Contact killing of Escherichia coli K12 and Staphylococcus cohnii on copper containing and alloyed materials

Kurzfassung

Maintaining human health during long-term space missions is of major concern at any time. It is well known that space missions influence the immune system of the explorers and that they become more susceptible to bacterial infections. At the same time, bacteria exposed to the space environment have been shown to get more aggressive and more resistant to antibiotics. This particular combination can lead to severe infections and may jeopardise the mission; therefore a strong and uncompromising strategy inhibiting the propagation of bacteria is urgently needed. To achieve this, antimicrobial surfaces may just offer the answer. They provide a promising alternative to conventional cleaning procedures with e.g. disinfectants and contain organic/inorganic substituents or antimicrobial metals such as copper or silver. Copper is an essential metal but toxic at higher concentrations. On the contrary, silver is not essential and therefore toxic even at low concentrations. Due to their antimicrobial properties, both metals were used for medical issues and the storage of drinking water since thousands of years. While the toxicity is well known, the exact mechanisms of the toxicity are still unclear. In this study the antimicrobial effect of pure copper and new, potentially antimicrobial eutectic alloys composed of aluminium, silver and/or copper was investigated under wet contact killing conditions. First, the survival of Gram-positive and Gram-negative cells after contact with antimicrobial materials was examined. Additionally, intracellular reactive oxygen species (ROS) production and damages of the membrane, DNA and RNA were determined for copper exposed samples. ROS production and membrane damages increased rapidly within one hour while the effect on cell survival was negligible even after two hours of exposure. Only longer exposure of up to four hours led to a rapid decrease in cell survival depending on the concentration of exposed cells/cm². Further investigations were conducted with selected strains of the Keio collection. This collection is composed of E. coli strains which exhibit knockouts in one gene whereby each deleted gene was exchanged by a kanamycin cassette. To determine metabolic influences of pure copper and eutectic alloy exposed samples, 21 strains which had either a direct link to copper and/or silver transport mechanisms or a general function in the microorganism were selected. After pre-testing, different survivability’s of E. coli ΔcopA, ΔrecA, ΔcutA, ΔcueR and ΔcueO were obtained. The survival was decreased in absence of CueO compared to ΔcopA strains when exposed to pure copper surfaces. In contrast to this, the survivability was vice versa when exposed to silver containing alloys whereas CueO had no function in silver oxidization. Finally, the release of metal ions and the cell associated metal concentration was determined by taking the example of Staphylococcus cohnii, Escherichia coli K12 and ΔcopA to identify possible connections between cell associated ions and the survival of cells. These measurements indicated that the higher release of ions after exposure of cells in buffer is caused by an indirect dissolution. Despite a relatively high cell associated ion concentration, the experiments in the current study (with ex vivo and in vivo DNA/RNA and the above mentioned Keio strains) pointed out that these ions were possibly primarily present in the periplasm. Additionally, investigations on ternary, eutectic alloys were conducted. They showed that special casting protocols need to be in progress to obtain antimicrobial effects. Due to the antimicrobial effect and light weight, these materials could be a great opportunity to be applied in hospitals or in spacecraft facilities.