In recent years several studies in laboratory settings and in hospital environments have demonstrated that surfaces of massive metallic copper have intrinsic antibacterial and antiviral properties. Microbes are rapidly inactivated by a quick, sharp shock known as contact killing. The underlying mechanism is not yet fully understood; however, in this process the cytoplasmic membrane is severely damaged. Pathogenic bacterial and viral high-consequence species able to evade the host immune system are among the most serious lethal microbial challenges to human health. Here, we investigated contact-killing mediated by copper surfaces of Gram-negative bacteria (Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis tularensis and Yersinia pestis) and of Gram-positive endospore-forming Bacillus anthracis. Additionally, we also tested inactivation of monkeypox virus and vaccinia virus on copper. This group of pathogens comprises biothreat species (or their close relatives) classified by the Center for Disease and Control and Prevention (CDC) as microbial select agents posing severe threats to public health and having the potential to be deliberately released. All agents were rapidly inactivated on copper between 30 s and 5 min with the exception of B. anthracis endospores. For vegetative bacterial cells prolonged contact to metallic copper resulted in the destruction of cell structure.

Discrimination of highly pathogenic bacteria, such as , from closely related species based on molecular biological methods is challenging. We applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to a collection of strains and close relatives in order to significantly improve the statistical confidence of identification results for this group of bacteria. Protein mass spectra of 189 verified and diverse strains of the group were generated using MALDI-TOF MS and subsequently analyzed with supervised and unsupervised statistical methods, such as shrinkage discriminant analysis (SDA) and principal-component analysis (PCA). We aimed at identifying specific biomarkers in the protein spectra of not present in closely related species. We could identify 7, 10, 18, and 14 -specific biomarker candidates that were absent in, ,, and strains, respectively. Main spectra (MSP) of a defined collection of strains were compiled using the Bruker Biotyper software and added to an in-house reference library. Reevaluation of this library with 15 hitherto untested strains of and yielded improved score values. The strains were identified with score values between 2.33 and 2.55 using the in-house database, while the same strains were identified with scores between 1.94 and 2.37 using the commercial database, and no false-positive identifications occurred using the in-house database.

Artificial laboratory evolution was used to produce mutant strains of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) able to survive on antimicrobial metallic copper surfaces. These mutants were 12- and 60-fold less susceptible to the copper-mediated contact-killing process than their respective parent strains. Growth of the mutant and its parent in complex growth medium was similar. Tolerance to copper ions of the mutants was unchanged. The mutant phenotype remained stable over about 250 generations under non-stress conditions. The mutants and their respective parental strains accumulated copper released from the metallic surfaces to a similar extent. Nevertheless, only the parental strains succumbed to copper stress when challenged on metallic copper surfaces, suffering complete destruction of the cell structure. Whole genome sequencing and global transcriptome analysis were used to decipher the genetic alterations in the mutant strains; however, these results did not explain the copper-tolerance phenotypes on the systemic level. Instead, the mutants shared features with those of stressed bacterial sub-populations entering the early or “shallow” persister state. In contrast to the canonical persister state, however, the ability to survive on solid copper surfaces was adopted by the majority of the mutant strain population. This indicated that application of solid copper surfaces in hospitals and elsewhere has to be accompanied by strict cleaning regimens to keep the copper surfaces active and prevent evolution of tolerant mutant strains. Significance Microbes are rapidly killed on solid copper surfaces by contact-killing. Copper surfaces thus have an important role to play in preventing the spread of nosocomial infections. Bacteria adapt to challenging natural and clinical environments through evolutionary processes, for instance by acquisition of beneficial spontaneous mutations. We wishes to address the question whether mutants can be selected that have evolved to survive contact-killing on solid copper surfaces. We isolated such mutants from E. coli and S. aureus MRSA by artificial laboratory evolution. The ability to survive on solid copper surfaces was a stable phenotype of the mutant population and not restricted to a small sub-population. As a consequence, standard operation procedures with strict hygienic measures are extremely important to prevent emergence and spread of copper-surface-tolerant persister-like bacterial strains if copper surfaces are to be sustainably used to limit the spread of pathogenic bacteria e.g., to curb nosocomial infections.