fermenters (E. coli, K. pneumoniae) form orange to yellow colonies due to acid production.

Pseudomonas aeruginosa Some strains appear mucoid Particularly common in patients with cystic fibrosis Some strains produce diffusible pigments Pyocyanin [blue] Fluorescein [yellow] Pyorubin [redbrown]

+ Laboratory Diagnosis Culture Identification Grow easily on common isolation media such as blood agar and MacConkey 37-42C Identification The colonial morphology (e.g., colony size, hemolytic activity, pigmentation, odor) + Biochemical tests (e.g., positive oxidase reaction)

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