Nonfermentative Gram-Negative Bacilli and Similar Bacteria

Chapter 13: Nonfermentative Gram-Negative Bacilli & Similar Bacteria

General Characteristics

  • Metabolic Properties

    • Nonfermentative bacteria metabolize carbohydrates through oxidative pathways.
    • These bacteria require oxygen as the final hydrogen ion acceptor for their metabolic processes.
    • They are unable to ferment glucose or other carbohydrates, evidenced by Triple Sugar Iron (TSI) test indicating K/K (alkaline/alkaline) results.

  • Oxygen Requirements

    • These organisms are classified as obligate aerobes, which means they cannot survive without oxygen.

  • Enzymatic Characteristics

    • Oxidase positive, confirming the presence of cytochrome c oxidase enzyme.

  • Growth Characteristics

    • Exhibit better growth on Blood Agar Plates (BAP) compared to MacConkey Agar (MAC) and Eosin Methylene Blue (EMB) agar.

Environmental Presence

  • Nonfermentative Gram-Negative Bacilli (GNB) are ubiquitous in various natural environments:

    • Commonly found in:
    • Soil
    • Plants
    • Decaying vegetation

  • Contaminants

    • Frequently identified as contaminants in:
    • Healthcare equipment
    • Water baths
    • Sinks

  • Colonization

    • These bacteria are not typical members of the human microbiota but may colonize skin and mucous membranes.

  • Clinical Significance

    • Associated with healthcare-associated infections and opportunistic infections including:
    • Urinary Tract Infections (UTIs)
    • Wound infections
    • Pneumonia
    • Sepsis
    • Meningitis
    • Common in patients with:
    • Prolonged antibiotic therapy
    • Invasive medical procedures
    • Trauma, burns, and wounds

Example: Pseudomonas aeruginosa

  • Exhibits high levels of antibiotic resistance, necessitating prolonged antibiotic treatment.

Oxidative Fermentative (OF) Media

  • Purpose: Designed for isolating non-fermenters.
  • Composition: High carbohydrate and low peptide concentrations; enhances acid end product production.
  • Indicator: Bromthymol blue is used alongside carbohydrates to indicate metabolic activity.
    • Results Interpretation:
    • Oxidative: Positive (yellow) in open tubes only.
    • Fermentative: Positive (yellow) in both open and closed tubes.
    • Asaccharolytic (nonoxidative): Negative (green or blue-green) in both open and closed tubes.

Nitrate Reduction Test

  • Processes involved: Reduction of nitrate (NO3) to nitrite (NO2) or nitrogen gas (N2).
  • Reagens Used: Sulfanilic acid and α-naphthylamine; a red color indicates positive reduction.
  • Procedure:
    • If no color change occurs, zinc is added:
    • Red = Negative for reduction.
    • If no change occurs, confirms the presence of nitrogen gas as the end product.

Identification Methods

  • Key Features in Identifying Non-Fermentative GNB:
    • Patterns of carbohydrate utilization (O-F test).
    • Motility patterns (cellular morphology, flagella type, number, arrangement).
    • Growth on MacConkey agar.
    • Decarboxylation reactions.
    • Modified indole test.
    • Nitrate reduction capabilities.
    • Nitrogen gas production.
    • Acetamide utilization.
    • Urease activity.

Pseudomonadaceae Family Characteristics

  • Classification: Continues to evolve, primarily grouped by ribosomal RNA (rRNA). Members include:

    • Pseudomonas aeruginosa
    • Pseudomonas fluorescens
    • Pseudomonas putida
    • Pseudomonas stutzeri

  • Historical Context: Isolated first in 1882 from soldier wounds showing green and blue pus.

  • Physical Characteristics:

    • Typically motile with polar flagella (except for Burkholderia mallei).
    • Capable of oxidizing glucose and other carbohydrates and are strict aerobes.
    • Generally cytochrome oxidase positive.

Specific Characteristics of Pseudomonas aeruginosa

  • Environmental Presence: Commonly found in natural water sources, taps, soaps, and disinfectants.
  • Infective Capacity: Major causative agent of non-fermentative GNR infections (75-80% of all such isolates) and accounting for 10% of healthcare-associated infections (HAIs).
  • Opportunistic Pathogen: Not part of normal human flora, yet can colonize skin and gastrointestinal tracts.
  • Antibiotic Resistance: Displays intrinsic and extrinsic resistance to many antibiotics; requires special consideration in treatment.

Unique Pigments

  • Pyocyanin: Blue pigment that is soluble in water and chloroform, unique to P. aeruginosa.
  • Pyoverdin: Yellow to yellow-green pigment that fluoresces under UV light.
  • Colony Morphology: Produces distinctive blue-green pigmented colonies on certain media, some strains may yield pyrubin (red) or pyomelanin (brown).
  • Odor: Characteristically emits a fruity odor reminiscent of overripe grapes.

Identification Methods for Pseudomonas aeruginosa

  • Culture Characteristics:

    • Large, rough, dull, greyish colonies on blood agar.
    • Shiny, opalescent colonies resembling ground lead with a feathery edge and narrow zone of beta-hemolysis.
    • Green pigment observation in cultures.
    • Characteristic odor noted during growth.

  • Additional Tests:

    • Oxidase positive test results.
    • TSI reaction yielding K/K and growth on slant.
    • Non-fermentation of maltose but capable of oxidizing glucose.
    • Mucoid strains produce alginate (capsule).
    • Positive nitrate reduction.

Virulence Factors of Pseudomonas aeruginosa

  • Adhesion Mechanisms: Includes flagella and fimbriae for attachment to host tissues.
  • Immune Evasion and Tissue Damage: Utilizes the following mechanisms:
    • Alginate capsule to evade immune response.
    • Exotoxin A (inhibits protein synthesis).
    • Elastase (degrades host tissues).
    • Pyoverdin and Pyocyanin (specific pigments aiding in pathogenicity).
    • Hemolysin and leukocidin affecting red and white blood cells, respectively.
    • Lipopolysaccharide (LPS) contributing to inflammatory response.

Infections Associated with Pseudomonas aeruginosa

  • Opportunistic & Healthcare-Associated Infections:

    • Common in patients with burns, cystic fibrosis, and leukemia; particularly at sites where moisture is abundant (e.g., indwelling catheters, tracheostomies).
    • Types of infections include:
    • Pneumonia
    • UTI
    • Keratitis
    • Endocarditis
    • Meningitis
    • Dermatitis from whirlpool use.
    • Mucoid Strain: Particularly harmful to patients with cystic fibrosis where it activates alginate gene expression.

  • Community-Acquired Infections: Includes keratitis/corneal ulcers (frequent in contact lens wearers), otitis externa (swimmer's ear), and folliculitis (hot tub-related).

Other Groups within Pseudomonadaceae

Fluorescent Group: Characteristics

  • Members: Produce pyoverdin pigments.
  • Identifying Agents:
    • Pseudomonas fluorescens: Associated with food spoilage; rare urinary tract infection.
    • Pseudomonas putida: Rarely causes septicemia; nonproteolytic.

Stutzeri Group: Soil Denitrifiers

  • Utilization of ammonium (NH4) and acetate as nitrogen and carbon sources, respectively.
  • Rare causes of infections such as otitis media and pneumonia in immunocompromised individuals.
  • Members include:
    • Pseudomonas stutzeri
    • Pseudomonas mendocina.

Pseudomallei Group

  • Contains Burkholderia species known for hospital-associated infections.
    • Typically Finds: Found in environmental sources such as soil and water.
    • Notable species include Burkholderia cepacia and Burkholderia pseudomallei, known for severe opportunistic infections, especially in cystic fibrosis patients and those with chronic granulomatous disease.

Burkholderia cepacia Complex

  • Growth Characteristics: Appears as yellow, serrated colonies on sheep blood agar; non-lactose fermenter on MacConkey agar.
  • Resistance: Contains intrinsic resistances to aminoglycosides and polymyxins, and acquired resistance to ß-lactams.

Burkholderia mallei

  • Associated Condition: Glanders, affecting primarily equids (horses, goats, etc.), presents as nodules in lungs and upper respiratory tract.
  • Transmission via direct contact or inhalation; recognized as a potential bioterrorism agent.

Burkholderia pseudomallei

  • Disease: Melioidosis or Whitmore's disease characterized by pneumonia, septic shock, and various skin infections; recognized as a potential bioterrorism agent due to aerosolized capability.

Ralstonia species

  • Pathogenicity: Generally non-pathogenic but can be clinically significant; includes:
    • R. picketii and R. insidiosa.

Stenotrophomonas maltophilia

  • Ecology: Found in various environments, primarily opportunistic pathogens, associated with HAIs.
  • Clinical significance in pneumonia, septicemia, and infections related to medical devices; resistant to multiple antibiotics.

Colony Morphology

  • Colonies appear large, smooth, and glistening on TSA, with a distinctive ammonia-like odor, indicative of Stenotrophomonas maltophilia.

Acinetobacter Species

  • Characteristics: GN coccobacilli, colonize human skin, and are non-motile but can cause various HAIs, including UTIs and respiratory infections.
  • Identification: Distinguishable by non-oxidative metabolism and specific growth patterns on MacConkey agar.

Achromobacter and Alcaligenes

  • General Notes: These genera share similar ecological niches and opportunistic pathogenicity, with specific identification criteria distinguishing them:
    • Achromobacter species: Found in water and GI tract but can be rare opportunistic pathogens.
    • Alcaligenes species: Typically associated with respiratory and wound infections; noted for distinct fruity odors.

Moraxella

  • Clinical Relevance: Includes species such as M. catarrhalis linked with respiratory tract infections and conjunctivitis.

Psychrobacter

  • Pathogenicity: Rarely isolated but can be associated with infections like bacteremia and meningitis; psychrotolerant microorganisms found in cold environments.

Flavobacteriaceae Family

  • Characteristics: Nonmotile, oxidase-positive bacteria typically found in a variety of wet environments; produce yellow pigments and include genera like Chryseobacterium and Elizabethkingia.

Chryseobacterium and Elizabethkingia

  • Clinical Significance: Opportunistic pathogens commonly associated with healthcare settings, known for their resistance profiles and ability to cause severe diseases like meningitis and endocarditis, especially in immunocompromised patients.

Summary of Identification and Characteristics

  • Tables and Illustrative Figures: Review of key differentiating characteristics, reagent tests, and cultivation requirements for the identification of significant non-fermentative Gram-negative rods is essential for precision in diagnostics and treatment.