Why are some organisms gram-variable?

Why Some Organisms Are Gram Variable

Some organisms are gram variable due to structural changes in their cell walls during different growth phases, antimicrobial exposure, or inherent cell wall composition that affects their ability to retain crystal violet dye during the Gram staining procedure. 1, 2

Mechanisms of Gram Variability

Cell Wall Structure and Growth Phase

• Gram variability occurs in bacteria that have cell wall structures of intermediate complexity between typical gram-positive and gram-negative organisms 1
• During different growth phases, these bacteria may demonstrate different staining characteristics:
  • In early growth phases (lag phase), many gram-variable organisms stain gram-positive
  • As cultures enter mid-exponential phase, 10-30% of cells may stain gram-negative 2
  • By stationary phase, the percentage of gram-negative staining cells increases to 15-40% 2

Two Main Patterns of Gram Variability

1. Actinomyces-Arthrobacter-Corynebacterium-Mycobacterium-Propionibacterium Group:

   • Cells become gram-negative primarily during cell division
   • Dividing cells are more fragile at the septum formation site
   • As cultures age, additional cells become gram-negative due to lesions in side walls 2

2. Bacillus-Butyrivibrio-Clostridium Group:

   • These bacteria have more complex cell walls with an S-layer
   • Initially gram-positive during lag and early exponential phases
   • As growth slows, the wall fabric beneath the S-layer becomes thinner and diffuse
   • By stationary phase, cultures may appear almost entirely gram-negative 2

Effect of Antimicrobial Therapy

• Antimicrobial treatment can significantly alter Gram staining characteristics
• Gram-positive bacteria may become gram-variable after exposure to antibiotics 3
• This can complicate identification in clinical specimens from patients receiving treatment

Clinical Implications

Diagnostic Challenges

• Gram-variable organisms can lead to misidentification and diagnostic errors
• In blood cultures, gram-variable organisms contributed to 0.7% of misinterpretations 4
• Misidentification can lead to inappropriate initial antibiotic selection

Examples of Clinically Important Gram-Variable Organisms

• Clostridioides difficile: An anaerobic, spore-forming organism that is technically gram-positive but may appear gram-variable 5
• Various Clostridium species: May appear gram-variable despite being phylogenetically gram-positive 2
• Some Bacillus species: Can appear gram-negative in later growth phases 2

Supplemental Testing

• The potassium hydroxide (KOH) test can help distinguish gram-positive from gram-negative anaerobes when Gram staining is ambiguous 6
• Antibiotic susceptibility patterns (vancomycin and colistin) can also help correctly classify gram-variable organisms 6

Common Pitfalls in Interpretation

• Failure to recognize that some organisms naturally display gram variability
• Misinterpreting gram-variable organisms as contamination or mixed cultures
• Not considering the effect of antimicrobial therapy on Gram stain results
• Overlooking the growth phase of the organism when interpreting Gram stain results

Best Practices for Handling Gram-Variable Organisms

• Consider the clinical context when interpreting Gram stain results
• Be aware of commonly gram-variable organisms in specific clinical scenarios
• Use supplementary tests when Gram stain results are ambiguous
• Recognize that some organisms (like Actinomyces, Arthrobacter, Bacillus, and Clostridium species) are inherently prone to gram variability 2
• Consider the impact of prior antimicrobial therapy on Gram stain interpretation 3

Understanding gram variability is essential for accurate microbiological diagnosis and appropriate antimicrobial therapy selection in clinical practice.