High-Resolution Microscopical Studies of Contact Killing Mechanisms on Copper-Based Surfaces

The mechanisms of bacterial contact killing induced by Cu surfaces were explored through high-resolution studies based on combinations of the focused ion beam (FIB), scanning transmission electron microscopy (STEM), high-resolution TEM, and nanoscale Fourier transform infrared spectroscopy (nano-FTIR) microscopy of individual bacterial cells of Gram-positive Bacillus subtilis in direct contact with Cu metal and Cu5Zn5Al1Sn surfaces after high-touch corrosion conditions. This approach permitted subcellular information to be extracted from the bioinorganic interface between a single bacterium and Cu/Cu5Zn5Al1Sn surfaces after complete contact killing. Early stages of interaction between individual bacteria and the metal/alloy surfaces include cell leakage of extracellular polymeric substances (EPSs) from the bacterium and changes in the metal/alloy surface composition upon adherence of bacteria. Three key observations responsible for Cu-induced contact killing include cell membrane damage, formation of nanosized copper-containing particles within the bacteria cell, and intracellular copper redox reactions. Direct evidence of cell membrane damage was observed upon contact with both Cu metal and Cu5Zn5Al1Sn surfaces. Cell membrane damage permits copper to enter into the cell interior through two possible routes, as small fragmentized Cu₂O particles from the corrosion product layer and/or as released copper ions. This results in the presence of intracellular copper oxide nanoparticles inside the cell. The nanosized particles consist primarily of CuO with smaller amounts of Cu₂O. The existence of two oxidation states of copper suggests that intracellular redox reactions play an important role. The nanoparticle formation can be regarded as a detoxification process of copper, which immobilizes copper ions via transformation processes within the bacteria into poorly soluble or even insoluble nanosized Cu structures. Similarly, the formation of primarily Cu(II) oxide nanoparticles could be a possible way for the bacteria to deactivate the toxic effects induced by copper ions via conversion of Cu(I) to Cu(II).