3. Family Enterobacteriaceae

Laboratory Diagnosis of Infectious Diseases

  • Focus: Bacteria from family Enterobacteriaceae

    • Genus Escherichia

    • Group KES

    • Group PPM

    • Genus Yersinia

  • Instructor: Assist. Prof. Denitsa Tsaneva-Damyanova, MD

Enterobacteriaceae

  • Largest family of Gram-negative rods

    • 50 genera and hundreds of species/subspecies

  • Ubiquitous; found in soil, water, vegetation, and as part of normal intestinal flora

  • Responsible for a variety of diseases:

    • 1/3 of all bacteremias

    • 70% of urinary tract infections (UTIs)

    • Many intestinal infections

Pathogen Classification

Primary Pathogens

  • Capable of causing disease in anyone

    • Examples: Shigella, Salmonella, Escherichia, Klebsiella, Yersinia

Opportunistic Pathogens

  • Cause disease under certain conditions or in specific hosts

    • Examples: Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis

Commensal Organisms

  • Normally non-pathogenic but can become pathogenic upon acquiring virulence genes

Medically Important Enterobacteriaceae

  • Citrobacter freundii

  • Citrobacter koseri

  • Enterobacter aerogenes

  • Enterobacter cloacae

  • Escherichia coli

  • Klebsiella pneumoniae

  • Klebsiella oxytoca

  • Morganella morganii

  • Proteus mirabilis

  • Salmonella enterica

  • Serratia marcescens

  • Shigella sonnei

  • Shigella flexneri

  • Yersinia pestis

  • Yersinia enterocolitica

  • Yersinia pseudotuberculosis

Morphological Characteristics

  • Size: 1-5 μm, Gram-negative rods

  • Common antigen: Enterobacterial common Ag

  • Flagella: many peritrichous, some genera non-motile

  • Non-spore forming

  • Appearance: mucoid colonies with prominent capsules (Klebsiella, some Enterobacter and Escherichia strains)

Physiological Characteristics

  • Grow rapidly, aerobically and anaerobically (facultative anaerobes)

  • Growth media:

    • Enriched blood agar

    • Selective: McConkey agar

    • Differential: Endo, Gasner, Levine, EMB agar

  • Sugar fermentation:

    • Lactose fermenters: pink-purple colonies (Escherichia, Klebsiella, Enterobacter, Citrobacter, and Serratia)

    • Non-fermenters: colorless colonies (Proteus, Salmonella, Shigella, Yersinia)

Growth Media

McConkey Agar

  • Selective and differential for Gram-negative bacteria

  • Inhibits growth of Gram-positive bacteria

  • Differentiates lactose fermenters (pink colonies) from non-lactose fermenters (translucent colonies)

EMB Agar

  • Contains dyes that inhibit Gram-positive bacteria

  • Color indicator for lactose fermentation (nucleated colonies for fermenters)

Biochemical Characteristics

  • Resistance to bile salts used for separation of enteric pathogens

  • Reduce nitrate to nitrite

  • Lack of cytochrome C oxidase distinguishes Enterobacteriaceae from other Gram-negative rods

  • Catalase reactions vary among species

Antigenic Structure

  • Main antigen: Heat-stable lipopolysaccharide (LPS)

    • Components:

      1. O polysaccharide (epidemiological classification)

      2. Core polysaccharide (common Ag)

      3. Lipid A (endotoxin activity)

  • Serologic classification based on:

    1. Somatic O antigens

    2. K antigens (capsule)

    3. H proteins (flagella)

Virulence Factors

  • Endotoxin: activity correlates with Lipid A of LPS

  • Capsule: protects against phagocytosis

  • Antigenic phase variation: gene expression control of surface antigens

  • Type III secretion system: delivers virulence factors to host cells

  • Resistance to serum killing and antimicrobial resistance (via plasmids)

Identification of Enterobacteriaceae

  • Series of tests for identification:

    • Phenol red test

    • Phenylalanine agar for deaminase detection

    • Methyl red and Voges-Proskauer tests

    • Citrate utilization test

    • Urease test

    • Catalase test

    • Oxidase test

    • Nutrient gelatin test

Clinical Importance

Common Species

  • E. coli, Klebsiella pneumoniae, and Proteus mirabilis account for 80-95% of isolates

  • Identification methods: biochemical tests or commercial identification systems

Carbohydrate Oxidation and Fermentation

  • Distinguishes fermentative metabolism (Enterobacteriaceae) from oxidative (e.g., Pseudomonas)

IMViC Tests

  • Methyl Red, Voges-Proskauer, Indol, and Citrate tests as part of identification

Example Results

  • Methyl Red:

    • A. Positive - Escherichia coli

    • B. Negative - Klebsiella pneumoniae

  • Voges-Proskauer:

    • A. Positive - Klebsiella pneumoniae

    • B. Negative - Escherichia coli

Enterobacteriaceae Overview

  • Morphology: rod-shaped, motile, non-spore forming

  • Physiology: grows at 4-45°C, ferments sugars

  • Antigen structure: diverse

Escherichia coli Specifics

Morphology

  • Rod-shaped, motile, 2 μm long

  • Non-spore forming but forms microcapsules and pili

Physiology

  • Grows on various media, ferments glucose, maltose, and lactose

  • Oxidase negative

Clinical Relevance

  • Major cause of UTIs, gastroenteritis, and other infections

  • Causes diverse clinical diseases:

    • Enteropathogenic (EPEC)

    • Enterotoxigenic (ETEC)

    • Enteroaggregative (EAEC)

    • Enterohemorrhagic (EHEC)

    • Enteroinvasive (EIEC)

Yersinia Overview

Pathogenesis and Epidemiology

  • Yersinia species cause zoonotic infections

  • Y.pestis causes plague

  • Infection can spread through bites or contaminated food

Diseases Caused by Yersinia

  • Bubonic plague, septicemic plague, yersiniosis, gastrointestinal infections

Diagnosis and Treatment

  • Specimens: feces, urine, blood, etc.

  • Challenges in culture; slow-growing at traditional temps

  • Antibiotic therapy must be susceptibility-guided

  • Common antibiotics: penicillins, cephalosporins, aminoglycosides

Conclusion

  • Enterobacteriaceae play crucial roles in both pathogenicity and normal flora

  • Accurate identification is essential for effective diagnosis and treatment of infections.