Common non-antibiotic drugs enhance selection for antimicrobial resistance in mixture with ciprofloxacin

Antimicrobial resistance (AMR) is a major health concern, and a range of antibiotic and non-antibiotic agents can select for AMR across a range of concentrations. Selection for AMR is often investigated using single compounds, however, in the natural environment and the human body, pharmaceuticals will be present as mixtures, including both non-antibiotic drugs (NADs), and antibiotics. Here, we assessed the effects of one of three NADs in combination with ciprofloxacin, a commonly used antibiotic that is often found at concentrations in global freshwaters sufficiently high to select for AMR. We used a combination of growth assays and qPCR to determine selective concentrations of mixtures and used metagenome sequencing to identify changes to the resistome and community composition. The selective concentration of ciprofloxacin was reduced from 40{micro}g/L to 10{micro}g/L in the mixtures, and mixtures showed a stronger selection for some AMR genes such as tolC and qnrB. The communities exposed to the mixtures also showed changed community compositions. These results demonstrate that NADs and ciprofloxacin are more selective than ciprofloxacin alone, and these mixtures can cause distinct changes to the community composition. This indicates that future work should consider combinations of antibiotics and NADs as drivers of AMR when considering its maintenance and acquisition.