Cu Nanoparticles Have Different Impacts in Escherichia coli and Lactobacillus brevis than Their Microsized and Ionic Analogues

Abstract

Copper formulations have been used for decades for antimicrobial and antifouling applications. With the development of nano-formulations of copper that are more effective than their ionic and micron-sized analogs, a key regulatory question is whether these materials should be treated as new or existing materials. To address this issue, here we compare the magnitude and mechanisms of toxicity of a series of Cu species (at concentration ranging from 2–250 µg/mL), including nano Cu, nano CuO, nano Cu(OH)2 (CuPro and Kocide), micro Cu, micro CuO, ionic Cu2+ (CuCl2 and CuSO4) in two species of bacteria (Escherichia coli and Lactobacillus brevis). The primary size of the particles studied ranged from 10 nm to 10 µm. Our results reveal that Cu and CuO nanoparticles (NPs) are more toxic than their micron-sized counterparts at the same Cu concentration, with toxicities approaching that of the ionic Cu species. Strikingly, these NPs showed distinct differences in their mode of toxicity when compared to the ionic and micron-sized Cu, highlighting the unique toxicity properties of materials at the nanoscale. In vitro DNA damage assays reveal that both nano Cu and micron-sized Cu are capable of causing complete degradation of plasmid DNA but electron tomography results show that only nanoformulations of Cu are internalized as intact intracellular particles. These studies suggest that nano Cu at the concentration of 50 µg/mL may have unique genotoxicity in bacteria compared to ionic and micron-sized Cu.

Comparative environmental fate and toxicity of copper nanomaterials

“…Although L. brevis showed similar trends, there was a stronger response to the Cu NPs and significant (p < 0.05) membrane damage from exposure to both nCu(OH) 2 and the copper salts (Kaweeteerawat et al, 2015). Exposure to Cu NPs resulted in oxidative stress (measured by biotic reactive oxygen species (ROS) generation) for E. coli and L. brevis, but there was no effect from CuCl 2 , CuSO 4 , μCu or μCuO at the concentrations studied ([Cu] up to 250 mg/L) (Kaweeteerawat et al, 2015). These results are in line with previous work indicating that exposure to Cu NPs caused cellular oxidative stress in bacterial cells (Li et al, 2013), yeast cells (Kasemets et al, 2013), and mammalian cells (Liu et al, 2014).…”

“…Commercial Cu nanopesticides were less toxic than nCu and nCuO, and toxicity was lowest for micron-scale Cu particles. Sub-lethal effect assays qualitatively measuring membrane potential, membrane damage, cellular ROS generation, and electron transport activity revealed that Cu ions and nanosized Cu resulted in significant (p < 0.05) membrane damage and a decrease in electron transport activity in E. coli, while micron sized particles had no effect (Kaweeteerawat et al, 2015). Although L. brevis showed similar trends, there was a stronger response to the Cu NPs and significant (p < 0.05) membrane damage from exposure to both nCu(OH) 2 and the copper salts (Kaweeteerawat et al, 2015).…”

“…Sub-lethal effect assays qualitatively measuring membrane potential, membrane damage, cellular ROS generation, and electron transport activity revealed that Cu ions and nanosized Cu resulted in significant (p < 0.05) membrane damage and a decrease in electron transport activity in E. coli, while micron sized particles had no effect (Kaweeteerawat et al, 2015). Although L. brevis showed similar trends, there was a stronger response to the Cu NPs and significant (p < 0.05) membrane damage from exposure to both nCu(OH) 2 and the copper salts (Kaweeteerawat et al, 2015). Exposure to Cu NPs resulted in oxidative stress (measured by biotic reactive oxygen species (ROS) generation) for E. coli and L. brevis, but there was no effect from CuCl 2 , CuSO 4 , μCu or μCuO at the concentrations studied ([Cu] up to 250 mg/L) (Kaweeteerawat et al, 2015).…”

“…Bacterial HTS growth inhibition assays performed on Escherichia coli dispersed in minimal medium ranked their toxicity (Cu concentration yielding 50% inhibition, in mg/L) as CuCl 2 (38 ± 8) > nCu (120 ± 14) = CuSO 4 (140 ± 23) = nCuO (160 ± 17) > nCu (OH) 2 -a, nCu(OH) 2 -b, μCu, μCuO (all > 250) (Kaweeteerawat et al, 2015). Similar assays performed on Lactobacillus brevis dispersed in Lactobacilli MRS broth ranked the various Cu species (in mg/L) as nCuO (3.6 ± 0.1) = nCu(OH) 2 -a (4.0 ± 0.1) > nCu (5.7 ± 0.2) = nCu(OH) 2 -b (6.2 ± 0.9) > CuCl 2 (7.8 ± 0.5) > μCu = μCuO (≥ 120) (Kaweeteerawat et al, 2015).…”

“…Similar assays performed on Lactobacillus brevis dispersed in Lactobacilli MRS broth ranked the various Cu species (in mg/L) as nCuO (3.6 ± 0.1) = nCu(OH) 2 -a (4.0 ± 0.1) > nCu (5.7 ± 0.2) = nCu(OH) 2 -b (6.2 ± 0.9) > CuCl 2 (7.8 ± 0.5) > μCu = μCuO (≥ 120) (Kaweeteerawat et al, 2015). Aggregation of the particles in the different media was significant (p < 0.05), with the nanoscale particles ranging from 250 to 1600 nm, and the microscale particles from 1300 to 1500 nm.…”