Comprehensive Study Guide to Enterobacteriaceae: Characteristics, Media, and Biochemical Identification

General Characteristics and Classification of Enterobacteriaceae

• Taxonomic Overview and Significance:

  • Enterobacteriaceae is the largest and most medically important family of Gram-negative bacilli.

  • While the family encompasses numerous genera and species, more than $90\%$ of medically significant isolates belong to only a small subset of genera.

  • Members of this family are distributed worldwide, serve as normal flora across all animals, and are recognized as frequent etiologic agents of nosocomial (hospital-acquired) infections.

• Core Defining Physiological Features:

  • All members ferment glucose.

  • All members reduce nitrates ($NO_3^-$) to nitrites ($NO_2^-$).

  • All members are oxidase-negative.

  • The negative oxidase reaction is of paramount diagnostic value for establishing a rapid distinction between Enterobacteriaceae and the majority of Gram-negative nonfermenting bacilli.

• Taxonomic Exception and Phylogenetics:

  • Plesiomonas shigelloides is an exception to the oxidase rule, presenting as oxidase-positive.

  • Formerly placed in the family Vibrionaceae, Plesiomonas shigelloides has been reclassified into the family Enterobacteriaceae.

  • Phylogenetic investigations demonstrate that Plesiomonas is closely related to members of the Enterobacteriaceae family, showing particular genetic proximity to the genus Proteus.

• Antigenic Structures:

  • $O$ Antigen (Somatic Antigen): Located structurally in the bacterial cell wall.

  • $H$ Antigen (Flagellar Antigen): Located on the bacterial flagella; associated with motility.

  • $K$ Antigen (Capsular Antigen): Located within the bacterial capsule.

Clinical Manifestations and Anatomical Sites of Infection

• Blood (Bacteremia / Sepsis):

  • Escherichia

  • Klebsiella

  • Enterobacter

• Central Nervous System (Meningitis):

  • Escherichia

• Gastrointestinal Tract (Enteritis / Gastroenteritis):

  • Escherichia

  • Salmonella

  • Shigella

  • Yersinia

• Lower Respiratory Tract (Pneumonia):

  • Klebsiella

  • Enterobacter

  • Escherichia

• Urinary Tract (UTIs):

  • Escherichia

  • Proteus

  • Klebsiella

Culture Media for Isolation, Selection, and Differentiation

• Media for Isolation and Selection of Salmonella and Shigella:

  • Bismuth Sulfite Agar:

  • Salmonella typhi produces distinct black colonies surrounded by a characteristic black zone of precipitate.

  • Brilliant Green Agar:

  • Proteus produces red to pink colonies.

  • Salmonella produces red to pink colonies.

  • Shigella is completely inhibited.

  • Gram-Negative (GN) Broth:

  • Inhibits the growth of the vast majority of Gram-negative rods.

  • Selectively enriches the growth of both Salmonella and Shigella.

  • Hektoen Enteric (HE) Agar:

  • Escherichia coli and other lactose/sucrose fermenters form orange to salmon pink colonies.

  • Proteus is generally inhibited.

  • Salmonella colonies appear blue to blue-green, developing black centers if hydrogen sulfide ($H_2S$) is generated.

  • Shigella colonies appear green.

  • Salmonella-Shigella (SS) Agar:

  • Escherichia coli produces red colonies.

  • Proteus forms colorless colonies with black centers.

  • Salmonella produces colorless colonies, with or without black centers.

  • Shigella forms colorless colonies.

  • Selenite Broth:

  • Serves as an enrichment medium for Salmonella recovered from stool specimens.

  • Completely inhibits the growth of Gram-positive organisms.

  • Xylose-Lysine-Deoxycholate (XLD) Agar:

  • Escherichia coli forms yellow colonies.

  • Proteus forms clear or yellow colonies.

  • Salmonella forms red colonies with prominent black centers.

  • Shigella forms clear colonies.

• Media for Isolation and Detection of Lactose Fermenters:

  • Eosin-Methylene Blue (EMB) Agar:

  • Inhibits Gram-positive organisms.

  • Escherichia coli develops characteristic dark colonies with a green metallic sheen.

  • Lactose fermenters present as pink to red colonies.

  • Lactose nonfermenters present as colorless colonies.

  • MacConkey Agar:

  • Inhibits Gram-positive organisms.

  • Lactose fermenters produce pink to red colonies.

  • Lactose nonfermenters produce colorless colonies.

Biochemical Identification Tests and Physiological Pathways

• Triple Sugar Iron (TSI) Agar:

  • Purpose: Differentiates Gram-negative bacilli by evaluating their capacity to ferment three specific carbohydrates (glucose, lactose, and sucrose), produce gas, and generate hydrogen sulfide ($H_2S$).

  • Formulation Components:

  • Glucose ($0.1\%$)

  • Lactose ($1\%$)

  • Sucrose ($1\%$)

  • Phenol red (pH indicator)

  • Sodium thiosulfate and ferrous sulfate (indicators of $H_2S$ production)

  • Reactions and Interpretation:

  • Glucose Fermentation Only: Alkaline (red) slant and acid (yellow) butt; glucose is present in limiting amounts ($0.1\%$) and is exhausted on the aerobic slant, causing reversion to red via peptone oxidation.

  • Lactose and/or Sucrose Fermentation: Acid (yellow) slant and acid (yellow) butt, indicating extensive acid generation from higher sugar concentrations ($1\%$).

  • Non-fermentation: Red slant and red or orange butt.

  • Gas Production: Indicated by the formation of bubbles, cracks, or disruption in the agar column.

  • $H_2S$ Production: Indicated by a black precipitate within the butt.

  • Diagnostic Utility: Differentiates lactose/sucrose fermenters (such as Escherichia coli) from non-fermenters (such as Salmonella and Shigella).

• Nitrate Reduction Test:

  • Purpose: Assesses the enzymatic capacity to reduce nitrate ($NO_3^-$) to nitrite ($NO_2^-$) or further into free nitrogen gas ($N_2$).

  • Medium: Nitrate broth formulated with potassium nitrate ($KNO_3$).

  • Procedural Steps:

  • Following standard incubation, add sulfanilic acid followed by $\alpha\text{-naphthylamine}$ to identify the presence of nitrite.

  • In the absence of an immediate color change, add elemental zinc powder to evaluate for residual, unreduced nitrate.

  • Reactions and Interpretation:

  • Positive for Nitrite Production: Development of a red color upon the addition of sulfanilic acid and $\alpha\text{-naphthylamine}$.

  • Complete Reduction to $N_2$ Gas: No color change after reagent addition, and remains colorless following zinc powder addition (signifies all nitrate was reduced past nitrite to $N_2$).

  • Negative Result: Medium remains colorless after initial reagents, but turns red following the addition of zinc powder (zinc chemically reduces unreacted nitrate to nitrite, confirming the organism failed to reduce nitrate).

  • Diagnostic Utility: Differentiates Enterobacteriaceae (which all reduce nitrates to nitrites) from other non-reducing Gram-negative bacilli.

• Oxidase Test:

  • Purpose: Detects the presence of bacterial cytochrome c oxidase functioning in the respiratory electron transport chain.

  • Reagent: Tetramethyl-p-phenylenediamine dihydrochloride.

  • Procedure: Bacterial colonies are smeared onto filter paper or an oxidase test strip pre-saturated with reagent.

  • Reactions and Interpretation:

  • Positive Result: Rapid transition to a dark purple or blue color within $10\text{--}30\text{ seconds}$.

  • Negative Result: Complete lack of color change.

  • Diagnostic Utility: Distinguishes oxidase-negative Enterobacteriaceae from oxidase-positive Gram-negative bacteria including Pseudomonas, Neisseria, and Vibrio species.

• Indole Test:

  • Purpose: Evaluates the presence of tryptophanase, which hydrolyzes the amino acid tryptophan into indole, pyruvic acid, and ammonia.

  • Medium: Tryptone broth or Sulfide Indole Motility (SIM) medium containing excess tryptophan.

  • Reagents: Kovac's reagent or Ehrlich's reagent.

  • Procedure: Add several drops of Kovac's reagent directly to the culture broth surface after incubation.

  • Reactions and Interpretation:

  • Positive Result: Formation of a red or pink ring at the surface meniscus (e.g., Escherichia coli).

  • Negative Result: Formation of a yellow ring or no visible color transition (e.g., Klebsiella).

  • Diagnostic Utility: Serves as a key differentiator between Escherichia coli (indole-positive) and indole-negative genera such as Klebsiella and Enterobacter.

• Methyl Red-Voges Proskauer (MR-VP) Tests:

  • Purpose: Distinguishes glucose fermentation end-products between stable mixed-acid pathways and neutral end-product pathways.

  • Medium: MR-VP broth, composed of glucose, peptone, and a phosphate buffer.

  • Methyl Red (MR) Test:

  • Principle: Detects stable, high-concentration organic acids that overcome the broth phosphate buffer, dropping the pH below $4.4$.

  • Positive Result: Red color development upon addition of methyl red indicator (e.g., Escherichia coli).

  • Negative Result: Yellow color (e.g., Enterobacter cloacae).

  • Voges-Proskauer (VP) Test:

  • Principle: Detects acetoin (acetylmethylcarbinol), a neutral precursor in the 2,3-butanediol fermentation pathway.

  • Positive Result: Red color development following the addition of Barritt's reagents (e.g., Enterobacter cloacae).

  • Negative Result: Absence of red color or development of a copper color.

  • Diagnostic Utility: Differentiates mixed-acid fermenters (E. coli: MR-positive, VP-negative) from 2,3-butanediol fermenters (Enterobacter: MR-negative, VP-positive).

• Citrate Utilization Test:

  • Purpose: Determines whether an organism can survive and reproduce using sodium citrate as its sole carbon source and inorganic ammonium salts as its sole nitrogen source.

  • Medium: Simmons Citrate Agar, containing sodium citrate, ammonium phosphate, and bromothymol blue as a pH indicator.

  • Procedure: Inoculate the agar slant lightly and incubate.

  • Reactions and Interpretation:

  • Positive Result: Visible bacterial growth on the slant surface accompanied by a medium color change from green (neutral) to blue (alkaline), caused by alkaline ammonium by-products (e.g., Enterobacter cloacae, Klebsiella pneumoniae).

  • Negative Result: Complete lack of bacterial growth; the medium remains green (e.g., Escherichia coli).

  • Diagnostic Utility: Key component of the IMViC profile (Indole, Methyl Red, Voges-Proskauer, Citrate) separating citrate-utilizing genera (Enterobacter, Klebsiella) from non-utilizers (Escherichia coli).

• Urease Test:

  • Purpose: Detects the enzyme urease, which hydrolyzes urea into carbon dioxide ($CO_2$) and alkaline ammonia ($NH_3$).

  • Medium: Urea broth or urea agar containing urea and phenol red.

  • Procedure: Inoculate medium and incubate.

  • Reactions and Interpretation:

  • Positive Result: Development of an intense, bright magenta/pink color caused by ammonia-induced alkalinization (e.g., Proteus mirabilis, Klebsiella pneumoniae).

  • Negative Result: Unchanged yellow or light orange color (e.g., Escherichia coli).

  • Diagnostic Utility: Rapidly identifies Proteus species, which are characteristic rapid urease producers, separating them from other Enterobacteriaceae.

• Hydrogen Sulfide ($H_2S$) Production Test:

  • Purpose: Identifies enzymatic degradation of sulfur-containing organic substrates (such as cysteine or sodium thiosulfate) yielding free $H_2S$ gas.

  • Media Utilized: Triple Sugar Iron (TSI) agar, Sulfide Indole Motility (SIM) medium, and Kligler Iron Agar (KIA).

  • Indicator Chemistry: Media contain iron salts (such as ferrous sulfate); released colorless $H_2S$ gas binds with iron salts to produce an insoluble, visible black ferrous sulfide precipitate.

  • Reactions and Interpretation:

  • Positive Result: Formation of a black precipitate within the deep/butt or along the inoculation stab line (e.g., Proteus mirabilis, Salmonella spp.).

  • Negative Result: No black precipitate formation.

  • Diagnostic Utility: Differentiates $H_2S$-producing genera (Salmonella, Proteus) from non-producing genera (Shigella, Escherichia coli).

• Motility Test:

  • Purpose: Determines flagellar-driven bacterial motility.

  • Media Utilized:

  • Semi-solid Motility Test Medium formulated with a low agar concentration of $0.4\%$ to facilitate locomotion.

  • SIM (Sulfide Indole Motility) Medium, functioning as a combined multi-test medium.

  • Procedure: Inoculate by performing a clean, single-needle vertical stab into the center of the medium, followed by incubation.

  • Reactions and Interpretation:

  • Positive Result: Diffuse, cloudy growth radiating laterally away from the stab line, creating turbidity throughout the agar (e.g., Escherichia coli, Proteus mirabilis).

  • Negative Result: Bacterial growth confined strictly to the original linear stab track with completely clear surrounding medium (e.g., Klebsiella pneumoniae, Shigella spp.).

  • Diagnostic Utility: Distinguishes motile Enterobacteriaceae (Proteus, E. coli) from non-motile genera (Klebsiella, Shigella).

• Additional Diagnostic Tests Utilized for Species-Level Differentiation:

  • Carbohydrate fermentation tests

  • DNase test

  • $\beta\text{-galactosidase}$ and orthonitrophenyl-$\beta\text{-D-galactopyranoside}$ (ONPG) test

  • Decarboxylase and dihydrolase tests

  • Phenylalanine deaminase test

  • Gelatin hydrolysis test