Could traces of fluoroquinolones in food induce ciprofloxacin resistance in Escherichia coli and Klebsiella pneumoniae? An in vivo study in Galleria mellonella with important implications for maximum residue limits in food

Abstract

We hypothesized that the residual concentrations of fluoroquinolones allowed in food (acceptable daily intake-ADIs) could select for ciprofloxacin resistance in our resident microbiota. We developed models of chronic Escherichia coli and Klebsiella pneumoniae infection in Galleria mellonella larvae and exposed them to ADI doses of ciprofloxacin via single dosing and daily dosing regimens. The emergence of ciprofloxacin resistance was assessed via isolation of the target bacteria in selective agar plates. Exposure to as low as one-tenth of the ADI dose of the single and daily dosing regimens of ciprofloxacin resulted in the selection of ciprofloxacin resistance in K. pneumoniae but not E. coli. This resistance was associated with cross-resistance to doxycycline and ceftriaxone. Whole genome sequencing revealed inactivating mutations in the transcription repressors, ramR and rrf2, as well as mutations in gyrA and gyrB. We found that ciprofloxacin doses 10-fold lower than those classified as acceptable for daily intake could induce resistance to ciprofloxacin in K. pneumoniae. These results suggest that it would be prudent to include the induction of antimicrobial resistance as a significant criterion for determining ADIs and the associated maximum residue limits in food.IMPORTANCEThis study found that the concentrations of ciprofloxacin/enrofloxacin allowed in food can induce de novo ciprofloxacin resistance in Klebsiella pneumoniae. This suggests that it would be prudent to reconsider the criteria used to determine "safe" upper concentration limits in food.

Keywords: AMR; Escherichia coli; Klebsiella pneumoniae; MSC; antimicrobial consumption; minimum selection concentration; quinolone; resistance.

Fig 1

Schematic overview of study methodology (figure produced with BioRender).

Fig 2

Ciprofloxacin MICs of bacterial colonies of E. coli, K. pneumoniae M14827, and K. pneumoniae M17125 after exposure to ciprofloxacin (ADI dose, 0.1 × ADI dose, or control/PBS) taken from selective plates with ciprofloxacin (0.125 µg/mL). Mean and SEM shown.

Fig 3

Selection of cross-resistance. Ceftriaxone and doxycycline MICs of bacterial colonies of K. pneumoniae M14827 after daily exposure to ciprofloxacin (ADI and 0.1 × ADI dose combined) versus baseline strain. Mean and SEM shown.