Antimicrobial resistance (AMR) has emerged as one of the most pressing threats to public health. AMR evolution occurs in the clinic but also in the environment, where antibiotics and heavy metals can select and co-select for AMR. While the selective potential of both antibiotics and metals is increasingly well-characterized, experimental studies exploring their combined effects on AMR evolution are rare. It has previously been demonstrated that fluoroquinolone antibiotics such as ciprofloxacin can chelate metal ions. To investigate how ciprofloxacin resistance is affected by the presence of metals, we quantified selection dynamics between a ciprofloxacin-susceptible and a ciprofloxacin-resistant Escherichia coli strain across a gradient of ciprofloxacin concentrations in presence and absence of zinc. The presence of zinc reduced growth of both strains, while ciprofloxacin inhibited exclusively the susceptible one. When present in combination zinc retained its inhibitory effect, while ciprofloxacin inhibition of the susceptible strain was reduced. Consequently, the minimal selective concentration for ciprofloxacin resistance increased up to five-fold in the presence of zinc. Environmental pollution usually comprises complex mixtures of antimicrobial agents. In addition to the usual focus on additive or synergistic interactions in complex selective mixtures, our findings highlight the importance of antagonistic selective interactions when considering resistance evolution.

Phone: (+44)7497497338 21 ORCID: 0000-0002-4169-6548 22 Graphical abstract 23 24One sentence summary 25The minimal selective concentration for a ciprofloxacin resistant E. coli strain increases up to 26 5-fold in the presence of Zinc cations. 27 3 Abstract 28 Antimicrobial resistance (AMR) has emerged as one of the most pressing global threats to 29 public health. AMR evolution occurs in the clinic but also in the environment, where low 30 concentrations of antibiotics and heavy metals can respectively select and co-select for 31 resistance. While the selective potential for AMR of both antibiotics and metals is 32 increasingly well-characterized, studies exploring the combined effect of both types of 33 selective agents are rare. It has previously been demonstrated that fluoroquinolone 34 antibiotics such as ciprofloxacin can chelate metal ions. To investigate how ciprofloxacin 35 resistance is affected by the presence of metals, we quantified selection dynamics between 36 a ciprofloxacin-susceptible and an isogenic ciprofloxacin-resistant Escherichia coli MG1655 37 strain across a gradient of ciprofloxacin concentrations in the presence and absence of Zinc 38 cations (Zn 2+ ). The minimal selective concentration (MSC) for ciprofloxacin resistance 39 significantly increased up to 5-fold in the presence of Zn 2+ . No such effect on the MSC was 40 found for gentamicin, an antibiotic not known to chelate zinc cations. Environmental pollution 41 usually consists of complex mixtures of antimicrobial agents. Our findings highlight the 42 importance of taking antagonistic as well as additive or synergistic interactions between 43 different chemical compounds into account when considering their effect on bacterial 44 resistance evolution. 45