Effect of Zinc Oxide and Copper Sulfate on Antibiotic Resistance Plasmid Transfer in Escherichia coli

Abstract

Heavy metals such as zinc (Zn) and copper (Cu) may be associated with antibiotic resistance dissemination. Our aim was to investigate whether sub-lethal dosage of Zn and Cu may enhance plasmid transfer and subsequently resistance genes dissemination. Plasmid conjugation frequencies (PCF) were performed with Escherichia coli strains bearing IncL-bla_OXA-48, IncA/C-bla_CMY-2, IncI1-bla_CTX-M-1, IncF-bla_CTX-M-1, and IncX3-bla_NDM-5 as donors. Mating-out assays were performed with sub-dosages of zinc oxide (ZnO) and Cu sulfate (CuSO₄). Quantification of the SOS response-associated gene expression levels and of the production of reactive oxygen species were determined. Increased PCF was observed for IncL, IncA/C, and IncX3 when treated with ZnO. PCF was only increased for IncL when treated with CuSO₄. The ROS production presented an overall positive correlation with PCF after treatment with ZnO for IncL, IncA/C, and IncX3. For CuSO₄ treatment, the same was observed only for IncL. No increase was observed for expression of SOS response-associated genes under CuSO₄ treatment, and under ZnO treatment, we observed an increase in SOS response-associated genes only for IncX3. Our data showed that sub-dosages of ZnO and CuSO₄ could significantly enhance PCF in E. coli, with a more marked effect observed with IncL, IncA/C, and IncX3 scaffolds. Our study suggested that use of certain heavy metals is not the panacea for avoiding use of antibiotics in order to prevent the dissemination of antibiotic resistance.

Keywords: Escherichia coli; One Health; copper; plasmid transfer; sub-dosage; zinc oxide.

Figure 1

Reactive species of oxygen (ROS) experiment by fluorimetry. (A) ROS production for five E. coli isolates carrying distinct plasmids after treated with sub-inhibitory concentration of zinc oxide (ZnO); (B) ROS production for the E. coli isolates carrying distinct plasmids after treated with sub inhibitory concentration of copper sulphate (CuSO₄). IF/OD, intensity of fluorescence/optical density; CTRL, control (no treated); ZnO (IncL and IncI1, 512 µg/mL; IncA/C, IncF, and IncX3, 128 µg/mL); CuSO₄ (all plasmids, 512 µg/mL). Data are presented in means and standard deviations. *, p < 0.05 (*, against the respective control); **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.