What is the impact of diluting antibiotics on their effectiveness and sensitivities?

Understanding Antibiotic Dilution in Susceptibility Testing

Dilution of antibiotics in laboratory susceptibility testing is a standardized technical process that creates a concentration gradient to determine the minimum inhibitory concentration (MIC), and has no direct impact on clinical effectiveness—the dilution occurs only in the laboratory setting, not in patient treatment. 1, 2

Laboratory Context: What Dilution Actually Means

The term "dilution" in antimicrobial susceptibility testing refers specifically to the laboratory methodology used to determine bacterial susceptibility, not to any clinical practice of diluting antibiotics before administration 1:

Technical Process of Dilution Testing

• Stock solutions are prepared at concentrations ≥1000 mg/L, then systematically diluted in geometric progressions (typically doubling dilutions) to create a range of concentrations 1, 2
• The dilution series is mixed with molten agar (19 mL added to each concentration) or broth containing a standardized bacterial inoculum 1
• This creates multiple test concentrations spanning from very low to very high antibiotic levels 1, 2

Purpose of the Dilution Method

• Determines the MIC: The lowest concentration that inhibits visible bacterial growth, which is the gold standard for reporting susceptibility 1
• Provides reproducible results across different laboratories when standardized protocols are followed 2
• Allows quality control by testing multiple concentration points rather than a single threshold 2

Clinical Interpretation: Impact on Sensitivities

What "Sensitive" Actually Means

When a laboratory reports a bacterium as "sensitive" or "resistant" to an antibiotic, this reflects:

• The MIC value determined through the dilution testing process 1
• Comparison of that MIC to established breakpoints that predict clinical success or failure 1
• The MIC represents the concentration between the reported value and the next lower concentration tested 2

Critical Caveat About Sub-MIC Concentrations

A major pitfall in clinical practice is assuming that antibiotic concentrations below the MIC are harmless—research demonstrates that even concentrations several hundred-fold below the MIC can select for resistant bacteria 3:

• Sub-inhibitory antibiotic concentrations in the environment or inadequate dosing can enrich resistant bacterial populations 3, 4
• This occurs through the mutant selection window, where concentrations are too low to kill all bacteria but high enough to provide selective pressure 4, 5
• De novo resistant mutants can be selected at sub-MIC concentrations 3

Clinical Application: Dosing Implications

Pharmacodynamic Principles Matter More Than Laboratory Dilutions

The effectiveness of antibiotics in patients depends on achieving appropriate concentrations at the infection site, not on the laboratory dilution process 5, 6:

• Concentration-dependent antibiotics (fluoroquinolones, aminoglycosides): Efficacy correlates with peak concentration/MIC ratio and area under the curve/MIC ratio—administer high doses less frequently 6
• Time-dependent antibiotics (beta-lactams): Efficacy correlates with the percentage of time serum concentrations exceed the MIC—administer more frequently or use continuous infusion 6
• Inadequately low dosing may contribute to resistance development by creating sub-optimal concentrations 5

Practical Dosing Strategy

Maximize antibiotic exposure by administering the highest recommended dose to ensure concentrations exceed not just the MIC of the isolated pathogen, but also the most resistant subpopulation in the colony 5:

• This prevents inadvertent selection pressure that favors resistant organisms 5
• For critically ill patients or those in septic shock, consider extended or continuous infusions of time-dependent antibiotics 7

Common Misunderstandings to Avoid

• Laboratory dilution ≠ Clinical dilution: The dilution process in susceptibility testing does not mean antibiotics should be diluted before patient administration 1, 2
• Stability concerns: Some antibiotics (particularly carbapenems and clavulanic acid) are unstable after reconstitution and cannot be stored—this is a pharmaceutical property, not related to the dilution testing method 2, 7
• MIC is not a single point: The reported MIC represents a range between tested concentrations due to the geometric dilution series used 2