13- University Medical Microbiology: Lab 13 - Wound Pathogens Study Guide

Lab 13: Wound Pathogens Introduction

  • Course Identification: Lab 13: Wound Pathogens, University Medical Microbiology.

  • Origin of Material: This lab content was adapted from Beth Behr.

  • Scope of Study: The lab focuses on distinguishing common wound pathogens, identifying enteric bacteria that cause wound infections, and understanding bacterial oxygen requirements.

Distinguishing Between Staphylococcus epidermidis and Staphylococcus aureus

This section outlines a comparative summary of diagnostic tests used to differentiate between two of the most common staphylococcal wound pathogens.

  • Hemolysis on Blood Agar (BAP):
      - Staphylococcus epidermidis: Typically exhibits a gamma reaction (non-hemolytic), though it may occasionally show beta hemolysis.
      - Staphylococcus aureus: Usually exhibits beta hemolysis (complete destruction of red blood cells).

  • Colony Color on Blood Agar:
      - Staphylococcus epidermidis: Colonies appear white.
      - Staphylococcus aureus: Colonies appear yellow or white.

  • Mannitol Fermentation (Mannitol Salt Agar - MSA):
      - Staphylococcus epidermidis: Does not ferment mannitol; the agar remains the color of "watermelon agar."
      - Staphylococcus aureus: Ferments mannitol; positive result is indicated by the agar turning yellow.

  • Coagulase Test:
      - Staphylococcus epidermidis: Negative result (−-), characterized by no clumping.
      - Staphylococcus aureus: Positive result (++), characterized by visible clumping.

  • DNase Production:
      - Staphylococcus epidermidis: Negative (no pink or purple color change).
      - Staphylococcus aureus: Positive (indicated by a pink or purple color reaction).

  • Catalase Production:
      - Staphylococcus epidermidis: Positive (visible bubbles when hydrogen peroxide is added).
      - Staphylococcus aureus: Positive (visible bubbles when hydrogen peroxide is added).
      - Critical Clinical Distinction: It is vital to remember that Streptococcus sp. do not produce catalase; therefore, they will result in "no bubbles" during this test.

Essential Microbiology Vocabulary

The following terms are fundamental to identifying and classifying microbial growth and environmental preferences:

  • Aerobic: Requires oxygen for growth.

  • Microaerophilic: Requires oxygen at lower levels than the atmosphere.

  • Anaerobic: Grows in the absence of oxygen.

  • Facultative: Capable of growing with or without oxygen (e.g., facultative anaerobes).

  • Obligate: Restricted to a specific characteristic (e.g., obligate aerobe must have oxygen).

  • Capnophilic: Requires elevated levels of carbon dioxide (CO2CO_2) to thrive.

  • Psychrophilic: Organisms that prefer cold temperatures.

  • Mesophilic: Organisms that grow best at moderate temperatures (typical human body temperature).

  • Thermophilic: Organisms that prefer heat or high temperatures.

  • Halophilic: Organisms that thrive in high-salt environments.

Detailed Profile of Staphylococcus epidermidis

  • Ecological Role: Part of the normal skin flora.

  • Pathogenicity: Can cause opportunistic infections, particularly in immunocompromised individuals.

  • Clinical Relevance: Known famously for its ability to form biofilms on medical devices such as catheters.

  • Gram Stain & Morphology: Gram-positive.

  • Catalase Status: Positive.

  • Coagulase Status: Negative.

  • Hemolysis: Gamma reaction on Blood Agar Plate (BAP).

  • DNase Production: Negative.

  • Mannitol Fermentation: Negative.

Detailed Profile of Staphylococcus aureus

  • Ecological Role: Frequent wound pathogen; however, it is not always pathogenic. Some individuals carry S.aureusS. aureus as a harmless commensal organism.

  • Gram Stain & Morphology: Gram-positive clusters of spheres.

  • Catalase Status: Positive.

  • Coagulase Status: Positive.

  • Hemolysis: Beta hemolysis on Blood Agar Plate (BAP).

  • DNase Status: Positive.

  • Mannitol Fermentation: Ferments mannitol (turns MSA yellow).

  • Selective Media Growth:
      - MacConkey (MAC): No growth.
      - Bile Esculin (BE): No growth.

Deoxyribonuclease (DNase) Test Evaluation

  • Media Title: DNase Test Agar with Toluidine Blue.

  • Purpose: Used for the detection of deoxyribonuclease activity in microorganisms.

  • Applicability: Specifically useful for identifying staphylococci.

  • Attribution: Documented by Dr. S. S. Khoramrooz on page 1313.

Enteric Bacteria and Wound Infection Profiles (Part 2)

This section covers bacteria often resident in the gastrointestinal tract that can transit to wounds to cause infection. The lab identifies organisms based on culturette numbers 33, 44, 55, and 66.

Escherichia coli

  • Ecological Role: Normal resident of the intestines.

  • Pathogenicity: Can cause wound infections if introduced to the site.

  • Gram Stain & Morphology: Gram-negative rod.

  • Bacterial Metabolism: Facultative anaerobe.

  • Media Reactions:
      - Blood Agar Plate (BAP): Gamma reaction.
      - Mannitol Salt Agar (MSA): No growth.
      - Bile Esculin (BE): No growth.
      - MacConkey (MAC): Ferments lactose.

Pseudomonas aeruginosa

  • Ecological Role: Opportunistic pathogen.

  • Clinical Concern: Noted for advanced antibiotic resistance.

  • Gram Stain & Morphology: Gram-negative rod.

  • Bacterial Metabolism: Obligate aerobe.

  • Media Reactions:
      - Blood Agar Plate (BAP): Beta hemolysis.
      - Mannitol Salt Agar (MSA): No growth.
      - Bile Esculin (BE): No growth.
      - MacConkey (MAC): Colorless growth (indicating no lactose fermentation).

Enterococcus faecalis

  • Ecological Role: Commensal bacteria living in the GI tract of humans.

  • Pathogenicity: Capable of causing disease.

  • Gram Stain & Morphology: Gram-positive chains of spheres.

  • Media Reactions:
      - Blood Agar Plate (BAP): Gamma reaction.
      - Mannitol Salt Agar (MSA): No growth.
      - Bile Esculin (BE): Growth is observed, and the bacteria blacken the agar.
      - MacConkey (MAC): No growth.

Bacterial Oxygen Requirements (Part 3)

  • Diagnostic Tool: Thioglycollate broth.

  • Mechanism of Action: Sodium thioglycolate acts as a reducing agent that "consumes" oxygen.

  • Oxygen Gradient: The concentration of oxygen varies along the depth of the medium due to the combination of chemical reduction and the diffusion of oxygen from the top of the broth. This allows researchers to determine the oxygen preference of an organism based on where it grows in the tube.

Anaerobic Wound Infections

  • Clostridium Species: These are anaerobic organisms known to cause serious wound infections. Because they are anaerobes, they thrive in environments where oxygen is depleted.