Study Notes on Enterobacteriales

Overview of Enterobacteriaceae

  • Large section of the course involving many clinically significant organisms.

  • Change in terminology: Previously referred to as Enterobacteriaceae, now classified under the order Enterobacteriales based on updated taxonomy.

  • Importance of understanding taxonomic classification for accurate terminology but does not substantially change laboratory identification methods.

Major Organisms in Enterobacteriales

  • Common genera:

    • Escherichia (E. coli)

    • Klebsiella

    • Enterobacter

    • Salmonella

    • Shigella

  • Plesiomonas shigelloides:

    • Oxidase positive, gram negative rod, not aligned with classic Enterobacterial profiles; often discussed due to similar clinical relevance.

Laboratory Identification Methods

  • Identifying organisms involves:

    • Culture characteristics

    • Selective and differential media

    • Biochemical testing

    • Susceptibility testing

  • Focus on patterns, identification algorithms, and the context of unexpected results.

Characteristics of Enterobacteriales

  • Morphology: Gram negative rods of varying shapes and sizes.

  • Cell wall structure:

    • Outer membrane present

    • Thin peptidoglycan layer

    • Porins act as channels across the membrane

  • Spore formation: Gram negative rods do not form spores.

Metabolic Characteristics

  • Classification as facultative anaerobes:

    • Ability to ferment glucose in anaerobic conditions (fermentation) and utilize oxygen in aerobic conditions (oxidation).

    • Acid byproducts from fermentation indicate a pH change (color change in pH indicators from red to yellow).

  • Oxidase test: Most Enterobacteriaceae are oxidase negative, except for Plesiomonas.

  • Nitrate reduction:

    • All Enterobacteriales can reduce nitrates to nitrites via nitrate reductase.

Motility

  • Most Enterobacteriales are motile except for:

    • Shigella species

    • Klebsiella species (except K. aerogenes)

    • Yersinia species

  • Mnemonic for non-motile: S.K.I. group (Shigella, Klebsiella, Yersinia).

Habitat and Pathogenicity

  • Ubiquitous presence in various environments (gastrointestinal tracts, hospital equipment, soil).

  • Pathogenic classifications:

    • Opportunistic pathogens: (e.g., Citrobacter, Enterobacter, Klebsiella, Proteus, Serratia)

    • Significant virulence factors but usually require special conditions (immunocompromised hosts, open wounds) to initiate disease.

    • Intestinal pathogens: (e.g., Shigella, Salmonella, Yersinia)

    • Generally have larger virulence factors, often not normal flora of humans.

  • Escherichia coli: Considered normal flora but can be opportunistic under certain circumstances.

Pathogenesis and Virulence Factors

  • Virulence factors include:

    • Production of endotoxins and enterotoxins.

    • Invasive enzymes that facilitate spread within the host.

    • Adhesive properties that make treatment difficult.

  • Antibiotic resistance:

    • Includes extended spectrum beta-lactamases (ESBLs).

  • Antigenic factors:

    • O antigen (somatic antigen): heat-stable.

    • H antigen (flagellar antigen): heat-stable.

    • K antigen (capsular antigen): heat-labile.

Media and Biochemical Tests

  • Various media used for growth and identification include:

    • McConkey's agar: Selective for gram negatives, differential for lactose fermentation.

    • EMB agar: Similar to McConkey's, contains methylene blue to inhibit gram positives.

    • Hektoen enteric agar: Selective and differential for stool cultures.

    • XLD agar: Displays fermentation ability with xylose, contains lysine for decarboxylation detection, can indicate H2S production.

    • Gram negative broth: For isolating Enterobacteriales from fecal specimens.

Five Tube Setup for Biochemical Testing

  1. Triple Sugar Iron Agar (TSI):

    • Tests for glucose, lactose, and sucrose fermentation; H2S production.

    • Reporting: Slant over butt (K/K, K/A, A/A), gas production status, H2S production status.

  2. Lysine Iron Agar (LIA):

    • Tests for lysine decarboxylase and deaminase and H2S production.

    • Reporting format similar to TSI.

  3. MIO Tube (Motility, Indole, Ornithine):

    • Motility determined by diffusion from stab line, indole production using tryptophanase, ornithine decarboxylation.

  4. MR-VP Test:

    • Methyl red: detects acid production after fermentation.

    • Voges-Proskauer: detects acetone production (butylene glycol fermentation).

  5. Urease Test:

    • Detects urea hydrolysis; pH change indicates urease activity.

Additional Identification Tests

  • Decarboxylase tubes: Detect decarboxylation of amino acids.

  • Phenylalanine deaminase agar (PDA): Differentiates Morganella, Proteus, and Providencia.

  • Nitrate reductase testing: Detects nitrate reduction and gas presence via durham tube.

Modern Identification Techniques

  • MALDI-TOF: Used for rapid identification in clinical labs but has limitations regarding antimicrobial susceptibility determination.

  • Molecular methods can identify resistance genes, but phenotypic antimicrobial susceptibility testing remains the gold standard.

  • Emphasis should be on culture characteristics, biochemical testing, and susceptibility interpretation for both exams and labs.