Health and Community 3: Identifying Antibiotic-Resistant Pseudomonas aeruginosa

Lecture Overview

  • Recap of Lecture 2: Antibiotic resistance, multi-drug resistance (MDR), superbugs, and drivers of antibiotic resistance.
  • Importance of identifying MDR strains using phenotypic methods (biotyping, antibiogram).
  • Genotypic methods are necessary for differentiating variants of MDR strains.
  • Example: Pseudomonas aeruginosa as a gram-negative superbug.
  • Focus on RFLP and PFGE as genotypic methods.
  • Resistance to carbapenem (beta-lactam antibiotic) and quinolones.
  • Clinical case study provided.

Clinical Case: Pseudomonas aeruginosa Infection

  • 38-year-old male with fever, chills, pelvic pain, and UTI symptoms.
  • No history of STDs or urinary trauma.
  • Developed septicemia after ten days in the hospital.
  • Pseudomonas aeruginosa identified in blood samples.
  • Questions: How was Pseudomonas aeruginosa identified, and what was the source of infection, given its antibiotic resistance?

Pseudomonas aeruginosa: General Information

  • Gram-negative bacilli.
  • Opportunistic pathogen: Infects immunocompromised individuals.
  • Causes pneumonia, septic shock, UTIs, GI infections, skin infections, etc.
  • Diffuse bronchopneumonia (lung infection).
  • Otitis externa (ear infection).
  • Colonization of burns (burns are susceptible to Pseudomonas aeruginosa).
  • Common cause of nosocomial infections (10% of cases).

Sources of Pseudomonas aeruginosa

  • Water sources (public toilets, washbasins, rivers).
  • Animals (paws and hairs of pets).
  • Farms (due to diesel and jet fuel usage, as it uses these as a carbon source).

Identification of Pseudomonas aeruginosa (Phenotypic Methods)

  • Gram stain: Gram-negative.
  • Distinguishing from other gram-negative bacteria:
    • Gram-Negative Cocci:
      • Neisseria meningitidis (meningitis)
      • Neisseria gonorrhoeae (gonorrhea)
    • Gram-Negative Bacilli:
      • Lactose Utilization:
        • Lactose-positive: Most Enterobacteriaceae
        • Lactose-negative: Further differentiated
      • Oxidase Test (for lactose-negative bacilli):
        • Oxidase-positive: Vibrio cholerae, Pseudomonas aeruginosa (strict aerobes)
        • Oxidase-negative: Bacteroides (anaerobes)
      • Differentiation of Pseudomonas aeruginosa from Vibrio cholerae: Pseudomonas aeruginosa cannot ferment glucose; Vibrio cholerae can.

Identifying the Infection Source (Genotypic Methods)

  • Samples taken from household supply, hot bathtub, and unrelated source.
  • All were positive for Pseudomonas aeruginosa using phenotypic methods.
  • Genotypic methods needed to identify the specific, resistant strain.
  • RFLP (Restriction Fragment Length Polymorphism) and PFGE (Pulsed-Field Gel Electrophoresis) used.

RFLP (Restriction Fragment Length Polymorphism)

  • Detection of variations in homologous DNA sequences.
  • Uses restriction enzymes to digest DNA.
  • Analysis of the number and size of resulting DNA fragments.
  • Restriction enzymes cleave DNA at specific recognition sites.
  • Digesting similar DNA fragments with restriction enzymes produces different fragment patterns due to variations in restriction sites.
  • Example:
  • Two DNA fragments with a single nucleotide difference (T instead of A).
  • Restriction enzyme digests the first DNA at five sites, producing six fragments.
  • The second DNA is digested at only four sites (due to the nucleotide difference), producing five fragments.

Gel Electrophoresis

  • Separates DNA fragments based on size.
  • DNA is negatively charged and migrates towards the positive electrode.
  • Smaller fragments move faster through the gel matrix.
  • Different-sized fragments line up according to size.

Combining RFLP and Gel Electrophoresis

  • DNA obtained from bacterial strain and digested with restriction enzymes (e.g., BAP H1, which recognizes GGATC sequences).
  • Digestion produces different fragment profiles based on nucleotide sequence differences.
  • DNA fragments are separated using gel electrophoresis.
  • DNA is loaded into a gel with a negative charge, and a current is applied.
  • DNA fragments migrate, with smaller fragments moving faster.
  • A stain (e.g., ethidium bromide) is used to visualize DNA fragments under UV light.

Limitations of Traditional Agarose Gel Electrophoresis

  • Good resolution for DNA fragments smaller than 20 kilobase pairs (kbps) or 20,000 base pairs.
  • Larger fragments migrate at similar rates, making differentiation difficult.
  • Alternative: Pulse Field Gel Electrophoresis (PFGE).

PFGE (Pulse Field Gel Electrophoresis)

  • Modified agarose gel electrophoresis.
  • Developed by Schwartz and Cantor in 1984.
  • Voltage applied in three directions: straight, 60 degrees left, and 60 degrees right.
  • The current is alternated between these directions; the duration and strength are equal.
  • DNA migrates in a net forward direction but follows a "straight, left, right" pattern.
  • Improves separation and resolution, allowing larger fragments to migrate at slightly different rates.
  • Takes longer than traditional gel electrophoresis.

Advantages and Disadvantages of PFGE

  • Advantages:
    • Good for analyzing recent evolutions and hospital outbreaks.
    • Distinguishes between different strains of the same species.
    • Faster than other genotypic methods.
  • Disadvantages:
    • Requires a lot of time for DNA to migrate.
    • Difficult to reproduce; results can be subjective.

Case Study Results

  • DNA from four isolates analyzed using RFLP and PFGE.
  • Isolates B and C were identical.
  • Isolate A (household water) was different (not the superbug).
  • Isolate D (patient's blood) matched the strain from the hot bathtub.
  • Source of infection: hot bathtub filled with water from a local stream (contaminated with Pseudomonas aeruginosa).
  • Patient purchased a new hot bathtub and used stream water.

Recommendations

  • Ensure hot tub water is clean.
  • Use clean water sources.
  • Maintain adequate chlorine concentration (2-3 parts per million) to kill Pseudomonas aeruginosa.

Importance of Identifying Antibiotic-Resistant Strains

  • Resistance against carbapenem (beta-lactam antibiotic) and quinolones.

Carbapenem Resistance

  • Carbapenem belongs to the beta-lactam antibiotic class.
  • Inhibits peptidoglycan synthesis.
  • Broad-spectrum antibiotic (effective against gram-negative and gram-positive bacteria).
  • Emergence and spread of carbapenem-resistant Pseudomonas aeruginosa strains.
  • Mechanisms of resistance:
    • Downregulation of pores (reduced antibiotic entry).
    • Upregulation of efflux pumps (increased antibiotic expulsion).
    • Acquisition of carbapenemases (enzymes that break down carbapenem).

Carbapenemases

  • Carbapenem is resistant to most beta-lactamases due to its structure.
  • Carbapenemases are beta-lactamases that can break down carbapenem.
  • Resistance to carbapenem is a major concern as it is the last line of defense.
  • Resistance can spread to other species (Enterobacteriaceae, Acinetobacter) via plasmids.

Detecting Carbapenemase-Producing Bacteria

  • Modified Hodge test (not a genetic method).
  • Similar to antibiotic susceptibility testing.
    1. A bacteria strain that is sensitive to carbapenem is spread all over the plate
    2. A filter disc that is soaked with the carbapenem antibiotic is placed in the middle
    3. Another bacteria, again, that is the the same bacteria that is sensitive to this antibiotic is streaked along a line through the middle of the antibiotic filter disc to the outside of the plate.
    4. A third bacteria, the test bacteria of concern is streaked along a line through the other side of the antibiotic filter disc.
    5. If after incubation the streak of test bacteria shows a clover-leaf shape then that indicates that the test is carbapenem resistant through the production of carbapenemases.
    6. If after incubation the test bacteria does not show a shape then that means there a mechanism that differs from the production of carbapenemases.

Quinolone Resistance

  • Resistance against quinolones (e.g., ciprofloxacin).
  • Mechanisms of resistance:
    • Mutations in DNA gyrase subunit A (target for ciprofloxacin).
    • Acquisition of plasmid-encoded efflux pumps.

Revision Points

  • Phenotypic methods for identifying Pseudomonas aeruginosa.
  • Principles of RFLP and PFGE.
  • Mechanisms of resistance against carbapenem and quinolones.
  • Importance of detecting carbapenemase-producing bacteria using the modified Hodge test.