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Enterobacteriaceae Overview

Jul 5, 2025

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Overview

This lecture covers Gram-negative enteric bacilli, focusing on the Enterobacteriaceae family. It details their classification, general characteristics, laboratory identification, key biochemical tests, antigenic markers, virulence factors, and clinical significance. The lecture is structured into three main parts: (1) methods of identification and biochemical tests, (2) opportunistic members, and (3) true enteric pathogens. The lecture also emphasizes the importance of recognizing exceptions and unique features among the Enterobacteriaceae, and provides guidance on laboratory procedures, result interpretation, and preparation for practical activities.

Classification of Gram-Negative Enteric Bacilli

  • Enterobacteriaceae are divided into two main groups:
    • Coliforms (Lactose Fermenters):
      • Normal inhabitants of the colon and abundant in the intestinal tract.
      • Includes genera such as Escherichia and Enterobacter.
    • Non-Lactose Fermenters:
      • Further subdivided into:
        • Invasive organisms: Salmonella, Shigella, Yersinia.
        • Opportunistic organisms: Proteus, Providencia, Morganella.
  • The family is organized into seven tribes, each containing specific genera and species (e.g., Escherichieae, Salmonelleae, Citrobactereae, etc.), which are important for identification and clinical diagnosis.
  • A table (not shown here) summarizes the seven tribes and their representative genera and species. Reviewing this table is essential for understanding the relationships and identification of Enterobacteriaceae.

General Characteristics of Enterobacteriaceae

  • Morphology:
    • Gram-negative, rod-shaped (bacilli), often short or filamentous.
    • Most are motile with peritrichous flagella, except Shigella, Klebsiella, and Yersinia (non-motile).
  • Habitat:
    • Primarily inhabit the intestinal tract, except Yersinia pestis.
  • Metabolism:
    • Facultative anaerobes (can grow with or without oxygen).
    • All ferment glucose anaerobically.
    • All reduce nitrate to nitrite, except a few (e.g., Pantoea agglomerans, some Arsenia).
  • Enzyme Activity:
    • Catalase positive (except Plesiomonas).
    • Oxidase negative.
  • Capsulation:
    • Most are non-encapsulated.
    • Exceptions: Klebsiella, some Enterobacter, and some Salmonella strains are encapsulated.
  • Temperature Tolerance:
    • Some can grow at low temperatures (1–5°C), such as Serratia and Yersinia.
  • Other Features:
    • Do not form spores.
    • Most are motile, except for the noted exceptions.
    • Some members can be differentiated by their ability to grow at lower temperatures or by their encapsulation status.

Laboratory Identification and Biochemical Tests

  • Primary Isolation Media:
    • Eosin Methylene Blue (EMB) Agar:
      • Inhibits non-enteric bacteria using eosin Y and methylene blue.
      • E. coli shows a green metallic sheen.
      • Lactose fermenters: colored colonies; non-fermenters: colorless.
    • MacConkey Agar:
      • Contains crystal violet and bile salts to inhibit non-enterics.
      • Lactose fermenters: pink/red colonies; non-fermenters: colorless.
    • Deoxycholate Citrate Agar (DCA):
      • Selective for enterics using sodium deoxycholate and citrate.
      • Lactose fermenters: pink colonies; non-fermenters: colorless.
  • Selective Media for Pathogen Isolation:
    • Hektoen Enteric Agar (HEA):
      • Bile salts inhibit non-enterics.
      • Lactose fermenters: yellow colonies; non-fermenters: green/blue-green.
    • Xylose Lysine Deoxycholate (XLD) Agar:
      • Detects fermentation of lactose, sucrose, and xylose.
      • Lactose fermenters: yellow colonies; non-fermenters: red.
    • Salmonella-Shigella (SS) Agar:
      • Selective for Salmonella and Shigella.
      • Lactose fermenters: pink/red colonies; non-fermenters: colorless.
  • Culture Media Use:
    • Specimens are first inoculated on primary isolation media.
    • Selective media are used to further isolate and identify specific pathogens.
    • Inhibitory agents in these media prevent the growth of non-target organisms, allowing for easier identification of enteric pathogens.
  • Key Biochemical Tests:
    • Oxidase Test:
      • Detects cytochrome c oxidase.
      • Enterobacteriaceae: oxidase negative (no color change).
      • Positive: blue or purple color within 10 seconds.
    • Nitrate Reduction Test:
      • Most reduce nitrate to nitrite.
      • Positive for most species except Pantoea agglomerans and some Arsenia.
      • Uses 0.1% nitrate in the medium; positive result is the formation of a red color after adding reagents.
    • Carbohydrate Utilization Tests:
      • Triple Sugar Iron Agar (TSIA):
        • Contains glucose (0.1%), lactose (1%), and sucrose (1%).
        • Differentiates bacteria by sugar fermentation, H₂S, and gas production.
        • Four main result groups based on slant/butt color and H₂S.
        • Yellow (acid) slant/butt: A/A; red (alkaline) slant: K/A; blackening indicates H₂S production.
        • Gas production is indicated by bubbles or cracks in the medium.
      • Kligler Iron Agar (KIA):
        • Contains glucose (0.1%) and lactose (1%).
        • Results interpreted by slant and butt color (alkaline/red or acid/yellow).
        • Similar to TSIA but does not contain sucrose.
    • Sugar Fermentation Test:
      • Determines ability to ferment specific carbohydrates.
      • Positive: yellow color (acid production).
      • All Enterobacteriaceae ferment glucose.
      • Uses peptone media with a pH indicator (phenol red).
    • Urease Test:
      • Detects hydrolysis of urea to ammonia.
      • Positive: pink/red color (commonly using Christensen’s urea agar).
      • Indicates the presence of urease enzyme.
    • Lysine Decarboxylation Test:
      • Detects ability to decarboxylate lysine, producing an alkaline (purple) result.
      • pH indicator: bromocresol purple.
      • Positive: purple color; negative: yellow or orange.
    • Phenylalanine Deamination (PAD) Test:
      • Detects production of phenylpyruvic acid.
      • Positive: green color after ferric chloride addition.
      • Differentiates among urease-positive Gram-negative bacilli.
      • Performed by adding 10% ferric chloride after incubation.
    • Enzyme Tests:
      • DNAse, lipase, and gelatinase tests.
      • Only Serratia is positive for all three.
  • Grouping by TSIA Results:
    • Bacteria are grouped into four categories based on TSIA results:
      • K/A, H₂S positive
      • A/A, H₂S positive
      • K/A, H₂S negative
      • A/A, H₂S negative
    • Further differentiation uses PAD, lysine decarboxylation, indole, and other tests.
    • For example, K/A H₂S positive may indicate Salmonella or Proteus; A/A H₂S positive may indicate Citrobacter; K/A H₂S negative may indicate Shigella or Providencia; A/A H₂S negative may indicate E. coli or Enterobacter.
    • Additional tests such as citrate utilization, motility, and indole help further distinguish between genera and species.
  • Interpretation and Differentiation:
    • After grouping by TSIA, further tests (PAD, lysine decarboxylation, indole, citrate, motility) are used to identify the specific genus and species.
    • Tables and charts summarizing these results are essential for quick reference and should be reviewed and memorized.
    • The lecture encourages taking screenshots or printouts of these tables for study and review.

IMViC Reactions

  • Indole Test:
    • Detects tryptophanase activity.
    • Positive: red ring after adding Kovac’s or Ehrlich’s reagent.
    • Media: tryptophan agar, SIM (sulfide-indole-motility) agar, or motility-indole-ornithine agar.
  • Methyl Red Test:
    • Detects stable acid production from glucose fermentation (mixed acid pathway).
    • Positive: red/pink after adding methyl red reagent.
    • Media: MR-VP broth.
  • Voges-Proskauer Test:
    • Detects butylene glycol (acetoin) production.
    • Positive: red/pink after adding VP reagents.
    • Media: MR-VP broth.
  • Citrate Utilization Test:
    • Detects ability to use citrate as sole carbon source.
    • Positive: blue color in the medium (Simmon’s citrate agar).
  • Interpretation:
    • IMViC patterns are essential for distinguishing closely related genera:
      • E. coli: ++--
      • Enterobacter: --++
      • Klebsiella: --++
      • Citrobacter: variable
    • These patterns help differentiate species within the Enterobacteriaceae.
    • IMViC results are often combined with other biochemical tests for accurate identification.
    • The lecture suggests memorizing these patterns and reviewing summary charts for practical use.

Antigenic Markers & Virulence Factors

  • O (Somatic) Antigen:
    • Heat-stable lipopolysaccharide (LPS) component.
    • Present in most species except some Proteus.
    • Detected by serologic testing.
  • H (Flagellar) Antigen:
    • Heat-labile protein.
    • Present only in motile species.
    • Absent in non-motile organisms (e.g., Shigella, Klebsiella).
  • K (Capsular) Antigen:
    • Found in encapsulated strains (e.g., Klebsiella, some E. coli, Salmonella).
    • Associated with increased virulence.
  • Virulence Factors:
    • Endotoxin (Lipid A):
      • Produced by all Enterobacteriaceae.
      • Triggers fever and inflammation by inducing pyrogen release from macrophages.
    • Capsule:
      • Protects against phagocytosis.
      • Present in encapsulated species.
    • Exotoxins:
      • Includes enterotoxins and Shiga toxin.
      • Cause fluid and electrolyte loss in the intestines.
    • Invasiveness:
      • Especially in non-lactose fermenters like Salmonella, Shigella, and Yersinia.
      • Enables bacteria to invade and damage host tissues.
    • Type III Secretion System:
      • Major virulence mechanism.
      • Allows bacteria to inject effector proteins into host cells, aiding infection and immune evasion.
      • Considered a key factor in the pathogenicity of many enteric bacteria.
  • Antigenic Testing:
    • Serologic methods are used to detect O, H, and K antigens for identification and epidemiological studies.

Key Terms & Definitions

  • Coliforms: Lactose-fermenting Enterobacteriaceae, normal flora of the intestine.
  • TSIA/KIA: Media used to differentiate bacteria by sugar fermentation and H₂S production.
  • IMViC: Set of four biochemical tests (Indole, Methyl Red, Voges-Proskauer, Citrate) for identifying enteric bacteria.
  • O Antigen: Somatic, heat-stable antigen of LPS.
  • H Antigen: Flagellar, heat-labile antigen, present in motile species.
  • K Antigen: Capsular antigen in encapsulated strains.
  • PAD Test: Phenylalanine deamination test, differentiates certain genera based on phenylpyruvic acid production.
  • Primary Isolation Media: Culture media used to initially grow and isolate enteric bacteria from clinical specimens.
  • Selective Media: Specialized media used to isolate specific pathogens by inhibiting the growth of non-target organisms.

Action Items / Next Steps

  • Review and memorize biochemical test results and patterns (TSIA/KIA, IMViC, PAD, urease, lysine decarboxylation) for key Enterobacteriaceae species.
  • Study tables and charts summarizing biochemical characteristics and test results for the seven tribes and common genera.
  • Prepare for laboratory activities, including the use of primary and selective media for isolation and identification.
  • Be ready for the next lecture, which will focus on opportunistic Enterobacteriaceae and true enteric pathogens.
  • Take screenshots or create summary tables from lecture slides for quick reference and review before practical exams or discussions.
  • Pay special attention to exceptions and unique features of certain genera (e.g., non-motility, encapsulation, temperature tolerance).
  • Practice interpreting laboratory results and differentiating species using the described biochemical tests and media.
  • Familiarize yourself with the antigenic markers and virulence factors, as these are important for understanding pathogenesis and for serologic identification.
  • Review the laboratory procedures for each test, including the expected results and their interpretation, to ensure accuracy during practical sessions.
  • Note any updates or additional recordings posted by the lecturer, and allocate time to review them before the next face-to-face or laboratory session.
  • Remember to check for any laboratory activities or discussions scheduled for the upcoming week, and be prepared to participate actively.

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