Laboratory studies to investigate the efficacy and mechanism of action of copper alloys to kill a range of bacterial pathogens and inactivate norovirus

Warnes, Sarah Louise (2014) Laboratory studies to investigate the efficacy and mechanism of action of copper alloys to kill a range of bacterial pathogens and inactivate norovirus. University of Southampton, Biological Sciences, Doctoral Thesis, 156pp.

Record type: Thesis (Doctoral)

Abstract

Contamination of dry surfaces with infectious pathogens can have a significant role in infection spread, particularly if the pathogen is resistant to environmental stressors and the infectious dose is low. The use of antimicrobial surfaces in high risk clinical and community environments could help to reduce cross contamination. Although copper alloys have been known to have medicinal properties for centuries it is only relatively recently that laboratory studies have demonstrated that alloys containing over 60% copper have antimicrobial properties.

This study encompasses work done 2008-2013 which has continued to investigate this premise, looking at efficacy of copper and copper alloys to kill newly emerging pathogens which are proving to be a significant risk to global healthcare and also determining the mechanism of pathogen destruction on copper surfaces. Stainless steel which is ubiquitous, partly because of resistance to corrosion, was used as a control surface throughout.

Initial work demonstrated that clinical isolates of vancomycin-resistant enterococci were rapidly killed on copper alloy surfaces within a few minutes to 2 hours dependant on the copper content of alloy, size of inoculum or aqueous content of the contamination (mimicking either wet droplet or dry fingertip touch contamination of fomites). In contrast, enterococci persisted on stainless steel for several months. Following increasing concerns about the emergence of infections caused by Gram-negative pathogenic bacteria these studies identified a rapid kill on copper alloys but not stainless steel of food-borne pathogens Escherichia coli O157 and Salmonella, and also multidrug-resistant E. coli and Klebsiella pneumoniae containing the β-lactamase genes bla_CTX-M-15 and bla_NDM-1, respectively (which are responsible for a wide range of community and hospital acquired infections worldwide with diminishing effective therapies).

Further studies identified that release of Cu(I) and Cu(II) ionic species was requisite for copper surface antibacterial toxicity but significant differences in killing mechanism was observed between Gram-positive and Gram-negative bacteria related to their structural dissimilarities. Exposure to copper alloys inhibited respiration in all bacteria tested. Bacterial genomic and plasmid DNA was rapidly destroyed in Gram-positive cells but the cell membrane was not compromised immediately; however in Gram-negative cells the inner cell membrane was immediately depolarised on contact with copper alloys but the DNA breakdown occurred more slowly. The outer membrane of Gram-negative bacteria remained intact upon initial contact with copper surfaces. Reactive oxygen species (ROS) are also generated so that in effect the bacteria ‘commit metabolic suicide’ on copper surfaces: hydroxyl radicals generated by Gram-negative bacteria suggested a role for a Fenton reaction although the importance varied between species. In enterococci short term production of superoxide was the principle ROS. The nucleic acid destruction observed in all bacteria tested could prevent the horizontal transfer of antibiotic resistance or virulence genes and allay concerns about the possibility of developing resistance to copper. This was supported when it was determined that transfer of β-lactamase genes from E. coli ST131 and K. pneumoniae to recipient E. coli did occur on stainless steel but not on copper dry surfaces and that transfer was immediate in the former. The incidence of carbapenemase gene bla_NDM-1 transfer increased with time on stainless steel, highlighting concerns that persistence of viable cells not only poses an infection risk but also that contamination of the environment with intact DNA also increases the risk of gene transfer. These results support the use of copper alloys as biocidal surfaces to kill pathogenic bacteria and prevent horizontal gene transfer (HGT).

The final investigation determined that copper alloys were efficacious in inactivating murine norovirus, a close surrogate for human virus, and exposure to copper surfaces destroyed the RNA genome. Copper ions were still responsible directly or indirectly for the inactivation but ROS were not part of the toxicity mechanism.

All the results suggest that copper alloys are effective at destroying a diverse range of pathogenic microorganisms although the mechanisms may be different and multi-faceted. Exposure to copper alloy dry surfaces also prevented the horizontal transfer of genes conferring drug resistance and virulence which has been responsible for the continuing evolution of some of the world’s most dangerous pathogens. The results support the use of copper alloys as constantly killing surfaces in healthcare and community environments in conjunction with regular and efficient cleaning and decontamination regimes using non-chelating reagents that could inhibit the copper ion activity. Recent hospital trials now support this thesis.