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

Tingru Chang,Inger Odnevall,Peter Hedström,Weijie Zhao,C Magnus Johnson,Christofer Leygraf,R Prasath Babu

doi:10.1021/acsami.1c11236

Tingru Chang, Inger Odnevall + Show 5 more

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https://doi.org/10.1021/acsami.1c11236

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Abstract

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).

Highlights

IntroductionThe antimicrobial functionality of metals and alloys has gained increasing interest due to the emergence of antibiotic-resistant bacterial and virus strains that threaten vital health and result in economic challenges.[1−3] Recent findings have highlighted the relatively rapid inactivation of SARS-CoV-2 on copper (Cu) and Cu-coated surfaces.[4−6] Owing to the desirable antimicrobial efficiency, Cu metal and Cu-based alloys surfaces are increasingly used as promising high-touch surfaces for hygienic applications to reduce the occurrence of healthcareassociated infections.[7−10] The ability of Cu metal and Cubased alloys to inactivate or kill 99.9% pathogenic bacteria within 2 h has been certified by the U.S Environmental Protection Agency (EPA).[11] As a result of the considerable reduction in the number of bacteria, the concept of “contact killing” induced by Cu and Cu-based alloys has been introduced
The mechanism of bacterial contact killing induced by copper was explored through high-resolution studies by means of FIBSEM, TEM, scanning transmission electron microscope (STEM), and nano-FTIR microscopy of individual bacteria of B. subtilis in direct contact with Cu metal and Cu5Zn5Al1Sn surfaces
The following conclusions could be drawn of relevance for contact killing: (1) Early stages of interaction between individual bacteria and the metal/alloy substrate include cell leakage of extracellular polymeric substances (EPS) from the bacterium and changes in matrix surface composition upon adherence of bacteria

Summary

Introduction

The antimicrobial functionality of metals and alloys has gained increasing interest due to the emergence of antibiotic-resistant bacterial and virus strains that threaten vital health and result in economic challenges.[1−3] Recent findings have highlighted the relatively rapid inactivation of SARS-CoV-2 on copper (Cu) and Cu-coated surfaces.[4−6] Owing to the desirable antimicrobial efficiency, Cu metal and Cu-based alloys surfaces are increasingly used as promising high-touch surfaces for hygienic applications to reduce the occurrence of healthcareassociated infections.[7−10] The ability of Cu metal and Cubased alloys to inactivate or kill 99.9% pathogenic bacteria within 2 h has been certified by the U.S Environmental Protection Agency (EPA).[11] As a result of the considerable reduction in the number of bacteria, the concept of “contact killing” induced by Cu and Cu-based alloys has been introduced It refers to lethal bacterial damage in contact with the metallic surface.[12,13] Even though the underlying main mechanism is not yet established, contact killing has been largely related to the release of copper ions from Cu-based surfaces.[14−17] Reported mechanisms for the antimicrobial efficiency of Cu involve cell membrane damage, protein inactivation, decay of DNA function, and suppression of respiration.[7,18]. A more detailed state-ofthe-art characterization of the bioinorganic interface between bacteria and Cu/Cu5Zn5Al1Sn surfaces is still lacking

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