Determination of the De Novo Minimum Selection Concentration of Trimethoprim In Vivo for Escherichia coli Using Galleria mellonella: A Pilot Study

We investigated whether the maximum residual levels of trimethoprim permitted in food (Acceptable Daily Intake-ADI) could select for de novo trimethoprim resistance in <i>Escherichia coli</i> in vivo. We designed chronic infection models of <i>E. coli</i> in <i>Galleria mellonella</i> and exposed them to sub-ADI doses of trimethoprim through a single-dosing regimen. The emergence of trimethoprim resistance was determined by isolating the target bacteria on selective agar plates, followed by species confirmation using MALDI-TOF mass spectrometry. The minimum inhibitory concentration (MIC) was assessed via the E-test to determine <i>E. coli</i> susceptibility to trimethoprim. Notably, exposure to as low as one-tenth of the ADI dose through a single-dosing regimen resulted in the selection of trimethoprim-resistant <i>E. coli</i>. Our findings indicate that trimethoprim doses ten-fold lower than the established ADI threshold could induce resistance to trimethoprim in <i>E. coli</i>. These results highlight the importance of considering antimicrobial resistance induction as a key factor when determining ADI levels in food.