In-vitro methods for determining minimal lethal concentrations of antimicrobial agents

Area of Science:

• Microbiology
• Pharmacology
• Clinical Diagnostics

Background:

• Antimicrobial susceptibility testing (AST) is crucial for guiding treatment decisions.
• Broth dilution methods determine the minimal lethal concentration (MLC) of antimicrobial agents.
• Accurate determination of MLC requires reliable subculturing techniques.

Purpose of the Study:

• To investigate the impact of antimicrobial drug carryover on subculture results in broth dilution tests.
• To identify conditions that lead to false-negative outcomes in determining minimal lethal concentration.
• To evaluate methods for improving the reliability of subculturing small sample volumes.

Main Methods:

• Broth dilution tests were performed to establish antimicrobial concentrations.
• Samples from tubes with no visible growth were subcultured onto drug-free agar.
• Viable cell counts were used to determine the concentration of drug killing 99.9% of cells.
• The effect of sample volume and spreading technique on drug carryover was assessed.
• Precision of four subculturing methods was evaluated using coefficients of variation.

Main Results:

• Drug carryover significantly reduced viable cell recovery, especially with larger sample volumes.
• The impact of drug carryover was minimal for sample volumes of 10 microliters or less.
• Spreading the sample over the agar surface mitigated the effect of drug carryover.
• Coefficients of variation for subculturing methods ranged from 16% to 25%.

Conclusions:

• False-negative results in antimicrobial testing can arise from drug carryover during subculturing.
• Optimizing sample volume and spreading technique enhances the accuracy of minimal lethal concentration determination.
• Awareness of sampling errors and use of replicate subcultures are essential for reliable AST results.