Although widespread antibiotic resistance has been mostly attributed to the selective pressure generated by overuse and misuse of antibiotics, recent growing evidence suggests that chemicals other than antibiotics, such as certain metals, can also select and stimulate antibiotic resistance via both co-resistance and cross-resistance mechanisms. For instance, tetL, merE, and oprD genes are resistant to both antibiotics and metals. However, the potential de novo resistance induced by heavy metals at environmentally-relevant low concentrations (much below theminimum inhibitory concentrations [MICs], also referred as sub-inhibitory) has hardly been explored. This study investigated and revealed that heavy metals, namely Cu(II), Ag(I), Cr(VI), and Zn(II), at environmentally-relevant and sub-inhibitory concentrations, promoted conjugative transfer of antibiotic resistance genes (ARGs) between E. coli strains. The mechanisms of this phenomenon were further explored, which involved intracellular reactive oxygen species (ROS) formation, SOS response, increased cell membrane permeability, and altered expression of conjugation-relevant genes. These findings suggest that sub-inhibitory levels of heavy metals that widely present in various environments contribute to the resistance phenomena via facilitating horizontal transfer of ARGs. This study provides evidence from multiple aspects implicating the ecological effect of low levels of heavy metals on antibiotic resistance dissemination and highlights the urgency of strengthening efficacious policy and technology to control metal pollutants in the environments.

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The widespread use of nanoparticles has triggered increasing concern and interest due to the adverse effects on global public health and environmental safety. Whether the presence of nano-metal oxides (NMOs) could facilitate the formation of new antimicrobial resistance genes (ARGs) via de novo mutation is largely unknown. Here, we proved that two widely used NMOs could significantly improve the mutation frequencies of CIP- and CHL-resistant E. coli isolates; however, the corresponding metal ions have weaker effects. Distinct concentration-dependent increases of 1.0–14.2 and 1.1–456.3 folds were observed in the resistance mutations after treatment with 0.16–100 mg/L nano-Al2O3 and 0.16–500 mg/L nano-ZnO, respectively, compared with those in the control. The resistant mutants showed resistance to multiple antibiotics and hereditary stability after sub-culturing for 5 days. We also explored the mechanism underlying the induction of antimicrobial resistance by NMOs. Whole-genome sequencing analysis showed that the mutated genes correlated with mono- and multidrug resistance, as well as undetected resistance to antibiotics. Furthermore, NMOs significantly promoted intracellular reactive oxygen species (ROS), which would lead to oxidative DNA damage and an error-prone SOS response, and consequently, mutation rates were enhanced. Our findings indicate that NMOs could accelerate the mutagenesis of multiple-antibiotic resistance and expanded the understanding of the mechanisms in nanoparticle-induced resistance, which may be significant for guiding the production and application of nanoparticles.

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