Enterobacteriaceae and Nonfermented

Week 6-7: Enterobacteriaceae and Nonfermented GNB

Competency and Learning Outcomes

  • Essential Procedures: Describe essential collection, transport, and handling procedures.
  • Media Determination: Determine appropriate media for use in isolating pathogenic microorganisms.
  • Gram Staining: Perform and read gram stains of clinical specimens and bacterial isolates.
  • Pathogens Relation: Relate pathogens to disease states.
  • Culture Characteristics: Discuss culture characteristics of selected clinically significant gram-negative bacilli.
  • Confirmatory Tests: Perform selected confirmatory tests for identifying clinically significant gram-negative bacilli.

Enterobacteriaceae Overview

  • Family Characteristics:

    • Large family of bacteria.
    • Approximately 100 recognized species belonging to over 30 genera.
    • Found in various environments: soil, water, and plants.
    • Some species are part of the normal flora in the gastrointestinal (GI) tract of humans and animals.
    • Some are strict pathogens, while others serve as opportunistic pathogens.

  • Classification Methods:

    • Phenotypical Characteristics: Early method of classification.
    • Biochemical Reactions: Used in laboratory diagnostics.
    • Molecular Classification: Current method involves evaluating DNA relatedness.

Characteristics of All Enterobacteriaceae

  • Morphology:
    • All are gram-negative bacilli and coccobacilli.
  • Fermentation:
    • All ferment glucose.
  • Oxidase Test:
    • All are cytochrome oxidase negative (with the exception of Plesiomonas).
  • Nitrate Reduction:
    • All reduce nitrates to nitrites.
  • Aerobic Conditions:
    • All are facultative anaerobes.
  • Growth Medium:
    • Grow well on blood agar, forming large, dull grey colonies.
    • Can be isolated on MacConkey or EMB agar.

Common Characteristics of Enterobacteriaceae

  • Morphological Characteristics:
    • Gram-negative straight rods found on standard microbiological media.
    • Oxidase negative and glucose fermenters.
    • Colony appearance varies—medium to large, entire, greyish-white, convex with potential foul odor, swarming on media, and may present color pigments.

Serological Characteristics

  • Members possess three types of antigenic determinants, useful for serotyping:
    • Somatic Antigen (O):
    • Located in the cell wall.
    • Releases lipopolysaccharide (LPS) or endotoxin, which can cause severe complications like shock, disseminated intravascular coagulation (DIC), fever, tissue injury, and multiple organ failure.
    • Heat stable and serves for serogroup identification (e.g., E. coli serotype 0157:H7).
    • Envelope Antigen (K):
    • Composed of capsular polysaccharide.
    • Heat labile.
    • Flagellar Antigen (H):
    • Present in motile species of Enterobacteriaceae; contributes to serotype determination alongside O antigen.

Isolation Techniques

  • Initial Isolation Media:
    • Selection depends on the specimen source:
    • Use blood agar along with MacConkey or EMB for specimens, excluding stool.
  • Stool Samples:
    • Use differential plates, selective plates, and enrichment broths like selenite.
    • Specific agars include XLD, HE, SS agars.
    • Normal flora coliforms produce lactose-positive colonies.
    • Specialized formulations such as Sorbitol MacConkey for verotoxic E. coli and CIN for Yersinia enterocolitica.
    • For urine samples, utilize differential plate and blood agar with common mediums SBAP/MacConkey biplate, emphasizing colony count.

Biochemical Identification

  • Carbohydrate Fermentation:
    • Testing involves individual sugars or sugar-alcohols.
    • A pH indicator changes color in response to acid production from fermentation (phenol red turns yellow at pH 6.8 or less).
    • Observe for gas production, evident via cracks in the media.
  • Triple Sugar Iron Agar (TSI):
    • Two-reaction chamber (aerobic slant and anaerobic deep).
    • Possible reactions:
    • If glucose is fermented alone, indicates K/A (Red/Yellow).
    • If glucose and lactose and/or sucrose are fermented, indicates A/A (Yellow/Yellow) with gas production.
    • H2S production leads to blackening in the media.
  • TSI Example Interpretations:
    • Different outcomes characterized distinct bacterial identities (e.g., Salmonella, Citrobacter).

IMVC Tests

  • Indole Test:
    • Detects enzyme tryptophanase, which converts tryptophan to indole, pyruvate, and ammonia.
    • Indole is indicated by a red color reaction with Kovach's reagent.
  • Methyl Red Test (MR):
    • Identifies glucose metabolic pathways: MR positive indicates production of mixed acid products (lactic, acetic, formic, and succinic acids).
    • A positive reaction yields a red color.
  • Voges-Proskauer Test (VP):
    • Identifies neutral end-product acetoin.
    • Pink-red color indicates a positive reaction upon adding KOH and alpha-naphthol.
  • Citrate Test:
    • Assesses if citrate can serve as the sole carbon source (positive if growth or blue coloration occurs).

Miscellaneous Biochemical Testing

  • Decarboxylase Activity:
    • Tests enzymatic removal of the carboxyl group from amino acids: lysine, ornithine, or arginine test indicative for specific genera.
    • Produces alkaline amines that change pH indicator color.
    • Validates family differentiation primarily within Klebsielleae.
    • Purple color signifies a positive result.
  • Deaminase Activity:
    • Uses amino acids such as lysine, tryptophan, or phenylalanine to detect amino group removal.
    • Positive deaminase results evident in species like Proteus, Providentia, and Morganella with significantly differing phenotypic expressions.

Specific Identifications

  • Klebsiella:
    • Majority present are non-motile, lactose positive, and display specific TSI responses.
    • Notable species include K. pneumoniae associated with various healthcare-associated infections.
  • Escherichia coli:
    • Frequenctly isolated organism, playing a role in various infections (urinary and neonatal).
    • Diarrheagenic forms include ETEC, EPEC, EIEC, EAEC, and EHEC (notably 0157:H7).
  • Salmonella:
    • Characterized by various serotypes with specific antigenic structures; infections result primarily from contaminated products.
  • Yersinia:
    • Y. pestis known for its pathogenicity in plague; other species associated with gastrointestinal infections.

Antibiotic Susceptibility and Testing

  • Enterobacteriaceae organisms can produce ESBL and AmpC beta-lactamases leading to increased resistance.
  • Testing Requirement: All clinical isolates require susceptibility testing, guiding treatment options including aminoglycosides, beta-lactams, and quinolones.

Summary of Enterobacteriaceae

  • Enterobacteriaceae encapsulates a diverse range of gram-negative rods, with significant pathogenic capacities across various human disease states, necessitating robust microbiological identification and susceptibility testing.