Copper-based fungicide copper hydroxide accelerates the evolution of antibiotic resistance via gene mutations in Escherichia coli

The extensive use of copper-based fungicides in orchards, especially in vineyards, leads to the accumulation of copper, which has caused growing concern. However, data on the acquisition of antibiotic resistance in opportunistic pathogens under copper-based fungicides are scarce. In this study, we investigated the potential development of antibiotic resistance in Escherichia coli K12 under selective copper hydroxide pressure. The results indicated that copper hydroxide at concentrations of 100 mg/L and 200 mg/L evolved resistance against chloramphenicol and tolerance against tetracycline to 4-8 and 2.00-2.67 times than the initial minimal inhibitory concentrations (MICs), respectively. Whole-genome sequencing analysis showed that the obtained resistant strains carried gene mutations including AcrAB-TolC multidrug efflux pump (acrB and marR), outer membrane porin (evZ), and another indirect pathways. Furthermore, the expression of multidrug efflux pump genes and oxidative stress-related genes were significantly upregulated, whereas outer membrane porin genes were downregulated. Thus, our results could well explain the emergence of antibiotic resistance and resistance mechanisms selected by copper-based fungicide, and provide a basis for the management of copper-based fungicide in agriculture to avoid the spread of antibiotic resistance.